RU2805085C2 - Target genes for targeted impact on nitrogen fixation to improve plant quality - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and agriculture and is a genetically engineered diazotrophic bacterium capable of increasing the amount of atmospheric nitrogen in a plant, having increased rpoN expression, and rpoN expression is increased by deleting the native rpoN promoter and replacing it with a constitutive promoter.

EFFECT: invention allows to increase the amount of nitrogen obtained from the atmosphere in a plant by bringing the plant into contact with a plurality of the said genetically engineered diazotrophic bacteria.

21 cl, 41 dwg, 8 tbl, 28 ex

Description

CROSS REFERENCE

[0001] This application claims benefit from U.S. Provisional Patent Application No. 62/577149, filed October 25, 2017, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE ART

[0002] Plants are connected to the microbiome through a shared metabolome. The multidimensional relationship between a given crop quality and the corresponding metabolome is characterized by a landscape with many local maxima. Optimization from a lower local maximum to another representing better quality by changing the influence of the microbiome on the metabolome may be desirable for a number of reasons, such as crop optimization. Meeting the needs of a growing global population requires economically, environmentally and socially sustainable approaches to agriculture and food production. The UN's Food and Agriculture Organization predicts that total food production will need to increase by 70% by 2050 to meet the needs of a growing population - a challenge compounded by multiple factors, including dwindling freshwater resources, increased competition for arable land, and rising prices. on energy, rising raw material costs and the likely need for crops to adapt to the impacts of a drier, hotter and more extreme global climate.

[0003] One area of interest is improving nitrogen fixation. Nitrogen gas (N ₂ ) is the main component of the Earth's atmosphere. In addition, elemental nitrogen (N) is an important component of many chemical compounds that make up living organisms. However, many organisms cannot directly use _N2 to synthesize chemicals used in physiological processes such as growth and reproduction. To be used _{, N2} must be combined with hydrogen. The combination of hydrogen with _N2 is called nitrogen fixation. Nitrogen fixation, regardless of whether it is carried out chemically or biologically, requires large amounts of energy. In biological systems, an enzyme known as nitrogenase catalyzes the reaction that leads to nitrogen fixation. An important goal of nitrogen fixation research is to extend this phenotype to non-legume plants, especially the agriculturally important cereals such as wheat, rice and corn. Despite enormous progress in understanding the development of nitrogen-fixing symbiosis between nodule bacteria and legumes, the path to using this knowledge to induce nitrogen-fixing nodules on non-legume crops remains unclear. Meanwhile, the challenge of ensuring sufficient supply of additional nitrogen sources, such as fertilizers, will continue to grow with the growing need for increased food production.

BRIEF DESCRIPTION OF THE INVENTION

[0004] The present invention relates to a genetically engineered bacterium containing a modification in glnD, wherein said modification is selected from the group consisting of deletion of the entire gene, deletion of substantially the entire gene, deletion of the ACT domain, deletion of more than 50% of the ACT domain, inactivation of the ACT domain, and inactivation of the UTase domain. In some embodiments, the genetically engineered bacterium contains a modification in glnA resulting in a change in the expression or activity of glnA. In some embodiments, the modification results in decreased expression of glnA. In some embodiments, the modification includes replacing the native glnA promoter with a promoter with lower activity. In some embodiments, the modification includes replacing the native glnA promoter with a promoter selected from the group consisting of a glnB promoter, a glnD promoter, and a glnE promoter. In some embodiments, the genetically engineered bacterium contains a modification in glnA resulting in decreased expression or activity of glnA.

[0005] The present invention relates to a genetically engineered bacterium having altered expression of rpoN. In some embodiments, the genetically engineered bacterium has increased expression of rpoN. In some embodiments, the genetically engineered bacterium has increased expression of rpoN. In some embodiments, a genetically engineered bacterium according to any one of claims is provided. 1-7 of the claims, where said genetically engineered bacterium is a genetically engineered diazotrophic bacterium. In some embodiments, rpoN expression is increased by deleting the native rpoN promoter and replacing it with a strong constitutive promoter. In some embodiments, the genetically engineered bacterium is intergeneric. In some embodiments, the genetically engineered bacterium is capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen. In some embodiments, the genetically engineered bacterium contains a modification in the nitrogen fixation genetic network. In some embodiments, the genetically engineered bacterium contains a modification in NifA. In some embodiments, the genetically engineered bacterium contains a modification that results in increased expression of NifA. In some embodiments, the genetically engineered bacterium contains a modification in NifL. In some embodiments, the genetically engineered bacterium contains a modification that results in decreased expression of NifL. In some embodiments, the genetically engineered bacterium contains a modification in NifH. In some embodiments, the genetically engineered bacterium contains a modification that results in increased expression of NifH. In some embodiments, said genetically engineered bacterium contains a modification that results in increased expression of Nif cluster genes. In some embodiments, the genetically engineered bacterium contains a modification in the nitrogen assimilation genetic network. In some embodiments, the genetically engineered bacterium contains a modification in GlnE. In some embodiments, the genetically engineered bacterium contains a modification that results in decreased GlnE activity. In some embodiments, the genetically engineered bacterium contains a modification that results in decreased amtB activity.

[0006] The present invention relates to a method of increasing the amount of atmospheric nitrogen in a plant, comprising bringing said plant into contact with a variety of said genetically engineered bacteria described herein. In some embodiments, the genetically engineered bacterium is applied to a seed of said plant. In some embodiments, the genetically engineered bacterium is applied to a seedling of said plant. In some embodiments, the genetically engineered bacterium is applied to the plant in a liquid formulation. In some embodiments, the genetically engineered bacterium is applied to said plant after planting but before harvesting of said plant. In some embodiments, the genetically engineered bacterium is applied to the plant as a nutritional supplement. In some embodiments, the genetically engineered bacterium is applied to the plant between one month and eight months after germination. In some embodiments, the genetically engineered bacterium is applied to the plant between two months and eight months after germination. In some embodiments, the genetically engineered bacterium is applied to the plant between one month and three months after germination. In some embodiments, the genetically engineered bacterium is applied to the plant between three months and six months after germination. In some embodiments, the plant is a cereal plant. In some embodiments, the plant is a corn plant. In some embodiments, the plant is a rice plant. In some embodiments, the plant is a wheat plant. In some embodiments, the plant is a soybean plant.

[0007] The present invention relates to a composition containing a seed and a seed; wherein said seed coating contains a plurality of said genetically engineered bacteria as described herein. In some embodiments, the seed is a cereal seed. In some embodiments, the seed is selected from the group consisting of corn seed, wheat seed, rice seed, soybean seed, rye seed, and sorghum seed. The present invention relates to a composition containing a plant and a variety of specified genetically engineered bacteria described herein. In some embodiments, the plant is a seedling. In some embodiments, the seed is a cereal seed. In some embodiments, the seed is selected from the group consisting of corn, rice, wheat, soybean, rye, and sorghum.

INCLUSION BY REFERENCE

[0008] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent or patent application had been specifically and individually indicated as being incorporated by reference.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[0009] The new features of the invention are set out in detail in the accompanying claims. A more complete understanding of the features and advantages of the present invention will be obtained with reference to the following detailed description, which sets forth illustrative embodiments employing the principles of the invention and the accompanying drawings, wherein:

[0010] Figure 1A-B depicts the enrichment and isolation of nitrogen-fixing bacteria. (A) A plate of Nfb medium containing agar was used to isolate single colonies of nitrogen-fixing bacteria. (B) Semisolid Nfb agar medium poured into a Balch tube. The arrow indicates a film of enriched nitrogen-fixing bacteria.

[0011] Figure 2 depicts an exemplary PCR screen for nifH. In this screen, positive bands were observed at ~350 bp. for two colonies. The lower bands represent primer dimers.

[0012] Figure 3 depicts an example of PCR screening of colonies from CRISPR-Cas-selected mutagenesis. CI006 colonies were screened using primers specific for the nifL locus. The wild-type PCR product is expected at ∼2.2 kb, whereas the mutant is expected at ∼1.1 kb. Seven out of ten colonies screened clearly showed the desired deletion.

[0013] Figures 4A-D depict the in vitro phenotypes of the various strains. Activities of strain CI010 mutants (Figure 4A) and strain CI006 mutants (Figure 4B) grown in nitrogen-fixing medium supplemented with 0 to 10 mM glutamine in an acetylene reduction assay (ARA). The ARA activities of additional strains are shown in Figure 4C, and the ammonium excretion profile over time for the two strains is shown in Figure 4D.

[0014] Figure 5 shows the culture expression profile of 9 different genes in CI006 strains involved in diazotrophic nitrogen fixation. The numbers represent the abundance of each transcript. Various conditions are indicated (0, 1, 10 mM glutamine and 0%, 10%, 20% N ₂ in ambient air).

[0015] Figure 6 depicts CI006 colonization of corn roots. Maize seedlings were inoculated with CI006 carrying the RFP expression plasmid. After two weeks of growth and maintenance of the plasmid by watering with an appropriate antibiotic, roots were harvested and visualized using fluorescence microscopy. Colonization of the intercellular space of the root is observed.

[0016] Figure 7 depicts nitrogen produced at the microbial level in the wild type strain (CI050) and the optimized strain (CM002).

[0017] Figure 8 shows the experimental conditions for Micro-Tom fruit weight analysis.

[0018] Figure 9 shows the screening of 10 strains for increased fruit weight of the Micro-Tom plant. Results from six parallel analyzes are presented. For column 3 p=0.07. For column 7 p=0.05.

[0019] Figures 10A-C show additional results for the ARA activity of candidate microorganisms and corresponding candidate mutants grown in nitrogen-fixing media supplemented with 0 to 10 mM glutamine.

[0020] Figure 11 depicts a double mutant that exhibits higher ammonia excretion than the single mutant from which it was derived.

[0021] Figure 12 shows the NDFA value obtained from the 15N gas uptake experiment (extrapolated back based on days of exposure) to measure NDFA in fertilized corn plants.

[0022] Figure 13 shows the NDFA value obtained from the 15N gas uptake experiment (extrapolated back based on days of exposure) to measure NDFA in setaria plants in the fertilized state.

[0023] Figure 14A shows the degree of inclusion of 15N gas. Plants inoculated with the evolved strain showed an increase in 15N gas incorporation compared to non-inoculated plants.

[0024] Figure 14B shows that 4 weeks after planting, up to 7% of the nitrogen in plants inoculated with the evolved strain comes from microbially fixed nitrogen.

[0025] Figure 14C shows that leaf area (and other biomass measurements, data not shown) is increased in plants inoculated with the evolved strain compared to unnoculated or wild-type inoculated plants.

[0026] Figure 15A depicts evolved strains showing significantly higher nifH production in root tissue as determined by an in planta transcriptomic study.

[0027] Figure 15B shows that the level of fixed nitrogen found in plant tissue correlates with the extent to which that particular plant is colonized by the HoME optimized strain.

[0028] Figure 16A depicts a soil texture map for various field soils tested for colonization. The soils from which some microorganisms were originally obtained are indicated by asterisks.

[0029] Figure 16B shows the colonization rates of strain 1 and strain 5, which were tested on four different soil types (circles). Both strains showed a relatively consistent colonization profile in different soil types.

[0030] Figure 16C shows the colonization of strain 1 tested in field trials during the growing season. Strain 1 persists in corn tissue up to 12 weeks after planting and after this time begins to show decreased colonization.

[0031] Figure 17 depicts a diagram of microorganism selection in accordance with embodiments.

[0032] Figure 18 depicts an expanded view of the microbiome composition measurement as shown in Figure 17.

[0033] Figures 19A and 19B show increased nitrogenase activity and ammonium excretion in mutants of the K. sacchari strain CI006 with modifications in the glycogen synthesis gene glgA.

[0034] Figures 20A and 20B show increased nitrogenase activity and ammonium excretion in mutants of the K. variicola strain CI137 with modifications in the glycogen synthesis gene glgA compared to parental strains with identical genotypes but without the glgA modification.

[0035] Figure 21 illustrates a diagram of the glnE gene, showing regions corresponding to the functional domains of the GlnE protein.

[0036] Figure 22 shows increased ammonium excretion in minimal nitrogen-free media in K. variicola CI137 mutants with modifications in the glnD gene.

[0037] Figure 23 shows increased ammonium excretion in mutants of the K. sacchari strain CI006 with modifications in the glnD gene.

[0038] Figure 24A shows increased ammonium excretion in minimal nitrogen-free media in K. variicola CI137 mutants with modifications in the glnK gene.

[0039] Figure 24B shows increased ammonium excretion in mutants of K. variicola strain CI137 with modifications in the glnA gene created by inserting a promoter from a gene constitutively expressed at a low level (glnE, glnD or glnB) upstream of transcription, and mutation of the start codon ATG to GTG in a strain with the glnE_KO2 mutation in minimal medium supplemented with 5 mM glutamine.

[0040] Figure 25 shows increased ammonium excretion in mutants of K. variicola strain CI137 with modifications in the glnA gene created by inserting a promoter from a gene constitutively expressed at a low level (glnE, glnD or glnB) upstream of transcription, and mutation of the ATG start codon to GTG in a strain with the glnE_KO2 mutation and ΔnifL::Prm1.2 in minimal medium supplemented with 5 mM glutamine.

[0041] Figure 26 shows increased ammonium excretion in mutants of K. sacchari strain CI006 with modifications in the glnA gene created by inserting a promoter from a gene constitutively expressed at a low level (glnE, glnD or glnB) upstream of transcription, and mutation of the ATG start codon to GTG in a strain with the glnE_KO2 mutation and ΔnifL::Prm1.2 in nitrogen-free minimal medium.

[0042] Figure 27 shows increased ammonium excretion in mutants of K. variicola strain CI137 with modifications to the GOGAT-encoding operon (glnBD) created by insertion of the glnD promoter upstream of the operon in the parental strain 137-2084.

[0043] Figure 28 shows the increase in nitrogenase activity, as measured by ARA, in mutants of K. variicola strain CI137 with modifications in the GOGAT-encoding operon (glnBD) created by insertion of the glnD promoter upstream of the operon, compared to the parental strain 137-2084.

[0044] Figure 29 shows increased ammonium excretion in mutants of K. sacchari strain CI006 with modifications in the expression of the glutaminase enzyme glsA2, created by insertion of a strong promoter upstream of the glsA2 gene, compared to the parental control.

[0045] Figure 30 shows increased ammonium excretion in mutants of K. variicola strain CI137 with modifications in the expression of the glutaminase enzyme glsA2, created by insertion of a strong promoter upstream of the glsA2 gene, compared to the parental control.

[0046] Figure 31 shows increased ammonium excretion in mutants of the K. variicola strain CI137 with modifications in the asnB gene compared to the parental control strain.

[0047] Figure 32 shows the increase in nitrogenase activity in mutants of the K. variicola strain CI137 with rpoN modifications compared to the parental control.

[0048] Figure 33 shows increased nitrogen excretion in K. variicola CI137 mutants with rpoN modifications compared to the parental control.

[0049] Figure 34 shows an increase in nitrogenase activity in mutants of the K. sacchari strain CI006 with altered expression of the otsB gene compared to the parental control.

[0050] Figure 35 shows increased nitrogen excretion in mutants of the K. sacchari strain CI006 with altered expression of the otsB gene compared to the parental control.

[0051] Figures 36A and 36B show unaffected nitrogen excretion and increased colonization in K. variicola CI137 mutants with modifications in phoB compared to the parental control.

[0052] Figure 37 shows increased colonization in K. sacchari strain CI006 mutants with modifications in yjbE compared to the parental control.

[0053] Figures 38A and B show increased nitrogenase activity and nitrogen excretion in mutants of K. sacchari strain CI006 with modifications in the expression of the sulfate transport gene otsB, created by insertion of a promoter immediately upstream of the gene, compared to the parental strain.

[0054] Figures 39A and 39B show increased nitrogenase activity in mutants of K. sacchari strain CI006 with modifications in the expression of the sulfate transport gene cysZ, created by insertion of a promoter immediately upstream of the gene, compared to the parental strain.

[0055] Figures 40A and 40B show increased ammonium excretion in mutants of the K. variicola strain CI137 with modifications in dctA1 and dctA2.

Figures 41A and 41B illustrate the ratio of the nifH transcript to the nifA transcript in several strains with different promoters and RBS sequences inserted upstream of nifA.

DETAILED DESCRIPTION OF THE INVENTION

[0056] The terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid" and "oligonucleotide" are used interchangeably. They refer to the polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogues. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) determined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNAs, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogues. Modification of the nucleotide structure, if any, can be made before or after assembly of the polynucleotide. The nucleotide sequence can be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, for example, by conjugation with a labeling moiety.

[0057] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized by hydrogen bonds between the bases of the nucleotide residues. Hydrogen bonding can occur by Watson-Crick base pairing, Hoogsteen bonding, or any other sequence-specific manner according to base complementarity. The complex may contain two chains forming a duplex structure, three or more chains forming a multistrand complex, one self-hybridizing chain, or any combination thereof. The hybridization reaction may represent a step in a larger process, such as initiation of PCR or enzymatic cleavage of a polynucleotide by an endonuclease. The second sequence, which is complementary to the first sequence, is called the "complement" of the first sequence. The term "hybridizable" when applied to a polynucleotide refers to the ability of the polynucleotide to form a complex that is stabilized by hydrogen bonding between the bases of nucleotide residues in the hybridization reaction.

[0058] “Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence, either of the traditional Watson-Crick type or other non-traditional types. Percent complementarity refers to the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 is 50%, 60%, 70%, 80%, 90% and 100%, respectively). “Perfectly complementary” means that all contiguous residues in a nucleic acid sequence will have a hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. “Substantially complementary” as used herein refers to a degree of complementarity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% within the area of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45 , 50 or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions. Sequence identity, for example to estimate percent complementarity, can be measured using any suitable alignment algorithm, including, but not limited to, the Needleman-Wunsch algorithm (see, for example, the EMBOSS Needle alignment tool, available at www.ebi.ac .uk/Tools/psa/emboss_needle/nucleotide.html, optional with default settings), BLAST algorithm (see for example the BLAST alignment tool available at blast.ncbi.nlm.nih.gov/Blast.cgi, optional with default settings) or Smith-Waterman algorithm (see for example the EMBOSS Water alignment tool, available at www.ebi.ac.uk/Tools/psa/emboss_water/nucleotide.html, optional with default settings) . Optimal alignment can be assessed using any appropriate parameters of the selected algorithm, including default parameters.

[0059] In general, “stringent conditions” of hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence preferentially hybridizes to the target sequence and substantially does not hybridize to non-target sequences. Severe conditions are generally sequence dependent and vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes, Part I, Chapter Two, “Overview of principles of hybridization and the strategy of nucleic acid probe assay,” Elsevier, N.Y.

[0060] In general, “sequence identity” refers to the exact nucleotide or amino acid match of two polynucleotide or polypeptide sequences, respectively. Typically, methods for determining sequence identity include determining the nucleotide sequence of a polynucleotide and/or determining the amino acid sequence it encodes and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotide or amino acid) can be compared by determining their "percentage of identity". The percentage identity of two sequences, whether nucleic acid sequences or amino acid sequences, can be calculated as the number of exact matches between the two aligned sequences divided by the length of the shorter sequences and multiplied by 100. In some cases, the percentage identity of the test sequence and the reference sequence, nucleic acid sequence acids or amino acid sequences, can be calculated as the number of exact matches between two aligned sequences divided by the length of the reference sequence and multiplied by 100. Percent identity can also be determined, for example, by comparing sequence information using the advanced computer program BLAST, including version 2.2.9, available from the National Institutes of Health. The BLAST program is based on the alignment algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and as discussed in Altschul, et al., J. Mol. Biol. 215:403-410 (1990); Karlin And Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25:3389–3402 (1997). Briefly, in the BLAST program, identity is defined as the number of identical aligned characters (usually nucleotides or amino acids) divided by the total number of characters in the shorter of two sequences. This program can be used to determine the percent identity across the entire length of proteins being compared. Default parameters are provided to optimize searches with short search sequences, such as in the blastp program. The program also allows the SEG filter to be used to mask segments of search sequences, as defined in the SEG program described by Wootton and Federhen, Computers and Chemistry 17: 149-163 (1993). Ranges of desired degrees of sequence identity are from approximately 80% to 100%, including integer values in between. Typically, the percentage identity between the disclosed sequence and the claimed sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%.

[0061] As used herein, the term “expression” refers to the process by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcript) and/or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. The transcripts and encoded polypeptides may be collectively referred to as the "gene product". If the polynucleotide is derived from genomic DNA, expression may involve splicing of the mRNA in the eukaryotic cell.

[0062] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by segments other than amino acids. These terms also include an amino acid polymer that has been modified; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation such as conjugation with a labeling moiety. As used herein, the term “amino acid” refers to natural and/or non-natural or synthetic amino acids, including glycine and both optical D- or L-isomers, amino acid analogues and peptidomimetics.

[0063] As used herein, the term "about" is used synonymously with the term "about". By way of illustration, the use of the term “about” in relation to quantities means that the values are slightly outside the values given, for example, plus or minus 0.1-10%.

[0064] The term “biologically pure culture” or “substantially pure culture” refers to a culture of the bacterial species described herein that does not contain other bacterial species in quantities sufficient to interfere with the replication of the culture or to be detected by conventional bacteriological methods.

[0065] “Plant productivity” generally refers to any aspect of the growth or development of a plant that is the reason why the plant is grown. For food crops such as grains or vegetables, "plant productivity" may refer to the grain or fruit yield harvested from a particular crop. As used herein, improving plant productivity generally refers to improving the yield of grains, fruits, flowers or other plant parts harvested for various purposes, improving the growth of plant parts including stems, leaves and roots, promoting plant growth, maintaining high chlorophyll content in leaves , increased number of fruits or seeds, increased unit weight of fruit or seeds, reduced NO ₂ emissions due to reduced use of nitrogen fertilizers and similar improvements in plant growth and development.

[0066] Microorganisms in and around food crops can influence the quality of those crops. Plant qualities that can be influenced by microorganisms include: yield (e.g. grain production, biomass production, fruit development, flowering); nutrition (for example, absorption of nitrogen, phosphorus, potassium, iron, microelements); control of abiotic stress (e.g. drought tolerance, salt tolerance, heat tolerance); and control of biotic stress (eg, pests, weeds, insects, fungi and bacteria). Strategies for changing crop quality include: increasing the concentration of key metabolites; changes in the time dynamics of the influence of microorganisms on key metabolites; linking the production/decomposition of microbial metabolites with new environmental factors; reduction of unwanted metabolites; and improving the balance of metabolites or parent proteins.

[0067] As used herein, the term “control sequence” refers to an operator, promoter, silencer, or terminator.

[0068] In some embodiments, the native or endogenous control sequences of genes according to the present invention are replaced by one or more infrageneric control sequences.

[0069] As used herein, the term “administered” refers to introduction through modern biotechnology, rather than introduction that occurs naturally.

[0070] In some embodiments, the bacteria of the present invention have been modified such that they are not naturally occurring bacteria.

[0071] In some embodiments, the bacteria of the present invention are present in the plant in quantities of at least 10 ³ CFU, 10 ⁴ CFU, 10 ⁵ CFU, 10 ⁶ CFU, 10 ⁷ CFU, 10 ⁸ CFU, 10 ⁹ CFU, 10 ¹⁰ CFU , 10 ¹¹ CFU or 10 ¹² CFU per gram of fresh or dry weight of the plant. In some embodiments, the bacteria of the present invention are present in the plant in an amount of at least about 10 ³ CFU, about 10 ⁴ CFU, about 10 ⁵ CFU, about 10 ⁶ CFU, about 10 ⁷ CFU, about 10 ⁸ CFU, about 10 ⁹ CFU , approximately 10 ¹⁰ CFU, approximately 10 ¹¹ CFU, or approximately 10 ¹² CFU per gram of fresh or dry weight of the plant. In some embodiments, the bacteria of the present invention are present in the plant in an amount of at least 10 ³ to 10 ⁹ , 10 ³ to 10 ⁷ , 10 ³ to 10 ⁵ , 10 ⁵ to 10 ⁹ , 10 ⁵ to 10 ⁷ , from 10 ⁶ to 10 ¹⁰ , from 10 ⁶ to 10 ⁷ CFU per gram of fresh or dry weight of the plant.

[0072] Fertilizers and exogenous nitrogen according to the present invention may contain the following nitrogen-containing molecules: ammonium, nitrate, nitrite, ammonia, glutamine, etc. The nitrogen sources of the present invention may include anhydrous ammonia, ammonium sulfate, urea, diammonium phosphate, formaldehyde urea, monoammonium phosphate, ammonium nitrate, ammonia water, calcium nitrate, potassium nitrate, sodium nitrate, etc.

[0073] As used herein, the term "exogenous nitrogen" refers to non-atmospheric nitrogen readily available in soil, field or growing media, which is present under non-limiting nitrogen conditions, including ammonia, ammonium, nitrate, nitrite, urea, uric acid, acid ammonium, etc.

[0074] As used herein, the term “non-limiting nitrogen conditions” refers to the availability of non-atmospheric nitrogen in a soil, field, or environment at concentrations greater than approximately 4 mM nitrogen as described in Kant et al. (2010). J. Exp. Biol. 62(4):1499-1509), incorporated herein by reference.

[0075] As used herein, the term “introduced genetic material” means genetic material that has been added to and remains a component of the recipient's genome.

[0076] In some embodiments, the genetic regulatory network for nitrogen fixation and assimilation includes polynucleotides encoding genes and non-coding sequences that direct, modulate, and/or regulate nitrogen fixation and/or assimilation by a microorganism and may comprise nif cluster polynucleotide sequences (e.g., nifA , nifB, nifC,…….nifZ), polynucleotides encoding nitrogen regulatory protein C, polynucleotides encoding nitrogen regulatory protein B, gln cluster polynucleotide sequences (eg, glnA and glnD), draT and ammonia transporters/permeases. In some cases, the Nif cluster may include NifB, NifH, NifD, NifK, NifE, NifN, NifX, hesa, and NifV. In some cases, the Nif cluster may contain part of NifB, NifH, NifD, NifK, NifE, NifN, NifX, hesa and NifV.

[0077] In some embodiments, the fertilizer according to the present invention contains at least 5 wt. %, 6 wt. %, 7 wt. %, 8 wt. %, 9 wt. %, 10 wt. %, 11 wt. %, 12 wt. %, 13 wt. %, 14 wt. %, 15 wt. %, 16 wt. %, 17 wt. %, 18 wt. %, 19 wt. %, 20 wt. %, 21 wt. %, 22 wt. %, 23 wt. %, 24 wt. %, 25 wt. %, 26 wt. %, 27 wt. %, 28 wt. %, 29 wt. %, 30 wt. %, 31 wt. %, 32 wt. %, 33 wt. %, 34 wt. %, 35 wt. %, 36 wt. %, 37 wt. %, 38 wt. %, 39 wt. %, 40 wt. %, 41 wt. %, 42 wt. %, 43 wt. %, 44 wt. %, 45 wt. %, 46 wt. %, 47 wt. %, 48 wt. %, 49 wt. %, 50 wt. %, 51 wt. %, 52 wt. %, 53 wt. %, 54 wt. %, 55 wt. %, 56 wt. %, 57 wt. %, 58 wt. %, 59 wt. %, 60 wt. %, 61 wt. %, 62 wt. %, 63 wt. %, 64 wt. %, 65 wt. %, 66 wt. %, 67 wt. %, 68 wt. %, 69 wt. %, 70 wt. %, 71 wt. %, 72 wt. %, 73 wt. %, 74 wt. %, 75 wt. %, 76 wt. %, 77 wt. %, 78 wt. %, 79 wt. %, 80 wt. %, 81 wt. %, 82 wt. %, 83 wt. %, 84 wt. %, 85 wt. %, 86 wt. %, 87 wt. %, 88 wt. %, 89 wt. %, 90 wt. %, 91 wt. %, 92 wt. %, 93 wt. %, 94 wt. %, 95 wt. %, 96 wt. %, 97 wt. %, 98 wt. %, 99 wt. % nitrogen.

[0078] In some embodiments, the fertilizer of the present invention contains at least about 5 wt. %, approximately 6 wt. %, approximately 7 wt. %, approximately 8 wt. %, approximately 9 wt. %, approximately 10 wt. %, approximately 11 wt. %, approximately 12 wt. %, approximately 13 wt. %, approximately 14 wt. %, approximately 15 wt. %, approximately 16 wt. %, approximately 17 wt. %, approximately 18 wt. %, approximately 19 wt. %, approximately 20 wt. %, approximately 21 wt. %, approximately 22 wt. %, approximately 23 wt. %, approximately 24 wt. %, approximately 25 wt. %, approximately 26 wt. %, approximately 27 wt. %, approximately 28 wt. %, approximately 29 wt. %, approximately 30 wt. %, approximately 31 wt. %, approximately 32 wt. %, approximately 33 wt. %, approximately 34 wt. %, approximately 35 wt. %, approximately 36 wt. %, approximately 37 wt. %, approximately 38 wt. %, approximately 39 wt. %, approximately 40 wt. %, approximately 41 wt. %, approximately 42 wt. %, approximately 43 wt. %, approximately 44 wt. %, approximately 45 wt. %, approximately 46 wt. %, approximately 47 wt. %, approximately 48 wt. %, approximately 49 wt. %, approximately 50 wt. %, approximately 51 wt. %, approximately 52 wt. %, approximately 53 wt. %, approximately 54 wt. %, approximately 55 wt. %, approximately 56 wt. %, approximately 57 wt. %, approximately 58 wt. %, approximately 59 wt. %, approximately 60 wt. %, approximately 61 wt. %, approximately 62 wt. %, approximately 63 wt. %, approximately 64 wt. %, approximately 65 wt. %, approximately 66 wt. %, approximately 67 wt. %, approximately 68 wt. %, approximately 69 wt. %, approximately 70 wt. %, approximately 71 wt. %, approximately 72 wt. %, approximately 73 wt. %, approximately 74 wt. %, approximately 75 wt. %, approximately 76 wt. %, approximately 77 wt. %, approximately 78 wt. %, approximately 79 wt. %, approximately 80 wt. %, approximately 81 wt. %, approximately 82 wt. %, approximately 83 wt. %, approximately 84 wt. %, approximately 85 wt. %, approximately 86 wt. %, approximately 87 wt. %, approximately 88 wt. %, approximately 89 wt. %, approximately 90 wt. %, approximately 91 wt. %, approximately 92 wt. %, approximately 93 wt. %, approximately 94 wt. %, approximately 95 wt. %, approximately 96 wt. %, approximately 97 wt. %, approximately 98 wt. % or approximately 99 wt. % nitrogen.

[0079] In some embodiments, the fertilizer according to the present invention contains from about 5 wt. % up to 50 wt. %, from approximately 5 wt. % up to 75 wt. %, from approximately 10 wt. % up to 50 wt. %, from approximately 10 wt. % up to 75 wt. %, from approximately 15 wt. % up to 50 wt. %, from approximately 15 wt. % up to 75 wt. %, from approximately 20 wt. % up to 50 wt. %, from approximately 20 wt. % up to 75 wt. %, from approximately 25 wt. % up to 50 wt. %, from approximately 25 wt. % up to 75 wt. %, from approximately 30 wt. % up to 50 wt. %, from approximately 30 wt. % up to 75 wt. %, from approximately 35 wt. % up to 50 wt. %, from approximately 35 wt. % up to 75 wt. %, from approximately 40 wt. % up to 50 wt. %, from approximately 40 wt. % up to 75 wt. %, from approximately 45 wt. % up to 50 wt. %, from approximately 45 wt. % up to 75 wt. % or approximately 50 wt. % up to 75 wt. % nitrogen.

[0080] In some embodiments, the increase in nitrogen fixation and/or production of 1% or more nitrogen in the plant is measured relative to control plants that were not exposed to the bacteria of the present invention. All increases or decreases in bacteria are measured relative to control bacteria. Any increases or decreases in plants are measured relative to control plants.

[0081] As used herein, a “constitutive promoter” is a promoter that is active under most conditions and/or at most stages of development. There are several advantages associated with the use of constitutive promoters in expression vectors used in biotechnology, such as: high levels of protein production used to select transgenic cells or organisms; high level of expression of reporter proteins or selectable markers, which ensures ease of detection and quantification; high level of production of a transcription factor, which is part of the transcription regulatory system; production of compounds requiring universal activity in the body; and production of compounds required at all stages of development. Non-limiting examples of constitutive promoters include the CaMV 35S promoter, opine promoters, ubiquitin promoter, alcohol dehydrogenase promoter, etc.

[0082] As used herein, a “non-constitutive promoter” is a promoter that is active under certain conditions, in certain cell types and/or at certain stages of development. For example, tissue-specific, tissue-preferred, cell-specific, cell-preferred, inducible, and developmental stage-controlled promoters are non-constitutive promoters. Examples of developmental stage-controlled promoters include promoters that selectively initiate transcription in certain tissues.

[0083] As used herein, an "inducible" or "repressible" promoter is a promoter that is under the control of chemical or environmental factors. Examples of environmental conditions that can influence transcription by inducible promoters include anaerobic conditions, certain chemicals, the presence of light, acidic or basic conditions, etc.

[0084] As used herein, a “tissue-specific” promoter is a promoter that initiates transcription only in certain tissues. In contrast to constitutive gene expression, tissue-specific expression is the result of the interaction of several levels of gene regulation. Accordingly, it is sometimes preferred in the art to use promoters from a homologous or closely related species to achieve efficient and reliable expression of transgenes in certain tissues. This is one of the main reasons for the significant number of tissue-specific promoters isolated from certain tissues described in both the scientific and patent literature.

[0085] As used herein, the term “operably linked” refers to the linkage of nucleic acid sequences on one nucleic acid fragment such that the function of one is controlled by the other. For example, a promoter is operably linked to a coding sequence if it is capable of regulating the expression of said coding sequence (ie, the coding sequence is under the transcriptional control of said promoter). Coding sequences may be operably linked to regulatory sequences in sense or antisense orientation. In another example, complementary regions of the RNA as described may be operably linked, directly or indirectly, to the 5' end of the target mRNA, or to the 3' end of the target mRNA, or within the target mRNA, or the first complementary region is 5' terminal, and its complement is 3'-terminal relative to the target mRNA.

Regulation of nitrogen fixation

[0086] One quality that may be targeted for regulation by the methods described herein is nitrogen fixation. Nitrogen fertilizer is the largest operating expense on a farm and a major driver of higher yields in row crops such as corn and wheat. This document describes microbial-based products that can deliver renewable forms of nitrogen to non-legume crops. Although some endophytes have the genetics necessary to fix nitrogen in pure crops, a fundamental technical problem is that wild-type endophytes of grains and cereals fail to fix nitrogen in fertilized fields. The use of chemical fertilizers and residual levels of nitrogen in field soils signal the microorganism to stop the biochemical nitrogen fixation pathway.

[0087] To develop a microorganism capable of fixing and transferring nitrogen to corn in the presence of fertilizer, changes in the transcriptional and post-translational levels of the nitrogen fixation regulatory network are required. To this end, this paper describes the technology of Host-Microbe Evolution (HoME) for the precise evolution of regulatory networks and the identification of new phenotypes. This paper also describes unique, proprietary libraries of nitrogen-fixing endophytes isolated from maize, coupled with extensive omics data on microbial-host interactions under various environmental conditions such as nitrogen deficiency and excess. This technology enables precise development of the genetic regulatory network of endophytes to produce microorganisms that actively fix nitrogen even in the presence of fertilizers in the field. This paper also describes evaluations of the technical potential of developing microorganisms that colonize corn root tissue and produce nitrogen for fertilized crops, as well as evaluations of endophyte compatibility with standard formulation methods and a variety of soils to determine the feasibility of integrating microorganisms into modern nitrogen management strategies.

[0088] To use elemental nitrogen (N) for chemical synthesis, life forms combine nitrogen gas (N ₂ ) available from the atmosphere with hydrogen in a process known as nitrogen fixation. Due to the energy-intensive nature of biological nitrogen fixation, diazotrophs (bacteria and archaea that fix atmospheric nitrogen gas) have evolved complex and tight regulation of the nif gene cluster in response to environmental oxygen and available nitrogen. The nif genes encode enzymes involved in nitrogen fixation (such as the nitrogenase complex) and proteins that regulate nitrogen fixation. Shamseldin (2013. Global J. Biotechnol. Biochem. 8(4):84-94) provides detailed descriptions of the nif genes and their products and is incorporated herein by reference. Disclosed herein are methods for producing a plant with improved quality, comprising isolating bacteria from a first plant, introducing a genetic change in the nif gene of the isolated bacteria, exposing a second plant to the variant bacteria, isolating bacteria from a second plant having improved quality relative to the first plant, and repeating the steps with bacteria isolated from the second plant.

[0089] In Proteobacteria, regulation of nitrogen fixation is centered around the _σ54 -dependent enhancer-binding protein NifA, a positive regulator of nif cluster transcription. Intracellular levels of active NifA are controlled by two key factors: transcription of the nifLA operon and inhibition of NifA activity by protein-protein interaction with NifL. Both of these processes are responsive to intracellular glutamine levels through the PII protein signaling cascade. This cascade is mediated by GlnD, which directly recognizes glutamine and catalyzes the uridylation or deuridylation of two PII regulatory proteins, GlnB and GlnK, in response to the absence or presence, respectively, of bound glutamine. Under excess nitrogen conditions, unmodified GlnB signals inactivation of the nifLA promoter. However, under nitrogen-limited conditions, GlnB is posttranslationally modified, which inhibits its activity and leads to transcription of the nifLA operon. Thus, nifLA transcription is tightly controlled in response to environmental nitrogen through the PII protein signaling cascade. At the post-translational level of regulation, NifA GlnK inhibits the NifL/NifA interaction depending on the total level of free GlnK in the cell.

[0090] NifA is transcribed from the nifLA operon, the promoter of which is activated by phosphorylated NtrC, another _σ54 -dependent regulator. The phosphorylation state of NtrC is mediated by the histidine kinase NtrB, which interacts with deuridylated GlnB but not uridylated GlnB. Under excess nitrogen conditions, high intracellular levels of glutamine lead to deuridylation of GlnB, which then interacts with NtrB, inactivating its phosphorylation activity and activating its phosphatase activity, resulting in dephosphorylation of NtrC and inactivation of the nifLA promoter. However, under nitrogen-limited conditions, low levels of intracellular glutamine lead to uridylation of GlnB, which inhibits its interaction with NtrB and allows phosphorylation of NtrC and transcription of the nifLA operon. Thus, nifLA expression is tightly controlled in response to environmental nitrogen through the PII protein signaling cascade. nifA, ntrB, ntrC and glnB are all genes that can be mutated in the methods described herein. These processes may also be susceptible to intracellular levels of ammonia, urea, or nitrate.

[0091] NifA activity is also regulated post-translationally in response to environmental nitrogen, most commonly through NifL-mediated inhibition of NifA activity. In general, the interaction between NifL and NifA is influenced by the PII protein signaling cascade through GlnK, although the nature of the interactions between GlnK and NifL/NifA varies significantly between diazotrophs. In Klebsiella pneumoniae, both forms of GlnK inhibit the NifL/NifA interaction, and the interaction between GlnK and NifL/NifA is determined by the total level of free GlnK in the cell. Under conditions of excess nitrogen, deuridylated GlnK interacts with the ammonium transporter AmtB, which serves to both block ammonium uptake by AmtB and sequester GlnK into the membrane, allowing inhibition of NifA by NifL. On the other hand, in Azotobacter vinelandii, interaction with deurilylated GlnK is required for NifL/NifA interaction and NifA inhibition, whereas uridylation of GlnK inhibits its interaction with NifL. There is evidence that in diazotrophs lacking the nifL gene, NifA activity is inhibited directly by interaction with deuridylated forms of both GlnK and GlnB under conditions of excess nitrogen. In some bacteria, the Nif cluster may be regulated by glnR, and in addition, in some cases this may involve negative regulation. Regardless of the mechanism, posttranslational inhibition of NifA is an important regulator of the nif cluster in most known diazotrophs. In addition, nifL, amtB, glnK and glnR are genes that can be mutated in the methods described herein.

[0092] In addition to regulating the transcription of nif cluster genes, many diazotrophs have evolved a mechanism for direct post-translational modification and inhibition of the nitrogenase enzyme itself, known as nitrogenase shutdown. This process is mediated by ADP-ribosylation of Fe protein (NifH) under conditions of excess nitrogen, which disrupts its interaction with the MoFe protein complex (NifDK) and eliminates nitrogenase activity. DraT catalyzes ADP-ribosylation of Fe protein and shutdown of nitrogenase, whereas DraG catalyzes removal of ADP-ribose and reactivation of nitrogenase. As with nifLA transcription and NifA inhibition, nitrogenase shutdown is also regulated through the PII protein signaling cascade. Under conditions of excess nitrogen, deuridylated GlnB interacts with and activates DraT, and deuridylated GlnK interacts with both DraG and AmtB to form a complex, sequestering DraG into the membrane. Under nitrogen-limited conditions, the uridylated forms of GlnB and GlnK do not interact with DraT and DraG, respectively, resulting in inactivation of DraT and diffusion of DraG to the Fe protein, where it removes ADP-ribose and activates nitrogenase. The methods described herein also involve introducing a genetic change in the nifH, nifD, nifK and draT genes.

[0093] Although some endophytes have the ability to fix nitrogen in vitro, often the genetics are suppressed in the field by high levels of exogenous chemical fertilizers. It is possible to decouple exogenous nitrogen sensing from nitrogenase enzyme expression to promote nitrogen fixation under field conditions. Improvement in integral nitrogenase activity over time further contributes to increased nitrogen production for use by the crop. Specific targets for genetic modification to promote nitrogen fixation in the field using the methods described herein include one or more genes selected from the group consisting of nifA, nifL, ntrB, ntrC, glnA, glnB, glnK, draT, amtB, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB and nifQ.

[0094] An additional target for genetic modification to promote nitrogen fixation in the field using the methods described herein is the NifA protein. The NifA protein is usually an activator for the expression of nitrogen fixation genes. Increased NifA production (either constitutively or under high ammonia conditions) bypasses the natural ammonia sensing pathway. In addition, decreased protein production of NifL, a known inhibitor of NifA, also leads to increased levels of free active NifA. In addition, increased transcription levels of the nifAL operon (either constitutively or under high ammonia conditions) also lead to an overall increase in NifA protein levels. Increased levels of nifAL expression are achieved by altering the promoter itself or by reducing NtrB expression (part of the ntrB and ntrC signaling cascade that initially led to the shutdown of the nifAL operon under high nitrogen conditions). High levels of NifA achieved by these or any other methods described herein enhance the nitrogen-fixing activity of endophytes.

[0095] Another target for genetic modification to promote nitrogen fixation in the field using the methods described herein is the GlnD/GlnB/GlnK PII signaling cascade. Intracellular glutamine levels are sensed through the GlnD/GlnB/GlnK PII signaling cascade. Active site mutations in GlnD that abolish the uridylyl scavenging activity of GlnD disrupt the nitrogen sensing cascade. In addition, a decrease in GlnB concentration causes a disruption in the glutamine sensing cascade. These mutations “trick” the cells into appearing to be in a nitrogen-limited state, thereby increasing the level of nitrogen-fixing activity. These processes may also be susceptible to intracellular, or extracellular, levels of ammonia, urea, or nitrate.

[0096] The amtB protein is also a target for genetic modification to promote nitrogen fixation in the field using the methods described herein. Ammonia uptake from the environment can be reduced by reducing amtB protein expression levels. Without intracellular ammonia, the endophyte is unable to perceive high levels of ammonia, which prevents the suppression of nitrogen fixation genes. Any ammonia that enters the intracellular compartment is converted to glutamine. Intracellular glutamine levels are the primary source of nitrogen sensing. Reducing intracellular glutamine levels prevents cells from accepting high levels of ammonium in the environment. This effect can be achieved by increasing the expression level of glutaminase, an enzyme that converts glutamine into glutamate. Additionally, intracellular glutamine levels can also be reduced by reducing glutamine synthase (the enzyme that converts ammonia to glutamine). In diazotrophs, fixed ammonia is rapidly assimilated into glutamine and glutamate for use in cellular processes. Disruption of ammonia assimilation may allow redirection of fixed nitrogen to be exported from the cell as ammonia. Fixed ammonia is preferentially assimilated into glutamine by glutamine synthetase (GS), encoded by glnA, and then into glutamine by glutamine oxoglutarate aminotransferase (GOGAT). In some examples, glnS encodes glutamine synthetase. GS is regulated posttranslationally by GS adenylyltransferase (GlnE), a bifunctional enzyme encoded by glnE that catalyzes both adenylation and deadenylation of GS through the activities of its adenylyltransferase (AT) and adenylyl-removing (AR) domains, respectively. Under nitrogen-limited conditions, glnA is expressed and the AR domain of GlnE deadenylates GS, mediating its activity. Under excess nitrogen conditions, glnA expression is turned off and the AT domain of GlnE is allosterically activated by glutamine, causing adenylation and inactivation of GS.

[0097] In addition, the draT gene can also be targeted for genetic modification to promote nitrogen fixation in the field using the methods described herein. After the cell produces nitrogen-fixing enzymes, nitrogenase shutdown is another level at which the cell suppresses nitrogen-fixing activity under high nitrogen conditions. This switch-off can be reversed by reducing the expression level of DraT.

[0098] Methods for imparting new phenotypes to microorganisms can be carried out at the transcriptional, translational and post-translational levels. The transcriptional level includes changes in the promoter (such as changes in sigma factor affinity or binding sites for transcription factors, including deletion of all or part of the promoter) or changes in transcription terminators and attenuators. The translational level includes changes in ribosome binding sites and changes in mRNA degradation signals. The post-translational level includes mutation of the active site of the enzyme and changes in protein-protein interactions. These changes can be achieved in many ways. Reducing the expression level (or completely eliminating it) can be achieved by replacing the native ribosome binding site (RBS) or promoter with one of lower strength/efficiency. ATG start sites may be replaced by a GTG, TTG, or CTG start codon, resulting in decreased translational activity of the coding region. Complete elimination of expression can be achieved by knockout (deletion) of the coding region of the gene. An open reading frame (ORF) shift is likely to result in a premature stop codon along the ORF, thus providing a nonfunctional truncated product. Insertion of stop codons within the reading frame will also provide a non-functional truncated product. To reduce the effective concentration of a particular gene, the addition of a degradation tag at the N- or C-terminus can also be carried out.

[0099] Conversely, the level of expression of the genes described herein can be achieved using a stronger promoter. To ensure high promoter activity under high nitrogen conditions (or any other conditions), a genome-wide transcriptional profile under high nitrogen conditions can be obtained, and from this data set, active promoters with the desired level of transcription can be selected to replace the weak promoter. Weak start codons can be replaced with an ATG start codon for better translation initiation efficiency. Weak ribosome binding sites (RBS) can also be replaced by other RBS with higher translation initiation efficiency. In addition, site-directed mutagenesis can also be performed to change enzyme activity.

[00100] Increasing the level of nitrogen fixation that occurs in a plant can lead to a reduction in the amount of chemical fertilizers needed for crop production and a reduction in greenhouse gas emissions (eg, nitrogen oxides).

Increased nitrogen-fixing activity

[00101] Nitrogenases are enzymes responsible for catalyzing nitrogen fixation. There are three types of nitrogenase found in various nitrogen-fixing bacteria: molybdenum-containing (Mo) nitrogenase, vanadium-containing (V) nitrogenase, and iron-only (Fe) nitrogenase. Nitrogenases are two-component systems consisting of component I (also known as dinitrogenase) and component II (also known as dinitrogenase reductase). Component I is the MoFe protein in molybdenum-containing nitrogenase, the VFe protein in vanadium-containing nitrogenase, and the Fe protein in iron-only nitrogenase. Component II is an Fe protein containing an iron-sulfur cluster (Fe-S).

[00102] In some cases, changing the amount of cofactors supplied can lead to increased nitrogen fixation. For example, increased sulfur uptake may provide a larger pool of cofactors for nitrogenase enzymes, thereby increasing the number of functional nitrogenase complexes. In some cases, sulfur uptake can be increased by activating sulfate transport genes. Examples of sulfate transport genes include, but are not limited to, cysPTWA, sbp, sbp, cysZK.

[00103] In some cases, changing the amount of cofactors supplied can lead to increased nitrogen fixation. For example, increased molybdenum (Mo) uptake may provide increased amounts of functional nitrogenase complexes. In some cases, Mo uptake can be increased by activating Mo transport genes. Examples of Mo transport genes include, but are not limited to, modEBA, modEB, and modA.

[00104] In some cases, cofactor supply may be influenced by iron absorption. Iron absorption may be influenced by the tonB transport system. In some cases, iron absorption can be influenced by activating the genes of the tonB transport system. Examples of tonB transport genes include, but are not limited to, tonB and exbAB. In some cases, iron absorption can be influenced by siderophores, which increase iron uptake by microorganisms and plants. In some cases, iron absorption can be influenced by activating siderophore biosynthetic genes. Examples of siderophore biosynthesis genes include, but are not limited to, yhfA, yusV, sbnA, fiu, yfiZ, and fur.

[00105] A change in metabolic flux to ATP can lead to increased nitrogen fixation. For example, metabolic flux to ATP can be increased by targeting glycogen biosynthesis. Glycogen biosynthesis may be influenced by carbon redirection to glycolysis, the TCA (tricarboxylic acid) cycle, and/or oxidative phosphorylation rather than glycogen synthesis. In some cases, glycogen biosynthesis can be influenced by deleting or suppressing the corresponding gene for glycogen synthase. An example of a glycogen synthase gene may include, but is not limited to, glgA.

[00106] Increased nitrogen fixation can be caused by changes in the amount of nitrogenase enzymes per cell. For example, the amount of nitrogenase enzymes per cell may be affected by nif derepression. Derepression of nif may be achieved through constitutive nitrogen deficiency signaling. In some cases, nif derepression can be achieved by deleting the UR domain of the corresponding genes. An example of a gene that may act as a target for derepression of nif genes includes, but is not limited to, glnD. In some cases, transcription of the nif cluster(s) can be enhanced by introducing strong promoters upstream of the nifHDK or nifDK operon.

[00107] Another way to increase nitrogen fixation would be to increase the amount of nitrogenase enzymes per cell by increasing transcription of the nif cluster. Transcription of the nif cluster can be increased by increasing transcription of NIFA. In some cases, transcription of the nif cluster may be influenced by an increase in the number of copies of the nifA gene in the genome.

[00108] Transcription of the nif cluster can also be increased by increasing translation of NifA. In some cases, NifA translation can be increased by increasing the strength of the ribosome binding site in the nifA gene.

[00109] Increased nitrogen fixation may result from changes in the sensitivity of nitrogenase to oxygen. Oxygen sensitivity may be affected by decreased oxygen perception. In some cases, decreased oxygen perception may be caused by a disorder in oxygen-sensing genes. Examples of oxygen sensing genes include, but are not limited to, nifT/fixU, fixJ, and fixL.

[00110] In some cases, oxygen sensitivity may be influenced by maintaining low levels of cytosolic oxygen through stimulation of cytochrome bd-mediated respiration. In some cases, oxygen sensitivity may be affected by activation of genes encoding cytochrome bd oxidase and/or knockout of alternative cytochrome systems. Examples of genes encoding cytochrome bd genes include, but are not limited to, cydABX, cydAB, and cydX. In some cases, nitrogenase can be protected from oxidation by changing the redox balance in the cell. The redox balance can be altered through the uptake of ROS (reactive oxygen species). One strategy to achieve ROS scavenging is to activate the corresponding genes. Examples of ROS scavenging genes include, but are not limited to, grxABCD, trxA, trxC, and tpx.

[00111] In some cases, oxygen sensitivity may be affected by free oxygen uptake. In some cases, uptake of free oxygen can be achieved by activating bacterial hemoglobin genes. An example of a hemoglobin gene is, but is not limited to, glbN. In some cases, the uptake of free oxygen can be achieved by activating the fixNOPQ genes encoding the high-affinity heme-copper oxidase type cbb3.

[00112] Modification of IHFa can lead to increased expression of nitrogenase. In some cases, nitrogenase expression can be increased by facilitating the interaction between nifA and σ54 at an activation sequence located upstream of certain genes. In particular, IHF activation can increase nitrogenase transcription. In some cases, activation of IHF in combination with nifA and σ54 can enhance transcription of the nitrogenase operon. In some cases, strains that may be used in this nitrogen expression enhancement process may include, but are not limited to, Rahnella aquatilis, Kosakonia sacchari, and/or Klebsiella variicola strains. In some cases, activation of the nitrogenase operon may be more effective if it is combined with a mutation in the gene encoding σ54.

[00113] Modification of the gene encoding σ54 can lead to increased expression of nitrogenase. In some cases, activation of the gene encoding σ54 can lead to increased transcription of nitrogenase. An example of a gene encoding σ54 includes, but is not limited to, rpoN. In some cases, strains that may be used in this nitrogen expression enhancement process may include, but are not limited to, Rahnella aquatilis, Kosakonia sacchari, and/or Klebsiella variicola strains. In some cases, activation of σ54 in combination with nifA and IHF can enhance transcription of the nitrogenase operon. In some cases, transcription of the nitrogenase operon can be further improved by combining σ54 activation with IHF mutation.

[00114] In some cases, removing a protein, such as DraT, from a strain, such as a kosakonia strain, can increase nitrogenase activity. In some cases, DraT can post-translationally modify the nitrogenase enzyme, inhibiting its activity. In some cases, strains that may be used in this process of increasing nitrogenase activity may include, but are not limited to, kosakonia strains.

[00115] In some cases, modification of the asnB gene can increase ammonium excretion. In particular, truncation and activation of the asnB gene can convert glutamine back to ammonium. AsnB enzymes contain two domains; one can deaminate glutamine to release ammonium, and the other uses ammonium to produce asparagine. Truncation of AsnB to delete the asparagine synthase domain and/or activation of the glutamine deaminase domain may promote the reconversion of cellular glutamine to ammonium, thereby increasing ammonium excretion. In some cases, strains that may be used in this process of increasing ammonium excretion may include, but are not limited to, strains of Rahnella aquatilis, Kosakonia sacchari and Klebsiella variicola.

[00116] In some cases, modification of the asnB gene can increase ammonium excretion. In particular, deletion of the asnB gene can reduce ammonium uptake in the cell. asnB can use cytosolic ammonium instead of glutamine as a nitrogen donor. In some cases, deletion, truncation, or activation of asnB can increase the amount of ammonium released from the cell. In some cases, strains that may be used in this process of increasing ammonium excretion may include, but are not limited to, strains of Rahnella aquatilis, Kosakonia sacchari and Klebsiella variicola.

In some cases, modification of glnD may be useful for altering the regulation of nitrogen assimilation. In particular, glnD modifications can be used to optimize the regulation of nitrogen assimilation pathways through the PII protein signaling pathway. The enzyme encoded by glnD modifies the PII proteins GlnK and GlnB. The GlnD enzyme reversibly uridylates and deuridylates PII proteins under nitrogen-limiting and nitrogen-abundant conditions, respectively. PII proteins communicate signaling cascades to nitrogen metabolic pathways. Examples of nitrogen metabolism genes affected by PII protein signaling include, but are not limited to, glnA encoding glutamine synthetase, ntrB/glnL encoding the sensor histidine kinase/phosphatase ntrB, glnG/ntrC DNA-binding transcriptional regulator ntrC, and the nifLA operon. In some cases, glnD can be deleted to reduce the transcription of nitrogen assimilation genes and the amount of nitrogen assimilated into the cell. In some cases, glnD may be modified by deletion of the ACT12 region, deletion of the UR region, and/or by inactivation of the UR region by mutating certain amino acid residues (eg, residues 90, 91, and/or 104). In some cases, strains that may be used in this nitrogen assimilation reduction process may include, but are not limited to, Rahnella aquatilis, Kosakonia sacchari, and Klebsiella variicola strains.

[00117] In some cases, modification of glnD may be useful for increasing nitrogenase activity, ammonium excretion and/or plant growth. In particular, removing a nitrogen-receptive area may increase nitrogenase activity and/or plant growth. In some cases, the ACT domain of glnD may be deleted. In some cases, the ACT domain is involved in determining nitrogen status through allosteric regulation by glutamine. Deletion of the ACT domain can reduce uridylyl transferase activity, thereby signaling excess nitrogen and suppressing nitrogen assimilation genes, leading to increased ammonium excretion. In some cases, strains that can be used in this process of increasing nitrogenase activity, ammonium excretion and/or plant growth may include, but are not limited to, Kosakonia sacchari and Klebsiella variicola strains.

[00118] In some cases, modification of glnD may be useful for increasing nitrogenase activity, ammonium excretion and/or plant growth. In particular, deletion or inactivation of the uridylyltransferase (UT) region in the glnD domain can increase nitrogenase activity, ammonium excretion and/or plant growth. Removal or inactivation of the UT domain can reduce uridylyl transferase activity, thereby signaling excess nitrogen and suppressing nitrogen assimilation genes, resulting in increased ammonium excretion. In some cases, strains that can be used in this process of increasing nitrogenase activity, ammonium excretion and/or plant growth may include, but are not limited to, strains of Rahnella aquatilis, Kosakonia sacchari and Klebsiella variicola.

[00119] In some cases, modification of GlnB may be useful for increasing the excretion of nitrogen compounds. In some cases, the uridylyltransferase (UTase) domain of GlnD modifies GlnB at tyrosine-51. In some cases, by modifying the UTase domain of GlnD, GlnB-UMP production can be reduced. In some cases, by removing the UTase domain of GlnD, GlnB-UMP production can be reduced. In some cases, by altering the UTase domain of GlnD, GlnB-UMP production can be prevented. In some cases, by removing the UTase domain of GlnD, GlnB-UMP production can be prevented. In some cases, GlnB can be modified by deletion of tyrosine-51. In some cases, GlnB can be modified by modifying GlnB at tyrosine-51. In some cases, strains that may be used in this process of increasing ammonium excretion may include, but are not limited to, strains of Rahnella aquatilis, Kosakonia sacchari and Klebsiella variicola.

[00120] In some cases, modification of GlnK may be useful for increasing ammonium excretion. In some cases, GlnK in a strain may function as a GlnB analogue due to the similarity in the structures of GlnK and GlnB. In some cases, modification of GlnK may increase ammonium excretion by eliminating inhibitory effects that may be due to GlnK. In some cases, by changing GlnK, the inhibitory effects on ammonium excretion can be reduced. In some cases, by removing GlnK, the inhibitory effects on ammonium excretion can be reduced. In some cases, by altering GlnK, the inhibitory effects on ammonium excretion due to GlnK can be prevented. In some cases, by deleting the glnK gene, the inhibitory effects on ammonium excretion caused by GlnK can be prevented. In some cases, strains that may be used in this process of increasing ammonium excretion may include, but are not limited to, strains of Rahnella aquatilis, Kosakonia sacchari and Klebsiella variicola.

[00121] In some cases, modification of glnK may be useful for increasing ammonium excretion. In some cases, the UTase domain of GlnD modifies glnK at tyrosine-51. In some cases, by modifying the UTase domain of GlnD, glnK-UMP production can be reduced. In some cases, by removing the UTase domain of GlnD, glnK-UMP production can be reduced. In some cases, by altering the UTase domain of GlnD, glnK-UMP production can be prevented. In some cases, by removing the UTase domain of GlnD, glnK-UMP production can be prevented. In some cases, GlnK can be modified by deletion of tyrosine-51. In some cases, GlnK can be modified by modifying GlnK at tyrosine-51. In some cases, strains that may be used in this process of increasing ammonium excretion may include, but are not limited to, strains of Rahnella aquatilis, Kosakonia sacchari and Klebsiella variicola.

[00122] In some cases, modification of glnL, which encodes the NtrB protein, may be useful to increase ammonium excretion. In particular, modification of NtrB may be useful for controlling glnA transcription independent of nitrogen status. In some cases, modification of ntrB can be achieved by deleting certain residues to dose activity. In some cases, strains that may be used in this process of increasing ammonium excretion may include, but are not limited to, strains of Rahnella aquatilis, Kosakonia sacchari and Klebsiella variicola.

[00123] In some cases, modification of glnA may be useful to increase ammonium excretion. In some cases, modification of NtrC may be useful for modifying the level of GlnA protein in the cell. In particular, modification of NtrC may be beneficial by preventing NtrC phosphorylation. Phosphorylated NtrC can lead to transcriptional activation of glnA. As such, modification of ntrC to prevent phosphorylation of ntrC may be beneficial in reducing glnA transcription. In some cases, modification of NtrC can be achieved by replacing aspartate 54. In some cases, strains that can be used in this process of increasing ammonium excretion may include, but are not limited to, strains of Rahnella aquatilis, Kosakonia sacchari and Klebsiella variicola.

[00124] In some cases, modification of glutaminase B may be useful to increase ammonium excretion. In particular, in some cases the conversion of glutamine back to glutamate and ammonia by glutaminase B may be activated to increase ammonium excretion. In some cases, strains that can be used in this process of increasing nitrogen excretion may include, but are not limited to, strains of Rahnella aquatilis, Kosakonia sacchari and Klebsiella variicola.

[00125] In some cases, modification of nifA may be useful to increase nitrogenase expression. In some cases, it may be useful to modify nifA to increase the number of copies of the nifA gene in the cell. The copy number of the nifA gene can be increased by inserting multiple copies of the nifA gene upstream of constitutively expressing promoters. In some cases, attaching a copy of the nifA gene to one or more housekeeping operons can increase the total number of nifA genes in the cell. In some cases, strains that may be used in this process of increasing nitrogenase expression may include, but are not limited to, Rahnella aquatilis and Klebsiella variicola strains.

[00126] In some cases, modification of the nitrogenase operon may be useful to increase nitrogenase expression. In some cases, it may be useful to activate nitrogenase operons to increase nitrogenase transcription. In some cases, promoters from the bacterium that are active when the bacterium colonizes the rhizosphere can be inserted before the nitrogenase operons to activate the nitrogenase operons. In some cases, nifL from the nitrogenase operons may be deleted to activate the nitrogenase operons. In some cases, nifA from the nitrogenase operons can be deleted to activate the nitrogenase operons. In some cases, nifA and nifL from the nitrogenase operons can be deleted to activate the nitrogenase operons. In some cases, multiple promoters can be placed immediately upstream of nifHDK genes to bypass nifA transcriptional control. In some cases, strains that may be used in this process of increasing nitrogenase expression may include, but are not limited to, Rahnella aquatilis and Klebsiella variicola strains.

[00127] In some cases, modification of glnE may be useful to increase ammonium excretion. In some cases, the conserved aspartate-amino acid-aspartate (DXD) motif on the AR domain of glnE can be altered. In some cases, alteration of the conserved DXD residue in the AR domain of glnE can be used to abolish glnE deadenylation activity. In some cases, residue D in the DXD motif in the AR region of glnE may be replaced. In some cases, replacement of residue D in the DXD motif in the AR region of glnE may not affect the GlnB binding site, allowing regulation of adenylation activity while reducing or preventing AR. activity. In some cases, strains that may be used in this process of increasing ammonium excretion may include, but are not limited to, strains of Rahnella aquatilis, Kosakonia sacchari and Klebsiella variicola.

[00128] In some cases, modification of glnA may be useful to increase AMM excretion. In some cases, glnA can be suppressed by inserting glnB, glnD, and/or glnE promoters upstream of the glnA gene. In some cases, modification of glnA may allow glnA expression to be uncoupled from the N-status signaling cascade and reduce expression to basal levels so that more fixed nitrogen remains unassimilated. In some cases, strains that may be used in this process of increasing ammonium excretion may include, but are not limited to, strains of Rahnella aquatilis, Kosakonia sacchari and Klebsiella variicola.

[00129] In some cases, modification of GOGAT may be useful to increase ammonium excretion. In some cases, GOGAT can be suppressed by insertion upstream of the GOGAT genes of the promoter controlling glnB, glnD and/or glnE. Suppression of GOGAT may in turn lead to decreased expression of glutamine oxyglutarate aminotransferase. In some cases, modification of GOGAT may allow GOGAT expression to be uncoupled from the N-status signaling cascade and reduce expression to basal levels so that more fixed nitrogen remains unassimilated. In some cases, strains that may be used in this process of increasing ammonium excretion may include, but are not limited to, strains of Rahnella aquatilis, Kosakonia sacchari and Klebsiella variicola.

[00130] In some cases, modification of GDH may be useful to increase ammonium excretion. In some cases, GDH can be suppressed by insertion upstream of the GDH promoter gene controlling glnB, glnD and/or glnE. Suppression of GDH may, in turn, lead to decreased expression of NAD-specific glutamate dehydrogenase. In some cases, modification of GDH may allow GDH expression to be uncoupled from the N-status signaling cascade and reduce expression to basal levels so that more fixed nitrogen remains unassimilated. In some cases, strains that may be used in this process of increasing ammonium excretion may include, but are not limited to, strains of Rahnella aquatilis, Kosakonia sacchari and Klebsiella variicola.

Increasing nitrogen fixation due to assimilation

[00131] The amount of nitrogen provided to the plant associated with the microorganisms can be increased by reducing the assimilation of nitrogen into the microorganism. In this case, assimilation may be influenced by the rate of ammonia excretion. By targeting ammonia assimilation, it is possible to increase nitrogen availability. In some cases, ammonia assimilation can be influenced by reducing the rate of ammonia reuptake after excretion. To reduce the degree of ammonia reuptake after excretion, any corresponding gene can be knocked out. An example of ammonia reuptake genes is, but is not limited to, amtB.

[00132] In some cases, assimilation may be influenced by the rate of uptake by the plant. By targeting genes and pathways responsible for nitrogen assimilation in plants, nitrogen availability can be increased. In some cases, plant ammonia assimilation can be altered by inoculation with microorganisms that promote the growth of N-fixing plants. Screening can be carried out to identify microorganisms causing ammonia assimilation in plants.

Increased nitrogen fixation due to colonization

[00133] Increasing the colonization ability of microorganisms may allow an increase in the amount of fixed nitrogen provided to the plant. Colonization may be influenced by changes in transportable volume (the prevalence of microorganisms on the root surface) and/or the adaptability of microorganisms. In some cases, the transport volume and adaptability of microorganisms can be influenced by changing the transport of organic acids. The transport of organic acids can be improved by activating the corresponding genes. An example of an organic acid transport gene is, but is not limited to, dctA.

[00134] For example, colonization ability may be influenced by the expression of agglutinins. Increased expression of agglutinins may promote adherence of microorganisms to plant roots. Examples of agglutinin genes may include, but are not limited to, fhaB and fhaC.

[00135] Colonization ability may be affected by increased endophyte infiltration. For example, endophyte penetration may be influenced by plant cell wall degrading enzymes (CDWE). Increased expression and/or secretion of CDWE may increase colonization and penetration of endophytic microorganisms. Examples of CDWE include, but are not limited to, polygalacturonases and cellulases. An example of a polygalacturonase gene is pehA. In some cases, the export of polygalacturonases and cellulases can be increased by providing an export signal together with the enzymes.

[00136] A change in the volume transferred can result in an increase in the amount of nitrogen provided to the corresponding plant. The volume carried may be affected by biofilm formation. In some cases, the volume transferred may be influenced by the small RNA rsmZ. The small RNA rsmZ is a negative regulator of biofilm formation. In some cases, biofilm formation may be promoted by deletion or suppression of rsmZ, leading to increased translation of rsmA (a positive regulator of secondary metabolism) and biofilm formation.

[00137] In some cases, biofilm formation may be influenced by increasing the ability of strains to adhere to the root surface. In some cases, biofilm formation may be facilitated by activation of large adhesion proteins. An example of a large adhesion protein includes, but is not limited to, lapA.

[00138] In some cases, the volume carried may be affected by quorum sensing. In some cases, quorum sensing can be enhanced by increasing the copy number of AHL biosynthetic genes.

[00139] In some cases, rhizosphere colonization may be influenced by root mass. For example, root mass may be influenced by the biosynthesis of IAA (indole-3-acetic acid) by microorganisms. An increase in IAA biosynthesis by a microorganism can stimulate the formation of root biomass. In some cases, IAA biosynthesis can be influenced by activating (at different levels) IAA biosynthesis genes. An example of an IAA biosynthesis gene is, but is not limited to, ipdC.

[00140] In some cases, ethylene signaling can cause systemic plant resistance and affect the colonization ability of the microorganism. Ethylene is a plant signaling molecule that causes a wide range of reactions depending on the plant tissue and ethylene level. The predominant model of root response to ethylene is that stressed plants respond quickly by producing a small peak of ethylene that initiates a plant defense response, such as the transcription of genes encoding defense proteins. If the stress persists or is intense, a second, much larger ethylene peak occurs, often several days later. This second peak of ethylene causes processes such as senescence, chlorosis and leaf drop, which can lead to significant inhibition of plant growth and survival. In some cases, plant growth promoting bacteria can stimulate root growth by producing the auxin IAA, which stimulates a small ethylene response in the roots. At the same time, bacteria can prevent the second major ethylene peak by producing an enzyme (ACC deaminase) that slows down the plant's ethylene production, thereby maintaining ethylene levels that help stimulate root growth. The induction of systemic resistance in plants can be influenced by bacterial IAA. In some cases, stimulation of IAA biosynthesis can be achieved by activating (at different levels) IAA biosynthesis genes. An example of a biosynthesis gene may include, but is not limited to, ipdC.

[00141] In some cases, colonization may be influenced by ACC deaminase. ACC deaminase can reduce ethylene production downstream by redirecting ACC into a byproduct. In some cases, ACC deaminase can be influenced by activating ACC deaminase genes. Examples of ACC deaminase genes include, but are not limited to, dcyD.

[00142] In some cases, colonization may be influenced by the volume tolerated and/or the adaptability of the microorganisms. For example, the tolerable volume and/or fitness of microorganisms may be affected by excess trehalose production. Excess trehalose production can increase drought tolerance. In some cases, excess trehalose production can be influenced by activating (at different levels) trehalose biosynthesis genes. Examples of trehalose biosynthetic genes include, but are not limited to, otsA, otsB, treZ, and treY. In some cases, activation of otsB can also increase nitrogen-fixing activity.

[00143] In some cases, the volume transferred may be influenced by root attachment. Attachment to roots may be influenced by the secretion of exopolysaccharides. In some cases, exopolysaccharide secretion can be influenced by increasing the expression of exopolysaccharide-producing proteins. Examples of exopolysaccharide-producing proteins include, but are not limited to, yjbE and pssM. In some cases, the secretion of exopolysaccharides can be influenced by activating cellulose biosynthesis. Examples of cellulose biosynthesis genes include, but are not limited to, the acs genes and the bcs gene clusters.

[00144] In some cases, the volume tolerated and/or adaptability of microorganisms may be affected by fungal inhibition. Fungal inhibition may be influenced by chitinases, which can degrade fungal cell walls and may lead to biocontrol of rhizosphere fungi. In some cases, fungal inhibition can be influenced by activating chitinase genes. Examples of chitinase genes include, but are not limited to, class 1 chitinase and chiA.

[00145] In some cases, efficient iron uptake can promote the survival of microorganisms in the rhizosphere, where they must compete with other soil microorganisms and the plant for iron uptake. In some cases, high-affinity chelation (siderophores) and transport systems may promote rhizosphere competence by 1) ensuring that microorganisms obtain sufficient iron, and 2) reducing the available iron for competing species. Increasing the ability of a microorganism to carry out these actions can increase its competitiveness in the rhizosphere. In some cases, iron absorption can be influenced by activating siderophore genes. Examples of siderophore genes include, but are not limited to, yhfA, yusV, sbnA, fiu, yfiZ, and fur. In some cases, iron absorption may be influenced by the tonB transport system. In some cases, iron absorption can be influenced by activating the genes of the tonB transport system. Examples of tonB transport genes include, but are not limited to, tonB and exbAB.

[00146] In some cases, the volume tolerated and/or adaptability of microorganisms may be influenced by redox equilibrium and/or ROS scavenging. Redox equilibrium and/or ROS scavenging may be influenced by bacterial glutathione (GSH) biosynthesis. In some cases, bacterial glutathione (GSH) biosynthesis can be influenced by activating bacterial glutathione biosynthetic genes. Examples of bacterial glutathione biosynthesis genes include, but are not limited to, gshA, gshAB, and gshB.

[00147] In some cases, redox equilibrium may be affected by ROS uptake. In some cases, ROS scavenging can be influenced by activation of catalases. Examples of catalase genes include, but are not limited to, katEG and Mn-catalase.

[00148] In some cases, biofilm formation may be influenced by phosphorus signaling. In some cases, phosphorus signaling can be influenced by changing the expression of genes responsible for phosphorus signaling. Examples of genes responsible for phosphorus signaling include, but are not limited to, phoR and phoB.

[00149] In some cases, the volume transferred may be influenced by root attachment. Attachment to roots may be influenced by surfactin biosynthesis. In some cases, surfactin biosynthesis can be influenced by activating surfactin biosynthesis to improve biofilm formation. An example of a surfactin biosynthesis gene includes, but is not limited to, srfAA.

[00150] In some cases, colonization and/or fitness of microorganisms may be affected by the volume carried, competition with other microorganisms, and/or protection of the crop from other microorganisms. In some cases, competition with other microorganisms and/or defense of the crop against other microorganisms may be affected by quorum sensing and/or quorum quenching. Quorum quenching may influence colonization by suppressing quorum sensing of potential pathogenic/competing bacteria. In some cases, quorum quenching can be influenced by insertion and/or activation of genes encoding quorum quenching enzymes. Examples of genes responsible for quorum quenching include, but are not limited to, ahlD, Y2-aiiA, aiiA, ytnP, and attM. In some cases, modification of enzymes involved in quorum quenching, such as Y2-aiiA and/or ytnP, may be beneficial for colonization. In some cases, activation of Y2-aiiA and/or ytnP can lead to hydrolysis of extracellular acyl-homoserine lactone (AHL). aiiA is an N-acyl-homoserine lactonase, which is an enzyme that cleaves homoserine lactone. AHL cleavage can stop or slow down the quorum signaling ability of competing Gram-negative bacteria. In some cases, strains that may be used in this colonization enhancement process may include, but are not limited to, Rahnella aquatilis, Kosakonia sacchari, and Klebsiella variicola strains.

[00151] In some cases, the tolerable volume and/or adaptability of microorganisms may be influenced by the biosynthesis of rhizobitoxin. Biosynthesis of rhizobitoxin can reduce ethylene production in the root by inhibiting ACC synthase. In some cases, rhizobitoxin biosynthesis can be influenced by activating rhizobitoxin biosynthesis genes.

[00152] In some cases, the volume transferred may be influenced by root attachment. Attachment to roots may be influenced by the secretion of exopolysaccharides. In some cases, exopolysaccharide secretion can be influenced by creating hypermucoid mutants by deleting mucA.

[00153] In some cases, root attachment may be influenced by phenazine biosynthesis. In some cases, phenazine biosynthesis can be influenced by activating phenazine biosynthesis genes to improve biofilm formation.

[00154] In some cases, root attachment may be influenced by cyclic lipopeptide (CLP) biosynthesis. In some cases, cyclic lipopeptide (CLP) biosynthesis can be influenced by activating CLP biosynthesis to improve biofilm formation.

In some cases, the volume tolerated and/or competition may be influenced by antibiotic synthesis. Antibiotic synthesis can increase the production of antibiotics to kill competing microorganisms. In some cases, increased antibiotic production can be achieved by examining genomes for antibiotic biosynthesis and activation pathways.

[00155] In some cases, colonization may be influenced by desiccation resistance. In some cases, modification of rpoE may be beneficial for colonization. In some cases, activation of rpoE can lead to increased expression of genes and pathways responsible for stress resistance. In some cases, rpoE can be activated by a unique switchable promoter. In some cases, rpoE can be activated using the arabinose promoter. rpoE is a sigma factor similar to phyR. When expressed, rpoE can cause the activation of multiple genes responsible for stress resistance. Since enzymes responsible for stress resistance may not be useful during the colonization cycle, a switchable promoter can be used. In some cases, the promoter may be active during biomass growth and/or during seed coating. In some cases, a switchable promoter may be used, where during the logarithmic growth phase of the biomass a sugar or chemical additive may be added, but where also during one or more other uses of the microorganism the promoter may not be switched on. In some cases, rpoE can be activated while rseA is suppressed. In some cases, strains that may be used in this colonization enhancement process may include, but are not limited to, Rahnella aquatilis, Kosakonia sacchari, and Klebsiella variicola strains.

[00156] In some cases, colonization may be influenced by desiccation resistance. In some cases, modification of rseA may be beneficial for colonization. In some cases, rseA can be repressed by a unique switchable promoter. In some cases, rseA can be repressed using the arabinose promoter. rseA is an anti-sigma factor coexpressed with rpoE. In some cases, the enzymes remain associated with each other, which can reduce or eliminate the ability of rpoE to act as a transcription factor. However, under stress conditions, resA can be cleaved off and rpoE can freely activate/repress genes responsible for stress resistance. By disrupting co-transcription with rpoE, rpoE and resA levels can be dosed independently, which may be useful for optimizing colonization of engineered strains. In some cases, strains that may be used in this colonization enhancement process may include, but are not limited to, Rahnella aquatilis, Kosakonia sacchari, and Klebsiella variicola strains.

Obtaining bacterial populations

Isolation of bacteria

[00157] Microorganisms useful for the methods and compositions disclosed herein can be obtained by extracting microorganisms from the surfaces or tissues of native plants. Microorganisms can be obtained by grinding the seeds to isolate the microorganisms. Microorganisms can be obtained by planting seeds in various soil samples and extracting microorganisms from tissues. In addition, microorganisms can be obtained by inoculating plants with exogenous microorganisms and determining which microorganisms appear in plant tissues. Non-limiting examples of plant tissues may include a seed, seedling, leaf, cut, plant, bulb or tuber.

[00158] The method for obtaining microorganisms can be carried out by isolating bacteria from soils. Bacteria can be collected from various types of soils. In some examples, soil may be characterized by qualities such as high or low fertility, moisture levels, mineral levels, and different farming practices. For example, soil may be involved in crop rotation, where different crops are planted in the same soil during alternating planting seasons. Consistently growing different crops in the same soil can prevent disproportionate depletion of certain minerals. Bacteria can be isolated from plants growing in selected soils. Plant seedlings can be harvested after 2-6 weeks of growth. For example, at least 400 isolates can be collected in one harvest cycle. Soil and plant types are indicative of both the plant's phenotype and the conditions that subsequently provide enrichment for certain phenotypes.

[00159] Microorganisms can be isolated from plant tissues to evaluate the qualities of the microorganisms. Parameters for processing tissue samples can be varied to isolate different types of associated microorganisms, such as rhizosphere bacteria, epiphytes, or endophytes. Isolates can be cultured in nitrogen-free media to enrich for nitrogen-fixing bacteria. Alternatively, microorganisms can be obtained from global strain banks.

[00160] To assess the qualities of microorganisms, in planta analytics are performed. In some embodiments, plant tissue may be processed for screening by high-throughput processing of DNA and RNA. In addition, non-invasive measurements can be used to assess plant characteristics such as colonization. Measurements of wild microorganisms can be obtained on each individual plant in turn. Measurements of wild microorganisms can also be made in the field using mid-throughput methods. Measurements can be taken sequentially over time. A model plant system may be used, including, but not limited to, Setaria.

[00161] Microorganisms in a plant system can be screened using transcriptional profiling of the microorganism in the plant system. Examples of screening using transcriptional profiling include quantitative polymerase chain reaction (qPCR) techniques, molecular barcodes for transcript detection, next-generation sequencing, and tagging of microorganisms with fluorescent markers. To assess colonization in a greenhouse, influencing factors may be measured including, but not limited to, the microbiome, abiotic factors, soil conditions, oxygen, moisture, temperature, inoculum conditions, and root location. Nitrogen fixation can be assessed in bacteria by measuring 15N gas/in the fertilizer (dilution) using IRMS (isotope mass spectrometry) or NanoSIMS, as described herein. NanoSIMS is high-resolution secondary ion mass spectrometry. The NanoSIMS method is a method for studying the chemical activity of biological samples. Catalysis of the reduction and oxidation reactions that control the metabolism of microorganisms can be studied at the cellular, subcellular, molecular and elemental levels. NanoSIMS can provide high spatial resolution greater than 0.1 μm. NanoSIMS is capable of detecting the use of isotopic tracers such as ¹³ C, ¹⁵ N and ¹⁸ O. Therefore, NanoSIMS can be used to analyze the chemical activity of nitrogen in the cell.

[00162] Automated greenhouses can be used for plant analytics. Measured plant controls in response to microorganisms include, but are not limited to, biomass, chloroplast analysis, CCD (charge-coupled device) camera, volumetric tomography measurements.

[00163] One way to enrich a population of microorganisms is to enrich according to genotype. For example, polymerase chain reaction (PCR) analysis with a target primer or a specific primer. Primers designed for the nifH gene can be used to identify diazotrophs, since diazotrophs express the nifH gene during nitrogen fixation. Microbial populations can also be enriched using single-cell culture-independent and chemotaxis-based isolation approaches. Alternatively, targeted isolation of microorganisms can be accomplished by culturing the microorganisms on selective media. Designed approaches to enrich microbial populations with desired traits can be based on bioinformatics data and are described herein.

Enrichment of microorganisms with nitrogen-fixing ability using bioinformatics

[00164] Bioinformatics tools can be used to identify and isolate plant growth promoting rhizobacteria (PGPR), which are selected based on their ability to fix nitrogen. Microorganisms with a high nitrogen-fixing ability can contribute to the appearance of favorable qualities in plants. Bioinformatic analysis methods for identifying PGPR include, but are not limited to, genomics, metagenomics, targeted isolation, gene sequencing, transcriptome sequencing, and modeling.

[00165] Genomic analysis can be used to identify PGPRs and confirm the presence of mutations using next generation sequencing techniques as described herein and microbial version control.

[00166] Metagenomics can be used to identify and isolate PGPR using a predictive algorithm for colonization. Metadata can also be used to detect the presence of an engineered strain in environmental and greenhouse samples.

[00167] Transcriptome sequencing can be used to predict genotypes leading to PGPR phenotypes. In addition, transcriptomic data are used to identify promoters to alter gene expression. Transcriptomic data can be analyzed in combination with whole genome sequence (WGS) to generate models of metabolism and gene regulatory networks.

Domestication of microorganisms

[00168] Microorganisms isolated from nature may undergo a domestication process in which the microorganisms are converted into a form that is genetically traceable and identifiable. One way to domesticate a microorganism is to make it resistant to antibiotics. The process of conferring antibiotic resistance can begin by testing the antibiotic susceptibility of a wild-type strain of the microorganism. If bacteria are sensitive to an antibiotic, then the antibiotic may be a good candidate for conferring antibiotic resistance. The antibiotic resistance gene or negative selection suicide vector can then be inserted into the genome of the microorganism using recombination engineering techniques. The negative selection suicide vector may consist of a deletion of the gene of interest, a selectable marker, and the negative selection marker sacB. Negative selection can be used to exchange native DNA sequences of a microorganism for genes that are resistant to antibiotics. The average throughput method can be used to evaluate multiple microorganisms simultaneously, allowing for parallel domestication. Alternative domestication methods include the use of homing nucleases to prevent suicide vector sequences from looping or to produce intermediate vector sequences.

[00169] DNA vectors can be introduced into bacteria by several methods, including electroporation and chemical transformations. For transformations, the standard vector library can be used. An example of a gene editing technique is CRISPR, which is preceded by Cas9 testing to ensure Cas9 activity in microorganisms.

Non-transgenic construction of microorganisms

[00170] A population of microorganisms with favorable qualities can be obtained through directed evolution. Directed evolution is an approach that mimics the process of natural selection to evolve proteins or nucleic acids toward a user-defined goal. An example of directed evolution is introducing random mutations into a population of microorganisms, selecting microorganisms with the most favorable qualities, and continuing to grow the selected microorganisms. The most favorable qualities of growth promoting rhizobacteria (PGPR) may be nitrogen fixation. The directed evolution method can be iterative and adaptive, based on a selection process after each iteration.

[00171] Plant growth promoting rhizobacteria (PGPR) with high nitrogen fixation ability can be obtained. Evolution of PGPR can be accomplished by introducing a genetic change. The genetic change can be introduced through polymerase chain reaction mutagenesis, oligonucleotide-directed mutagenesis, saturation mutagenesis, fragment shuffling mutagenesis, homologous recombination, CRISPR/Cas9 systems, chemical mutagenesis, and combinations thereof. These approaches allow the introduction of random mutations into a population of microorganisms. For example, mutants can be generated using synthetic DNA or RNA through oligonucleotide-directed mutagenesis. Mutants can be generated using tools contained in plasmids, which are subsequently eliminated. Genes of interest can be identified using libraries derived from other species with improved properties, including, but not limited to, improved PGPR properties, improved cereal colonization, increased oxygen sensitivity, increased nitrogen fixation, and increased ammonia excretion. Intragenus genes can be designed from these libraries using software such as Genious or Platypus development software. Mutations can be developed using machine learning. Mutations can be designed using a metabolic model. Automated mutation engineering can be modeled after Platypus and will guide the RNA for Cas-directed mutagenesis.

[00172] Intrageneric genes can be transferred into a host microorganism. In addition, reporter systems can also be transferred into the microorganism. Reporter systems characterize promoters, determine the success of transformation, screen for mutants, and act as negative screening tools.

[00173] Microorganisms carrying the mutation can be cultured through serial passaging. A colony of a microorganism contains one variant of the microorganism. Microbial colonies are checked using an automatic colony picker and a liquid handler. Mutants with gene duplication and increased copy number express a higher genotype of the desired quality.

Selection of microorganisms that promote plant growth based on nitrogen fixation

[00174] Microbial colonies can be screened using various assays to assess nitrogen fixation. One way to measure nitrogen fixation is to perform a single enzymatic assay that measures nitrogen excretion. An alternative method is the acetylene reduction assay (ARA) with built-in sampling over time. ARA can be performed in high-throughput microtube array plates. ARA can be performed on living plants and plant tissues. Media composition and media oxygen concentration in ARA assays may vary. Another way to screen variants of microorganisms is to use biosensors. The use of NanoSIMS and Raman microspectroscopy can be used to study the activity of microorganisms. In some cases, bacteria can also be cultured and propagated using bioreactor fermentation techniques. Bioreactors are designed to increase the persistence of bacterial growth and reduce the sensitivity of bacteria to oxygen. Plate-based microfermenters with medium to high throughput are used to assess oxygen sensitivity, nutritional requirements, nitrogen fixation, and nitrogen excretion. Bacteria can also be co-cultured with competitive or beneficial microorganisms to explore hidden pathways. Flow cytometry can be used to screen for bacteria that produce high levels of nitrogen using chemical, colorimetric, or fluorescent indicators. Bacteria can be cultured in the presence or absence of a nitrogen source. For example, bacteria can be cultured with glutamine, ammonia, urea or nitrates.

Selection of microorganisms

[00175] Microbial breeding is a method for systematically identifying and improving the role of species in the microbiome of a crop. The method consists of three stages: 1) selection of candidate species by mapping plant-microbe interactions and predicting regulatory networks associated with a particular phenotype, 2) pragmatic and predictive improvement of microbial phenotypes through intraspecific crossing of regulatory networks and gene clusters, and 3) screening and selection of new genotypes of microorganisms that provide the desired phenotypes of crops. To systematically evaluate strain improvement, a model is created that relates microbial community colonization dynamics to genetic activity through key species. This model is used to predict selection of genetic targets and increase the frequency of selection for improvements in microbiome-encoded traits of agronomic relevance.

[00176] The production of bacteria to improve plant qualities (eg, nitrogen fixation) can be achieved through sequential passaging. The production of these bacteria can be accomplished by selecting plants that have a particular improved quality that is influenced by the microbiological flora, as well as identifying bacteria and/or compositions capable of imparting one or more improved qualities to one or more plants. One method of obtaining bacteria for plant improvement involves the following steps: (a) isolating the bacteria from the tissue or soil of the first plant; (b) introducing a genetic change into one or more bacteria to produce one or more variant bacteria; (c) exposure of multiple plants to variant bacteria; (d) isolating bacteria from the tissue or soil of one of the plurality of plants, wherein the plant from which the bacteria are isolated is of improved quality compared to other plants in the plurality of plants; and (e) repeating steps (b)-(d) with bacteria isolated from the improved plant (step (d)). Steps (b)-(d) may be repeated any number of times (eg, once, twice, three times, four times, five times, ten times or more) until the improved quality in the plant reaches the desired level. In addition, the plurality of plants may be more than two plants, such as 10-20 plants, or 20 or more, 50 or more, 100 or more, 300 or more, 500 or more, or 1000 or more plants.

[00177] In addition to obtaining improved quality plants, a bacterial population is obtained containing bacteria containing one or more genetic changes introduced into one or more genes (eg, genes regulating nitrogen fixation). By repeating the steps described above, it is possible to obtain a population of bacteria comprising the most suitable members of that population that correlate with the plant quality of interest. Bacteria in this population can be identified and their beneficial properties can be determined, for example, using genetic and/or phenotypic analysis. Genetic analysis can be performed on the isolated bacteria in step (a). Phenotypic and/or genotypic information can be obtained using methods such as: high-throughput screening of plant chemical components, sequencing methods including high-throughput sequencing of genetic material, differential display methods (including DDRT-PCR and DD-PCR), nucleic acid microarray methods , RNA-sequencing (shotgun sequencing of the entire transcriptome) and qRT-PCR (quantitative real-time PCR). The information obtained can be used for community profiling regarding the identity and activity of the bacteria present, such as phylogenetic analysis or microarray-based screening of nucleic acids encoding components of rRNA operons or other taxonomically informative loci. Examples of loci containing taxonomic information include the 16S rRNA gene, 23S rRNA gene, 5S rRNA gene, 5.8S rRNA gene, 12S rRNA gene, 18S rRNA gene, 28S rRNA gene, gyrB gene, rpoB gene, fusA gene, recA gene, coxl gene, nifD gene. Examples of taxonomic profiling processes to determine the taxa present in a population are described in US 20140155283. Identification of bacteria may involve characterizing the activity of one or more genes or one or more signaling pathways, such as genes associated with the nitrogen fixation pathway. In bacterial populations, there may also be synergistic interactions (where two components, through their combination, increase the desired effect by more than an additive amount) between different bacterial species.

[00178] The genetic change may be a gene selected from the group consisting of: nifA, nifL, ntrB, ntrC, glnA, glnB, glnK, draT, amtB, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE , nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB and nifQ. The genetic change may be a change in a gene encoding a protein with functionality selected from the group consisting of: glutamine synthetase, glutaminase, glutamine synthetase-adenylyltransferase, transcriptional activator, anti-transcriptional activator, pyruvate:flavodoxin oxidoreductase, flavodoxin or ADP-D-ribosyltransferase NAD+:dinitrogenase reductase. The genetic change may be a mutation resulting in one or more of: increased expression or activity of NifA or glutaminase; decreased expression or activity of NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT, AmtB; reduced adenylate-removing activity of GlnE; or reduced uridylyl scavenging activity of GlnD. Introduction of a genetic change may involve the insertion and/or deletion of one or more nucleotides at the target site, for example, 1, 2, 3, 4, 5, 10, 25, 50, 100, 250, 500 or more nucleotides. The genetic change introduced into one or more bacteria in the methods disclosed herein may be a knockout mutation (eg, promoter deletion, insertion or deletion to produce a premature stop codon, deletion of an entire gene), or it may be an elimination or eliminating the activity of a protein domain (eg, a point mutation affecting the active site, or deletion of part of a gene encoding the corresponding part of the protein product), or it may alter or eliminate the regulatory sequence of the target gene. One or more regulatory sequences may also be inserted, including heterologous regulatory sequences and regulatory sequences contained in the genome of the bacterial species or genus corresponding to the bacteria into which the genetic change is introduced. In addition, regulatory sequences can be selected based on the level of gene expression in the bacterial culture or plant tissue. The genetic change may be a predetermined genetic change intentionally introduced into a target site. The genetic change may be a random mutation within the target site. The genetic change may be an insertion or deletion of one or more nucleotides. In some cases, many different genetic changes (eg, 2, 3, 4, 5, 10 or more) are introduced into one or more isolated bacteria before exposing the plants to the bacteria to evaluate improvement. The plurality of genetic changes may be of any of the above types, the same or different types, and in any combination. In some cases, a plurality of different genetic changes are introduced sequentially, introducing a first genetic change after the first isolation step, a second genetic change after the second isolation step, and so on, to accumulate in the bacteria a plurality of genetic changes that impart successively improved qualities to the associated plants.

[00179] In general, the term “genetic change” refers to any change introduced into a polynucleotide sequence relative to a reference polynucleotide, such as a reference genome or part thereof, or a reference gene or part thereof. A genetic change may be called a "mutation", and the sequence or organism containing the genetic change may be called a "genetic variant" or "mutant". Genetic changes can have any number of effects, such as increasing or decreasing some biological activity, including gene expression, metabolism, and cell signaling. Genetic changes can be intentionally introduced into a target site or introduced randomly. A range of molecular tools and techniques are available to introduce a genetic change. For example, the genetic change can be introduced through polymerase chain reaction mutagenesis, oligonucleotide-directed mutagenesis, saturation mutagenesis, fragment shuffling mutagenesis, homologous recombination, recombination engineering, lambda Red-dependent recombination, CRISPR/Cas9 systems, chemical mutagenesis, and combinations thereof. . Chemical methods of introducing a genetic change include exposing the DNA to a chemical mutagen, such as ethyl methane sulfonate (EMS), methyl methane sulfonate (MMS), N-nitrosourea (EN U), N-methyl-N-nitro-N'-nitrosoguanidine, 4-nitroquinoline-N -oxide, diethyl sulfate, benzopyrene, cyclophosphamide, bleomycin, triethylmelamine, acrylamide monomer, nitrogen mustard, vincristine, diepoxyalkanes (e.g. diepoxybutane), ICR-170, formaldehyde, procarbazine hydrochloride, ethylene oxide, dimethylnitrosamine, 7,12-dimethylbenz(a)anthracene , chlorambucil, hexamethylphosphoramide, bisulfan and the like. Agents that cause radiation mutations include ultraviolet radiation, γ-irradiation, X-rays, and fast neutron bombardment. A genetic change can also be introduced into the nucleic acid using, for example, trimethylpsoralen with ultraviolet light. Random or targeted insertion of a transposable DNA element, such as a transposable element, is another suitable method for producing a genetic change. Genetic changes can be introduced into the nucleic acid during amplification in a cell-free in vitro system, for example, using a polymerase chain reaction (PCR) technique such as error-prone PCR. Genetic changes can be introduced into nucleic acid in vitro using DNA shuffling techniques (eg, exon shuffling, domain swapping, and the like). Genetic changes can also be introduced into the nucleic acid as a result of a deficiency of a DNA repair enzyme in the cell; for example, the presence in a cell of a mutant gene encoding a mutant DNA repair enzyme is expected to generate mutations at a high frequency (i.e., approximately 1 mutation/ 100 genes - 1 mutation/10,000 genes) in the cell genome. Examples of genes encoding DNA repair enzymes include, but are not limited to, Mut H, Mut S, Mut L, and Mut U and their homologs in other species (e.g., MSH 1 6, PMS 1 2, MLH 1, GTBP, ERCC-1 etc). Illustrative descriptions of various methods for introducing genetic changes are contained, for example, in Stemple (2004) Nature 5:1-7; Chiang et al. (1993) PCR Methods Appl 2(3): 210-217; Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; and US Patent Nos. 6,033,861 and 6,773,900.

[00180] Genetic changes introduced into microorganisms can be classified as transgenic, cisgenic, intragenomic, intrageneric, intergeneric, synthetic, evolved, rearranged, or SNP (single nucleotide polymorphisms).

[00181] A genetic change can be introduced into a variety of metabolic pathways in microorganisms to produce improvements in the qualities described above. Exemplary pathways include sulfur uptake pathway, glycogen biosynthesis, glutamine regulatory pathway, molybdenum uptake pathway, nitrogen fixation pathway, ammonia assimilation, ammonia excretion or secretion, nitrogen uptake, glutamine biosynthesis, annamox, phosphate solubilization, organic acid transport, organic acid production, agglutinin production , reactive oxygen radical scavenging genes, indolylacetic acid biosynthesis, trehalose biosynthesis, enzymes or pathways responsible for plant cell wall breakdown, genes responsible for root attachment, exopolysaccharide secretion, glutamate synthase pathway, iron uptake pathways, siderophore pathway, chitinase pathway, ACC deaminase, glutathione biosynthesis, genes responsible for phosphorus signaling, quorum quenching pathway, cytochrome pathways, hemoglobin pathway, bacterial hemoglobin-like pathway, small RNA rsmZ, rhizobitoxin biosynthesis, adhesion protein lapA, AHL quorum sensing pathway, phenazine biosynthesis, cyclic lipopeptide biosynthesis and production of antibiotics.

[00182] CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats)/CRISPR-associated (Cas) systems can be used to introduce desired mutations. CRISPR/Cas9 provides bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNA (crRNA) to drive the silencing of invasive nucleic acids. The Cas9 protein (or its functional equivalent and/or variant, i.e., a Cas9-like protein) inherently possesses DNA endonuclease activity dependent on the association of the protein with two natural or synthetic RNA molecules called crRNA and tracrRNA (also called guide RNA). In some cases, the two molecules are covalently linked to form a single molecule (also called single guide RNA (“sgRNA”). Thus, Cas9 or Cas9-like protein binds to DNA-targeting RNA (this term includes both the two-molecule configuration of guide RNA, and a single-molecule guide RNA configuration) that activates Cas9 or Cas9-like protein and directs the protein to the target nucleic acid sequence. If Cas9 or Cas9-like protein retains its natural enzymatic function, it will cleave the target DNA, creating a double-strand break that may result in genome modification (i.e. editing: deletion, insertion (when a donor polynucleotide is present), substitution, etc.), thereby altering gene expression.Some Cas9 variants (which are covered by the term Cas9-like) have been modified to such such that they have reduced DNA cleavage activity (in some cases they cleave one strand instead of both strands of the target DNA, while in other cases they have DNA cleavage activity ranging from greatly reduced to absent). Additional illustrative descriptions of CRISPR systems for introducing a genetic change are contained, for example, in US8795965.

[00183] As a cyclic amplification method in polymerase chain reaction (PCR) mutagenesis, mutagenic primers are used to introduce desired mutations. PCR involves cycles of denaturation, annealing, and extension. After PCR amplification, selection of mutant DNA and removal of parental plasmid DNA can be accomplished by: 1) replacing dCTP with hydroxymethylated-dCTP during PCR followed by restriction enzyme digestion to remove only non-hydroxymethylated parental DNA; 2) simultaneous mutagenesis of both the antibiotic resistance gene and the gene under study, changing the plasmid to another antibiotic resistance, with the new antibiotic resistance then facilitating the selection of the desired mutation; 3) after introducing the desired mutation, cleavage of the parent methylated template DNA with the restriction enzyme Dpnl, which cleaves only methylated DNA, with the help of which mutagenized unmethylated chains are detected; or 4) circularization of mutated PCR products in an additional ligation reaction to increase the efficiency of transformation of mutated DNA. Additional description of illustrative methods is contained, for example, in US 7132265, US 6713285, US 6673610, US 6391548, US 5789166, US 5780270, US 5354670, US 5071743 and US 20100267147.

[00184] Oligonucleotide-directed mutagenesis, also called site-directed mutagenesis, typically uses a synthetic DNA primer. This synthetic primer contains the desired mutation and is complementary to the template DNA around the mutation site so that it can hybridize with DNA in the gene of interest. The mutation may be a single base change (point mutation), a multiple base change, a deletion or insertion, or a combination thereof. The single-stranded primer is then extended using DNA polymerase, which copies the rest of the gene. The gene thus copied contains the mutated site and can then be introduced into a host cell as a vector and cloned. Finally, mutants can be selected by DNA sequencing to verify that they contain the desired mutation.

[00185] Genetic changes can be introduced using error-prone PCR. In this technique, the gene of interest is amplified using DNA polymerase under conditions that are insufficient for fidelity of sequence replication. As a result, the amplification products contain at least one sequence error. When a gene is amplified and the resulting reaction product(s) contain one or more changes in sequence from the template molecule, the resulting products are mutated from the template. Another means of introducing random mutations is to expose the cells to a chemical mutagen such as nitrosoguanidine or ethyl methane sulfonate (Nestmann, Mutat Res 1975 June; 28(3):323-30), and the vector containing the gene is then isolated from the host.

[00186] Saturation mutagenesis is another form of random mutagenesis that attempts to generate all or nearly all possible mutations at a specific site or narrow region of a gene. In general, saturation mutagenesis involves subjecting to mutagenesis the entire set of mutagen cassettes (where each cassette is, for example, 1-500 bases in length) at a specific polynucleotide sequence to be mutated (wherein the sequence to be mutated is, for example, 15 to 100 bases in length) 000 bases). Therefore, a group of mutations (eg, ranging from 1 to 100 mutations) is introduced into each cassette to be mutated. The group of mutations to be introduced into one cassette may be different from or the same as a second group of mutations to be introduced into a second cassette during the application of one cycle of saturating mutagenesis. Examples of such groupings are deletions, additions, groupings of certain codons, and groupings of certain nucleotide cassettes.

[00187] Fragment shuffling mutagenesis, also called DNA shuffling, is a method for the rapid spread of beneficial mutations. An example shuffling process uses DNase to fragment a set of parental genes into sections of, for example, approximately 50-100 bp in length. A polymerase chain reaction (PCR) is then carried out without primers - DNA fragments with sufficient overlapping homologous sequence will anneal to each other and then be extended by DNA polymerase. Several cycles of this PCR extension are allowed after some DNA molecules have reached the size of the parent genes. These genes can then be amplified by another PCR, this time adding primers designed to be complementary to the ends of the strands. Primers may have additional sequences added to their 5' ends, such as restriction enzyme recognition site sequences necessary for ligation into a cloning vector. Additional examples of permutation techniques are provided in US 20050266541.

[00188] Homologous recombination mutagenesis involves recombination between an exogenous DNA fragment and a target polynucleotide sequence. After a double-strand break occurs, sections of DNA around the 5' ends of the break are trimmed in a process called resection. In the next stage of strand invasion, the protruding 3' end of the broken DNA molecule is then “incorporated” into a similar or identical DNA molecule that is not broken. This method can be used for gene deletion, exon removal, gene addition, and introduction of point mutations. Homologous recombination mutagenesis can be constant or dependent on certain conditions. Typically, a recombination matrix is also provided. The recombination template may be a component of another vector, contained in a separate vector, or provided as a separate polynucleotide. In some embodiments, the recombination template is for use as a template in homologous recombination, for example, within or near a target sequence nicked or cleaved by a site-specific nuclease. The template polynucleotide may be of any suitable length, for example, about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000 nucleotides or more. In some embodiments, the template polynucleotide is complementary to a portion of the polynucleotide containing the target sequence. When optimally aligned, the template polynucleotide may overlap one or more nucleotides of the target sequences (e.g., approximately 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 nucleotides or more). In some embodiments, when the template sequence and the polynucleotide containing the target sequence are optimally aligned, the closest nucleotide of the template polynucleotide is within about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400 , 500, 1000, 5000, 10000 or more nucleotides from the target sequence. Non-limiting examples of site-directed nucleases useful in homologous recombination methods include zinc finger nucleases, CRISPR nucleases, TALE nucleases and meganuclease. For a more detailed description of the use of such nucleases, see, for example, US 8795965 and US 20140301990.

[00189] Mutagens can be used to create genetic changes, creating primarily point mutations and short deletions, insertions, transversions and/or transitions, including chemical mutagens or radiation. Mutagens include, but are not limited to, ethyl methane sulfonate, methyl methane sulfonate, N-ethyl-N-nitrosourea, triethylmelamine, N-methyl-N-nitrosourea, procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitrosamine, N -methyl-N'-nitro-nitrosoguanidine, nitrosoguanidine, 2-aminopurine, 7,12-dimethylbenz(a)anthracene, ethylene oxide, hexamethylphosphoramide, bisulfane, diepoxyalkanes (diepoxyoctane, diepoxybutane and the like), 2-methoxy-6-chloro- 9[3-(ethyl-2-chloroethyl)aminopropylamino]acridine dihydrochloride and formaldehyde.

[00190] Introduction of a genetic change may be an incomplete process such that some bacteria in the population of treated bacteria carry the desired mutation and others do not. In some cases, it is desirable to apply selection pressure to enrich for bacteria carrying the desired genetic variation. Traditionally, selection for successful genetic variants has involved selection for or against a specific functionality conferred or eliminated by a genetic change, such as the insertion of an antibiotic resistance gene or the elimination of a metabolic activity capable of converting a non-lethal compound into a lethal metabolite. It is also possible to apply selection pressure based on the polynucleotide sequence itself, such that only the desired genetic change needs to be introduced (eg, without the simultaneous need for a selectable marker). In this case, selection pressure may involve the segregation of genomes lacking the genetic change introduced at the target site, so that selection is effectively directed against the reference sequence into which the genetic change is to be introduced. Typically, cleavage occurs within 100 nucleotides of the target site (eg, within 75, 50, 25, 10 or less nucleotides of the target site, including cleavage adjacent to or within the target site). Cleavage may be directed by a site-specific nuclease selected from the group consisting of a zinc finger nuclease, a CRISPR nuclease, a TALE nuclease (TALEN), or a meganuclease. This process is similar to processes for enhancing homologous recombination at a target site, except that no template for homologous recombination is provided. As a result, bacteria lacking the desired genetic change are more likely to undergo breakdown, which, if not repaired, results in cell death. The bacteria that survive the selection can then be isolated for use on plants to evaluate for improved quality.

[00191] A CRISPR nuclease can be used as a site-specific nuclease to direct cleavage to a target site. Improved selection of mutant microorganisms can be achieved by using Cas9 to kill unmutated cells. The plants are then inoculated with the mutated microorganisms to reaffirm the symbiosis and create evolutionary selection pressure to select for effective symbionts. The microorganisms can then be re-isolated from plant tissues. CRISPR nuclease systems used for selection against non-variant organisms may use elements similar to those described above for introducing a genetic change, except that no template for homologous recombination is provided. Cleavage directed at the target site thus enhances the death of affected cells.

[00192] Other options for specifically inducing target site cleavage are available, such as zinc finger nucleases, TALE nuclease (TALEN) systems, and meganuclease. Zinc finger nucleases (ZFNs) are artificial DNA endonucleases produced by fusing the DNA binding domain of a zinc finger with a DNA cleavage domain. ZFNs can be designed to target desired DNA sequences, and this allows zinc finger nucleases to cleave unique target sequences. When introduced into a cell, ZFNs can be used to edit target DNA in the cell (eg, the cell's genome) by inducing double-strand breaks. Transcription activator-like effector nucleases (TALENs) are artificial DNA endonucleases produced by fusing the TAL effector DNA-binding domain (transcription activator-like) with a DNA cleavage domain. TALENs can be rapidly engineered to bind virtually any desired DNA sequence, and when introduced into a cell, TALENs can be used to edit target DNA in a specified cell (eg, the cell's genome) by inducing double-strand breaks. Meganucleases (homing endonuclease) are endodeoxyribonucleases characterized by a large recognition site (double-stranded DNA sequences of 12-40 nucleotide pairs). Meganucleases can be used to replace, eliminate or modify highly targeted sequences. By modifying their recognition sequence through protein engineering, the target sequence can be altered. Meganucleases can be used to modify all types of genome, be it bacterial, plant or animal, and they are generally grouped into four families: LAGLIDADG family, GIY-YIG family, His-Cyst box family and HNH family. Exemplary homing endonucleases include I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I -TevI, I-TevII and I-TevIII.

[00193] The methods of the present invention can be used to introduce or improve one or more of a variety of desirable qualities. Examples of traits that can be introduced or improved include: root biomass, root length, height, shoot length, leaf number, water efficiency, total biomass, yield, fruit size, grain size, photosynthetic rate, drought tolerance, heat tolerance, salt tolerance, tolerance to nematode stress, fungal pathogen resistance, bacterial pathogen resistance, viral pathogen resistance, metabolite level and proteome expression. Desirable qualities including height, total biomass, root and/or shoot biomass, seed germination, seedling survival, photosynthetic efficiency, transpiration rate, seed/fruit number or weight, grain or fruit yield of the plant, leaf chlorophyll content, photosynthetic rate, length roots, or any combination thereof, can be used to measure growth and compared with the growth rate of control crop plants (eg plants without improved traits) grown under identical conditions.

[00194] A preferred quality to be introduced or improved is nitrogen fixation, as described herein. In some cases, a plant produced by the methods described herein exhibits a difference in quality that is at least about 5%, e.g., at least about 5%, at least about 8%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50% by at least about 60%, at least about 75%, at least about 80%, at least about 80%, at least about 90%, or at least 100%, according to at least about 200%, at least about 300%, at least about 400% or more greater than a control crop plant grown under the same soil conditions. In additional examples, a plant produced by the methods described herein exhibits a difference in quality that is at least about 5% greater, such as at least about 5%, at least about 8%, at least about by 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50 %, at least about 60%, at least about 75%, at least about 80%, at least about 80%, at least about 90%, or at least 100%, at least about 200%, at least about 300%, at least about 400% or more greater than a control crop plant grown under similar soil conditions.

[00195] The quality to be improved may be assessed under conditions involving the application of one or more biotic or abiotic stressors. Examples of stressors include abiotic stresses (such as heat stress, salt stress, drought stress, cold stress, and nutrient stress) and biotic stresses (such as nematode stress, stress, insect pest stress, fungal pathogen stress, bacterial pathogen stress, and viral pathogen stress).

The quality improved by the methods and compositions of the present invention may be nitrogen fixation, including in a plant previously incapable of nitrogen fixation. In some cases, bacteria isolated in accordance with the method described herein produce 1% or more (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or more) of plant nitrogen, which may represent an increase in nitrogen fixation capacity of at least 2 times (e.g., 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 50 times, 100 times, 1000 times or more) compared to bacteria isolated from the first plant before any genetic changes were introduced. In some cases, bacteria produce 5% or more of the plant's nitrogen. The desired level of nitrogen fixation can be achieved after repeating the steps of introducing a genetic change, exposing multiple plants, and isolating bacteria from the plants with improved quality one or more times (e.g., 1, 2, 3, 4, 5, 10, 15, 25 or more once). In some cases, increased levels of nitrogen fixation are achieved in the presence of a fertilizer supplemented with glutamine, ammonia, or another chemical source of nitrogen. There are known methods for assessing the degree of nitrogen fixation, examples of which are described herein.

[00196] Microbial breeding is a method for systematically identifying and improving the role of species in the microbiome of a crop. The method consists of three stages: 1) selection of candidate species by mapping plant-microbe interactions and predicting regulatory networks associated with a particular phenotype, 2) pragmatic and predictive improvement of microbial phenotypes through intraspecific crossing of regulatory networks and gene clusters, and 3) screening and selection of new genotypes of microorganisms that provide the desired phenotypes of crops. To systematically evaluate strain improvement, a model is created that relates microbial community colonization dynamics to genetic activity through key species. This model is used to predict selection of genetic targets and increase the frequency of selection for improvements in microbiome-encoded traits of agronomic relevance.

Nitrogen fixation

[00197] Described herein are methods for increasing nitrogen fixation in a plant, comprising exposing the plant to bacteria containing one or more genetic changes introduced into one or more nitrogen fixation regulating genes, wherein the bacteria produce 1% or more of the plant's nitrogen (e.g., 2 %, 5%, 10% or more), which may represent an increase in nitrogen fixation capacity of at least 2 times compared to the plant in the absence of bacteria. Bacteria can produce nitrogen in the presence of fertilizer containing glutamine, urea, nitrates or ammonia. Genetic changes may be any of the genetic changes described herein, including the examples presented above, in any number and in any combination. The genetic change may be introduced into a gene selected from the group consisting of nifA, nifL, ntrB, ntrC, glutamine synthetase, glnA, glnB, glnK, draT, amtB, glutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifY , nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB and nifQ. The genetic change may be a mutation resulting in one or more of: increased expression or activity of nifA or glutaminase; decreased expression or activity of nifL, ntrB, glutamine synthetase, glnB, glnK, draT, amtB; reduced adenylate-removing activity of GlnE; or reduced uridylyl scavenging activity of GlnD. The genetic change introduced into one or more bacteria in the methods described herein may be a knockout mutation, or it may eliminate the regulatory sequence of the target gene, or it may involve the insertion of a heterologous regulatory sequence, for example, the insertion of a regulatory sequence contained in genome of the same species or genus of bacteria. The regulatory sequence may be selected based on the level of gene expression in the bacterial culture or plant tissue. Genetic change can be caused by chemical mutagenesis. Plants grown in stage (c) may be exposed to biotic or abiotic stressors.

[00198] The amount of nitrogen fixation occurring in the plants described herein can be measured in several ways, for example, using an acetylene reduction (AR) assay. The acetylene reduction assay can be performed in vitro or in vivo. Evidence that a particular bacterium is providing fixed nitrogen to the plant may include: 1) the plant's total N supply increases significantly upon inoculation, preferably with a concomitant increase in plant N concentration; 2) symptoms of nitrogen deficiency are reduced under N-limiting conditions during inoculation (which should include an increase in dry mass); 3) _N2 fixation is documented using a ^15N approach ( _which may be isotope dilution experiments, ^15N2 reduction assays, or ^15N natural abundance assays); 4) fixed N is included in the plant protein or metabolite; and 5) all of these effects are not observed in uninoculated plants or in plants inoculated with a mutant inoculum strain.

[00199] The wild-type nitrogen fixation regulatory cascade can be represented as a digital logic circuit where incoming O ₂ and NH ₄ ⁺ pass through the NOR gate, the effluent of which enters the AND gate in addition to ATP. In some embodiments, the methods disclosed herein disrupt the influence of NH ₄ ⁺ on this chain at multiple points in the regulatory cascade so that microorganisms can produce nitrogen even in fertilized fields. However, the methods disclosed herein also involve changing the influence of ATP or O ₂ on the chain, or replacing the chain with other regulatory cascades in the cell, or changing genetic circuits other than nitrogen fixation. Gene clusters can be rearranged to create functional products under the control of a heterologous regulatory system. By eliminating native regulatory elements outside and within the coding sequences of gene clusters and replacing them with alternative regulatory systems, the functional products of complex genetic operons and other gene clusters can be controlled and/or translocated into heterologous cells, including cells of different species other than the species from which the native ones were derived. genes. Once rearranged, synthetic gene clusters can be controlled by genetic circuits or other inducible regulatory systems, thereby controlling the expression of products in the desired manner. Expression cassettes can be designed to function as logic gates, pulse generators, oscillators, switches, or storage devices. The control expression cassette may be coupled to a promoter such that the expression cassette functions as an environmental sensor, such as an oxygen sensor, a temperature sensor, a tactile sensor, an osmotic stress sensor, a membrane stress sensor, or a redox sensor.

[00200] For example, the nifL, nifA, nifT, and nifX genes may be deleted from the nif gene cluster. Synthetic genes can be constructed by randomizing the codons of the DNA encoding each amino acid sequence. Codon selection is performed so that codon usage is as different as possible from codon usage in the native gene. The proposed sequences are scanned for any undesirable features such as restriction enzyme recognition sites, transposon recognition sites, repetitive sequences, sigma-54 and sigma-70 promoters, cryptic ribosome binding sites and rho-independent terminators. Synthetic ribosome binding sites are selected to match the strength of each corresponding native ribosome binding site, for example by constructing a fluorescent reporter plasmid in which the 150 bp surrounding the gene start codon (-60 to +90) is fused to fluorescent gene. This chimera can be expressed under the control of the Ptac promoter and fluorescence can be measured by flow cytometry. To create synthetic ribosome binding sites, a reporter plasmid library is generated using 150 bp. (-60 to +90) synthetic expression cassette. Briefly, a synthetic expression cassette may consist of a random DNA spacer, a degenerate sequence encoding an RBS library, and a coding sequence for each synthetic gene. Several clones are screened to identify a synthetic ribosome binding site that best matches the native ribosome binding site. In this way, synthetic operons consisting of the same genes as native operons are constructed and tested for functional complementation. A more detailed illustrative description of synthetic operons is provided in US 20140329326.

Types of bacteria

[00201] Microorganisms useful in the methods and compositions disclosed herein can be obtained from any source. In some cases, the microorganisms may be bacteria, archaea, protozoa, or fungi. The microorganisms of the present invention may be nitrogen-fixing microorganisms, such as nitrogen-fixing bacteria, nitrogen-fixing archaea, nitrogen-fixing fungi, nitrogen-fixing yeasts or nitrogen-fixing protozoa. The microorganisms used in the methods and compositions disclosed herein may be spore-forming microorganisms, such as spore-forming bacteria. In some cases, bacteria suitable for the methods and compositions disclosed herein may be gram-positive bacteria or gram-negative bacteria. In some cases, the bacteria may be endospore-forming bacteria belonging to the phylum Firmicute. In some cases, the bacteria may be diazotrophic. In some cases, the bacteria may not be diazotrophic.

[00202] The methods and compositions of this invention can be used with archaea, such as, for example, Methanothermobacter thermoautotrophicus.

[00203] In some cases, bacteria that may be suitable include, but are not limited to, Agrobacterium radiobacter, Bacillus acidocaldarius, Bacillus acidoterrestris, Bacillus agri, Bacillus aizawai, Bacillus albolactis, Bacillus alcalophilus, Bacillus alvei, Bacillus aminoglucosidicus, Bacillus aminovorans, Bacillus amylolyticus (also known as Paenibacillus amylolyticus) Bacillus amyloliquefaciens, Bacillus aneurinolyticus, Bacillus atrophaeus, Bacillus azotoformans, Bacillus badius, Bacillus cereus (synonyms: Bacillus endorhythmos, Bacillus medusa), Bacillus chitinosporus, Bacillus circulans, Bacillus coagul ans, Bacillus endoparasiticus Bacillus fastidiosus, Bacillus firmus, Bacillus kurstaki, Bacillus lacticola, Bacillus lactimorbus, Bacillus lactis, Bacillus laterosporus (also known as Brevibacillus laterosporus), Bacillus lautus, Bacillus lentimorbus, Bacillus lentus, Bacillus licheniformis, Bacillus maroccanus, Bacillus megaterium, Bacillus metiens, Bacillus mycoides, Bacillus natto, Bacillus nematocida, Bacillus nigrificans, Bacillus nigrum, Bacillus pantothenticus, Bacillus popillae, Bacillus psychrosaccharolyticus, Bacillus pumilus, Bacillus siamensis, Bacillus smithii, Bacillus sphaericus, Bacillus subtilis, Bacillus thuringiensis, Bacillus uniflagellatus, Bradyrhizobium jap onicum, Brevibacillus brevis Brevibacillus laterosporus (formerly - Bacillus laterosporus), Chromobacterium subtsugae, Delftia acidovorans, Lactobacillus acidophilus, Lysobacter antibioticus, Lysobacter enzymogenes, Paenibacillus alvei, Paenibacillus polymyxa, Paenibacillus popilliae (formerly Bacillus popilliae), Pantoea agglomerans, Pasteuria penetrans (formerly Bacillus penetrans), Pasteur ia usgae, Pectobacterium carotovorum (formerly Erwinia carotovora), Pseudomonas aeruginosa, Pseudomonas aureofaciens, Pseudomonas cepacia (formerly known as Burkholderia cepacia), Pseudomonas chlororaphis, Pseudomonas fluorescens, Pseudomonas proradix, Pseudomonas putida, Pseudomonas sy ringae, Serratia entomophila, Serratia marcescens, Streptomyces colombiensis, Streptomyces galbus, Streptomyces goshikiensis, Streptomyces griseoviridis, Streptomyces lavendulae, Streptomyces prasinus, Streptomyces saraceticus, Streptomyces venezuelae, Xanthomonas campestris, Xenorhabdus luminescens, Xenorhabdus nematophila, Rhodococcus globerulus AQ719 (register NRRL accession number B-21663), Bacillus sp. AQ175 (registration number ATCC 55608), Bacillus sp. AQ 177 (registration number ATCC 55609), Bacillus sp. AQ178 (ATCC registration number 53522) and Streptomyces sp. strain, NRRL registration number B-30145. In some cases, the bacterium may be Azotobacter chroococcum, Methanosarcina barkeri, Klesiella pneumoniae, Azotobacter vinelandii, Rhodobacter spharoides, Rhodobacter capsulatus, Rhodobcter palustris, Rhodosporillum rubrum, Rhizobium leguminosarum or Rhizobium etli.

[00204] In some cases, the bacterium may be a Clostridium species, for example, Clostridium pasteurianum, Clostridium beijerinckii, Clostridium perfringens, Clostridium tetani, Clostridium acetobutylicum.

[00205] In some cases, the bacteria used in the methods and compositions of the present invention may be cyanobacteria. Examples of cyanobacterial genera include Anabaena (eg Anagaena sp. PCC7120), Nostoc (eg Nostoc punctiforme) or Synechocystis (eg Synechocystis sp. PCC6803).

[00206] In some cases, the bacteria used in the methods and compositions of the present invention may be of the Chlorobi phylum, such as Chlorobium tepidum.

[00207] In some cases, microorganisms used in the methods and compositions of the present invention may contain a gene homologous to the known NifH gene. Sequences of known NifH genes can be found, for example, in the Zehr laboratory NifH database (https://wwwzehr.pmc.ucsc.edu/nifH_Database_Public/, April 4, 2014) or in the Buckley laboratory NifH database (http://www .css.cornell.edu/faculty/buckley/nifh.htm, and Gaby, John Christian, and Daniel H. Buckley, "A comprehensive aligned nifH gene database: a multipurpose tool for studies of nitrogen-fixing bacteria." Database 2014 (2014): bau001.). In some cases, microorganisms used in the methods and compositions of the present invention may contain a sequence encoding a polypeptide having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 96%, 98 %, 99%, or greater than 99% sequence identity from the Zehr laboratory NifH database (https://wwwzehr.pmc.ucsc.edu/nifH_Database_Public/, April 4, 2014). In some cases, microorganisms used in the methods and compositions of the present invention may contain a sequence encoding a polypeptide having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 96%, 98 %, 99%, or greater than 99% sequence identity from the NifH database of the Buckley laboratory (Gaby, John Christian, and Daniel H. Buckley. "A comprehensive aligned nifH gene database: a multipurpose tool for studies of nitrogen-fixing bacteria." Database 2014 (2014): bau001.).

[00208] Microorganisms useful for the methods and compositions described herein can be obtained by extracting microorganisms from the surfaces or tissues of native plants; grinding seeds to isolate microorganisms; planting seeds in various soil samples and isolating microorganisms from tissues; or inoculating plants with exogenous microorganisms and determining which microorganisms appear in plant tissues. Non-limiting examples of plant tissues include a seed, seedling, leaf, cut, plant, bulb or tuber. In some cases, bacteria are isolated from the seed. Parameters for sample processing can be varied to isolate different types of associated microorganisms, such as rhizosphere microorganisms, epiphytes, or endophytes. Bacteria can also be obtained from a repository, such as collections of environmental strains, rather than initially being isolated from the first plant. Microorganisms can be genotyped and phenotyped by sequencing the genomes of isolated microorganisms; in planta community profiling; characterization of transcriptomic functionality of communities or isolated microorganisms; or screening for microbial traits using selective or phenotypic media (e.g., nitrogen fixation or phosphate solubilization phenotypes). Selected strains or candidate populations can be obtained using sequence data; phenotypic data; plant data (e.g. genomic, phenotypic and/or yield data); soil data (e.g. pH, N/P/K content and/or major soil biotic communities); or any combination thereof.

[00209] The bacteria and methods for producing bacteria described herein can be applied to bacteria that can effectively self-propagate on leaf surfaces, root surfaces, or within plant tissues without causing a destructive plant defense response, or to bacteria that are resistant to plant defense responses . The bacteria described herein can be isolated by culturing a plant tissue extract or leaf surface wash in a medium without added nitrogen. However, the bacteria may be non-culturable, that is, it may not be known whether they are culturable or difficult to culture using standard methods known in the art. The bacteria described herein may be an endophyte, an epiphyte, or a bacterium that inhabits the rhizosphere of a plant (rhizosphere bacteria). Bacteria obtained after repeating the steps of introducing a genetic change, affecting a plurality of plants, and isolating bacteria from plants with improved quality one or more times (for example, 1, 2, 3, 4, 5, 10, 15, 25 or more times), may be endophytic, epiphytic or rhizosphere. Endophytes are organisms that enter plants without causing disease symptoms or causing the formation of symbiotic structures, and are of agronomic interest because they can enhance plant growth and improve plant nutrition (e.g., through nitrogen fixation). The bacteria may be a seed-borne endophyte. Seed-borne endophytes include bacteria associated with or derived from grass or plant seeds, such as seed-borne bacterial endophytes, found in mature, dry, intact (e.g., no cracks, visible fungal infections, or premature germination) seeds. The seed-borne bacterial endophyte may be associated with or derived from the seed surface; alternatively or in addition, it may be associated with or derived from the interior of the seed (eg, from a surface-sterilized seed). In some cases, a seed-borne bacterial endophyte is able to multiply in plant tissue, such as inside a seed. Additionally, in some cases, the seed-borne bacterial endophyte is able to survive desiccation.

[00210] The bacterium isolated in accordance with the methods of the invention, or used in the methods or compositions of the invention, may contain many different bacterial taxa in combination. By way of example, bacteria may include Proteobacteria (such as Pseudomonas, Enterobacter, Stenotrophomonas, Burkholderia, Rhizobium, Herbaspirillum, Pantoea, Serratia, Rahnella, Azospirillum, Azorhizobium, Azotobacter, Duganella, Delftia, Bradyrhizobiun, Sinorhizobium and Halomonas), Firmicutes (such as Bacillus, Paenibacillus, Lactobacillus, Mycoplasma and Acetabacterium) and Actinobacteria (such as Streptomyces, Rhodacoccus, Microbacterium and Curtobacterium). The bacteria used in the methods and compositions of the invention may include nitrogen-fixing bacterial consortia of two or more bacterial species. In some cases, one or more bacterial species in bacterial consortia may be capable of nitrogen fixation. In some cases, one or more species within bacterial consortia may promote or enhance the nitrogen-fixing ability of other bacteria. Nitrogen-fixing bacteria and bacteria that enhance the nitrogen-fixing ability of other bacteria may be the same or different. In some examples, a bacterial strain may be able to fix nitrogen in combination with another bacterial strain or in certain bacterial consortia, but may be unable to fix nitrogen in a monoculture. Examples of bacterial genera that may occur in nitrogen-fixing bacterial consortia include, but are not limited to, Herbaspirillum, Azospirillum, Enterobacter, and Bacillus.

Bacteria that can be produced by the methods disclosed herein include Azotobacter sp., Bradyrhizobium sp., Klebsiella sp. and Sinorhizobium sp. In some cases, the bacteria may be selected from the group consisting of: Azotobacter vinelandii, Bradyrhizobium japonicum, Klebsiella pneumoniae and Sinorhizobium meliloti. In some cases, the bacteria may belong to the genus Enterobacter or Rahnella. In some cases, the bacteria may belong to the genus Frankia or Clostridium. Examples of bacteria of the genus Clostridium include, but are not limited to, Clostridium acetobutilicum, Clostridium pasteurianum, Clostridium beijerinckii, Clostridium perfringens and Clostridium tetani. In some cases, the bacteria may belong to the genus Paenibacillus, for example Paenibacillus azotofixans, Paenibacillus borealis, Paenibacillus durus, Paenibacillus macerans, Paenibacillus polymyxa, Paenibacillus alvei, Paenibacillus amylolyticus, Paenibacillus campinasensis, Paenibacillus chibensis, Paenibacillus glucanolyticus, Paenibacillus illinoisensis, Paenibacillus larvae, subspecies Larvae , Paenibacillus larvae, subspecies Pulvifaciens, Paenibacillus lautus, Paenibacillus macerans, Paenibacillus macquariensis, Paenibacillus macquariensis, Paenibacillus pabuli, Paenibacillus peoriae or Paenibacillus polymyxa.

[00211] In some examples, bacteria isolated in accordance with the methods of the invention may belong to one or more of the following taxa: Achromobacter, Acidithiobacillus, Acidovorax, Acidovoraz, Acinetobacter, Actinoplanes, Adlercreutzia, Aerococcus, Aeromonas, Afipia, Agromyces, Ancylobacter, Arthrobacter, Atopostipes, Azospirillum, Bacillus, Bdellovibrio, Beijerinckia, Bosea, Bradyrhizobium, Brevibacillus, Brevundimonas, Burkholderia, Candidatus Haloredivivus, Caulobacter, Cellulomonas, Cellvibrio, Chryseobacterium, Citrobacter, Clostridium, Coraliomargarita, Corynebacterium, Cupriavidus , Curtobacterium, Curvibacter, Deinococcus, Delftia , Desemzia, Devosia, Dokdonella, Dyella, Enhydrobacter, Enterobacter, Enterococcus, Erwinia, Escherichia, Escherichia/Shigella, Exiguobacterium, Ferroglobus, Filimonas, Finegoldia, Flavisolibacter, Flavobacterium, Frigoribacterium, Gluconacetobacter, Hafnia, Halobaculum, Halomonas, Halosimplex, Herbaspirillum , Hymenobacter , Klebsiella, Kocuria, Kosakonia, Lactobacillus, Leclercia, Lentzea, Luteibacter, Luteimonas, Massilia, Mesorhizobium, Methylobacterium, Microbacterium, Micrococcus, Microvirga, Mycobacterium, Neisseria, Nocardia, Oceanibaculum, Ochrobactrum, Okibacterium, Oligotropha, Oryzihumus, Oxalophagus, Pa enibacillus, Panteoa , Pantoea, Pelomonas, Perlucidibaca, Plantibacter, Polynucleobacter, Propionibacterium, Propioniciclava, Pseudoclavibacter, Pseudomonas, Pseudonocardia, Pseudoxanthomonas, Psychrobacter, Ralstonia, Rheinheimera, Rhizobium, Rhodococcus, Rhodopseudomonas, Roseateles, Ruminococcus, Sebaldella, Sedi minibacillus, Sediminibacterium, Serratia, Shigella, Shinella , Sinorhizobium, Sinosporangium, Sphingobacterium, Sphingomonas, Sphingopyxis, Sphingosinicella, Staphylococcus, 25 Stenotrophomonas, Strenotrophomonas, Streptococcus, Streptomyces, Stygiolobus, Sulfurisphaera, Tatumella, Tepidimonas, Thermomonas, Thiobacillus, Variovorax, genus WPS-2 incertae sedis, Xanthomonas and Zimmermannella.

[00212] The bacterium can be obtained from any general terrestrial environment, including its soils, plants, fungi, animals (including invertebrates) and other biota, including sediments, water and biota of lakes and rivers; from the marine environment, its biota and sediments (e.g. seawater, sea mud, marine plants, marine invertebrates (e.g. sponges), marine vertebrates (e.g. fish)); geosphere of land and seas (regolith and stone, for example, crushed underground rocks, sands and clays); cryosphere and its meltwater; atmosphere (eg filtered airborne dust, clouds and raindrops); urban, industrial and other artificial environments (for example, accumulated organic and mineral substances on concrete, roadside ditches, roof surfaces and road surfaces).

[00213] The plants from which the bacteria are derived may be a plant that has one or more desirable qualities, for example, a plant that grows naturally in a particular environment or under certain conditions of interest. For example, a particular plant may grow naturally in sandy soil or sand with high salt content, or in extreme temperatures, or with little water, or it may be resistant to certain pests or diseases present in the environment, and this may be desirable for growing a commercial crop under such conditions, especially if they are, for example, the only conditions available in a particular geographic location. As another example, bacteria may be collected from industrial crops grown in such environmental conditions, or more specifically from individual plants of the crop that best demonstrate the quality of interest among the crop grown in any particular environment: e.g. , the fastest growing plants in a crop grown in salt-limited soils, or the least damaged plants in crops susceptible to severe insect damage or epidemic disease, or plants that have desired amounts of certain metabolites and other compounds, including fiber content, oil content and the like, or plants exhibiting desirable colors, tastes or odors. The bacteria can be collected from the plant of interest or any material found in the environment of interest, including fungi and other animal and plant biota, soil, water, sediment and other environmental elements, as mentioned previously.

[00214] Bacteria can be isolated from plant tissue. This release may occur from any suitable plant tissue, including, for example, the root, stem and leaves, as well as the reproductive tissues of the plant. For example, traditional isolation methods from plants typically involve sterile separation of the plant material of interest (e.g., root or stem length, leaves), sterilization of the surface with a suitable solution (e.g., 2% sodium hypochlorite), and then placing the plant material on a microbial growth medium. . Alternatively, the plant material, the surface of which has been sterilized, can be ground in a sterile liquid (usually water), and a liquid suspension comprising small pieces of ground plant material can be spread over the surface of a suitable solid agar medium or media, which may be or not be selective (for example, contain only phytic acid as a source of phosphorus). This approach is particularly suitable for bacteria that form isolated colonies, and these can be collected individually on separate culture plates and further purified to a single species by well known methods. Alternatively, samples of plant roots or foliage may not be subjected to surface sterilization, but only thoroughly washed to allow surface-dwelling epiphytic microorganisms to be included in the isolation process, or epiphytic microorganisms may be isolated separately by imprinting pieces of plant roots, stems, or leaves. on the surface of the agar medium and removing them, after which individual colonies are isolated as described above. This approach is particularly suitable for bacteria, for example. Alternatively, the roots can be treated without washing away small amounts of soil adhering to the roots, allowing the inclusion of microorganisms that colonize the plant's rhizosphere. Otherwise, soil adhering to the roots can be removed, diluted and spread on suitable selective and non-selective agar media to isolate individual colonies of rhizosphere bacteria.

[00215] Biologically pure cultures of Rahnella aquatilis and Enterobacter sacchari were deposited on July 14, 2015 at the American Type Culture Collection (ATCC; international depository authority), Manassas, Virginia, USA, and were assigned patent deposit number PTA-122293 and PTA-122294, respectively. These deposits were made in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and its Regulations (Budapest Treaty).

Compositions

[00216] Compositions containing bacteria or bacterial populations prepared in accordance with the methods described herein and/or having the characteristics described herein may be in the form of a liquid, foam or dry product. In some examples, the composition containing the bacterial populations may be in the form of a dry powder, a suspension of powder and water, or a fluid seed treatment composition.

[00217] The composition can be manufactured in bioreactors such as continuous stirred tank reactors, batch reactors, and on a farm. In some examples, the compositions may be stored in a container, such as a bottle, or in small bulk quantities. In some examples, the compositions may be stored in an object selected from the group consisting of a bottle, a can, an ampoule, a packaging container, a vessel, a bag, a box, a bin, an envelope, a carton, a container, a silo, a shipping container, a truck bed, and/or packaging .

[00218] The compositions can also be used to improve plant performance. In some examples, one or more compositions may be applied as a coating to the seed. In some examples, one or more compositions may be applied as a coating to the sprout. In some examples, one or more compositions may be applied as a coating to the surface of the seed. In some examples, one or more may be applied as a layer above the surface of the seed. In some examples, the composition applied as a coating to the seed may be in liquid form, in dry product form, in foam form, in the form of a powder and water suspension, or a flowable seed treatment composition. In some examples, one or more compositions may be applied to the seed and/or sprout by spraying, dipping, coating, encapsulating and/or coating the seed and/or sprout with one or more compositions. In some examples, a plurality of bacteria or bacterial populations may be coated on a plant seed and/or seedling. In some examples, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more than ten bacteria in the bacterial combination may be selected from one of the following genera: Acidovorax, Agrobacterium, Bacillus, Burkholderia, Chryseobacterium, Curtobacterium, Enterobacter, Escherichia, Methylobacterium, Paenibacillus, Pantoea, Pseudomonas, Ralstonia, Saccharibacillus, Sphingomonas and Stenotrophomonas.

[00219] In some examples, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or more than ten bacteria and bacterial populations in a combination of endophytes selected from one of the following families: Bacillaceae, Burkholderiaceae, Comamonadaceae, Enterobacteriaceae, Flavobacteriaceae, Methylobacteriaceae, Microbacteriaceae, Paenibacillileae, Pseudomonnaceae, Rhizobiaceae, Sphingomonadaceae, Xanthomonadaceae, Cladosporiaceae, Gnomon iaceae, Incertae sedis, Lasiosphaeriaceae , Netriaceae and Pleosporaceae.

[00220] In some examples, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or more than ten bacteria and bacterial populations in a combination of endophytes selected from one of the following families: Bacillaceae, Burkholderiaceae, Comamonadaceae, Enterobacteriaceae, Flavobacteriaceae, Methylobacteriaceae, Microbacteriaceae, Paenibacillileae, Pseudomonnaceae, Rhizobiaceae, Sphingomonadaceae, Xanthomonadaceae, Cladosporiaceae, Gnomon iaceae, Incertae sedis, Lasiosphaeriaceae , Netriaceae, Pleosporaceae.

[00221] Examples of the compositions may include seed coatings for commercially important crops, such as sorghum, canola, tomatoes, strawberries, barley, rice, maize and wheat. Examples of the compositions may also include seed coatings for corn, soybeans, canola, sorghum, potatoes, rice, vegetables, grains and oilseeds. The seeds offered herein may be genetically modified organism (GMO), non-GMO, organic, or conventional. In some examples, the compositions may be sprayed onto aerial parts of the plant or applied to the roots by insertion into furrows in which plant seeds are sown, watering the soil, or dipping the roots into a suspension of the composition. In some examples, the compositions may be dehydrated in a suitable manner that preserves cell viability and the ability to artificially inoculate and colonize host plants. Bacterial species may be present in the compositions at concentrations ranging from 10 ⁸ to 10 ¹⁰ CFU/ml. In some examples, the compositions may be supplemented with micronutrient ions, such as molybdenum ions, iron ions, manganese ions, or combinations of these ions. The ion concentration in example compositions as described herein can range from about 0.1 to about 50 mM. Some examples of compositions may also include a carrier such as beta-glucan, carboxymethylcellulose (CMC), bacterial extracellular polymeric substance (EPS), sugar, animal milk, or other suitable carriers. In some examples, peat or planting materials may be used as the carrier, or biopolymers in which the composition is embedded may be used as the carrier.

[00222] Compositions containing the bacterial populations described herein can be applied as a coating to the surface of the seed. Also contemplated as such are compositions containing seeds coated with one or more of the bacteria described herein. The seed coating can be formed by mixing the bacterial population with a porous, chemically inert granular carrier. Alternatively, the compositions may be inserted directly into the furrows in which the seeds are sown, or sprayed onto the leaves of plants, or applied by dipping the roots into a suspension of the composition. An effective amount of the composition can be used to colonize the subsoil adjacent to the roots of a plant with viable, reproducing bacteria, or to colonize the leaves of a plant with viable, reproducing bacteria. Generally, an effective amount is an amount sufficient to produce plants with improved qualities (eg, a desired level of nitrogen fixation).

[00223] The bacterial compositions described herein can be formulated using an agriculturally acceptable carrier. A formulation suitable for these embodiments may include at least one component selected from the group consisting of a tackifier, a microbiological stabilizer, a fungicide, an antibacterial agent, a preservative, a stabilizer, a surfactant, an anti-caking agent, a herbicide, a nematicide, insecticide, plant growth regulator, fertilizer, rodenticide, desiccant, bactericide, nutrient or any combination thereof. In some examples, the compositions may be suitable for long-term storage. For example, any of the compositions described herein may include an agriculturally acceptable carrier (e.g., one or more of a fertilizer, such as a non-natural fertilizer, a binder, such as a non-natural binder, and a pesticide, such as a non-natural pesticide) . The non-natural binder may be, for example, a polymer, copolymer or synthetic wax. For example, any of the seeds, seedlings or coated plants described herein may contain such an agriculturally acceptable carrier in the seed coating. In any of the compositions or methods described herein, the agriculturally acceptable carrier may be or may include a non-natural compound (e.g., a non-natural fertilizer, a non-natural binder such as a polymer, copolymer or synthetic wax, a non-natural pesticide ). Non-limiting examples of agriculturally acceptable carriers are described below. Additional examples of agriculturally acceptable carriers are known in the art.

[00224] In some cases, the bacteria are mixed with an agriculturally acceptable carrier. The carrier may be a solid carrier or a liquid carrier and be present in various forms, including microspheres, powders, emulsions and the like. The carrier may be any one or more carriers imparting various properties such as increased stability, wettability or dispersibility. Wetting agents may be included in the composition, such as natural or synthetic surfactants, which may be nonionic or ionic surfactants, or a combination thereof. Water-in-oil emulsions can also be used to prepare a composition comprising isolated bacteria (see, for example, US Pat. No. 7,485,451). Suitable preparations that may be formulated include wettable powders, granules, gels, agar plates or pellets, thickeners and the like, microencapsulated particles and the like, liquids such as aqueous fluids, aqueous suspensions, water-in-oil emulsions and etc. The preparation may include grain or legume products, such as ground grains or beans, broth or flour made from grains or beans, starch, sugar or oil.

[00225] In some embodiments, the agricultural carrier may be soil or a growing medium for plant growth. Other agricultural carriers that may be used include water, fertilizers, vegetable oils, wetting agents, or combinations thereof. Alternatively, the agricultural carrier may be a solid such as diatomaceous earth, loam, silica, alginate, clay, bentonite, vermiculite, bolls, other plant and animal products, or combinations thereof, including granules, pellets or slurries. Mixtures of any of the above ingredients are also contemplated as carriers, such as, but not limited to, pesta (flour and kaolin clay), agar or flour-based granules in loam, sand or clay, etc. Preparations may include bacterial food sources such as barley, rice or other biological materials such as seeds, plant parts, sugar cane bagasse, husks or stems from grain processing, shredded plant material or wood from construction waste, sawdust or fine fibers from recycling paper, fabric or wood.

[00226] For example, a fertilizer may be used to promote growth or provide nutrients to a seed, seedling, or plant. Non-limiting examples of fertilizers include nitrogen, phosphorus, potassium, calcium, sulfur, magnesium, boron, chloride, manganese, iron, zinc, copper, molybdenum and selenium (or salts thereof). Additional examples of fertilizers include one or more amino acids, salts, carbohydrates, vitamins, glucose, NaCl, yeast extract, NH ₄ H ₂ PO ₄ , (NH ₄ ) ₂ SO ₄ , glycerol, valine, L-leucine, lactic acid, propionic acid , succinic acid, malic acid, citric acid, potassium hydrogen tartrate, xylose, lyxose and lecithin. In one embodiment, the formulation may include a tackifier or sticky substance (called a binder) to help bind other active agents to the substance (eg, the surface of the seed). Such agents are used to combine bacteria with carriers, which may contain other compounds (eg, control agents that are not biological), to produce a coating composition. Such compositions help create coatings around the plant or seed to maintain contact of the microorganism and other agents with the plant or plant part. In one embodiment, the binders are selected from the group consisting of: alginate, gums, starches, lecithins, formononetin, polyvinyl alcohol, alkali metal formononetinate, hesperetin, polyvinyl acetate, cephalins, gum arabic, xanthan gum, mineral oil, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), arabinogalaktan, methyl cellulose, PAG-400, chitosan, polyacrylamide, polyacrylate, polyacrylonitril, glycerin, trietilenlycol, vinyl acetate, gelllan gellania, polyvinyl, polyvinyl, polyvinil, gkhatti and block-pllock-polymosopolim moat polyoxyethylene and polyoxbulene.

[00227] In some embodiments, the binders may be, for example, a wax such as carnauba wax, beeswax, Chinese wax, shellac wax, spermaceti wax, candelilla wax, castor wax, ouricuri wax and rice bran wax, polysaccharide ( eg starch, dextrins, maltodextrins, alginate and chitosans), fat, oil, protein (eg gelatin and zeins), acacias and shellacs. Binders may be non-natural compounds, such as polymers, copolymers and waxes. For example, non-limiting examples of polymers that can be used as a binder include: polyvinyl acetates, polyvinyl acetate copolymers, ethylene vinyl acetate (EVA) copolymers, polyvinyl alcohols, polyvinyl alcohol copolymers, celluloses (e.g., ethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, and carboxymethylcellulose), polyvinylpyrrolidones, vinyl chloride, vinylidene chloride copolymers, calcium lignosulfonates, acrylic copolymers, polyvinyl acrylates, polyethylene oxide, acylamide polymers and copolymers, polyhydroxyethyl acrylate, methyl acrylamide monomers and polychloroprene.

[00228] In some examples, one or more of the binders, antifungal agents, growth-regulating agents, and pesticides (eg, insecticide) are non-naturally occurring compounds (eg, in any combination). Additional examples of agriculturally acceptable carriers include dispersants (eg, polyvinylpyrrolidone/vinyl acetate PVPIVA S-630), surfactants, binders and fillers.

[00229] The formulation may also contain a surfactant. Non-limiting examples of surfactants include nitrogen/surfactant mixtures such as Prefer 28 (Cenex), Surf-N(US), Inhance (Brandt), P-28 (Wilfarm) and Patrol (Helena); esterified vegetable oils include Sun-It II (AmCy), MSO (UAP), Scoil (Agsco), Hasten (Wilfarm), and Mes-100 (Drexel); and organosilicone surfactants include Silwet L77 (UAP), Silikin (Terra), Dyne-Amic (Helena), Kinetic (Helena), Sylgard 309 (Wilbur-Ellis), and Century (Precision). In one embodiment, the surfactant is present at a concentration of 0.01 vol. % up to 10 vol. %. In yet another embodiment, the surfactant is present at a concentration of 0.1 vol. % up to 1 vol. %.

[00230] In some cases, the drug includes a microbiological stabilizer. Such agent may include a drying agent, which may include any compound or mixture of compounds that can be classified as a drying agent, regardless of whether the compound or compounds are used in such concentrations that they actually have a drying effect on the liquid inoculum. Such desiccants are ideally compatible with the bacterial population used and should promote the ability of the microbial population to survive application to the seed and survive drying. Examples of suitable drying agents include one or more of trehalose, sucrose, glycerol and methylene glycol. Other suitable drying agents include, but are not limited to, non-reducing sugars and sugar alcohols (eg, mannitol or sorbitol). The amount of drying agent included in the formulation may be from about 5% to about 50% (w/v), such as from about 10% to about 40%, from about 15% to about 35%, or from about 20% to approximately 30%. In some cases, it is preferred that the formulation contains agents such as a fungicide, antibacterial agent, herbicide, nematicide, insecticide, plant growth regulator, rodenticide, bactericide, or nutrient. In some examples, the agents may include protectants that provide protection against pathogens on the surface of the seeds. In some examples, controls may provide some level of control of soil-borne pathogens. In some examples, protectants may be effective primarily on the surface of the seed.

[00231] In some examples, the fungicide may include a compound or agent, chemical or biological, that can inhibit the growth of a fungus or kill a fungus. In some examples, the fungicide may include compounds that may be fungistatic or fungicidal. In some examples, the fungicide may be a protectant, or agents, that are effective primarily on the seed surface, providing protection against pathogens on the seed surface and providing some level of control of soil-borne pathogens. Non-limiting examples of protective fungicides include captan, maneb, thiram or fludioxonil.

[00232] In some examples, the fungicide may be a systemic fungicide that can be taken up by the emerging seedling and inhibit or kill the fungus within the tissues of the host plant. Systemic fungicides used for seed treatment include, but are not limited to, the following: azoxystrobin, carboxin, mefenoxam, metalaxyl, thiabendazole, trifloxystrobin, and various triazole fungicides including difenoconazole, ipconazole, tebuconazole, and triticonazole. Mefenoxam and metalaxyl are mainly used to control the aquatic molds Pythium and Phytophthora. Some fungicides are preferred depending on the plant species, either due to minor differences in the sensitivity of pathogenic fungal species or differences in fungicide distribution or plant sensitivity. In some examples, the fungicide may be a biological control agent such as a bacterium or fungus. Such organisms may parasitize pathogenic fungi or secrete toxins or other substances that may kill or otherwise interfere with fungal growth. Any type of fungicide, especially fungicides commonly used on plants, can be used as a control agent in a seed formulation.

[00233] In some examples, the seed coating composition contains a control agent having antibacterial properties. In one embodiment, the control agent with antibacterial properties is selected from compounds described elsewhere herein. In yet another embodiment, the compound is streptomycin, oxytetracycline, oxolinic acid, or gentamicin. Other examples of antibacterial compounds that can be used in a seed coating composition include compounds based on dichlorophen and benzyl alcohol semiformal (Proxel® from ICI or Acticide® RS from Thor Chemie and Kathon® MK 25 from Rohm & Haas), and isothiazolinone derivatives such as alkyl isothiazolinones and benzisothiazolinones (Acticide® MBS from Thor Chemie).

[00234] In some examples, the growth regulator is selected from the group consisting of: abscisic acid, amidochlor, ancymidol, 6-benzylaminopurine, brassinolide, butralin, chlormequat (chlormequat chloride), choline chloride, cyclanilide, daminozide, dikegulac, dimetipine, 2,6-dimethylpuridine , ethephon, flumetraline, flurprimidol, flutiacet, forchlorfenuron, gibberellic acid, inabenfide, indole-3-acetic acid, maleic acid hydrazide, mefluidide, mepiquat (mepiquat chloride), naphthylacetic acid, N-6-benzyladenine, paclobutrazol, prohexadione triti ophosphate, 2, 3,5-tri-iodobenzoic acid, trinexapac-ethyl and uniconazole. Additional non-limiting examples of growth regulators include brassinosteroids, cytokinins (e.g., kinetin and zeatin), auxins (e.g., indolylacetic acid and indolylacetylaspartate), flavonoids and isoflavonoids (e.g., formononetin and diosmetin), phytoalexins (e.g., glyceollin), and phytoalexin-inducing oligosaccharides (e.g., formononetin and diosmetin). e.g. pectin, chitin, chitosan, polygalacuronic acid and oligogalacturonic acid), and gibellerins. Such agents are ideally compatible with the agricultural seeds or sprouts to which the drug is applied (for example, they should not have a detrimental effect on the growth or health of the plant). In addition, the agent ideally does not pose safety concerns when used in humans, animals, or industry (e.g., there are no safety concerns or the compound is sufficiently unstable that a commercial herbal product derived from the plant will contain negligible amounts of the compound).

[00235] Some examples of nematode antagonist biological control agents include ARF18; 30 Arthrobotrys spp.; Chaetomium spp.; Cylindrocarpon spp.; Exophilia spp.; Fusarium spp.; Gliocladium spp.; Hirsutella spp.; Lecanicillium spp.; Monacrosporium spp.; Myrothecium spp.; Neocosmospora spp.; Paecilomyces spp.; Pochonia spp.; Stagonospora spp.; vesicular-arbuscular mycorrhizal fungi, Burkholderia spp.; Pasteuria spp., Brevibacillus spp.; Pseudomonas spp.; and Rhizobacteria. Particularly preferred nematode antagonist biocontrol agents include ARF18, Arthrobotrys oligospora, Arthrobotrys dactyloides, Chaetomium globosum, Cylindrocarpon heteronema, Exophilia jeanselmei, Exophilia pisciphila, Fusarium aspergilus, Fusarium solani, Gliocladium catenulatum, Gliocladium roseum, Gliocladium vix ens, Hirsutella rhossiliensis, Hirsutella minnesotensis, Lecanicillium lecanii, Monacrosporium drechsleri, Monacrosporium gephyropagum, Myrotehcium verrucaria, Neocosmospora vasinfecta, Paecilomyces lilacinus, Pochonia chlamydosporia, Stagonospora heteroderae, Stagonospora phaseoli, vesicular-arbuscular mycorrhizal fungi, Burkholderia cepacia, Pasteuria penetrans, Pasteuria thornei, Pasteuria nishizawae, Pasteuria ramosa, Pastrueia usage , Brevibacillus laterosporus strain G4, Pseudomonas fluorescens and Rhizobacteria.

[00236] By way of example, the nutrients may be selected from the group consisting of nitrogen fertilizer, including, but not limited to, urea, ammonium nitrate, ammonium sulfate, non-pressurized nitrogen solutions, ammonia water, anhydrous ammonia, ammonium thiosulfate , sulfur coated urea, formaldehyde urea, IBDU (isobutylidene diurea), polymer coated urea, calcium nitrate, formaldehyde urea and methylene urea, phosphate fertilizers such as diammonium phosphate, monoammonium phosphate, ammonium polyphosphate, concentrated superphosphate and triple superphosphate, and potassium fertilizers such as potassium chloride , potassium sulfate, potassium magnesium sulfate, potassium nitrate. Such compositions may exist as free salts or ions in the seed coating composition. Alternatively, the nutrients/fertilizers can be complexed or chelated to provide sustained release over time.

[00237] Some examples of rodenticides may include substances selected from the group consisting of 2-isovalerylindane-1,3-dione, 4-(quinoxalin-2-ylamino)benzenesulfonamide, alpha-chlorohydrin, aluminum phosphate, alpha-naphthylthiourea, arsenic trioxide , barium carbonate, bisthiosem, brodifacoum, bromadiolone, bromethalin, calcium cyanide, chloralose, chlorophacinone, cholecalciferol, coumachlor, coumafuryl, coumatetral, crimidine, difenacoum, difethialone, diphacinone, ergocalciferol, flocumafen, fluoroacetamide, flupropadine, flupropadine hydrox chloride, hydrogen cyanide, iodomethane , lindane, magnesium phosphide, methyl bromide, norbormide, fosacetim, phosphine, phosphorus, pindone, potassium arsenite, pyrinuron, scyliroside, sodium arsenite, sodium cyanide, sodium fluoroacetate, strychnine, thallium sulfate, warfarin and zinc phosphide.

[00238] In liquid form, such as solutions or suspensions, bacterial populations can be mixed or suspended in water or aqueous solutions. Suitable liquid diluents or carriers include water, aqueous solutions, petroleum distillates or other liquid carriers.

[00239] Solid compositions can be prepared by dispersing bacterial populations in and on a suitably partitioned solid carrier such as peat, wheat, bran, vermiculite, clay, talc, bentonite, diatomaceous earth, Fuller's earth, pasteurized soil, and the like. When such preparations are used in the form of wettable powders, biocompatible dispersing agents such as nonionic, anionic, amphoteric or cationic dispersing and emulsifying agents may be used.

[00240] Solid carriers used in the preparation of preparations include, for example, mineral carriers such as kaolin clay, pyrophyllite, bentonite, montmorillonite, diatomaceous earth, acid light soil, vermiculite and perlite, and inorganic salts such as ammonium sulfate, phosphate ammonium, ammonium nitrate, urea, ammonium chloride and calcium carbonate. Organic fine powders such as wheat flour, wheat bran and rice bran can also be used. Liquid carriers include vegetable oils such as soybean oil and cottonseed oil, glycerin, ethylene glycol, polyethylene glycol, propylene glycol, polypropylene glycol, etc.

Application of bacterial populations on crops

[00241] The bacterial or bacterial population composition described herein can be applied in-furrow, in talc, or as a seed treatment formulation. The composition may be applied to the seed mass as a bulk batch, as a small bulk batch, in a bag, or in talcum powder.

[00242] The sower can sow the treated seeds and grow the crop according to traditional methods, double row planting or no-till methods. Seed can be distributed using a control seed meter or a separate seed meter. Seeds can also be distributed using compressed air or by hand. Sowing seeds can be done using variable rate technology. In addition, the use of bacteria or bacterial populations described herein can be accomplished using variable rate techniques. In some examples, the bacteria can be applied to the seeds of corn, soybeans, canola, sorghum, potatoes, rice, vegetables, grains, pseudocereals and oilseeds. Examples of grains may include barley, fonio, oats, bristles, rye, pearl millet, sorghum, spelt, teff, triticale and wheat. Examples of pseudograins may include brosimum, buckwheat, cattail, chia, flax, amaranth, boscia senegal, quinoa, and sesame. In some examples, the seeds may be genetically modified organism (GMO), non-GMO, organic, or conventional.

[00243] Additional additives such as microfertilizer, PGR (Plant Growth Regulator), herbicide, insecticide and fungicide can be used to treat crops. Examples of additives include crop protection agents such as insecticides, nematicides, fungicides, enhancers such as dyes, polymers, granulating agents, priming and disinfectants, and other agents such as inoculant, PGR, softener and trace elements. PGRs can be natural or synthetic plant hormones that affect root growth, flowering, or stem elongation. PGRs may include auxins, gibberellins, cytokinins, ethylene, and abscisic acid (ABA).

[00244] The composition can be applied in a furrow in combination with a liquid fertilizer. In some examples, liquid fertilizer may be stored in tanks. NPK fertilizers contain macroelements sodium, phosphorus and potassium.

[00245] The composition can improve plant qualities, such as promoting plant growth, maintaining high chlorophyll content in leaves, increasing the number of fruits or seeds, and increasing the weight of fruits or seeds. The methods of the present invention can be used to introduce or improve one or more of a variety of desirable qualities. Examples of traits that can be introduced or improved include: root biomass, root length, height, shoot length, leaf number, water efficiency, total biomass, yield, fruit size, grain size, photosynthetic rate, drought tolerance, heat tolerance, salt tolerance, tolerance to low nitrogen stress, nitrogen use efficiency, nematode stress resistance, fungal pathogen resistance, bacterial pathogen resistance, viral pathogen resistance, metabolite level, metabolite level modulation and proteome expression. Desirable qualities including height, total biomass, root and/or shoot biomass, seed germination, seedling survival, photosynthetic efficiency, transpiration rate, seed/fruit number or weight, grain or fruit yield of the plant, leaf chlorophyll content, photosynthetic rate, length roots, or any combination thereof, can be used to measure growth and compared with the growth rate of control crop plants (eg, plants without the introduced and/or improved traits) grown under identical conditions. In some examples, desirable qualities including height, total biomass, root and/or shoot biomass, seed germination, seedling survival, photosynthetic efficiency, transpiration rate, seed/fruit number or weight, grain or fruit yield of the plant, leaf chlorophyll content, intensity photosynthesis, root length, or any combination thereof, can be used to measure growth and compared with the growth rate of control crop plants (e.g., plants without the improved and/or introduced traits) grown under similar conditions.

[00246] Agronomic quality of the host plant may include, but is not limited to, the following: altered oil content, altered protein content, altered seed carbohydrate composition, altered seed oil composition, and altered seed protein composition, chemical resistance, cold tolerance, delayed senescence , disease resistance, drought tolerance, ear weight, improved growth, improved health, heat tolerance, herbicide resistance, herbivore resistance, improved nitrogen fixation, improved nitrogen utilization, improved root architecture, improved water use efficiency, increased biomass, increased root length, increase in seed weight, increase in shoot length, increased yield, increased yield under conditions of water shortage, grain weight, grain moisture content, metal resistance, number of ears, number of grains per ear, number of pods, improved nutrition, pathogen resistance, pest resistance , improved photosynthetic capacity, salt tolerance, delayed leaf senescence, improved vigor, increased dry weight of mature seeds, increased wet weight of mature seeds, increased number of mature seeds per plant, increased chlorophyll content, increased number of pods per plant, increased length of pods per plant, decreased number of wilted leaves per plant, decrease in the number of significantly wilted leaves per plant and increase in the number of non-wilted leaves per plant, detectable modulation of metabolite level, detectable modulation of transcript level and detectable modulation of proteome, compared with a plant of the same line grown from seeds without the use of the indicated drug for seed treatment.

[00247] In some cases, plants are inoculated with bacteria or bacterial populations that are isolated from the same plant species as the plant element of the plant being inoculated. For example, a bacterium or bacterial population that is commonly found in one variety of maize (corn) is associated with a plant element of a plant of another variety of maize that in its natural state lacks said bacteria and bacterial populations. In one embodiment, the bacteria and bacterial populations are obtained from a plant of a related species as a plant element of the plant being inoculated. For example, bacteria and bacterial populations that are commonly found in Zea diploperennis (Iltis et al.), (Diploperennial teosinte) are applied to maize, or vice versa. In some cases, plants are inoculated with bacteria and bacterial populations heterologous to the plant element of the plant being inoculated. In one embodiment, the bacteria and bacterial populations are obtained from a plant of another species. For example, bacteria and bacterial populations that are typically found in dicotyledonous plants are applied to a monocotyledonous plant (eg, to inoculate corn with bacteria and bacterial populations derived from soybeans), or vice versa. In other cases, the bacteria and bacterial populations to be inoculated onto the plant are obtained from species related to the plant being inoculated. In one embodiment, the bacteria and bacterial populations are derived from a related taxon, such as a related species. The plant of another species may be an agricultural plant. In yet another embodiment, the bacteria and bacterial populations are part of the formulated composition inoculated into any element of the host plant.

[00248] In some examples, the bacterium or bacterial population is exogenous, where the bacteria and bacterial population are isolated from a plant other than the plant being inoculated. For example, in one embodiment, the bacteria or bacterial population may be isolated from another plant of the same species as the plant being inoculated. In some cases, the bacteria or bacterial population may be isolated from a species related to the plant being inoculated.

[00249] In some examples, bacteria and bacterial populations described herein are capable of moving from one tissue type to another. For example, the detection and isolation of bacteria and bacterial populations in mature plant tissues of the present invention after coating the outer surface of seeds demonstrates their ability to move from the outer surface of the seed into the vegetative tissues of the maturing plant. Therefore, in one embodiment, the population of bacteria and bacterial populations are capable of moving from the outer surface of the seed into the vegetative tissues of the plant. In one embodiment, the bacteria and bacterial populations coated on a plant seed are capable of becoming concentrated in another tissue of the plant when the seed germinates into the vegetative state. For example, bacteria and bacterial populations may be able to concentrate in any of the plant tissues, including root, adventitious root, embryonic root, root hair, shoot, leaf, flower, bud, panicle, meristem, pollen, pistil, ovary, stamen, fruit, creeping shoot, rhizome, nodule, tuber, trichome, stomatal cells, hydathodes, petal, sepal, scale, awn, vascular cambium, phloem and xylem. In one embodiment, the bacteria and bacterial populations are able to concentrate in the root and/or root hairs of the plant. In yet another embodiment, the bacteria and bacterial populations are capable of concentrating in photosynthetic tissues, such as leaves and shoots of a plant. In other cases, bacteria and bacterial populations are concentrated in the conducting tissues of the plant, such as the xylem and phloem. In yet another embodiment, the bacteria and bacterial populations are capable of concentrating in the reproductive tissues (flower, pollen, pistil, ovary, stamen, fruit) of the plant. In yet another embodiment, the bacteria and bacterial populations are capable of concentrating in the root, shoots, leaves, and reproductive tissues of the plant. In yet another embodiment, the bacteria and bacterial populations colonize the fruit or seed tissue of the plant. In yet another embodiment, the bacteria and bacterial populations are capable of colonizing a plant such that they are present on the surface of the plant (ie, their presence can be detected on the outer surface of the plant or in the episphere of the plant). In other embodiments, the bacteria and bacterial populations are capable of being concentrated in substantially all or all tissues of the plant. In certain embodiments, the bacteria and bacterial populations are not concentrated in the root of the plant. In other cases, bacteria and bacterial populations are not concentrated in photosynthetic plant tissues.

[00250] The effectiveness of the compositions can also be assessed by measuring the relative maturity of the crop or heat storage units (CHU). For example, the bacterial population may be applied to corn, and corn growth may be assessed according to the relative maturity of the corn grain or the time at which the corn grain reaches maximum weight. Heat storage units (CHU) can also be used to predict corn crop maturity. CHUs determine the amount of heat stored by measuring maximum daily temperatures during plant growth.

[00251] For example, bacteria can be concentrated in any of the plant tissues, including root, adventitious root, embryonic root, root hair, shoot, leaf, flower, bud, panicle, meristem, pollen, pistil, ovary, stamen, fruit, creeper , rhizome, node, tuber, trichome, stomatal cells, hydathodes, petal, sepal, scale, awn, vascular cambium, phloem and xylem. In yet another embodiment, the bacteria or bacterial population is capable of concentrating in photosynthetic tissues, such as leaves and shoots of a plant. In other cases, bacteria and bacterial populations are concentrated in the conducting tissues of the plant, such as the xylem and phloem. In yet another embodiment, the bacteria or bacterial population is capable of concentrating in the reproductive tissues (flower, pollen, pistil, ovary, stamen or fruit) of the plant. In yet another embodiment, the bacteria and bacterial populations are capable of concentrating in the root, shoots, leaves, and reproductive tissues of the plant. In yet another embodiment, the bacteria or bacterial population colonizes the fruit or seed tissue of the plant. In yet another embodiment, the bacteria or bacterial population is capable of colonizing the plant such that it is present on the surface of the plant. In yet another embodiment, the bacteria or bacterial population is capable of being concentrated in substantially all or all tissues of the plant. In certain embodiments, the bacteria or bacterial population is not concentrated in the root of the plant. In other cases, bacteria and bacterial populations are not concentrated in photosynthetic plant tissues.

[00252] The effectiveness of bacterial compositions applied to crops can be assessed by measuring various crop growth characteristics, including, but not limited to, planting rate, emergence vigor, root vigor, drought tolerance, plant height, drying, and bulk weight.

Plant species

[00253] The methods and bacteria described herein are suitable for any of a variety of plants, such as plants of the genera Hordeum, Oryza, Zea and Triticeae. Other non-limiting examples of suitable plants include mosses, lichens and algae. In some cases, plants have economic, social and/or environmental value, such as food crops, textile crops, oilseeds, forestry or pulp and paper plants, biofuel feedstocks and/or ornamental plants. In some examples, plants can be used to produce economically valuable products such as grain, feed meal, starch, syrup, flour, oil, film, packaging, nutraceutical product, pulp, animal feed, fish feed, base material for industrial chemicals, grain product, processed human food, sugar, alcohol and/or protein. Non-limiting examples of crops include maize, rice, wheat, barley, sorghum, millet, oats, triticale, buckwheat, sweet corn, sugar cane, onions, tomatoes, strawberries and asparagus.

[00254] In some examples, plants that can be produced or improved using the methods and compositions disclosed herein may include plants that are important or of interest to agriculture, horticulture, biomass for the production of biofuel molecules, and other chemicals and/or forestry. Some examples of these plants may include pineapple, banana, coconut, lily, china, alfalfa, tomatillo, melon, chickpeas, chicory, clover, kale, lentils, soybeans, tobacco, potatoes, sweet potatoes, radishes, cabbage, canola, apple trees , grapes, cotton, sunflowers, Arabidopsis, canola, citrus fruits (including orange, tangerine, kumquat, lemon, lime, grapefruit, tangerine, tangelo, citron and pomelo), pepper, beans, lettuce, Panicum virgatum, Sorghum bicolor (sorghum sudanese), Miscanthus giganteus (miscanthus), Saccharum sp. (energy cane), Populus balsamifera (poplar), Zea mays (corn), Glycine max (soybean), Brassica napus (canola), Triticum aestivum (wheat), Gossypium hirsutum (cotton), Oryza sativa (rice), Helianthus annuus ( sunflower), Medicago sativa (alfalfa), Beta vulgaris (sugar beet), Pennisetum glaucum (millet), Panicum spp. Sorghum spp., Miscanthus spp., Saccharum spp., Erianthus spp., Populus spp., Secale cereale (rye), Salix spp. (willow), Eucalyptus spp. (eucalyptus), Triticosecale spp. (hybrid triticum - wheat and rye), bamboo, Carthamus tinctorius (safflower), Jatropha curcas (jatropha), Ricinus communis (castor oil), Elaeis guineensis (oil palm), Phoenix dactylifera (date palm), Archontophoenix cunninghamiana (king palm), Syagrus romanzoffiana (Rumyantsev's siagrus), Linum usitatissimum (flax), Brassica juncea, Manihot esculenta (cassava), Lycopersicon esculentum (tomato), Lactuca saliva (lettuce), Musa paradisiaca (banana), Solanum tuberosum (potato), Brassica oleracea (broccoli , cauliflower, Brussels sprouts), Camellia sinensis (tea), Fragaria ananassa (strawberry), Theobroma cacao (cocoa), Coffea arabica (coffee), Vitis vinifera (grape), Ananas comosus (pineapple), Capsicum annum (spicy and sweet pepper), Allium cepa (onion), Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita maxima (pumpkin), Cucurbita moschata (pumpkin), Spinacea oleracea (spinach), Citrullus lanatus (watermelon), Abelmoschus esculentus (okra) , Solanum melongena (eggplant), Papaver somniferum (opium poppy), Papaver orientale, Taxus baccata, Taxus brevifolia, Artemisia annua, Cannabis saliva, Camptotheca acuminate, Catharanthus roseus, Vinca rosea, Cinchona officinalis, Coichicum autumnale, Veratrum californica, Digitalis lanata, Digitalis purpurea, Dioscorea spp., Andrographis paniculata, Atropa belladonna, Datura stomonium, Berberis spp., Cephalotaxus spp., Ephedra sinica, Ephedra spp., Erythroxylum coca, Galanthus wornorii, Scopolia spp., Lycopodium serratum (Huperzia serrata), Lycopodium spp. ., Rauwolfia serpentina, Rauwolfia spp., Sanguinaria canadensis, Hyoscyamus spp., Calendula officinalis, Chrysanthemum parthenium, Coleus forskohlii, Tanacetum parthenium, Parthenium argentatum (guayule), Hevea spp. (hevea), Mentha spicata (mint), Mentha piperita (mint), Bixa orellana, Alstroemeria spp., Rosa spp. (rose), Dianthus caryophyllus (carnation), Petunia spp. (petunia), Poinsettia pulcherrima (poinsettia), Nicotiana tabacum (tobacco), Lupinus albus (lupin), Uniola paniculata (oats), Hordeum vulgare (barley) and Lolium spp. (rye).

[00255] In some examples, a monocotyledonous plant may be used. Monocots belong to the orders Alismatales, Arales, Arecales, Bromeliales, Commelinales, Cyclanthales, Cyperales, Eriocaulales, Hydrocharitales, Juncales, Lilliales, Najadales, Orchidales, Pandanales, Poales, Restionales, Triuridales, Typhales and Zingiberales. Plants belonging to the class Gymnospermae are Cycadales, Ginkgoales, Gnetales and Pinales. In some examples, the monocotyledonous plant may be selected from the group consisting of maize, rice, wheat, barley, and sugarcane.

[00256] In some examples, a dicotyledonous plant may be used, including plants belonging to the orders Aristochiales, Asterales, Batales, Campanulales, Capparales, Caryophyllales, Casuarinales, Celastrales, Cornales, Diapensales, Dilleniales, Dipsacales, Ebenales, Ericales, Eucomiales, Euphorbiales, Fabales, Fagales, Gentianales, Geraniales, Haloragales, Hamamelidales, Middles, Juglandales, Lamiales, Laurales, Lecythidales, Leitneriales, Magniolales, Malvales, Myricales, Myrtales, Nymphaeales, Papeverales, Piperales, Plantaginales, Plumb aginales, Podostemales, Polemonia les, Polygalales , Polygonales, Primulales, Proteales, Rafflesiales, Ranunculales, Rhamnales, Rosales, Rubiales, Salicales, Santales, Sapindales, Sarraceniaceae, Scrophulariales, Theales, Trochodendrales, Umbellales, Urticales and Violates. In some examples, the dicotyledonous plant may be selected from the group consisting of cotton, soybean, pepper, and tomato.

[00257] In some cases, the plant to be improved does not always readily respond to experimental conditions. For example, growing a crop plant sufficiently to allow improved quality to be assessed consistently over multiple iterations may take too long. Accordingly, the first plant from which the bacteria are initially isolated, and/or the plurality of plants to which the genetically modified bacteria are applied, may be a model plant, such as a plant better suited to evaluation under the desired conditions. Non-limiting examples of model plants include Setaria, Brachypodium and Arabidopsis. The ability of bacteria isolated according to the method of the invention using a model plant can then be applied to a different type of plant (eg a crop plant) to confirm the conferment of improved quality.

[00258] Qualities that can be improved by the methods disclosed herein include any observable characteristic of the plant, including, for example, growth rate, height, weight, color, taste, odor, changes in the plant's production of one or more compounds (including, e.g. metabolites, proteins, drugs, carbohydrates, oils and any other compounds). It is also contemplated to select plants based on genotypic information (eg, including the expression profile of plant genes in response to bacteria or identifying the presence of genetic markers, such as markers associated with increased nitrogen fixation). Plants may also be selected based on the absence, suppression or inhibition of a particular trait or quality (such as an undesirable trait or quality) rather than on the basis of the presence of a certain trait or quality (such as a desirable trait or quality).

EXAMPLES

[00259] The examples provided herein describe methods for isolating bacteria, analyzing bacteria and plants, and improving plant qualities. The examples are provided for illustrative purposes only and should not be construed as limiting the invention in any way.

Example 1. Isolation of microorganisms from plant tissue

[00260] Topsoil was obtained from various agricultural areas of Central California. Twenty soil types with varying structural characteristics were collected, including heavy clay, peaty clay loam, silty clay and sandy loam. Seeds of a variety of field corn, sweet corn, non-hybrid corn, and tomatoes were sown in each soil type, as shown in Table 1.

Type of crop Field corn Sweet corn Non-hybrid corn Tomato Varieties Mo17 'Golden Cross Bantam T-51' by Ferry-Morse 'Moseby Prolific' by Victory Seeds Roma VF by Ferry-Morse B73 'Silver Queen Hybrid' by Ferry-Morse 'Reid's Yellow Dent' by Victory Seeds Stover Roma DKC 66-40 'Sugar Dots' by Ferry-Morse 'Hickory King' by Victory Seeds 'Micro Tom Hybrid' from Totally Tomatoes DKC 67-07 Heinz 1015 DKC 70-01 Heinz 2401 Heinz 3402 Heinz 5508 Heinz 5608 Heinz 8504

Table 1. Crop type and varieties planted in different soil characteristics

[00261] Plants were dug up after 2-4 weeks of growth and excess soil was removed from the root surfaces with deionized water. After removing the soil, the surface of the plants was sterilized with a disinfectant solution and washed vigorously in sterile water. A cleaned 1 cm section of root was cut from the plant and placed in phosphate buffered saline containing 3 mm steel beads. A suspension was prepared by vigorously shaking the solution using a TissueLyser II from Qiagen.

[00262] A suspension of root and saline solution was diluted and inoculated onto various types of culture media to isolate rhizosphere, endophytic, epiphytic and other plant-associated microorganisms. R2A and Nfb agar media were used to obtain single colonies, and Nfb semisolid slants were used to obtain populations of nitrogen-fixing bacteria. After 2-4 weeks of incubation in semi-solid Nfb slants, microbial populations were harvested and streaked to obtain single colonies on R2A agar as shown in Figures 1A-B. Single colonies were resuspended in a mixture of R2A and glycerol, subjected to PCR analysis, and frozen at -80°C for subsequent analysis. Approximately 1000 individual colonies were obtained and designated as “isolated microorganisms.”

[00263] The isolates were then subjected to colony PCR screening to detect the presence of the nifH gene to identify diazotrophs. The previously described Ueda 19F/388R primer set, which has been shown to detect more than 90% of diazotrophs in screens, was used to examine the presence of the nif cluster in each isolate (Ueda et al. 1995; J. Bacteriol. 177: 1414-1417) . Single colonies of purified isolates were picked, resuspended in PBS, and used as a template for colony PCR, as shown in Figure 2. Colonies of isolates that produced positive bands in the PCR were streaked again, and the process of PCR and streaked colonies was repeated twice. to prevent false positive identification of diazotrophs. The purified isolates were then named “candidate microorganisms.”

Example 2. Determination of characteristics of isolated microorganisms

Sequencing, analysis and phylogenetic characterization

[00264] 16S rDNA sequencing with primer set 515f-806r was used to generate tentative phylogenetic identifiers for the isolated and candidate microorganisms (see, e.g., Vernon et al.; BMC Microbiol. Dec. 23, 2002;2:39.). Microorganisms include various genera, including: Enterobacter, Burkholderia, Klebsiella, Bradyrhizobium, Rahnella, Xanthomonas, Raoultella, Pantoea, Pseudomonas, Brevundimonas, Agrobacterium, and Paenibacillus, as shown in Table 2.

Genus Isolates Genus Isolates Achromobacter 7 Paenibacillus 1 Agrobacterium 117 Paenisporosarcina 3 Agromyces 1 Pantoea 14 Alicyclobacillus 1 Pedobacter 16 Asticcacaulis 6 Pimelobacter 2 Bacillus 131 Pseudomonas 212 Bradyrhizobium 2 Rhizobium 4 Brevibacillus 2 Rhodoferax 1 Burkholderia 2 Sphingobacterium 13 Caulobacter 17 Sphingobium 23 Chryseobacterium 42 Sphingomonas 3 Comamonas 1 Sphingopyxis 1 Dyadobacter 2 Stenotrophomonas 59 Flavobacterium 46 Streptococcus 3 Halomonas 3 Variovorax 37 Leptothrix 3 Xylanimicrobium 1 Lysobacter 2 unidentified 75 Neisseria 13

Table 2. Diversity of microorganisms isolated from tomato plants, according to deep sequencing of 16S rDNA.

[00265] The genomes of 39 candidate microorganisms were then sequenced using the Illumina Miseq platform. Genomic DNA from pure cultures was extracted using the QIAmp DNA mini kit (QIAGEN), and total DNA libraries for sequencing were prepared through a third party supplier (SeqMatic, Hayward). The genome was then assembled using the A5 pipeline (Tritt et al. 2012; PLoS One 7(9):e42304). Genes were identified and annotated, and genes associated with the regulation and expression of nitrogen fixation were annotated as targets for mutagenesis.

Transcriptomic profiling of candidate microorganisms

[00266] Transcriptomic profiling of strain CI010 was performed to identify promoters active in the presence of environmental nitrogen. Strain CI010 was cultivated in a nitrogen-free medium with a specific composition and supplemented with 10 mM glutamine. Total RNA was extracted from these cultures (QIAGEN RNeasy kit) and subjected to RNASeq sequencing using Illumina HiSeq (SeqMatic, Fremont, CA). Sequencing reads were mapped to CI010 genome data using Geneious, and highly expressed genes under the control of proximal transcriptional promoters were identified. Tables 3A–C list the genes and their relative expression levels as measured by RNASeq sequencing of total RNA. Proximal promoter sequences were recorded for use in mutagenesis of nif pathways, nitrogen utilization pathways, or other genes with the desired level of expression.

Assessing the ease of genetic manipulation

[00267] Candidate microorganisms have been characterized on the basis of transformability and ease of genetic manipulation. First, optimal carbon source utilization was determined by growth on a small panel of appropriate media, as well as the growth curve in both nitrogen-free and enriched media. Second, the natural antibiotic resistance of each strain was determined by spot plating and growth in liquid culture containing a panel of antibiotics used as selective mutagenesis markers. Third, each strain was tested for its transformability by electroporation of a set of plasmids. The plasmid set includes a combined expansion of seven origins of replication, i.e., p15a, pSC101, CloDF, colA, RK2, pBBR1, and pRO1600, and four antibiotic resistance markers, i.e., CmR, KmR, SpecR, and TetR. This systematic assessment of the compatibility of origins of replication and resistance markers was used to identify vectors for plasmid mutagenesis in candidate microorganisms.

Example 3: Mutagenesis of Candidate Microorganisms

Lambda Red-dependent knockouts

[00268] Several candidate microbial mutants have been generated using plasmid pKD46 or a derivative containing a kanamycin resistance marker (Datsenko et al. 2000; PNAS 97(12): 6640-6645). Knockout cassettes were designed with 250 bp of homology flanking the target gene and generated by PCR with overlapping primers. Candidate microorganisms were transformed with pKD46, cultured in the presence of arabinose to induce expression of the lambda Red machinery, prepared for electroporation, and transformed with knockout cassettes to generate candidate mutant strains. Four candidate microorganisms and one laboratory strain, Klebsiella oxytoca M5A1, were used to generate thirteen candidate mutants of the nitrogen fixation regulatory genes nifL, glnB, and amtB, as shown in Table 4.

Strain nifL glnB amtB M5A1 X X X CI006 X X X CI010 X X X CI019 X X CI028 X X

Table 4. List of single-knockout mutants generated by lambda Red-dependent mutagenesis

Oligo-directed mutagenesis with Cas9 selection

[00269] Oligo-directed mutagenesis was used to target genomic changes to the rpoB gene in E. coli DH10B, and mutants were selected using the CRISPR-Cas system. A mutagenic oligonucleotide (ss1283: “G*T*T*G*ATCAGACCGATGTTCGGACCTTCcaagGTTTCGATCGGACATACGCGACCGTAGTGGGTCGGGTGTACGTCTCGAACTTCAAAGCC”, where * denotes a thiophosphate linkage) was designed to confer rifampicin resistance via a 4-bp mutation. in the rpoB gene. Cells containing a plasmid encoding Cas9 were induced to express Cas9, prepared for electroporation, and then electroporated with both a mutagenic oligonucleotide and a plasmid encoding the constitutive expression of a guide RNA (gRNA) targeting Cas9 for cleavage of the wild-type rpoB sequence. Electroporated cells were recovered in nonselective medium overnight to ensure sufficient segregation of the resulting mutant chromosomes. After plating to select a plasmid encoding a gRNA, two of the ten colonies screened contained the desired mutation, while the remainder were shown to be escape mutants generated by protospacer mutation in the gRNA plasmid or loss of the Cas9 plasmid .

Lambda Red-dependent mutagenesis with selection by Cas9

[00270] Candidate microbial mutants CI006 and CI010 were generated by lambda Red-dependent mutagenesis with CRISPR-Cas selection. The knockout cassettes contained an endogenous promoter identified by transcriptional profiling (as described in Example 2 and shown in Table 3) and ~250 bp homology regions flanking the deletion target. CI006 and CI010 were transformed with plasmids encoding the lambda Red-dependent recombination system (exo, beta, gam genes) under the control of the inducible arabinose promoter and Cas9 under the control of the inducible IPTG promoter. Red-dependent recombination and Cas9 systems were induced in the resulting transformants, and the strains were prepared for electroporation. The knockout cassettes and the plasmid-encoded gRNA of choice were then transformed into competent cells. After plating with antibiotics selective for both the Cas9 plasmid and the gRNA plasmid, a putative knockout mutation was detected in 7 of 10 colonies screened, as shown in Figure 3 .

Example 4 In Vitro Phenotyping of Candidate Molecules

[00271] The effect of exogenous nitrogen on nitrogenase biosynthesis and activity in various mutants was assessed. An acetylene reduction assay (ARA) was used to measure nitrogenase activity under pure culture conditions (Temme et al. 2012; 109(18): 7085-7090). Strains were grown in sealed tubes and the reduction of acetylene to ethylene was quantified using an Agilent 6890 gas chromatograph. The activity of candidate microorganisms and corresponding candidate mutants grown in nitrogen-fixing media supplemented with 0 to 10 mM glutamine in the ARA assay is shown in Figures 4A- B and Figures 10A-C.

[00272] A range of glutamine and ammonia concentrations were tested under anaerobic culture conditions to quantify the effect on nitrogen-fixing activity. In wild-type cells, activity decreased rapidly as glutamine concentration increased. However, in a series of initial knockout mutations, a class of mutations was confirmed that allows genes responsible for nitrogen fixation to be expressed at glutamine concentrations that would otherwise abolish activity in the wild type. This profile was generated in four different diazotroph species, as can be seen in Figure 4C. In addition, reconfiguring the regulatory network using identified genetic parts allowed for predictable tuning of the level of nitrogen-fixing activity. This can be seen in Figure 4B, which illustrates strains CM023, CM021, CM015 and CI006. Strain CM023 is a low-level evolved strain; strain CM021 is a high-level evolved strain; strain CM015 is an evolved mid-level strain; strain CI006 is wild type (strain 2). Ammonia excreted into the culture supernatants was analyzed using an enzyme-based assay (MEGAZYME). The assay measured the amount of NADPH consumed at 340 nm absorbance. The analysis was performed on bacterial cultures grown in a nitrogen-free anaerobic environment with an initial density of 1E9 CFU/ml. In a panel of six evolved strains, one strain excreted up to 100 μM ammonia within 48 hours, as can be seen in Figure 4D. In addition, the double mutant showed higher ammonia excretion than the single mutant from which it was derived, as shown in Figure 11. This demonstrates the ability of microorganisms to produce ammonia beyond their physiological requirements.

Transcriptional profiling of pure cultures

[00273] CI006 transcriptional activity was measured using the Nanostring Elements platform. Cells were grown in nitrogen-free medium, and 10E8 cells were harvested after 4-hour incubation. Total RNA was extracted using the Qiagen RNeasy kit. Purified RNA was sent to Core Diagnostics in Palo Alto, California, for probe hybridization and digital analyzer analysis as shown in Figure 5.

Example 5 Phenotyping of candidate microorganisms in planta

Colonization of plants by candidate microorganisms

[00274] Colonization of desired plant hosts by a candidate microorganism was quantified using short-term plant growth experiments. Maize plants were inoculated with strains expressing RFP (red fluorescent protein), either from a plasmid or from a Tn5-integrated RFP expression cassette. Plants were grown in both sterilized sand and non-sterile peat media, and inoculation was carried out by pipetting 1 ml of cell culture directly over the developing coleoptile of the plant three days after germination. Plasmids were maintained by watering the plants with a solution containing a suitable antibiotic. After three weeks, plant roots were harvested, washed three times with sterile water to remove visible soil, and divided into two samples. One root sample was analyzed using fluorescence microscopy to determine localization patterns of candidate microorganisms. Microscopy was carried out on the thinnest intact roots of the plant, 10 mm long, as shown in Figure 6.

[00275] A second quantitative method for assessing colonization has been developed. Quantitative PCR analysis was performed on whole DNA preparations from plant roots inoculated with endophytes. Corn seeds (Dekalb DKC-66-40) were germinated in pre-autoclaved sand in a 2.5-by-2.5-inch by 10-inch pot. One day after planting, 1 ml of overnight cultured endophyte (SOB medium) was poured directly into the seed location. 1 ml of this overnight culture is approximately equivalent to approximately 10 ⁹ CFU, varying by up to 3 times depending on the strain used. Each seedling was fertilized 3 times a week with 50 ml of modified Hoagland's solution supplemented with either 2.5 mM or 0.25 mM ammonium nitrate. Four weeks after planting, root samples were collected for DNA extraction. The remaining soil was washed off with a jet of water under pressure. These tissue samples were then homogenized using the QIAGEN Tissuelyzer, followed by DNA extraction using the QIAmp DNA Mini Kit (QIAGEN) according to the recommended protocol. These DNA extracts were analyzed by qPCR using RT-PCR Stratagene Mx3005P with primers designed (using Primer BLAST from NCBI) to be specific to loci in the genome of each endophyte. The presence of endophyte genomic copies was quantified. To further confirm the identity of the endophytes, the PCR amplification products were sequenced and confirmed to have the correct sequence. A summary of the colonization profile of strains CI006 and CI008 from candidate microorganisms is presented in Table 5. Strain CI008 has demonstrated colonization rates of up to 10 ⁷ CFU/g root fresh weight.

Strain Colonization rate (CFU/g wet weight) CI006 1.45×10 ⁵ CI008 1.24×10 ⁷

Table 5. Colonization of maize measured by qPCR

RNA profiling in planta

[00276] Biosynthesis of nif pathway components in planta was assessed by measuring transcription of nif genes. Total RNA was obtained from root tissue of plants inoculated with CI006 (planting methods as described previously). RNA extraction was performed using the RNEasy mini kit according to the recommended protocol (QIAGEN). Total RNA from these plant tissues was then analyzed using Nanostring Elements kits (NanoString Technologies, Inc.) using probes that are specific for the nif genes in the genome of strain CI006. A summary of nif gene expression in planta is given in Table 6. Expression of nifH genes was detected in plants inoculated with strains CM013, whereas nifH expression was not detected in plants inoculated with CI006. Strain CM013 is a derivative of strain CI006, in which the nifL gene was knocked out.

[00277] Highly expressed CM011 genes ranked by transcript per kb. per million (TPM) were measured in planta under fertilized conditions. The promoters that control the expression of some of these highly expressed genes have been used as templates for homologous recombination into target loci for nitrogen fixation and assimilation. RNA samples from a CM011-inoculated greenhouse-grown plant were extracted, rRNA was removed using a Ribo-Zero kit, sequenced using Illumina's Truseq platform, and mapped back to the CM011 genome. Highly expressed genes from CM011 are listed in Table 7.

Strains Relative transcript expression CI006 9.4 CM013 103.25

Table 6. Expression of nifH in planta

Gene name Gene localization Direction Raw number of reads TPM (transcripts per kb per million) CDS rpsH 18196-18588 the opposite 4841.5 27206.4 CDS rplQ 11650-12039 the opposite 4333 24536.2 CDS rpsJ 25013-25324 the opposite 3423 24229 CDS rplV 21946-22278 the opposite 3367.5 22333 CDS rpsN 18622-18927 the opposite 2792 20150.1 CDS rplN 19820-20191 the opposite 3317 19691.8 CDS rplF 17649-18182 the opposite 4504.5 18628.9 CDS rpsD 13095-13715 the opposite 5091.5 18106.6 CDS rpmF 8326-8493 direct 1363.5 17923.8 CDS rplW 23429-23731 the opposite 2252 16413.8 CDS rpsM 14153-14509 the opposite 2269 14036.2 CDS rplR 17286-17639 the opposite 2243.5 13996.1 CDS rplC 24350-24979 the opposite 3985 13969.2 CDS rplK 25526-25954 the opposite 2648.5 13634.1 CDS rplP 20807-21217 the opposite 2423 13019.5 CDS rplX 19495-19809 the opposite 1824 12787.8 CDS rpsQ 20362-20616 the opposite 1460.5 12648.7 CDS bhsA 3 79720-79977 the opposite 1464 12531.5 CDS rpmC 20616-20807 the opposite 998.5 11485 CDS rpoA 12080-13069 the opposite 4855 10830.2 CDS rpoA 23728-24333 the opposite 2916.5 10628.5 CDS bhsA 1 78883-79140 the opposite 1068 9141.9 CDS rpsS 22293-22571 the opposite 1138.5 9011.8 CDS rpmA 2210-2467 direct 1028.5 8803.7 CDS rpmD 16585-16764 the opposite 694.5 8520.8 CDS rplB 22586-23410 the opposite 3132 8384 CDS rpsC 21230-21928 the opposite 2574.5 8133.9 CDS rplE 18941-19480 the opposite 1972.5 8066.9 CDS rplO 16147-16581 the opposite 1551 7874.2 CDS subunit of SecY preprotein translocase 14808-16139 the opposite 4657 7721.2 rpsM CDS 16771-17271 the opposite 1671.5 7368 CDS rpsK 13746-14135 the opposite 1223.5 6928.2 CDS tufA 27318-28229 the opposite 2850 6901.3 CDS rpmI 38574-38771 direct 615 6859.5 CDS rplU 1880-2191 direct 935.5 6621.7 CDS rplT 38814-39170 direct 1045 6464.4 CDS bhsA 2 79293-79550 the opposite 754 6454.1 CDS rpmB 8391-8627 the opposite 682 6355.1 CDS rplJ 23983-24480 the opposite 1408 6243.9 CDS fusA 2 481-2595 the opposite 5832 6089.6 CDS rpsA 25062-26771 the opposite 4613 5957.6 CDS rpmJ 14658-14774 the opposite 314 5926.9 CDS rpsR 52990-53217 direct 603 5840.7 CDS rpsG 2692-3162 the opposite 1243 5828.2 CDS rpsI 11354-11746 the opposite 980.5 5509.8 CDS cspC 1 8091-8300 the opposite 509 5352.8 CDS rpsF 52270-52662 direct 916 5147.4 CDS rpsT 55208-55471 the opposite 602 5035.9 CDS infC 38128-38478 direct 755 4750.3 CDS cspG 30148-30360 direct 446 4624.2

Table 7. Highly expressed genes from CM011

Analysis ¹⁵ N

[00278] The primary method for demonstrating fixation uses the nitrogen isotope 15N, which is present in the atmosphere at a fixed ratio relative to 14N. By introducing elevated levels of 15N into fertilizer or into the atmosphere, fixation in plant tissues can be observed either directly, in the form of increased amounts of 15N fixed from the atmosphere with the addition of 15N2 gas (Yoshida 1980), or inversely through dilution of the enriched fertilizer with N2 gas from the atmosphere ( Iniguez 2004). The dilution method allows observation of the cumulative fixed nitrogen during plant growth, while the _15N2 gas method is limited to measuring fixation that occurs during the short interval during which the plant can be grown in an isolated atmosphere (intensity measurement). Therefore, the gas method has greater specificity (since any elevated levels of _15N2 in the plant above atmospheric levels can be clearly attributed to fixation), but it cannot show cumulative activity.

[00279] Both assays were performed to measure the fixation activity of the improved strains relative to wild-type and uninoculated corn plants, and increased in planta fixation rates were observed for several of the improved strains (Figure 12, Figure 14A and Figure 14B). These assays demonstrate that strain activity observed in vitro translates to in vivo results. In addition, these assays allow the measurement of the effect of fertilizers on the activity of the strain and provide evidence for the appropriate functionality of the strain for agricultural conditions. Similar results were observed when setaria plants were inoculated with wild types and improved strains (Figure 13). The in planta fixation activity shown in Figures 14A-14C is further supported by transcriptomic data. The evolved strains exhibit increased levels of nifH transcript compared to their wild-type counterparts. Additionally, the level of microbially derived nitrogen in planta also correlates with the level of colonization for each individual plant. These results (Figure 12, Figure 13, Figures 14A-14C, Figure 15A and Figure 15B) support the hypothesis that the microorganism, through improved regulation of the nif gene cluster, is the likely cause of the increase in atmospheric nitrogen levels observed in plant tissue. In addition to directly measuring fixation, the effect of plant inoculation with improved strains was measured in a biomass assay of plants exposed to nitrogen deficiency stress. Although plant biomass can be related to many possible microorganism-plant interactions, addition of fixed nitrogen can be expected to affect plant phenotype when nitrogen is limited. Inoculated plants were grown in the complete absence of nitrogen, and inoculated plants showed significant increases in leaf area, shoot fresh and dry weight, and root fresh and dry weight compared to untreated controls (Figure 14C). Although these differences cannot be attributed solely to nitrogen fixation, they support the conclusion that the improved strains actively provide nitrogen to the plant. Corn and setaria plants were grown and inoculated as described above. Plants were regularly supplied with fertilizer containing 1.2% ¹⁵ N through irrigation. Microbial nitrogen fixation was determined by measuring ^15N levels in plant tissue. Fourth leaf tissue was collected and dried 4 weeks after planting. Dried leaf samples were homogenized using beads (QIAGEN Tissuelyzer) and aliquoted into tin capsules for IRMS (MBL Stable Isotope Laboratory at The Ecosystems Center, Woods Hole, MA). Nitrogen derived from atmosphere (NDFA) was calculated, and the nitrogen production of CI050 and CM002 is shown in Figure 7.

Analysis of phytohormone production

[00280] The dwarf tomato (Solanum lycopersicum) cultivar 'Micro-Tom' has previously been used to study the effects of indole-3-acetic acid on fruit ripening in an in vitro assay (Cohen 1996; J Am Soc Hortic Sci 121: 520-524). To evaluate the production and secretion of phytohormones by candidate microorganisms, a plate-based screening assay was developed using Micro-Tom immature fruits. Twelve-well tissue culture plates were prepared by filling the wells with agar medium, allowing it to solidify, and spot-plating 10 μl of overnight cultures of microorganisms onto the agar surface as shown in Figure 8. Agar wells containing increasing amounts of gibberellic acid (GA), but without bacterial culture, were used as positive controls and standards. Flowers the day after bloom, cut from growing Micro-Tom plants, were inserted stem-wise into agar at the point of a bacterial spot culture. These flowers were observed for 2-3 weeks, after which the fruits were collected and weighed. An increase in fruit weight in several parallel assays indicates production of a plant hormone by the inoculating microorganism, as shown in Figure 9.

Example 6. Cyclic evolution of host-microorganism

[00281] Corn plants were inoculated with CM013 and grown for 4 weeks to approximately V5 growth stage. Plants showing improved nitrogen accumulation from microbial sources in the ^15N assay were dug up and the roots were pressure washed with water to remove the bulk of the soil. A 0.25 g root segment was cut and washed in PBS to remove fine soil particles and non-adherent microorganisms. Tissue samples were homogenized using 3 mm steel beads in a QIAGEN TissueLyser II. The homogenate was diluted and plated on SOB agar medium. Single colonies were resuspended in liquid media and subjected to PCR analysis of 16s rDNA and mutations unique to the inoculating strain. The process of isolation, mutagenesis, inoculation and re-isolation of microorganisms can be repeated many times to improve the quality of the microorganism, the quality of the plant and the colonization ability of the microorganism.

Example 7: Cross-Geography Compatibility

[00282] The ability of improved microorganisms to colonize an inoculated plant is critical to the plant's success in the field. Although the isolation methods described are designed to select from soil microorganisms that may have close associations with crop plants such as corn, many strains may not colonize effectively under different plant genotypes, environments, soil types, or inoculation conditions. Because colonization is a complex process requiring a series of interactions between the microorganism strain and the host plant, screening for colonization competence has become a central way to select priority strains for further development. Early attempts to assess colonization used fluorescent labeling of strains, which was effective but time-consuming and did not scale to each strain. Since colonization activity cannot be directly improved, it is critical that potential product candidates are selected from strains that are natural colonizers.

[00283] An assay has been developed to test for persistent colonization of wild-type strains in any single host plant using qPCR and primers designed to be specific for the strain in the community sample. This assay is designed to quickly measure the degree of microbial colonization in maize tissue samples. Initial tests using strains assessed as likely colonizers using fluorescence microscopy and plate-based techniques indicated that a qPCR approach would be both quantitative and scalable.

[00284] A typical assay is conducted as follows: plants, primarily corn and wheat varieties, are grown in a peat-based greenhouse soil mixture in six parallel assays per strain. Four or five days after planting, a 1 ml dose of early stationary phase bacterial cultures diluted to an optical density at 590 nm of 0.6-1.0 (approximately 5E+08 CFU/ml) is pipetted onto the developing coleoptile. Plants are watered only with tap water and allowed to grow for four weeks before sampling, after which the plants are dug up and the roots are thoroughly washed to remove most peat residues. Clean root samples are cut and homogenized to create a suspension of plant cell detritus and associated bacterial cells. The authors developed a high-throughput DNA extraction protocol that efficiently provided a mixture of plant and bacterial DNA for use as a template for qPCR. Based on experiments with bacterial cell additions, this DNA extraction process provides a quantitative sample of bacterial DNA relative to root wet weight. Each strain is evaluated using strain-specific primers designed using Primer BLAST (Ye 2012) and compared to background amplification from uninoculated plants. Because some primers exhibit off-target amplification in uninoculated plants, colonization is determined by either the presence of amplification or increased amplification of the correct product compared to background levels.

[00285] This assay was used to measure the compatibility of a microbiological product with different soil geographies. Field soil qualities and field conditions can have a huge impact on the impact of a microbial product. Soil pH, water holding capacity, and competitive microorganisms are just a few examples of soil factors that can influence inoculum survival and colonization ability. Colonization assays were conducted using three different soil types collected from agricultural fields in California as plant growing media (Figure 16A). Intermediate inoculation densities were used to simulate realistic agricultural conditions. Within 3 weeks, strain 5 colonized all plants at 1E+06 to 1E+07 CFU/g fresh weight. After 7 weeks of plant growth, the evolved version of strain 1 showed a high degree of colonization (1E+06 CFU/g fresh weight) in all soil types. (Figure 16B).

[00286] Additionally, to evaluate colonization under complex field conditions, a 1-acre field trial was initiated in San Luis Obispo in June 2015 to evaluate the impact and colonization of seven wild-type strains on two varieties of field corn. Agricultural planning and trial execution were performed by contracted field research organization Pacific Ag Research. For inoculation, the same peat seed coating technology was used that was tested in the inoculation methods experiment. Plant samples were collected throughout the growing season to assess colonization within the root and stem. Samples were collected from three parallel assays of each treatment at four and eight weeks after planting and from all six parallel assays of each treatment shortly before harvest at 16 weeks. Additional samples were collected from all six parallel treatment assays inoculated with strain 1 and strain 2, as well as untreated controls, after 12 weeks. The number of cells per gram of fresh weight of washed roots was assessed as in other colonization assays using qPCR and strain-specific primers. Two strains, strain 1 and strain 2, showed consistent and widespread root colonization that peaked at 12 weeks and then declined sharply (Figure 16C). Although strain 2 appeared to be present in quantities an order of magnitude lower than strain 1, it was found in more consistent quantities across different plants. Apparently, none of the strains efficiently colonized the interior of the stem. In support of the colonization data obtained by qPCR, both strains were successfully reisolated from root samples using culture and 16S sequencing to identify isolates having matching sequences.

[00287] Examples of microbial selection can be summarized in the diagram shown in Figure 17 and Figure 18. Figure 17 depicts microbial selection where the composition of the microbiome can first be measured and species of interest identified. Microbiome metabolism can be mapped and linked to genetics. The targeted genetic change can then be introduced using techniques including, but not limited to, conjugation and recombination, chemical mutagenesis, adaptive evolution, and gene editing. Derived microorganisms are used to inoculate crops. In some examples, crops with better phenotypes are selected.

[00288] As shown in Figure 17, the composition of the microbiome can first be measured and species of interest identified. Microbiome metabolism can be mapped and linked to genetics. Nitrogen metabolism may involve the entry of ammonia (NH ₄ ⁺ ) from the rhizosphere into the bacterial cytosol via the AmtB transporter. Ammonia and L-glutamate (L-Glu) are catalyzed by glutamine synthetase and ATP into glutamine. Glutamine can lead to biomass production (plant growth) and can also inhibit the expression of the nif operon. The targeted genetic change can then be introduced using techniques including, but not limited to, conjugation and recombination, chemical mutagenesis, adaptive evolution, and gene editing. Derived microorganisms are used to inoculate crops. Crops with the best phenotypes are selected.

[00289] As used herein (especially in the context of the following claims), the singular terms refer to both the singular and plural unless otherwise indicated, indicated herein, or clearly contrary to the context. The terms “comprising,” “having,” and “including” are to be construed as non-limiting (i.e., to mean “including but not limited to”) unless otherwise noted. The reference to ranges of values herein is intended solely for the purpose of facilitating individual reference to each value within the range, unless otherwise indicated herein, and each individual value is included in the description as if it were mentioned separately. For example, if the range 10-15 is disclosed, then 11, 12, 13 and 14 are also disclosed. All methods described herein can be performed in any suitable order unless otherwise indicated or clearly inconsistent with context. Any possible examples or exemplary references (eg, “such as”) herein are intended solely to better disclose the invention and do not limit the scope of the present invention unless otherwise stated in the claims. Nothing in the specification should be construed as indicating any element missing from the claims as being essential to the practice of the invention.

[00290] Although preferred embodiments of the present invention have been shown and described herein, those skilled in the art will appreciate that such embodiments are provided by way of example only. Many variations, changes and substitutions will be apparent to those skilled in the art without departing from the scope of the invention. It should be understood that various alternatives to the embodiments described herein may be used in the practice of the invention. The following claims are intended to define the scope of the invention and thus cover methods and structures within the scope of these claims and their equivalents.

[00291] Table 3A

Name Minimum value Maximum value Length Direction Murein lipoprotein CDS 2 929 898 2 930 134 237 direct membrane protein CDS 5 217 517 5 217 843 327 direct CDS of zinc/cadmium binding protein 3 479 979 3 480 626 648 direct CDS of acyl carrier protein 4 563 344 4 563 580 237 the opposite CDS ompX 4 251 002 4 251 514 513 direct CDS of DNA binding protein HU-beta 375 156 375 428 273 direct CDS sspA 629 998 630 636 639 the opposite CDS tatE 3 199 435 3,199,638 204 the opposite LexA repressor CDS 1 850 457 1 851 065 609 direct CDS hisS <3999979 4 001 223 >1245 direct

[00292] Table 3B

Name Absolute confidence in differential expression Differential expression ratio RNASeq_nifL - raw read count RNASeq_nifL - raw number of transcripts RNASeq_WT - raw read count RNASeq_WT - raw number of transcripts Murein lipoprotein CDS 1000 -1.8 12950.5 10078.9 5151.5 4106.8 membrane protein CDS 1000 -1.3 9522.5 5371.3 5400 3120 CDS of zinc/cadmium binding protein 3.3 1.1 6461 1839.1 5318 1550.6 CDS of acyl carrier protein 25.6 1.6 1230.5 957.6 1473.5 1174.7 CDS ompX 1.7 1.1 2042 734.2 1687.5 621.5 CDS of DNA binding protein HU-beta 6.9 -1.3 1305 881.7 725 501.8 CDS sspA 0.2 1 654 188.8 504.5 149.2 CDS tatE 1.4 1.3 131 118.4 125 115.8 LexA repressor CDS 0.1 -1.1 248 75.1 164 50.9 CDS hisS 0 -1.1 467 69.2 325 49.3

Example 8: Deletion of glgA increases nitrogen excretion

[00293] Glycogen synthesis redirects carbon away from glycolysis and the TCA cycle. Decreased or eliminated glycogen synthesis can result in increased carbon flux toward glycolysis and the TCA cycle, resulting in greater ATP availability in the cell. This may lead to increased nitrogenase activity since nitrogen fixation requires 16 ATP to reduce one molecule of N2 and may be limited by ATP availability. Glycogen synthase is encoded by the glgA gene. Suppression or deletion of glgA may result in increased nitrogen-fixing activity.

[00294] To this end, Kosakonia sacchari CI006 and Klebsiella variicola strains were constructed in which glgA was deleted. Strains 6-1694, 6-1744, and 137-1893 were subjected to an ammonium excretion assay (AMM), which measures ammonia excretion over time under nitrogen-fixation conditions by culturing cells in nitrogen-free media, pelleting the cells by centrifugation, and measuring free ammonium in cell-free media. broth; higher levels of ammonium excretion indicate higher nitrogen fixation and excretion. Strains 6-1681 and 137-1893 were subjected to the ARA assay as previously described in medium supplemented with 5 mM ammonium phosphate. Strain 6-1694 corresponds to the parent strain 6-342. Strain 6-1744 corresponds to the parent strain 6-923. Strain 6-1681 corresponds to the parent strain 6-346. Strain 137-1893 corresponds to the parent strain 137-1586.

[00295] AMM analysis data is shown in Figures 19A-20B. Strains 6-1694 and 6-1744 exhibit increased nitrogen excretion capacity compared to their parent strains (Figure 19A). Strain 6-1681 showed increased activity in ARA compared to its parent strain (Figure 19B). Strain 137-1893 showed both increased ammonium excretion (Figure 20A) and increased nitrogenase activity by ARA (Figure 20B). Thus, by reducing or eliminating glycogen synthesis in bacteria, the amount of nitrogen excreted can be increased. Details of the strains described herein are contained in Tables 8 and 9.

Example 9: glnD Mutations Increase Nitrogen Excretion

[00296] GlnD is a bifunctional uridylyl transferase/uridylyl scavenging enzyme that serves as a central "switch" for nitrogen sensing in the cell. It contains three distinct domains: a UTase domain that uridylates the PII proteins GlnB and GlnK; uridylyl-removing domain (UR), also known as the HD domain, which removes the uridylyl group from PII proteins; and two ACT domains that regulate the activity of two other domains in response to glutamine binding (Figure 21). When there is excess nitrogen, glutamine binds the ACT domain and the UR domain is activated; during nitrogen starvation, ACT is not bound and the UTase domain is active. Subsequent PII protein signaling influences the transcription of several gene clusters, including nitrogenase and nitrogen assimilation genes.

[00297] Various truncations and deletions were performed to see how they affected nitrogen fixation and excretion. Several glnD mutations modifying the UTase, UR, and ACT1/2 (glutamine-responsive) domains resulted in increased activity in ARA and ammonium excretion assays. Figure 22 demonstrates several glnD modifications resulting in increased ammonium excretion in K. variicola CI137; Figure 23 demonstrates several glnD modifications leading to increased ammonium excretion in K. sacchari.

Example 10: Deletion of glnK increases nitrogen excretion

[00298] GlnK is a PII protein that may act as a post-translational regulator and may be involved in glutamine synthesis, which may represent a competing pathway for ammonium excretion. Higher levels of GlnK may reduce the amount of NH4 excreted. Removal of glnK may reduce or eliminate the inhibitory effect on NH4 excretion.

[00299] GlnK was deleted in a strain that already contained a nifL::1.2 deletion and a glnE deletion. Thus, the glnK deletion was introduced into a strain that already had an increased ability to fix nitrogen. The glnK deletion strain is designated 137-2225 and the parent strain is designated 137-2084 in Figure 24A. An AMM assay was performed on these cells, in which the amount of NH4+ excreted per OD per hour (in mM) was measured. In this assay, greater NH4+ excretion may indicate higher nitrogen-fixing activity. Specifically, strain 137-2225 exhibited nitrogen-fixing activity that was 1.32-fold higher than that of the parent strain, as determined by AMM assay.

Example 11: Suppression of the glnA gene increases ammonium excretion

[00300] Glutamine synthetase (GS) is encoded by the glnA gene. A decrease in GS activity may lead to a decrease in the degree of nitrogen assimilation. Under conditions of nitrogen fixation, this can lead to intracellular ammonium accumulation and ammonium excretion.

[00301] Several strains have been constructed in which the native promoter for glnA has been replaced with a promoter known to be constitutively expressed at low levels: a promoter from the glnB, glnD, or glnE genes. This is expected to result in decreased glnA transcription. In some cases, the ATG start codon of the glnA gene has also been mutated to GTG to reduce the extent of translation initiation of the GS protein. Figure 24B, Figure 25 and Figure 26 describe several mutants CI137 and CI006 in which suppression of the glnA gene resulted in increased ammonium excretion.

Example 12: Inhibition of glutamate synthase results in increased ammonium excretion

[00302] Glutamate serves as the main source of nitrogen in the cell, and the GS-GOGAT pathway is the main pathway for nitrogen assimilation. The gltBD operon encodes GOGAT. A strain (137-2215) was constructed in which the native promoter for the gltBD operon was deleted and replaced with a strong constitutive promoter. Figure 27 demonstrates how 137-2215 exhibits increased ammonium excretion compared to parental control; Figure 28 demonstrates that 137-2215 exhibited increased nitrogen-fixing activity according to the ARA assay.

Example 13. Activation of the glutaminase gene glsA2 leads to increased excretion of ammonium

[00303] Glutaminase enzymes cleave the C-N bond in glutamine, releasing ammonium and glutamate. Increased glutaminase activity in the cell can lead to increased release of ammonium from glutamine and excretion of ammonium. The glsA2 gene encodes glutaminase. Strains were constructed in which the native promoter of the glsA2 gene was deleted and replaced with a strong promoter sequence. Figure 29 and Figure 30 demonstrate increased ammonium excretion by these strains compared to control parental strains.

Example 14 Activation of the glutaminase domain of asnB leads to increased ammonium excretion

[00304] The asnB gene encodes a transaminase enzyme that has two domains: a glutaminase domain and an asparagine synthase domain. Figure 31 shows how deletion of the asparagine synthase domain or activation of the asnB gene can lead to increased ammonium excretion.

Example 15: Activation of rpoN increases nitrogen fixation

[00305] rpoN (sigma-54) is an initiation factor used to promote the attachment of RNA polymerase to specific initiation sites. Along with nifA and ihfA/B, it transcribes nifH. Activation of rpoN may lead to increased levels of nifH, which may lead to increased nitrogen fixation.

[00306] Two strains of K. variicola CI137 were constructed in which rpoN was activated by deleting its native promoter and replacing it with a strong constitutive promoter. The data are shown in Figure 32 and Figure 33. The rpoN modified strains are designated 137-2251 and 137-2285, and the parent strain is designated 137-2084.

[00307] In Figure 32, the data is plotted as the decimal logarithm of the amount of ethylene produced per colony forming unit per hour, such that a higher amount of ethylene produced indicates greater nitrogen fixation. The assay was performed with 5 mM ammonium phosphate provided as a nitrogen source. Strain 137-2251 fixed nitrogen 1.23 times better than the parent strain (p=0.028), while strain 137-2285 fixed nitrogen 1.95 times better than the parent strain (p=0.031).

[00308] These results suggest that nitrogen fixation can be enhanced by increasing the amount of the polymerase responsible for transcription of the RNA encoding the nifH enzyme.

[00309]

Example 16: Activation of otsB leads to increased nitrogen-fixing activity

[00310] otsB is involved in trehalose biosynthesis in the cell, and modifications to otsB can improve strain stability. In this example, modifications to otsB were created and tested to determine whether these modifications had a negative effect on nitrogen fixation in cells.

[00311] Three strains of nitrogen-fixing bacteria have been modified to increase expression of the otsB gene. The native promoter of the otsB gene was deleted and replaced by a strong constitutive promoter. The modified strains were 6-921 and 6-922 (parent strain 6-435) and 6-1697 (parent strain 6-923). The ARA assay was performed on modified strains 6-921 and 6-922 (Figure 34). Data are presented as the decimal logarithm of the amount of ethylene formed per colony-forming unit per hour, so that higher amounts of ethylene formed indicate greater nitrogen fixation. The assay was performed with 5 mM ammonium phosphate provided as a nitrogen source. CI006 was used as a negative control. Unexpectedly, the nitrogen-fixing activity of the modified strain was increased compared to the parent strain.

[00312] Ammonium excretion assay was performed on strain 6-1697. Data are presented as NH4+ excreted (mM/OD) per hour, such that a higher value indicates greater nitrogen excretion. Data refer to experiments performed on different days. The data is shown in Figure 35. The nitrogen excretion activity was higher compared to the parent. Surprisingly, the ammonium excretion activity of the modified strain was increased compared to the parent strain.

Example 17: phoB can be modified to increase rhizosphere colonization

[00313] Colonization of some strains may be associated with biofilm formation, which may be influenced by phosphorus signaling, which in some cases may be influenced by changes in the expression of genes responsible for phosphorus signaling, such as phoB. Thus, changes in phoB may affect the colonization of some bacterial strains.

[00314] A modification of phoB (strain 137-1901) was created in a bacterial strain with high nitrogen excretion according to the results of the AMM analysis. The parent strain was designated 137-1586.

[00315] An AMM assay was performed on the cells to determine whether the modifications had a negative effect on the nitrogen excretion activity of the cells. The modifications did not reduce cellular nitrogen excretion activity (Figure 36A). Thus, phoB can be modified without reducing nitrogen excretion.

[00316] A corn root colonization assay was conducted to determine whether modifications affected the strain's ability to colonize plant roots. Briefly, germinated plants were inoculated with approximately 109 cells and allowed to grow for 4 weeks before being harvested. The combined embryonic root and first node were sampled approximately 1 to 2 inches below the plant canopy. After collection, samples were lysed and qPCR was performed to determine the number of cells present per gram of root tissue (CFU/g fresh weight). Strain 137-1901 showed increased colonization compared to parental strain 137-1586, as can be seen in Figure 36B. Thus, phoB modifications may increase cell colonization, since root colonization by the mutant strain was higher in this case than that of the parental strain.

Example 18: yjbE2 can be modified to increase rhizosphere colonization

[00317] The yjbE genes can stimulate the secretion of exopolysaccharides, which may enhance root attachment of certain strains.

[00318] A maize root colonization assay was performed to determine whether modifications to yjbE affected the strain's ability to colonize plant roots. Briefly, germinated plants were inoculated with approximately 10 ⁹ cells and allowed to grow for 4 weeks before being harvested. The combined embryonic root and first node were sampled approximately 1 to 2 inches below the plant canopy. After collection, samples were lysed and qPCR was performed to determine the number of cells present per gram of root tissue (CFU/g fresh weight). Strain 6-1779 showed increased colonization compared to parental strain 6-848, as can be seen in Figure 37.

Example 19. otsB

otsB is involved in trehalose biosynthesis in the cell, and modifications to otsB can improve strain stability. In this example, modifications to otsB were created and tested to determine whether these modifications had a negative effect on nitrogen fixation in cells.

[00319] A strain of nitrogen-fixing bacteria has been modified to increase expression of the otsB gene. ARA assay was performed on modified cells. Data are presented as the decimal logarithm of the amount of ethylene formed per colony-forming unit per hour, so that higher amounts of ethylene formed indicate greater nitrogen fixation. The assay was performed with 0 mM (top section) or 5 mM (bottom section) ammonium phosphate provided as a nitrogen source. Modified strains 6-1697 and 6-1698 (parental strain 6-923). CI006 was used as a negative control. The data is shown in Figure 38A. The nitrogen-fixing activity of the modified strain was increased compared to the parent strain. Thus, modifications to otsB can be made without negatively affecting the nitrogen-fixing activity of cells.

[00320] An ammonium excretion assay was performed on the modified cells. Data are presented as NH4+ excreted (mM/OD) per hour, such that a higher value indicates greater nitrogen excretion. Data refer to experiments performed on different days. The data is shown in Figure 38B. The activity of nitrogen excretion was higher compared to the parent. Thus, modifications to otsB can be made without negatively affecting the nitrogen-fixing activity of cells.

Example 20. CysZ mutation increases nitrogen fixation

[00321] CysZ mediates sulfate uptake, providing sulfur, which is then used to synthesize sulfur-containing compounds in bacterial cells, including cofactors required for nitrogenase activity. Increasing the expression of a sulfur transport gene such as cysZ may increase the number of functional nitrogenase complexes. For this purpose, two Kosakonia sacchari strains with cysZ modifications were created, designated 6-1691 and 6-916. Acetylene reduction assays (ARAs) were performed and data were compared to a parental strain without mutations in cysZ (strains 6-346 and 6-435 were the parents of 6-1691 and 6-916, respectively). Strain CI006 was used as a negative control, and ARA assays were performed in the same manner for both strains, except that 6-1691 and its parent were assayed in 5 mM ammonium phosphate (Figure 39A), while 6-916 and its parent was analyzed in 5 mM glutamine (Figure 39B). Both strains exhibit increased nitrogenase activity compared to the wild type and parental strains. The increase demonstrated by 6-1691 was approximately 14-fold, while strain 6-916 had a nitrogen-fixing activity approximately 6-fold higher than its parent.

Example 21 Biofilm Formation

[00322] Biofilm formation can influence the amount of nitrogen provided to the associated plant. Increasing biofilm formation on the plant root can increase the amount of nitrogen provided to the plant.

[00323] Certain genes may regulate biofilm formation. For example, smZ may act as a negative regulator of biofilm regulation. An smZ mutation that results in a reduction or elimination of smZ function can increase biofilm formation in bacteria. Mutagenesis can be performed to mutate smZ in strains with increased nitrogen excretion and nitrogen fixation activities to create a library of smZ mutant bacteria.

[00324] Certain proteins may promote biofilm formation. For example, large adhesion proteins, including lapA, may contribute to biofilm formation. One or more of the promoters that regulate lapA expression may be activated so that more lapA is produced, as determined by Western blot analysis.

[00325] Strains with smZ mutations and lapA activation can be generated to create a library of smZ/lapA strains for testing. smZ/LapA strains may be characterized by increased biofilm formation and may have biofilm formation capabilities greater than strains with only the smZ mutation or the lapA mutation.

[00326] Strains can be grown in lysogeny buffer (LB) and inoculated into soil with the seedling. The seedling, soil, and bacterial strain may be in a pot, in a field, indoors, or outdoors. The seedling can be planted in spring, summer, autumn or winter. The air may have a humidity of approximately 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. The weather may be rainy, foggy, cloudy or sunny. The temperature may be approximately 4°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C or 45°C. The seedling can be planted at dawn, morning, noon, afternoon, dusk, evening or night. The seedling can be grown for approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 or 30 days. After time has passed, you can dig up the plant and measure the amount of biofilm on the roots.

Example 22: Oxygen Sensitivity

[00327] The oxygen sensitivity of nitrogenase enzymes may affect nitrogen fixation, nitrogen excretion, or both. Decreasing the oxygen sensitivity of the nitrogenase enzyme may increase nitrogen-fixing activity, increase nitrogen excretion activity, or both. A bacterial strain can be created containing one or more nitrogenase enzymes with a reduced oxygen content, such that the strain exhibits increased nitrogen-fixing activity or increased nitrogen excretion activity.

[00328] Bacterial cells with wild type or genetically improved nitrogen-fixing activity, nitrogen-containing activity, or both, can be genetically modified. Genetic modifications can be introduced into cells such that one or more nitrogenase enzyme genes are mutated through a mutagenesis protocol, and a library of such strains can be generated.

[00329] Each strain in the library can be subjected to an oxygen sensitivity assay. To achieve this, AMM and ARA assays can be performed under conditions with different oxygen concentrations. The oxygen concentration can vary from 1 µM to 6 µM. Data can be analyzed to determine whether strains exhibit reduced nitrogen excretion or nitrogen-fixing activity as a function of oxygen concentration. Some strains with modified nitrogenase enzymes may show little or no reduction in nitrogen excretion or nitrogen-fixing activity when oxygen concentrations increase.

Example 23. Changing modA, modB, modE

[00330] The molybdenum uptake pathway may refer to a metabolic pathway in bacteria that allows the uptake of molybdenum into the cell. Molybdenum can act as a cofactor in nitrogen fixation. Increasing cellular uptake of molybdenum may increase nitrogen fixation, nitrogen excretion, or both.

[00331] Key enzymes in the molybdenum transport pathway may include modA, modB and modE. Modification or activation of any one, two, or three of these genes in a bacterial strain can increase molybdenum uptake into cells and can increase nitrogen fixation, nitrogen excretion, or both, in cells.

[00332] Genetic modifications can be made to these genes in bacterial cells to increase molybdenum transport. To identify such modifications, mutagenesis can be carried out in and around the active sites of genes encoding modA, modB or modC. For each gene, a library of mutants can be constructed. These libraries can then be screened for increased molybdenum scavenging activity. These libraries can be generated from wild-type cells or from cells that have already been modified to exhibit increased nitrogen fixation, increased nitrogen excretion, or both.

[00333] Genetic modifications can be made in these cells such that one or more genes in the molybdenum uptake pathway are activated. A library of variants can also be constructed to increase promoter activity for one or more genes in the molybdenum uptake pathway. For example, the modABC promoter can be genetically tuned to increase transcription. Western blotting can be used to determine which strains have increased expression of one or more molybdenum uptake genes, and these strains may be of interest as variants.

[00334] To screen for increased molybdenum scavenging activity, each mutant or variant of interest, as well as the unmutated parent strain for each strain, can be inoculated and grown in three wells of a 96-well plate in LB medium. Once the cells reach an optical density of 0.8, material from one of the wells for each mutant can be collected, the cells are pelleted by centrifugation, excess LB is washed off, and the cells are resuspended, lysed, and prepared for spectroscopy in a 96-well plate. Molybdenum content can be measured using spectroscopy to determine the original molybdenum content of samples. The remaining samples may be supplemented with molybdenum in the form of molybdate tetraoxyanion in LB or LB only, so that the amount added is 10% or less of the total volume in each well. The final concentration of molybdenum in each well in the assay may be one of 10 pM, 100 pM, 1 nM, 10 nM, or 100 nM, and more than one concentration may be assayed. The assay may be conducted for one or more of 1 second, 10 seconds, 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, and 60 minutes.

[00335] First, for each concentration, the amount of molybdenum transported into each cell can be plotted against time to determine the optimal duration of the assay. The optimal duration may be a time interval in which steady state has not been reached and the reaction rate remains linear. The amount of molybdenum transported into each cell can then be plotted against concentration for the optimal duration and a Michaelis-Menten analysis can be performed. Mutant strains of interest or variants showing a significant increase (p<0.05) in molybdenum transport compared to the unmutated and unmodified parental strain, as determined by Km comparison, can be selected for further analyses.

[00336] Each strain selected for further analyzes can then be subjected to an AMM assay as well as an ARA assay as described herein to measure nitrogen excretion and nitrogen fixation, respectively. One or more of these mutations or variants may increase AMM, ARA, or both, of one or more of these bacterial strains.

Example 24: Increasing Iron Transport Can Increase Nitrogen Fixation

[00337] The iron uptake pathway may refer to a metabolic pathway in bacteria that mediates the uptake of iron into the cell. Iron may act as a cofactor to stimulate nitrogen fixation. Increasing cellular iron uptake may increase nitrogen fixation, nitrogen excretion, or both.

[00338] Key enzymes in the iron transport pathway may include exbA, exbB and tonB. Modification or activation of any one, two or three of these genes in a bacterial strain can increase iron uptake into cells and can increase nitrogen fixation, nitrogen excretion, or both, in cells.

[00339] Genetic modifications can be made to these genes in bacterial cells to increase iron transport. To identify such modifications, mutagenesis can be performed in and around the active sites of genes encoding exbA, exbB, or tonB. For each gene, a library of mutants can be constructed. These libraries can then be screened for increased iron uptake activity. These libraries can be generated from wild-type cells or from cells that have already been modified to exhibit increased nitrogen fixation, increased nitrogen excretion, or both.

[00340] Genetic modifications can be made in these cells such that one or more genes in the iron uptake pathway are activated. The library of variants can also be constructed to increase promoter activity for one or more genes in the iron uptake pathway. Western blotting can be used to determine which strains have increased expression of one or more iron uptake genes, and these strains may be of interest as variants.

[00341] To screen for increased iron uptake activity, each mutant or variant of interest, as well as the unmutated parent strain for each strain, can be inoculated and grown in three wells of a 96-well plate in LB medium. Once the cells reach an optical density of 0.8, material from one of the wells for each mutant can be collected, the cells are pelleted by centrifugation, excess LB is washed off, and the cells are resuspended, lysed, and prepared for spectroscopy in a 96-well plate. Iron content can be measured using spectroscopy to determine the initial iron content of samples. The remaining samples may be supplemented with iron in the form of FeSO ₄ in LB or LB only, so that the amount added is 10% or less of the total volume in each well. 0.1 mM acetic acid (final concentration) may be added to improve iron solubility. The final concentration of iron in each well in the assay may be one of 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, or 100 μM, and more than one concentration may be assayed. The assay may be conducted for one or more of 1 second, 10 seconds, 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, and 60 minutes.

[00342] First, the amount of iron transported into each cell can be plotted against time for each concentration to determine the optimal duration of the assay. The optimal duration may be a time interval in which steady state has not been reached and the reaction rate remains linear. The amount of iron transported into each cell can then be plotted against concentration for the optimal duration and a Michaelis-Menten analysis can be performed. Mutant strains of interest or variants showing a significant increase (p<0.05) in iron transport compared to the unmutated and unmodified parental strain, as determined by Km comparison, can be selected for further analyses.

[00343] Each strain selected for further analyzes can then be subjected to an AMM assay as well as an ARA assay as described herein to measure nitrogen excretion and nitrogen fixation, respectively. One or more of these mutations or variants may increase AMM, ARA, or both, of one or more of these bacterial strains.

Example 25. Increasing the number of siderophore biosynthetic genes can increase nitrogen fixation

[00344] Yhf sidrophores, or iron chelate molecules, can influence iron uptake by bacteria. Increased production of yhf siderophores may enhance iron uptake by bacteria. Modifications of siderophore genes, including yhfA, yusV, sbnA, yfiZ, fiu, and fur, can increase the production of siderophores yhf, which in turn can increase cellular iron uptake. These modifications may result in increased nitrogen fixation or excretion.

[00345] Genetic modifications can be made to these genes in bacterial cells to increase siderophore production. To identify such modifications, mutagenesis can be carried out in and around the active sites of genes encoding yhfA, yusV, sbnA, yfiZ, fiu or fur. For each gene, a library of mutants can be constructed. These libraries can then be screened for increased siderophore production. These libraries can be generated from wild-type cells or from cells that have already been modified to exhibit increased nitrogen fixation, increased nitrogen excretion, or both. To screen for increased siderophore production, each mutant can be grown to an optical density of 0.8 and then an excess, but not toxic, amount of siderophore precursors can be added and incubated for 30 minutes or 60 minutes. The amount of siderophores produced after a designated period of time can be normalized, and strains exhibiting increased siderophore production can be screened for increased iron transport.

[00346] To screen for increased iron uptake activity, each mutant or variant of interest, as well as the unmutated parent strain for each strain, can be inoculated and grown in three wells of a 96-well plate in LB medium. Once the cells reach an optical density of 0.8, material from one of the wells for each mutant can be collected, the cells are pelleted by centrifugation, excess LB is washed off, and the cells are resuspended, lysed, and prepared for spectroscopy in a 96-well plate. Iron content can be measured using spectroscopy to determine the initial iron content of samples. The remaining samples may be supplemented with iron in the form of FeSO ₄ in LB or LB only, so that the amount added is 10% or less of the total volume in each well. 0.1 mM acetic acid (final concentration) may be added to improve iron solubility. The final concentration of iron in each well in the assay may be one of 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, or 100 μM, and more than one concentration may be assayed. The assay may be conducted for one or more of 1 second, 10 seconds, 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, and 60 minutes.

[00347] First, the amount of iron transported into each cell can be plotted against time for each concentration to determine the optimal duration of the assay. The optimal duration may be a time interval in which steady state has not been reached and the reaction rate remains linear. The amount of iron transported into each cell can then be plotted against concentration for the optimal duration and a Michaelis-Menten analysis can be performed. Mutant strains of interest or variants showing a significant increase (p<0.05) in iron transport compared to the unmutated and unmodified parental strain, as determined by Km comparison, can be selected for further analyses.

[00348] Each strain selected for further analyzes can then be subjected to an AMM assay as well as an ARA assay as described herein to measure nitrogen excretion and nitrogen fixation, respectively. One or more of these mutations or variants may increase AMM, ARA, or both, of one or more of these bacterial strains.

Example 26. dctA1 and dctA2

[00349] dctA1 and dctA2 are responsible for the aerobic transport of the dicarboxylates fumarate, L- and D-malate and, to a lesser extent, succinate, from the periplasm across the inner membrane. Modifications of these genes can influence the colonization of microorganisms.

Modifications in dctA1 (strain 137-1861) and dctA2 (strain 137-1905) were created in a strain with high nitrogen excretion as determined by AMM analysis. The parent strain was designated 137-1905.

[00350] An AMM assay was performed on the cells to determine whether the modifications had a negative effect on the nitrogen excretion activity of the cells. The modifications did not reduce cellular nitrogen excretion activity (Figure 40A). Thus, dctA1 and dctA2 can be modified without reducing nitrogen excretion.

[00351] Colony formation assays were performed on cells using qPCR to determine whether the modifications had an effect on the colony-forming properties of the cells. Briefly, germinated plants were inoculated with approximately 10 ⁹ cells and allowed to grow for 4 weeks before being harvested. The combined embryonic root and first node were sampled approximately 1 to 2 inches below the plant canopy. After collection, samples were lysed and qPCR was performed to determine CFU/g fresh weight. The data is shown in Figure 40B. Both strains showed increased colonization. Thus, modifications of dctA1 and dctA2 may increase colonization in cells.

Example 27. Increasing resistance to drying

[00352] Improving desiccation tolerance may enhance colonization of a strain used for seed coating or other dry application to a plant or field. A genetically engineered microorganism will be created containing an arabinose promoter functionally linked to the rpoE gene. The specified genetically engineered microorganism and non-engineered parental control will be cultured in arabinose supplemented medium for 48 hours and then dried. After drying, a portion of each of the engineered and unengineered strains will be regenerated in arabinose-free medium, and the number of microorganisms in the medium will be analyzed after 24 hours. A medium containing the engineered strain will contain more microorganisms than a medium containing the non-engineered parent strain.

[00353] The dried microorganisms will be applied to corn seeds and planted in arabinose-free soil in a greenhouse under conditions suitable for corn seed germination. After 4 weeks, the seedlings will be harvested and the number of colony forming units of both engineered and non-engineered microorganisms on the plant roots will be assessed. Plants exposed to engineered microorganisms will associate with more microorganisms than plants exposed to the nonengineered parent strain.

Example 28: Alteration of the nifA RBS increases nifH transcription

[00354] Promoter-RBS sequences inserted upstream of nifA result in increased nifH:nifA transcription ratios, indicating an increase in the amount of active NifA protein due to the increased strength of the inserted RBS, resulting in a higher rate of NifA translation initiation.

[00355]

Table 8. Strains described herein

Variety First mention Current name Universal name Line Description of mutagenic DNA Genotype 1 Application text CI006 CI006 Isolated strain from the genus Enterobacter Absent Wild type 2 Application text CI008 CI008 Isolated strain from the genus Burkholderia Absent Wild type 3 Application text CI010 CI010 Isolated strain from the genus Klebsiella Absent Wild type 4 Application text CI019 CI019 Isolated strain from the genus Rahnella Absent Wild type 5 Application text CI028 CI028 Isolated strain from the genus Enterobacter Absent Wild type 6 Application text CI050 CI050 Isolated strain from the genus Klebsiella Absent Wild type 7 Application text CM002 CM002 Mutant CI050 Disruption of the nifL gene by an inserted kanamycin resistance (KanR) expression cassette encoding the aminoglycoside O-phosphotransferase gene aph1. ΔnifL::KanR 8 Application text CM011 CM011 Mutant CI019 Disruption of the nifL gene by an inserted spectinomycin resistance expression cassette (SpecR), encoding the streptomycin 3''-O-adenylyltransferase gene aadA. ΔnifL::SpecR 9 Application text CM013 CM013 Mutant CI006 Disruption of the nifL gene by an inserted kanamycin resistance (KanR) expression cassette encoding the aminoglycoside O-phosphotransferase gene aph1. ΔnifL::KanR 10 Figure 4A CM004 CM004 Mutant CI010 Disruption of the amtB gene by an inserted kanamycin resistance (KanR) expression cassette encoding the aminoglycoside O-phosphotransferase gene aph1. ΔamtB::KanR eleven Figure 4A CM005 CM005 Mutant CI010 Disruption of the nifL gene by an inserted kanamycin resistance (KanR) expression cassette encoding the aminoglycoside O-phosphotransferase gene aph1. ΔnifL::KanR 12 Figure 4B CM015 CM015 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the ompX gene (Prm5). ΔnifL::Prm5 13 Figure 4B CM021 CM021 Mutant CI006 Disruption of the nifL gene by an inserted fragment of the region upstream of the unannotated gene and the first 73 bp. this gene (Prm2). ΔnifL::Prm2 14 Figure 4B CM023 CM023 Mutant CI006 Disruption of the nifL gene by an inserted fragment of the region upstream of the acpP gene and the first 121 bp. acpP gene (Prm4). ΔnifL::Prm4 15 Figure 10A CM014 CM014 Mutant CI006 Disruption of the nifL gene by an inserted fragment of the region upstream of the lpp gene and the first 29 bp. lpp gene (Prm1). ΔnifL::Prm1 16 Figure 10A CM016 CM016 Mutant CI006 Disruption of the nifL gene by an inserted fragment of the region upstream of the lexA gene 3 and the first 21 bp. lexA 3 gene (Prm9). ΔnifL::Prm9 17 Figure 10A CM022 CM022 Mutant CI006 Disruption of the nifL gene by an inserted fragment of the region upstream of the mntP 1 gene and the first 53 bp. mntP 1 (Prm3) gene. ΔnifL::Prm3 18 Figure 10A CM024 CM024 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the sspA gene (Prm7). ΔnifL::Prm7 19 Figure 10A CM025 CM025 Mutant CI006 Disruption of the nifL gene by an inserted fragment of the region upstream of the hisS gene and the first 52 bp. hisS gene (Prm10). ΔnifL::Prm10 20 Figure 10B CM006 CM006 Mutant CI010 Disruption of the glnB gene by an inserted kanamycin resistance (KanR) expression cassette encoding the aminoglycoside O-phosphotransferase gene aph1. ΔglnB::KanR 21 Figure 10C CI028 nifL:KanR CM017 Mutant CI028 Disruption of the nifL gene by an inserted kanamycin resistance (KanR) expression cassette encoding the aminoglycoside O-phosphotransferase gene aph1. ΔnifL::KanR 22 Figure 10C CI019 nifL:SpecR CM011 Mutant CI019 Disruption of the nifL gene by an inserted spectinomycin resistance expression cassette (SpecR), encoding the streptomycin 3''-O-adenylyltransferase gene aadA. ΔnifL::SpecR 23 Figure 10C CI006 nifL:KanR CM013 Mutant CI006 Disruption of the nifL gene by an inserted kanamycin resistance (KanR) expression cassette encoding the aminoglycoside O-phosphotransferase gene aph1. ΔnifL::KanR 24 Figure 10C CI010 nifL:KanR CM005 Mutant CI010 Disruption of the nifL gene by an inserted kanamycin resistance (KanR) expression cassette encoding the aminoglycoside O-phosphotransferase gene aph1. ΔnifL::KanR 25 Figure 4C Strain 2 CI006 Isolated strain from the genus Enterobacter Absent Wild type 26 Figure 4C Strain 4 CI010 Isolated strain from the genus Klebsiella Absent Wild type 27 Figure 4C Strain 1 CI019 Isolated strain from the genus Rahnella Absent Wild type 28 Figure 4C Strain 3 CI028 Isolated strain from the genus Enterobacter Absent Wild type 29 Figure 4B Strain 2 CI006 Isolated strain from the genus Enterobacter Absent Wild type thirty Figure 4B High CM014 Mutant CI006 Disruption of the nifL gene by an inserted fragment of the region upstream of the lpp gene and the first 29 bp. lpp gene (Prm1). ΔnifL::Prm1 31 Figure 4B Average CM015 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the ompX gene (Prm5). ΔnifL::Prm5 32 Figure 4B Short CM023 Mutant CI006 Disruption of the nifL gene by an inserted fragment of the region upstream of the acpP gene and the first 121 bp. acpP gene (Prm4). ΔnifL::Prm4 33 4D figure Strain 2 CI006 Isolated strain from the genus Enterobacter Absent Wild type 34 4D figure Evolved CM029 Mutant CI006 Disruption of the nifL gene by an inserted fragment of the region upstream of the ompX gene (Prm5) and a deletion of 1287 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO1). ΔnifL::Prm5 ΔglnE-AR_KO1 35 Figure 14C Wild CI006 Isolated strain from the genus Enterobacter Absent Wild type 36 Figure 14C Evolved CM014 Mutant CI006 Disruption of the nifL gene by an inserted fragment of the region upstream of the lpp gene and the first 29 bp. lpp gene (Prm1). ΔnifL::Prm1 37 Figure 14B Wild CI019 Isolated strain from the genus Rahnella Absent Wild type 38 Figure 14B Evolved CM011 Mutant CI019 Disruption of the nifL gene by an inserted spectinomycin resistance expression cassette (SpecR), encoding the streptomycin 3''-O-adenylyltransferase gene aadA. ΔnifL::SpecR 39 Figure 14A Evolved CM011 Mutant CI019 Disruption of the nifL gene by an inserted spectinomycin resistance expression cassette (SpecR), encoding the streptomycin 3''-O-adenylyltransferase gene aadA. ΔnifL::SpecR 40 Figure 15A Wild CI006 Isolated strain from the genus Enterobacter Absent Wild type 41 Figure 15A Evolved CM013 Mutant CI006 Disruption of the nifL gene by an inserted kanamycin resistance (KanR) expression cassette encoding the aminoglycoside O-phosphotransferase gene aph1. ΔnifL::KanR 42 Figure 15B Has no name CM011 Mutant CI019 Disruption of the nifL gene by an inserted spectinomycin resistance expression cassette (SpecR), encoding the streptomycin 3''-O-adenylyltransferase gene aadA. ΔnifL::SpecR 43 Figure 16B Strain 5 CI008 Isolated strain from the genus Burkholderia Absent Wild type 44 Figure 16B Strain 1 CM011 Mutant CI019 Disruption of the nifL gene by an inserted spectinomycin resistance expression cassette (SpecR), encoding the streptomycin 3''-O-adenylyltransferase gene aadA. ΔnifL::SpecR 34 4D figure Evolved CM029 Utant CI006 Disruption of the nifL gene by an inserted fragment of the region upstream of the ompX gene (Prm5) and a deletion of 1287 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO1). ΔnifL::Prm5 ΔglnE-AR_KO1

Table 9. Other strains described herein

Strain ID Line Description of mutagenic DNA Chromosomal genotype Cure status CI006 CI006 Parent Kosakonia saccari, wild type Wild type N/A CI137 CI137 Parental K. variicola, wild type Wild type N/A 137-1036 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the infC gene (PinfC) upstream of nifA. Deletion 1647 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO2). ΔnifL::PinfC uncured 6-412 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the ompX gene (Prm5) upstream of nifA. Deletion 1287 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO1). ΔnifL::Prm5 ΔglnE-AR_KO1 Healed 6-435 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the lpp gene (Prm1) upstream of nifA. Deletion 1287 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO1). Deletion of the entire amtB CDS. ΔnifL::Prm1 ΔglnE-AR_KO1, ΔamtB 6-1691 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the lpp gene (Prm1) upstream of nifA. Deletion 1287 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO1). Deletion 150 bp. upstream of the cysZ gene and the Prm1 insertion directly upstream of cysZ. ΔnifL::Prm,1 ΔglnE-AR_KO1, cysZ::Prm1 Uncured 6-346 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the lpp gene (Prm1) upstream of nifA. Deletion 1287 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO1). ΔnifL::Prm1, ΔglnE-AR_KO1 Uncured 6-923 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the lpp gene (Prm1) upstream of nifA. Deletion 1287 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO1). Deletion of the entire amtB CDS. Deletion of the native treZ promoter and insertion of a fragment (Prm2) from the region upstream of PROKKA_04751 (a hypothetical protein), immediately upstream of treZ. ΔnifL::Prm1 ΔglnE-AR_KO1, ΔamtB, treZ::Prm2 Uncured 6-1694 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the ompX gene (Prm5) upstream of nifA. Deletion 1287 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO1). Deletion of the entire amtB CDS. ΔnifL::Prm5 ΔglnE-AR_KO1, ΔglgA Uncured 6-342 Mutant CI006 Disruption of the nifL gene by an inserted fragment of the region upstream of the ompX gene (Prm5) and a deletion of 1287 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO1). ΔnifL::Prm5 ΔglnE-AR_KO1 Uncured 6-1681 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the lpp gene (Prm1) upstream of nifA. Deletion 1287 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO1). Deletion of the entire amtB CDS. ΔnifL::Prm1 ΔglnE-AR_KO1, ΔglgA Uncured 6-404 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the lpp gene (Prm1) upstream of nifA. Deletion 1287 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO1). ΔnifL::Prm1 ΔglnE-AR_KO1 Healed 6-921 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the lpp gene (Prm1) upstream of nifA. Deletion 1287 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO1). Deletion of the entire amtB CDS. Deletion of the native otsB promoter and insertion of Prm1 immediately upstream of ostB. ΔnifL::Prm1 ΔglnE-AR_KO1, ΔamtB, otsB::Prm1 Uncured 6-922 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the lpp gene (Prm1) upstream of nifA. Deletion 1287 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO1). Deletion of the entire amtB CDS. Deletion of the native otsB promoter and insertion of a fragment (Prm2) from the region upstream of PROKKA_04751 (a hypothetical protein), immediately upstream of otsB. ΔnifL::Prm1 ΔglnE-AR_KO1, ΔamtB, otsB::Prm2 Uncured 6-1744 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the lpp gene (Prm1) upstream of nifA. Deletion 1287 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO1). Deletion of the entire amtB CDS. Deletion of the entire amtB CDS. Deletion of the native treZ promoter and insertion of a fragment (Prm2) from the region upstream of PROKKA_04751 (a hypothetical protein), immediately upstream of treZ. ΔnifL::Prm1 ΔglnE-AR_KO1, ΔamtB, ΔglgA, treZ::Prm2 uncured 6-322 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the lpp gene (Prm1) upstream of nifA. ΔnifL::Prm1 uncured 6-400 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the lpp gene (Prm1) upstream of nifA. ΔnifL::Prm1 healed 6-848 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the lpp gene (Prm1) upstream of nifA. Deletion 1647 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO2). Deletion of the entire amtB CDS. ΔnifL::Prm1, ΔglnE-AR_KO2, ΔamtB Healed 137-2084 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the cspE gene (Prm1.2) upstream of nifA. Deletion 1647 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO2). ΔnifL::Prm1.2 ΔglnE-AR_KO2 healed 137-2251 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the cspE gene (Prm1.2) upstream of nifA. Deletion 1647 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO2). Deletion of the native rpoN promoter and insertion of a fragment (Prm8.2) located from 77 bp. after the nlpI start codon up to 376 bp. after the nlpI start codon, immediately upstream of rpoN. ΔnifL::Prm1.2 ΔglnE-AR_KO2, rpoN::Prm8.2 healed 137-2285 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the cspE gene (Prm1.2) upstream of nifA. Deletion 1647 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO2). Deletion of the native rpoN promoter and insertion of a fragment (Prm1.2) immediately upstream of the rpoN CDS. ΔnifL::Prm1.2 ΔglnE-AR_KO2, rpoN::Prm1.2 healed 137-2225 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the cspE gene (Prm1.2) upstream of nifA. Deletion 1647 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO2). Deletion of the entire glnK CDS. ΔnifL::Prm1.2 ΔglnE-AR_KO2, ΔglnK healed 137-1586 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the infC gene (PinfC) upstream of nifA. Deletion 1647 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO2). ΔnifL::PinfC, ΔglnE-AR_KO2 healed 137-1860 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the infC gene (PinfC) upstream of nifA. Deletion 1647 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO2). Deletion of the native dctA1 promoter and insertion of a fragment (Prm8.2) located from 77 bp. after the nlpI start codon up to 376 bp. after the nlpI start codon, immediately upstream of dctA1. The native start codon of dctA1 was also changed from GTG to ATG. ΔnifL::PinfC, ΔglnE-AR_KO2, dctA1::Prm8.2_ATG-start healed 137-1905 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the infC gene (PinfC) upstream of nifA. Deletion 1647 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO2). Deletion of the native dctA1 promoter and insertion of a fragment (Prm1.2) immediately upstream of dctA1. The native start codon of dctA1 was also changed from GTG to ATG. ΔnifL::PinfC, ΔglnE-AR_KO2, dctA1::Prm1.2_ATG-start uncured 6-1697 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the lpp gene (Prm1) upstream of nifA. Deletion 1287 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO1). Deletion of the entire amtB CDS. Deletion of the native treZ promoter and insertion of a fragment (Prm2) from the region upstream of PROKKA_04751 (a hypothetical protein), immediately upstream of treZ. Deletion of the native otsB promoter and insertion of Prm1 immediately upstream of ostB. ΔnifL::Prm1 ΔglnE-AR_KO1, ΔamtB, treZ::Prm2, otsB::Prm1 Uncured 137-1901 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the cspE gene (Prm1.2) upstream of nifA. Deletion 1647 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO2). Deletion of the native phoB1 promoter and insertion of a fragment (Prm1.2) immediately upstream of the phoB1 CDS. ΔnifL::Prm1.2 ΔglnE-AR_KO2, phoB1::Prm1.2 Healed 6-1779 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the lpp gene (Prm1) upstream of nifA. Deletion 1647 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO2). Deletion of the entire amtB CDS. Deletion of the native bcsII promoter and insertion of a fragment (Prm2) from the region upstream of PROKKA_04751 (a hypothetical protein), immediately upstream of bcsII. Deletion of the native yjbE2 promoter and insertion of Prm1 immediately upstream of yjbE2. ΔnifL::Prm1, ΔglnE-AR_KO2, ΔamtB, bcsII::Prm2, yjbE2::Prm1 Uncured 6-1782 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the ompX gene (Prm5) upstream of nifA. Deletion 1287 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO1). Deletion of the native glnA promoter and insertion of a fragment (PrmglnB) from a region upstream of the glnB gene, immediately upstream of glnA. ΔnifL::Prm5 ΔglnE-AR_KO1, glnA::PrmglnB_ATG-start Uncured 6-1783 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the ompX gene (Prm5) upstream of nifA. Deletion 1287 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO1). Deletion of the native glnA promoter and insertion of a fragment (PrmglnB) from a region upstream of the glnB gene, immediately upstream of glnA. The native glnA start codon was also changed from ATG to GTG. ΔnifL::Prm5 ΔglnE-AR_KO1, glnA::PrmglnB_GTG-start Uncured 6-1906 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the lpp gene (Prm1) upstream of nifA. Deletion 1287 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO1). Deletion of the native glnA promoter and insertion of a fragment (PrmglnB) from a region upstream of the glnB gene, immediately upstream of glnA. The native glnA start codon was also changed from ATG to GTG. ΔnifL::Prm1 ΔglnE-AR_KO1, glnA::PrmglnB_GTG-start Uncured 6-1907 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the lpp gene (Prm1) upstream of nifA. Deletion 1287 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO1). Deletion of the native glnA promoter and insertion of a fragment (PrmglnE) from a region upstream of the glnE gene, immediately upstream of glnA. ΔnifL::Prm1 ΔglnE-AR_KO1, glnA::PrmglnE_ATG-start Uncured 6-1908 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the lpp gene (Prm1) upstream of nifA. Deletion 1287 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO1). Deletion of the native glnA promoter and insertion of a fragment (PrmglnE) from a region upstream of the glnE gene, immediately upstream of glnA. The native glnA start codon was also changed from ATG to GTG. ΔnifL::Prm1 ΔglnE-AR_KO1, glnA::PrmglnE_GTG-start Uncured 6-1912 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the lpp gene (Prm1) upstream of nifA. Deletion of the native glnA promoter and insertion of a fragment (PrmglnE) from a region upstream of the glnE gene, immediately upstream of glnA. ΔnifL::Prm1, glnA::PrmglnE_ATG-start Uncured 137-2656 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the cspE gene (Prm1.2) upstream of nifA. Deletion of the native glnA promoter and insertion of a fragment (PrmglnB) from a region upstream of the glnB gene, immediately upstream of glnA. ΔnifL::Prm1.2, glnA::PrmglnB_ATG-start Healed 137-2657 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the cspE gene (Prm1.2) upstream of nifA. Deletion of the native glnA promoter and insertion of a fragment (PrmglnB) from a region upstream of the glnB gene, immediately upstream of glnA. The native glnA start codon was also changed from ATG to GTG. ΔnifL::Prm1.2, glnA::PrmglnB_GTG-start Healed 137-2658 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the cspE gene (Prm1.2) upstream of nifA. Deletion of the native glnA promoter and insertion of a fragment (PrmglnD) from a region upstream of the glnD gene, immediately upstream of glnA. ΔnifL::Prm1.2, glnA::PrmglnD_ATG-start Healed 137-2659 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the cspE gene (Prm1.2) upstream of nifA. Deletion of the native glnA promoter and insertion of a fragment (PrmglnD) from a region upstream of the glnD gene, immediately upstream of glnA. The native glnA start codon was also changed from ATG to GTG. ΔnifL::Prm1.2, glnA::PrmglnD_GTG-start Healed 137-2660 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the cspE gene (Prm1.2) upstream of nifA. Deletion of the native glnA promoter and insertion of a fragment (PrmglnE) from a region upstream of the glnE gene, immediately upstream of glnA. ΔnifL::Prm1.2, glnA::PrmglnE_ATG-start Healed 137-2661 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the cspE gene (Prm1.2) upstream of nifA. Deletion of the native glnA promoter and insertion of a fragment (PrmglnE) from a region upstream of the glnE gene, immediately upstream of glnA. The native glnA start codon was also changed from ATG to GTG. ΔnifL::Prm1.2, glnA::PrmglnE_GTG-start Healed 137-2257 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the cspE gene (Prm1.2) upstream of nifA. Deletion 1647 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO2). Deletion of the native glnA promoter and insertion of a fragment (PrmglnB) from a region upstream of the glnB gene, immediately upstream of glnA. ΔnifL::Prm1.2 ΔglnE-AR_KO2, glnA::PrmglnB_ATG-start Healed 137-2259 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the cspE gene (Prm1.2) upstream of nifA. Deletion 1647 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO2). Deletion of the native glnA promoter and insertion of a fragment (PrmglnB) from a region upstream of the glnB gene, immediately upstream of glnA. The native glnA start codon was also changed from ATG to GTG. ΔnifL::Prm1.2 ΔglnE-AR_KO2, glnA::PrmglnB_GTG-start Healed 137-2261 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the cspE gene (Prm1.2) upstream of nifA. Deletion 1647 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO2). Deletion of the native glnA promoter and insertion of a fragment (PrmglnD) from a region upstream of the glnD gene, immediately upstream of glnA. ΔnifL::Prm1.2 ΔglnE-AR_KO2, glnA::PrmglnD_ATG-start Healed 137-2263 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the cspE gene (Prm1.2) upstream of nifA. Deletion 1647 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO2). Deletion of the native glnA promoter and insertion of a fragment (PrmglnD) from a region upstream of the glnD gene, immediately upstream of glnA. The native glnA start codon was also changed from ATG to GTG. ΔnifL::Prm1.2 ΔglnE-AR_KO2, glnA::PrmglnD_GTG-start Healed 137-2281 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the cspE gene (Prm1.2) upstream of nifA. Deletion 1647 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO2). Deletion of the native glnA promoter and insertion of a fragment (PrmglnE) from a region upstream of the glnE gene, immediately upstream of glnA. ΔnifL::Prm1.2 ΔglnE-AR_KO2, glnA::PrmglnE_ATG-start Healed 137-2219 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the cspE gene (Prm1.2) upstream of nifA. Deletion 1647 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO2). Deletion 546 bp. before the stop codon of the glnD gene containing the ACT1/2 domain of the bifunctional uridylyltransferase/uridyl-removing enzyme (ΔglnD-ACT12_truncation) ΔnifL::Prm1.2 ΔglnE-AR_KO2, ΔglnD_ACT12_truncation Healed 137-2221 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the cspE gene (Prm1.2) upstream of nifA. Deletion 1647 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO2). Deletion 975 bp. after the start codon of the glnD gene containing the uridylyltransferase (UT) domain of the bifunctional uridylyltransferase/uridyl-removing enzyme (ΔglnD-UT_truncation) ΔnifL::Prm1.2 ΔglnE-AR_KO2, ΔglnD_UT_truncation Healed 137-2223 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the cspE gene (Prm1.2) upstream of nifA. Deletion 1647 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO2). Deletion of the entire glnD CDS. ΔnifL::Prm1.2 ΔglnE-AR_KO2, ΔglnD Healed 137-2245 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the cspE gene (Prm1.2) upstream of nifA. Deletion 1647 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO2). Deletion of 66 bp, starting from nucleotide pair 1519 to nucleotide pair 1586 of the glnD gene containing the uridylyl-removing (UR) domain of the bifunctional uridylyltransferase/uridyl-removing enzyme (ΔglnD-UR_truncation) ΔnifL::Prm1.2 ΔglnE-AR_KO2, ΔglnD_UR_truncation Healed 137-2227 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the infC gene (PinfC) upstream of nifA. Deletion 546 bp. before the stop codon of the glnD gene containing the ACT1/2 domain of the bifunctional uridylyltransferase/uridyl-removing enzyme (ΔglnD-ACT12_truncation) ΔnifL::PinfC, ΔglnD_ACT12_truncation Healed 137-2229 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the infC gene (PinfC) upstream of nifA. Deletion of the entire glnD CDS. ΔnifL::PinfC, ΔglnD Healed 137-2253 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the infC gene (PinfC) upstream of nifA. Deletion 975 bp. after the start codon of the glnD gene containing the uridylyltransferase (UT) domain of the bifunctional uridylyltransferase/uridyl-removing enzyme (ΔglnD-UT_truncation) ΔnifL::PinfC, ΔglnD_UT_truncation Healed 137-2283 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the infC gene (PinfC) upstream of nifA. Inactivation of the uridylyltransferase (UT) domain of the bifunctional uridylyltransferase/uridyl scavenging enzyme, glnD, by mutating amino acid residues 90 and 91 from GG to DV and residue 104 from D to A. ΔnifL::PinfC, ΔglnD_UT_deactivation Healed 137-2237 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the cspE gene (Prm1.2) upstream of nifA. Deletion 1647 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO2). Deletion of the native glsA2 promoter and insertion of a fragment (Prm1.2) immediately upstream of the glsA2 CDS. ΔnifL::Prm1.2, ΔglnE-AR_KO2, glsaA2::Prm1.2 Healed 6-2552 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the lpp gene (Prm1) upstream of nifA. Deletion 1287 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO1). Deletion 549 bp. before the stop codon of the glnD gene containing the ACT1/2 domain of the bifunctional uridylyltransferase/uridyl-removing enzyme (ΔglnD-ACT12_truncation) ΔnifL::Prm1, ΔglnE-AR_KO1, ΔglnD-ACT12_truncation Healed 6-2597 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the lpp gene (Prm1) upstream of nifA. Deletion 1287 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO1). Deletion 987 bp. after the start codon of the glnD gene containing the uridylyltransferase (UT) domain of the bifunctional uridylyltransferase/uridyl-removing enzyme (ΔglnD-UT_truncation) ΔnifL::Prm1, ΔglnE-AR_KO1, ΔglnD_UT_truncation Healed 6-2587 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the lpp gene (Prm1) upstream of nifA. Deletion 1287 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO1). Deletion of the entire glnD CDS. ΔnifL::Prm1, ΔglnE-AR_KO1, ΔglnD Healed 6-2636 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the lpp gene (Prm1) upstream of nifA. Deletion 1287 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO1). Inactivation of the uridylyltransferase (UT) domain of the bifunctional uridylyltransferase/uridyl scavenging enzyme, glnD, by mutating amino acid residues 90 and 91 from GG to DV and residue 104 from D to A. ΔnifL::Prm1, ΔglnE-AR_KO1, ΔglnD_UT_deactivation Healed 6-2090 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the ompX gene (Prm5) upstream of nifA. Deletion 1287 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO1). Deletion 549 bp. before the stop codon of the glnD gene containing the ACT1/2 domain of the bifunctional uridylyltransferase/uridyl-removing enzyme (ΔglnD-ACT12_truncation) ΔnifL::Prm5, ΔglnE-AR_KO1, ΔglnD-ACT12_truncation Healed 6-2122 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the ompX gene (Prm5) upstream of nifA. Deletion 1287 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO1). Deletion of the entire glnD CDS. ΔnifL::Prm5, ΔglnE-AR_KO1, ΔglnD Healed 6-2411 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the ompX gene (Prm5) upstream of nifA. Deletion 1287 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO1). Deletion 987 bp. after the start codon of the glnD gene containing the uridylyltransferase (UT) domain of the bifunctional uridylyltransferase/uridyl-removing enzyme (ΔglnD-UT_truncation) ΔnifL::Prm5, ΔglnE-AR_KO1, ΔglnD_UT_truncation Healed 6-2615 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the ompX gene (Prm5) upstream of nifA. Deletion 1287 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO1). Inactivation of the uridylyltransferase (UT) domain of the bifunctional uridylyltransferase/uridyl scavenging enzyme, glnD, by mutating amino acid residues 90 and 91 from GG to DV and residue 104 from D to A. ΔnifL::Prm5, ΔglnE-AR_KO1, ΔglnD_UT_deactivation Healed 6-2627 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the ompX gene (Prm5) upstream of nifA. Deletion 1287 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO1). Inactivation of the uridylyl-removal (UR) domain of the bifunctional uridylyltransferase/uridyl-removing enzyme, glnD, by mutating amino acid residues 515 and 516 from HD to AA. ΔnifL::Prm5, ΔglnE-AR_KO1, ΔglnD_UR_deactivation Healed 6-916 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the lpp gene (Prm1) upstream of nifA. Deletion 1287 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO1). Deletion of the entire amtB CDS. Deletion of the native cysZ promoter and insertion of Prm1 directly upstream of cysZ. ΔnifL::Prm1 ΔglnE-AR_KO1, ΔamtB, cysZ::Prm1 healed 137-1893 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the infC gene (PinfC) upstream of nifA. Deletion 1647 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO2). Deletion of the entire amtB CDS. ΔnifL::PinfC, ΔglnE-AR_KO2, ΔglgA healed 6-2118 Mutant CI006 Disruption of the nifL gene by an inserted fragment of a region upstream of the lpp gene (Prm1) upstream of nifA. Deletion of the native glsA2 promoter and insertion of Prm1 immediately upstream of glsA2. ΔnifL::Prm1, glsA2::Prm1 healed 137-2502 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the cspE gene (Prm1.2) upstream of nifA. Deletion 1647 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO2). Deletion 1011 bp. before the stop codon of the asnB gene, encoding asparagine synthetase B. ΔnifL::Prm1.2 ΔglnE-AR_KO2, ΔasnB-truncation healed 137-2504 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the cspE gene (Prm1.2) upstream of nifA. Deletion 1647 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO2). Deletion of the native asnB promoter and insertion of a fragment (Prm1.2) immediately upstream of the asnB CDS. ΔnifL::Prm1.2, ΔglnE-AR_KO2, asnB::Prm1.2 healed 137-2215 Mutant CI137 Disruption of the nifL gene by an inserted fragment of a region upstream of the cspE gene (Prm1.2) upstream of nifA. Deletion 1647 bp. after the start codon of the glnE gene containing the adenylyl-removing domain of glutamate ammonium ligase adenylyltransferase (ΔglnE-AR_KO2). Deletion of the native promoter of the gltBD operon and insertion of a fragment (PrmglnD) from the region upstream of the glnD gene, immediately upstream of the gltBD operon. ΔnifL::Prm1.2 ΔglnE-AR_KO2, gltBD::PrmglnD_ATG-start healed

Notwithstanding the appended claims, the invention set forth herein is also defined by the following paragraphs:

1. A strain of microorganism with improved resistance to desiccation.

2. A microorganism strain where the expression of rpoE is regulated by a switchable promoter.

3. The microorganism strain according to paragraph 2, wherein said switchable promoter is an arabinose promoter.

4. The microorganism strain according to point 2, where the rseA gene is suppressed.

5. A strain of microorganism according to paragraph 2, wherein said switchable promoter is a promoter that can be activated by a chemical substance.

6. The microorganism strain according to paragraph 2, wherein said switchable promoter is a promoter that can be activated by sugar.

7. The microorganism strain according to paragraph 2, wherein said switchable promoter is a promoter that can be activated by arabinose.

8. A method for increasing the resistance of a microorganism to desiccation, comprising designing said microorganism so that it contains the rpoE gene operably linked to a switchable promoter, activating said switchable promoter during the logarithmic phase of biomass growth, thereby increasing the resistance of said microorganism to desiccation.

9. A method of increasing the resistance of a microorganism to desiccation during seed coating, comprising designing said microorganism to contain an rpoE gene operably linked to a switchable promoter, activating said switchable promoter before drying, thereby increasing the resistance of said microorganism to desiccation.

10. The method according to paragraph 9, wherein said switchable promoter is a promoter that can be activated by a chemical substance.

11. The method according to paragraph 9, wherein said switchable promoter is a promoter that can be activated by sugar.

12. The method according to paragraph 9, wherein said switchable promoter is a promoter that can be activated by arabinose.

13. The method according to paragraph 9, wherein said switchable promoter is activated during the logarithmic phase of biomass growth.

14. The method according to paragraph 9, wherein said switchable promoter is activated during seed coating.

15. The method according to paragraph 9, wherein said microorganism is also designed to reduce the expression of rseA.

16. A genetically engineered bacterium containing a heterologous regulatory element functionally associated with cysP.

17. A genetically engineered bacterium according to paragraph 16, wherein said heterologous regulatory element is a promoter.

18. The genetically engineered bacterium according to claim 16, wherein said heterologous regulatory element is native to said genetically engineered bacterium.

19. A genetically engineered bacterium containing a genetic change that results in increased expression of a protein that catalyzes the transport of nitrogenase cofactor components into the cell.

20. A genetically engineered bacterium according to paragraph 19, where said protein, which catalyzes the transport of nitrogenase cofactor components into the cell, is a sulfur transporter gene.

21. The genetically engineered bacterium according to paragraph 20, wherein said sulfur transporter gene is cysZ.

22. The genetically engineered bacterium according to claim 20, wherein said sulfur transporter gene is cysK.

23. The genetically engineered bacterium according to paragraph 20, wherein said sulfur transporter gene is cysT.

24. The genetically engineered bacterium according to claim 20, wherein said sulfur transporter gene is cysW.

25. The genetically engineered bacterium according to claim 20, wherein said sulfur transporter gene is cysA.

26. The genetically engineered bacterium according to claim 20, wherein said sulfur transporter gene is sbp.

27. A genetically engineered bacterium according to paragraph 19, where the specified genetic change leads to increased expression of the cysZK operon.

28. A genetically engineered bacterium according to paragraph 19, where the specified genetic change leads to increased expression of the cysPTWA operon.

29. A genetically engineered bacterium according to paragraph 19, where said protein, which catalyzes the transport of nitrogenase cofactor components into the cell, is a molybdenum transporter gene.

30. The genetically engineered bacterium according to paragraph 29, wherein said molybdenum transporter gene is modE.

31. The genetically engineered bacterium according to claim 29, wherein said molybdenum transporter gene is modB.

32. The genetically engineered bacterium according to paragraph 29, wherein said molybdenum transporter gene is modA.

33. A genetically engineered bacterium according to paragraph 19, where the specified genetic change leads to increased expression of the modEB operon.

34. A genetically engineered bacterium according to paragraph 19, where the specified genetic change leads to increased expression of the modEBA operon.

35. A genetically engineered bacterium according to paragraph 19, where said protein, which catalyzes the transport of nitrogenase cofactor components into the cell, is an iron transporter gene.

36. The genetically engineered bacterium according to claim 35, wherein said iron transporter gene is exbA.

37. The genetically engineered bacterium according to claim 35, wherein said iron transporter gene is exbB.

38. The genetically engineered bacterium according to claim 35, wherein said iron transporter gene is tonB.

39. A genetically engineered bacterium according to paragraph 19, where said genetic change results in increased expression of the exbAB operon.

40. A genetically engineered bacterium according to any one of provisions 19-39, wherein said genetic change involves insertion of a promoter.

41. A genetically engineered bacterium with altered expression of the siderophore biosynthesis gene.

42. The genetically engineered bacterium according to claim 41, wherein said siderophore biosynthesis gene is yhfA.

43. The genetically engineered bacterium according to claim 41, wherein said siderophore biosynthesis gene is yusV.

44. The genetically engineered bacterium according to claim 41, wherein said siderophore biosynthesis gene is sbnA.

45. The genetically engineered bacterium according to claim 41, wherein said siderophore biosynthesis gene is yfiZ.

46. The genetically engineered bacterium according to paragraph 41, wherein said siderophore biosynthesis gene is fiu.

47. The genetically engineered bacterium according to paragraph 41, wherein said siderophore biosynthesis gene is fur.

48. A genetically engineered bacterium containing a genetic change in glgA; wherein said genetically engineered bacterium has increased nitrogen-fixing activity compared to a bacterium of the same species without said genetic change in glgA.

49. A genetically engineered bacterium according to paragraph 47, where the specified genetic change leads to a decrease in the expression of glgA.

50. A genetically engineered bacterium containing a non-native ribosome binding site, resulting in increased translation of NifA.

51. A genetically engineered bacterium containing at least two copies of the NifA gene.

52. A genetically engineered bacterium according to paragraph 51, containing at least three copies of the NifA gene.

53. A genetically engineered bacterium according to paragraph 51, containing at least four copies of the NifA gene.

54. A genetically engineered bacterium according to paragraph 51, containing at least five copies of the NifA gene.

55. A genetically engineered bacterium according to paragraph 51, containing at least six copies of the NifA gene.

56. A genetically engineered bacterium according to paragraph 51, containing at least seven copies of the NifA gene.

57. A genetically engineered bacterium according to paragraph 51, containing more than ten copies of the NifA gene.

58. A genetically engineered bacterium containing a heterologous promoter operably linked to at least one gene selected from the group consisting of nifH, nifD and nifK; wherein said heterologous promoter is derived from the genetic material of the host microorganism.

59. A genetically engineered bacterium that contains a genetic variant in the gene responsible for the uptake of reactive oxygen species.

60. The genetically engineered bacterium according to paragraph 59, wherein said gene responsible for the uptake of reactive oxygen species is grxA.

61. The genetically engineered bacterium according to claim 59, wherein said gene responsible for scavenging reactive oxygen species is grxB.

62. The genetically engineered bacterium according to paragraph 59, wherein said gene responsible for the uptake of reactive oxygen species is grxC.

63. The genetically engineered bacterium according to paragraph 59, wherein said gene responsible for the uptake of reactive oxygen species is grxD.

64. The genetically engineered bacterium according to paragraph 59, wherein said gene responsible for the uptake of reactive oxygen species is trxA.

65. The genetically engineered bacterium according to claim 59, wherein said gene responsible for scavenging reactive oxygen species is trxC.

66. The genetically engineered bacterium according to paragraph 59, wherein said gene responsible for the uptake of reactive oxygen species is tpx.

67. A genetically engineered bacterium containing a genetic structure that results in altered expression of the grxABCD operon.

68. A genetically engineered bacterium containing a genetic variant that results in increased levels of nitrogenase activity under conditions of at least 0.05% oxygen compared to a non-engineered diazotrophic bacterium of the same species.

69. A genetically engineered bacterium according to paragraph 68, wherein said genetic variant results in increased levels of nitrogenase activity under conditions of at least 0.1% oxygen compared to a non-engineered diazotrophic bacterium of the same species.

70. A genetically engineered bacterium as defined in paragraph 68, wherein said genetic variant results in increased levels of nitrogenase activity under conditions of at least 0.5% oxygen compared to a non-engineered diazotrophic bacterium of the same species.

71. A genetically engineered bacterium as defined in paragraph 68, wherein said genetic variant results in increased levels of nitrogenase activity under conditions of at least 1% oxygen compared to a non-engineered diazotrophic bacterium of the same species.

72. A genetically engineered bacterium according to paragraph 68, where the specified genetic variant leads to a change in the expression of the cydABX operon.

73. A genetically engineered bacterium according to paragraph 68, where the specified genetic variant leads to a change in the expression of the cydAB operon.

74. A genetically engineered bacterium according to paragraph 68, where the specified genetic variant leads to a change in the expression of the cydX gene.

75. A genetically engineered bacterium according to paragraph 68, where the specified genetic variant leads to a change in the expression of the cydA gene.

76. A genetically engineered bacterium according to paragraph 68, where the specified genetic variant leads to a change in the expression of the cydB gene.

77. A genetically engineered bacterium according to paragraph 68, where the specified genetic variant leads to a change in the expression of the glbN gene.

78. A genetically engineered bacterium according to paragraph 68, where the specified genetic variant leads to a change in the expression of the fixNOPQ operon.

79. A genetically engineered bacterium according to paragraph 68, where the specified genetic variant leads to a change in the expression of the fixN gene.

80. A genetically engineered bacterium according to paragraph 68, where the specified genetic variant leads to a change in the expression of the fixO gene.

81. A genetically engineered bacterium according to paragraph 68, where the specified genetic variant leads to a change in the expression of the fixP gene.

82. A genetically engineered bacterium according to paragraph 68, where the specified genetic variant leads to a change in the expression of the fixQ gene.

83. Genetically engineered bacterium containing a modification in asnB.

84. A genetically engineered bacterium containing a modification in a gene selected from the group consisting of asnB, asnA, glnL, uridylyltransferase, amtB and GDH.

85. A genetically engineered bacterium according to paragraph 84, where the specified genetic variant leads to a change in the expression of the asnB gene.

86. A genetically engineered bacterium according to paragraph 84, where the specified genetic variant leads to a change in the expression of the asnA gene.

87. A genetically engineered bacterium according to paragraph 84, where the specified genetic variant leads to a change in the expression of the glnL gene.

88. A genetically engineered bacterium according to paragraph 84, where the specified genetic variant leads to a change in the expression of the uridylyl transferase gene.

89. A genetically engineered bacterium according to paragraph 84, where the specified genetic variant leads to a change in the expression of the GDH gene.

90. A genetically engineered bacterium containing a modification in glnK, wherein said modification is selected from the group consisting of deletion of the entire gene, deletion of substantially the entire gene, deletion of the ACT domain, deletion of more than 50% of the ACT domain, inactivation of the ACT domain, and inactivation of the UTase domain.

91. A genetically engineered bacterium containing a modification in glnD, wherein said modification is selected from the group consisting of deletion of the entire gene, deletion of substantially the entire gene, deletion of the ACT domain, deletion of more than 50% of the ACT domain, inactivation of the ACT domain, and inactivation of the UTase domain.

92. A genetically engineered bacterium with improved survival in the rhizosphere compared to a non-engineered bacterium of the same species, wherein the bacterium contains a genetic change resulting in increased expression of a sugar transporter.

93. The genetically engineered bacterium according to claim 92, wherein said sugar transporter is dctA.

94. A genetically engineered bacterium that contains a genetic change that results in increased expression of a sugar transporter.

95. The genetically engineered bacterium according to claim 94, wherein said sugar transporter is dctA.

96. A genetically engineered bacterium with improved survival in the rhizosphere compared to a non-engineered bacterium of the same species, where the bacterium contains a genetic change that results in altered quorum signaling.

97. The genetically engineered bacterium of claim 96, wherein said genetic change resulting in a change in quorum signaling results in a change in quorum quenching.

98. The genetically engineered bacterium of claim 96, wherein said genetic change resulting in an alteration in quorum signaling results in an alteration in the expression of Y2-aiiA.

99. The genetically engineered bacterium of claim 96, wherein said genetic change resulting in an alteration in quorum signaling results in an alteration in the expression of ytnP.

100. A genetically engineered bacterium with improved survival in the rhizosphere compared to a non-engineered bacterium of the same species, where the bacterium contains a genetic change resulting in increased root attachment.

101. The genetically engineered bacterium of paragraph 100, wherein said genetic change resulting in increased root attachment includes a genetic change resulting in a change in phenazine biosynthesis.

102. The genetically engineered bacterium of paragraph 100, wherein said genetic change resulting in increased root attachment includes a genetic change resulting in a change in the biosynthesis of cyclic lipopeptides.

103. The genetically engineered bacterium of paragraph 100, wherein said genetic change resulting in increased root attachment includes a genetic change resulting in a change in the expression of agglutinins.

104. The genetically engineered bacterium of claim 100, wherein said genetic change resulting in increased root attachment includes a genetic change resulting in a change in the expression of fhaB.

105. The genetically engineered bacterium of paragraph 100, wherein said genetic change resulting in increased root attachment includes a genetic change resulting in a change in the expression of fhaC.

106. The genetically engineered bacterium of paragraph 100, wherein said genetic change resulting in increased root attachment includes a genetic change resulting in a change in the expression of yjbE.

107. The genetically engineered bacterium of paragraph 100, wherein said genetic change resulting in increased root attachment includes a genetic change resulting in a change in the expression of pssM.

108. The genetically engineered bacterium of paragraph 100, wherein said genetic change resulting in increased root attachment includes a genetic change resulting in a change in the expression of acs genes.

109. The genetically engineered bacterium of claim 100, wherein said genetic change resulting in increased root attachment includes a genetic change resulting in a change in the expression of otsA.

110. The genetically engineered bacterium of paragraph 100, wherein said genetic change resulting in increased root attachment includes a genetic change resulting in a change in the expression of the otsA-containing operon.

111. The genetically engineered bacterium of claim 100, wherein said genetic change resulting in increased root attachment includes a genetic change resulting in altered expression of treY.

112. The genetically engineered bacterium of paragraph 100, wherein said genetic change resulting in increased root attachment includes a genetic change resulting in a change in the expression of the treY-containing operon.

113. A genetically engineered bacterium containing a genetic change that results in increased biofilm formation.

114. The genetically engineered bacterium of claim 113, wherein said genetic change resulting in increased biofilm formation includes a genetic change resulting in altered expression of smZ.

115. The genetically engineered bacterium of claim 113, wherein said genetic change resulting in increased biofilm formation includes a genetic change resulting in altered expression of lapA.

116. The genetically engineered bacterium of paragraph 113, wherein said genetic change resulting in increased biofilm formation includes a genetic change resulting in a change in the expression of AHL biosynthetic genes.

117. The genetically engineered bacterium of claim 113, wherein said genetic change resulting in increased biofilm formation includes a genetic change resulting in altered expression of phoB.

118. The genetically engineered bacterium of claim 113, wherein said genetic change resulting in increased biofilm formation includes a genetic change resulting in altered expression of phoR.

119. The genetically engineered bacterium of claim 113, wherein said genetic change that results in increased biofilm formation includes a genetic change that results in a change in the expression of MucA.

120. A genetically engineered bacterium with improved survival in the rhizosphere compared to a non-engineered bacterium of the same species, wherein the bacterium contains a genetic change resulting in increased expression of a cell wall degrading enzyme.

121. The genetically engineered bacterium according to paragraph 120, wherein said cell wall degrading enzyme includes polygalacturonase.

122. The genetically engineered bacterium according to paragraph 120, wherein said cell wall degrading enzyme includes cellulase.

123. The genetically engineered bacterium of claim 120, wherein said cell wall degrading enzyme includes pehA.

124. A genetically engineered bacterium according to any one of provisions 19-123, which is a genetically engineered diazotrophic bacterium.

125. A genetically engineered bacterium according to any one of provisions 19-123, wherein said genetically engineered bacterium is not intergeneric.

126. A genetically engineered bacterium according to any one of provisions 19-123, wherein said genetically engineered bacterium is intergeneric.

127. A genetically engineered bacterium according to any one of provisions 19-123, wherein said genetically engineered bacterium is capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen.

128. The genetically engineered bacterium according to any one of provisions 19-123, wherein the genetically engineered bacterium contains a modification in the nitrogen fixation genetic network.

129. The genetically engineered bacterium according to any one of provisions 19-123, wherein the genetically engineered bacterium contains a modification in NifA.

130. The genetically engineered bacterium according to any one of provisions 19-123, wherein the genetically engineered bacterium contains a modification that results in increased expression of NifA.

131. The genetically engineered bacterium according to any one of provisions 19-123, wherein the genetically engineered bacterium contains a modification in NifL.

132. The genetically engineered bacterium according to any one of provisions 19-123, wherein the genetically engineered bacterium contains a modification resulting in decreased expression of NifL.

133. The genetically engineered bacterium according to any one of provisions 19-123, wherein the genetically engineered bacterium contains a modification in NifH.

134. The genetically engineered bacterium according to any one of provisions 19-123, wherein the genetically engineered bacterium contains a modification resulting in increased expression of NifH.

135. The genetically engineered bacterium according to any one of provisions 19-123, wherein the genetically engineered bacterium contains a modification resulting in increased expression of the Nif gene cluster.

136. The genetically engineered bacterium according to any one of provisions 19-123, wherein the genetically engineered bacterium contains a modification in the nitrogen assimilation genetic network.

137. The genetically engineered bacterium according to any one of provisions 19-123, wherein the genetically engineered bacterium contains a modification in GlnE.

138. The genetically engineered bacterium according to any one of provisions 19-123, wherein the genetically engineered bacterium contains a modification resulting in decreased GlnE activity.

139. The genetically engineered bacterium according to any one of provisions 19-123, wherein the genetically engineered bacterium contains a modification that results in reduced amtB activity.

140. A method of increasing the amount of atmospheric nitrogen in a plant, comprising bringing said plant into contact with a plurality of said genetically engineered bacteria according to any one of provisions 19-139.

141. The method according to paragraph 140, wherein said genetically engineered bacterium is applied to the seed of said plant.

142. The method according to paragraph 140, wherein said genetically engineered bacterium is applied to a seedling of said plant.

143. The method according to paragraph 140, where the specified genetically engineered bacterium is applied to the specified plant in the composition of a liquid preparation.

144. The method of claim 140, wherein said genetically engineered bacterium is applied to said plant after planting but before harvesting of said plant.

145. The method according to paragraph 140, where the specified genetically engineered bacterium is applied to the specified plant as a fertilizer.

146. The method according to paragraph 140, wherein said genetically engineered bacterium is applied to said plant within a period of one month to eight months after germination.

147. The method according to paragraph 140, wherein said genetically engineered bacterium is applied to said plant within a period of two months to eight months after germination.

148. The method according to paragraph 140, wherein said genetically engineered bacterium is applied to said plant within a period of one month to three months after germination.

149. The method according to paragraph 140, wherein said genetically engineered bacterium is applied to said plant within a period of three months to six months after germination.

150. The method according to paragraph 140, wherein said plant is a cereal plant.

151. The method according to paragraph 140, wherein said plant is a corn plant.

152. The method according to paragraph 140, wherein said plant is a rice plant.

153. The method according to paragraph 140, wherein said plant is a wheat plant.

154. The method according to paragraph 140, wherein said plant is a soybean plant.

155. A composition comprising a seed and a seed coating, wherein said seed coating contains a plurality of said genetically engineered bacteria according to any one of provisions 19-39.

156. The composition according to paragraph 155, wherein said seed is a cereal seed.

157. The composition of claim 155, wherein said seed is selected from the group consisting of corn seed, wheat seed, rice seed, soybean seed, rye seed and sorghum seed.

158. A composition comprising a plant and a plurality of said genetically engineered bacteria according to any one of provisions 19-39.

159. The composition according to paragraph 158, wherein said plant is a seedling.

160. The composition according to paragraph 158, wherein said seed is a cereal seed.

161. The composition of claim 158, wherein said seed is selected from the group consisting of corn, rice, wheat, soybean, rye and sorghum.

162. A method for increasing nitrogen fixation in a microorganism, including the introduction of genetic modification leading to a change in the activity of the sulfur transporter.

163. A method for increasing nitrogen fixation in a microorganism, including the introduction of a genetic modification leading to a change in the expression of the sulfur transport gene.

164. A method for increasing nitrogen fixation in a microorganism, including the introduction of a genetic modification leading to a change in the activity of the molybdenum transporter.

165. A method for increasing nitrogen fixation in a microorganism, including the introduction of a genetic modification leading to a change in the expression of the molybdenum transport gene.

166. A method for increasing nitrogen fixation in a microorganism, including the introduction of a genetic modification leading to a change in the activity of the iron transporter.

167. A method for increasing nitrogen fixation in a microorganism, including the introduction of a genetic modification leading to a change in the expression of the iron transport gene.

168. A method for increasing nitrogen fixation in a microorganism, including the introduction of genetic modification leading to a change in the biosynthesis of siderophores.

169. A method for increasing nitrogen fixation in a microorganism, including the introduction of a genetic modification leading to a change in the expression of the gene that controls the biosynthesis of siderophores.

170. A method for increasing nitrogen fixation in a microorganism, including the introduction of a genetic modification leading to a change in the activity of a scavenger of reactive oxygen species.

171. A method for increasing nitrogen fixation in a microorganism, including the introduction of a genetic modification leading to a change in the expression of the gene responsible for the absorption of reactive oxygen species.

172. A method for increasing nitrogen fixation in a microorganism, including the introduction of a genetic modification leading to a change in the activity of a protein involved in quorum signaling.

173. A method for increasing nitrogen fixation in a microorganism, including the introduction of a genetic modification leading to a change in the expression of the gene responsible for quorum signaling.

174. A method of increasing nitrogen fixation in a microorganism, including the introduction of genetic modification leading to a change in the activity of agglutinins.

175. A method for increasing nitrogen fixation in a microorganism, including the introduction of genetic modification leading to a change in the expression of agglutinins.

176. A method for increasing nitrogen fixation in a microorganism, including the introduction of a genetic modification leading to a change in the activity of a protein involved in the formation of a biofilm.

177. A method for increasing nitrogen fixation in a microorganism, including the introduction of a genetic modification leading to a change in the expression of the gene responsible for the formation of biofilm.

178. A method for increasing nitrogen fixation in a microorganism, including the introduction of a genetic modification leading to a change in the activity of the AHL biosynthesis protein.

179. A method for increasing nitrogen fixation in a microorganism, including the introduction of a genetic modification leading to a change in the expression of the AHL biosynthesis gene.

180. A method for increasing nitrogen fixation in a microorganism, including the introduction of a genetic modification leading to a change in the activity of an enzyme that destroys the cell wall.

181. A method for increasing nitrogen fixation in a microorganism, including the introduction of a genetic modification leading to a change in the expression of the gene for an enzyme that destroys the cell wall.

182. A method for increasing nitrogen fixation in a microorganism, including the introduction of genetic modification leading to a change in the genetic network of nitrogen fixation.

183. A method of increasing nitrogen fixation in a microorganism, including the introduction of genetic modification leading to a change in the genetic network of nitrogen assimilation.

184. A method for increasing nitrogen fixation by a microorganism, including introducing a genetic modification into a gene from the group consisting of rpoE, rseA, cysP, cysZ, cysK, cysT, cysW, cysA, sbp, cysZK operon, cysPTWA operon, modE, modB, modA, modEB operon , operon modEBA, exbA, exbB, tonB, operon exbAB, isyhfA, yusV, sbnA, yfiZ, fiu, fur, glgA, NifA, nifH, nifD, nifK, grxA, grxB, grxC, grxD, trxA, trxC, tpx, operon grxABCD, cydABX operon, cydX from the cydAB operon, cydA, cyddB, glbN, fixNOPQ, fixN, fixO, foxP, fixQ, asnB, asnA, glnL, uridylyltransferase, amtB, glnK, GDH and glnD.

186. A method of increasing ammonium excretion by a microorganism, including the introduction of a genetic modification leading to a change in the activity of the sulfur transporter.

187. A method for increasing ammonium excretion by a microorganism, including the introduction of a genetic modification leading to a change in the expression of the sulfur transport gene.

188. A method of increasing ammonium excretion by a microorganism, including the introduction of a genetic modification leading to a change in the activity of the molybdenum transporter.

189. A method for increasing ammonium excretion by a microorganism, including the introduction of a genetic modification leading to a change in the expression of the molybdenum transport gene.

190. A method of increasing ammonium excretion by a microorganism, including the introduction of a genetic modification leading to a change in the activity of the iron transporter.

191. A method of increasing ammonium excretion by a microorganism, including the introduction of a genetic modification leading to a change in the expression of the iron transport gene.

192. A method of increasing ammonium excretion by a microorganism, including the introduction of genetic modification leading to a change in the biosynthesis of siderophores.

193. A method for increasing ammonium excretion by a microorganism, including the introduction of a genetic modification leading to a change in the expression of a gene that controls the biosynthesis of siderophores.

194. A method of increasing ammonium excretion by a microorganism, including the introduction of a genetic modification leading to a change in the activity of a scavenger of reactive oxygen species.

195. A method of increasing ammonium excretion by a microorganism, including the introduction of a genetic modification leading to a change in the expression of the gene responsible for the absorption of reactive oxygen species.

196. A method of increasing ammonium excretion by a microorganism, including the introduction of a genetic modification leading to a change in the activity of a protein involved in quorum signaling.

197. A method of increasing ammonium excretion by a microorganism, including the introduction of a genetic modification leading to a change in the expression of the gene responsible for quorum signaling.

198. A method of increasing ammonium excretion by a microorganism, including the introduction of a genetic modification leading to a change in the activity of agglutinins.

199. A method of increasing ammonium excretion by a microorganism, including the introduction of a genetic modification leading to a change in the expression of agglutinins.

200. A method of increasing ammonium excretion by a microorganism, including the introduction of a genetic modification leading to a change in the activity of a protein involved in the formation of a biofilm.

201. A method of increasing ammonium excretion by a microorganism, including the introduction of a genetic modification leading to a change in the expression of the gene responsible for the formation of biofilm.

202. A method for increasing ammonium excretion by a microorganism, including the introduction of a genetic modification leading to a change in the activity of the AHL biosynthesis protein.

203. A method for increasing ammonium excretion by a microorganism, including the introduction of a genetic modification leading to a change in the expression of the AHL biosynthesis gene.

204. A method of increasing ammonium excretion by a microorganism, including the introduction of a genetic modification leading to a change in the activity of an enzyme that destroys the cell wall.

205. A method of increasing ammonium excretion by a microorganism, including the introduction of a genetic modification leading to a change in the expression of the gene for an enzyme that destroys the cell wall.

206. A method of increasing ammonium excretion by a microorganism, including the introduction of genetic modification leading to a change in the genetic network of nitrogen fixation.

207. A method of increasing ammonium excretion by a microorganism, including the introduction of genetic modification leading to a change in the genetic network of nitrogen assimilation.

208. A method for increasing ammonium excretion by a microorganism, including introducing a genetic modification into a gene from the group consisting of rpoE, rseA, cysP, cysZ, cysK, cysT, cysW, cysA, sbp, cysZK operon, cysPTWA operon, modE, modB, modA, operon modEB, modEBA operon, exbA, exbB, tonB, exbAB operon, isyhfA, yusV, sbnA, yfiZ, fiu, fur, glgA, NifA, nifH, nifD, nifK, grxA, grxB, grxC, grxD, trxA, trxC, tpx, grxABCD operon, cydABX operon, cydX from the cydAB operon, cydA, cyddB, glbN, fixNOPQ, fixN, fixO, foxP, fixQ, asnB, asnA, glnL, uridylyltransferases, amtB, glnK, GDH and glnD.

209. A method of increasing the survival of a microorganism in the rhizosphere, including the introduction of a genetic modification leading to a change in the activity of a protein involved in attachment to roots.

210. A method of increasing the survival of a microorganism in the rhizosphere, including the introduction of a genetic modification leading to a change in the expression of a protein gene involved in attachment to roots.

211. A method of increasing the survival of a microorganism in the rhizosphere, including the introduction of genetic modification leading to a change in the activity of the molybdenum transporter.

212. A method of increasing the survival of a microorganism in the rhizosphere, including the introduction of genetic modification leading to a change in the expression of the molybdenum transport gene.

213. A method of increasing the survival of a microorganism in the rhizosphere, including the introduction of genetic modification leading to a change in the activity of the iron transporter.

214. A method of increasing the survival of a microorganism in the rhizosphere, including the introduction of genetic modification leading to a change in the expression of the iron transport gene.

215. A method of increasing the survival of a microorganism in the rhizosphere, including the introduction of genetic modification leading to a change in the biosynthesis of siderophores.

216. A method of increasing the survival of a microorganism in the rhizosphere, including the introduction of genetic modification leading to a change in the expression of the gene that controls the biosynthesis of siderophores.

217. A method of increasing the survival of a microorganism in the richosphere, including the introduction of genetic modification leading to a change in the activity of the absorber of reactive oxygen species.

218. A method of increasing the survival of a microorganism in the rhizosphere, including the introduction of genetic modification leading to a change in the expression of the gene responsible for the absorption of reactive oxygen species.

219. A method for increasing the survival of a microorganism in the rhizosphere, including the introduction of a genetic modification leading to a change in the activity of a protein involved in quorum signaling.

220. A method for increasing the survival of a microorganism in the rhizosphere, including the introduction of a genetic modification leading to a change in the expression of the gene responsible for quorum signaling.

221. A method of increasing the survival of a microorganism in the rhizosphere, including the introduction of genetic modification leading to a change in the activity of agglutinins.

222. A method of increasing the survival of a microorganism in the rhizosphere, including the introduction of genetic modification leading to a change in the expression of agglutinins.

223. A method of increasing the survival of a microorganism in the rhizosphere, including the introduction of a genetic modification leading to a change in the activity of a protein involved in the formation of a biofilm.

224. A method of increasing the survival of a microorganism in the rhizosphere, including the introduction of genetic modification leading to a change in the expression of the gene responsible for the formation of biofilm.

225. A method of increasing the survival of a microorganism in the rhizosphere, including the introduction of genetic modification leading to a change in the activity of the AHL biosynthesis protein.

226. A method of increasing the survival of a microorganism in the rhizosphere, including the introduction of genetic modification leading to a change in the expression of the AHL biosynthesis gene.

227. A method of increasing the survival of a microorganism in the rhizosphere, including the introduction of a genetic modification leading to a change in the activity of an enzyme that destroys the cell wall.

228. A method for increasing the survival of a microorganism in the rhizosphere, including the introduction of a genetic modification leading to a change in the expression of the gene for an enzyme that destroys the cell wall.

229. A method of increasing the survival of a microorganism in the rhizosphere, including the introduction of genetic modification leading to a change in the genetic network of nitrogen fixation.

230. A method of increasing the survival of a microorganism in the rhizosphere, including the introduction of genetic modification leading to a change in the genetic network of nitrogen assimilation.

231. A method for increasing the survival of a microorganism in the rhizosphere, including introducing a genetic modification into a gene from the group consisting of fhaB, fhaC, yjbE, pssM, otsA, treY, smZ, lapA, phoB, phoR, MucA and pehA.

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LIST OF SEQUENCES

<110> PIVOT BIO, INC.

<120> TARGETED GENES FOR DIRECTED INFLUENCE ON NITROGEN FIXATION

TO IMPROVE PLANT QUALITY

<130> 47736-715.601

<140> PCT/US18/57613

<141> 2018-10-25

<150> 62/577.149

<151> 2017-10-25

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 9

<212>PRT

<213> Unknown

<220>

<223> Description of the unknown:

Sequence of the "LAGLIDADG" family protein motif

<400> 1

Leu Ala Gly Leu Ile Asp Ala Asp Gly

15

<210> 2

<211> 90

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: Synthetic

oligonucleotide

<400> 2

gttgatcaga ccgatgttcg gaccttccaa ggtttcgatc ggacatacgc gaccgtagtg 60

ggtcgggtgt acgtctcgaa cttcaaagcc 90

<---

Claims (23)

1. A genetically engineered diazotrophic bacterium capable of increasing the amount of atmospheric nitrogen in a plant, having increased expression of rpoN, and the expression of rpoN is increased by deletion of the native rpoN promoter and replacement with a constitutive promoter. 2. The genetically engineered diazotrophic bacterium according to claim 1, wherein said genetically engineered diazotrophic bacterium further comprises a genetic modification in glnD, wherein said genetic modification is selected from the group consisting of: a deletion of the entire gene, a deletion of substantially the entire gene, a deletion of the ACT domain, a deletion more than 50% of the ACT domain, inactivation of the ACT domain, reduction of the uridyl scavenging activity of glnD compared to the wild type bacterium, removal of the uridyl scavenging activity of glnD and inactivation of the Utase domain. 3. The genetically engineered diazotrophic bacterium of claim 2, wherein said genetic modification to glnD includes deletion of the ACT domain, deletion of more than 50% of the ACT domain, or inactivation of the ACT domain. 4. Genetically engineered diazotrophic bacterium according to any one of paragraphs. 1-3, wherein said genetically engineered diazotrophic bacterium is intergeneric. 5. Genetically engineered diazotrophic bacterium according to any one of paragraphs. 1-3, wherein said genetically engineered diazotrophic bacterium further comprises a modification in the nitrogen fixation genetic network or a modification in the nitrogen assimilation genetic network. 6. Genetically engineered diazotrophic bacterium according to any one of paragraphs. 1-3, wherein said genetically engineered diazotrophic bacterium further comprises a genetic modification that results in increased expression of NifA, a genetic modification that results in decreased expression of NifL, a genetic modification that results in increased expression of NifH, a genetic modification that results in decreased GlnE activity or a genetic modification that results in decreased AmtB activity. 7. Genetically engineered diazotrophic bacterium according to any one of paragraphs. 1-3, where said genetically engineered diazotrophic bacterium additionally contains: a genetic modification that results in increased expression of NifA; And a genetic modification that results in decreased expression of NifL. 8. A method for increasing the amount of nitrogen obtained from the atmosphere in a plant, comprising bringing said plant into contact with a plurality of said genetically engineered diazotrophic bacteria according to any one of claims. 1-7. 9. The method of claim 8, wherein said genetically engineered diazotrophic bacterium is applied to a seed of said plant, a seedling of said plant, a plant after planting but before harvesting of said plant, or the plant as a top dressing. 10. The method according to claim 8, where the specified genetically engineered diazotrophic bacterium is applied to the specified plant as a liquid preparation or is applied in a period from one month to eight months, in a period from two months to eight months, in a period from one month to three months , or in the period from three months to six months after germination. 11. The method of claim 8, wherein said plant is selected from the group consisting of: a cereal plant, a corn plant, a rice plant, a wheat plant or a soybean plant. 12. A composition for application to a seed, where the composition contains a plurality of the specified genetically engineered diazotrophic bacteria according to any one of paragraphs. 1-7. 13. The composition of claim 12, wherein said seed is a cereal seed, a corn seed, a wheat seed, a rice seed, a soybean seed, a rye seed, or a sorghum seed. 14. A composition for application to a plant, where the composition contains a plurality of the specified genetically engineered diazotrophic bacteria according to any one of paragraphs. 1-7, optionally, wherein said plant is a cereal plant, a corn plant, a rice plant, a wheat plant, a soybean plant, a rye plant, or a sorghum plant. 15. The composition according to claim 14, wherein said plant is a seedling. 16. A seed for producing a plant having an increased amount of atmospheric nitrogen, wherein said seed is coated with the composition of claim 12. 17. The seed according to claim 16, characterized in that said seed is a seed of a cereal crop, corn, wheat, rice, soybean, rye or sorghum. 18. A plant with an increased amount of atmospheric nitrogen, containing a variety of genetically engineered diazotrophic bacteria according to any one of claims 1-7. 19. A plant having an increased amount of atmospheric nitrogen, wherein said plant is coated with the composition of claim 14. 20. The plant according to claim 18 or 19, wherein said plant is a shoot. 21. The plant according to any one of claims 18 to 20, wherein said plant is a cereal plant, a corn plant, a rice plant, a wheat plant, a soybean plant, a rye plant or a sorghum plant.

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