JPS643475B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel lysozyme-sensitive microorganism, a method for preparing protoplasts using the microorganism, and a method for transforming the microorganism. The present invention will be explained in detail below. The present invention provides novel lysozyme-sensitive microorganisms and methods for preparing protoplasts and transforming microorganisms using the microorganisms. The novel lysozyme-sensitive microorganisms of the present invention include microorganisms that belong to the genus Corynebacterium or Brevibacterium and are sensitive to lysozyme at a concentration of less than 25 μg/ml. The microorganisms of the present invention are treated with lysozyme to simply dissolve and remove their cell walls without pretreatment with antibiotics or other drugs during culture, and are converted into protoplasts under hypertonic conditions. Genetic engineering techniques that incorporate deoxyribonucleic acid (DNA) into vector plasmid DNA in vitro and introduce it into microbial cells have recently attracted much attention. Transformed cells obtained by genetic engineering techniques can be used as a means of producing heterologous DNA accompanying the autonomous replication of vectors, and can be valuable to cells by allowing the heterologous DNA to exist within the cell. It is also important as a means of imparting properties. Most of the research in this field has been conducted using E. coli as the host, but other industrially useful microorganisms such as Bacillus subtilis, which is a producer of amylase, and bacteria that produce antibiotics, etc. Attempts are also being made to establish ``recombinant DNA techniques'' with actinomycetes and yeast, which are bacteria that produce brewing alcohol. Genetic engineering techniques require isolation of plasmids from cultured cells in order to isolate vector plasmids or analyze hybrid plasmids that have incorporated foreign DNA, but this requires first destroying the cells. No. Its destruction is
Since it is necessary to perform the process under mild conditions to prevent shearing of the DNA, mechanical destruction methods such as ultrasound and French press cannot be applied, and a method using a cell wall lytic enzyme is usually used. Recombination of the above
All bacterial species whose DNA has been studied have lytic enzymes that serve the purpose, and these are used. The cell walls of many bacteria, such as Escherichia coli and Bacillus subtilis, can be easily dissolved and removed by the bacterial cell wall lytic enzyme lysozyme, but some bacteria are difficult to lyse with lysozyme. For such bacterial species with poorly soluble cell walls, methods are used to make the cells sensitive to lysozyme by exposing them to high concentrations of amino acids such as glycine or antibiotics such as penicillin during culture, and then treating the cells with lysozyme. . Bacterial species belonging to the genus Corynebacterium and Brevibacterium, which are used for the industrial production of many amino acids such as glutamic acid and lysine, usually have cell walls that are poorly soluble in lysozyme. The cell wall removal method described above has been used in which cultured cells treated with lysozyme have been treated with antibiotics such as penicillin. However, this method is not only complicated;
The cell wall-removed cells (protoplasts) generated by lysozyme treatment under hypertonic conditions have drawbacks such as low ability to return to normal cells (regeneration ability). In order to establish recombinant DNA techniques for useful bacteria such as Bacteria, we have searched for methods for removing the cell walls of these Bacteria.As one method, the present applicant disclosed in Japanese Patent Application Laid-Open No. 122794/1983. We have investigated cell wall removal methods using lysozyme using lysozyme-sensitive strains of Corynebacterium and Brevibacterium species, but these strains, like lysozyme-insensitive strains, did not require pretreatment with penicillin, etc. The cell wall was not dissolved and removed by lysozyme.As a result of further investigation, it was found that the cell wall of a lysozyme supersensitive strain that has a higher sensitivity to lysozyme than the lysozyme-sensitive strain described in the publication was pretreated with penicillin, etc. Furthermore, the present inventors found that by using such a strain, plasmids can be easily extracted from inside cells, and that the regenerative ability of protoplasts produced by lysozyme treatment can be improved. The present inventors discovered that lysozyme-sensitive strains of the genus Corynebacterium or Brevibacterium can efficiently transform DNA using protoplasts, leading to the completion of the present invention. It is obtained by mutagenesis treatment using a non-susceptible strain (for example, a wild strain) or a lysozyme-low sensitivity strain as a parent strain.Mutation induction methods include conventional methods such as ultraviolet irradiation, radiation irradiation, and mutagen treatment. To select a strain that is hypersensitive to lysozyme, such as the microorganism of the present invention (hereinafter, the lysozyme-sensitive strain of the present invention may be referred to as a lysozyme hypersensitive strain) from mutagenized mutant strains, the parent strain is What is necessary is to select a microorganism that cannot grow completely in a medium containing an extremely low concentration of lysozyme (less than about 25 μg/ml), but can grow in a lysozyme-free medium.Specific examples of the microorganisms of the present invention include: L-15 strain (FEI 5946) derived from Corynebacterium glutamicum strain 225-106 (FEI 5945) as a parent strain, and L- induced using Corynebacterium herkyulis ATCC13868 as a parent strain. Strain 103 (Feikoken Bacillus 5947), L derived from Brevibacterium deivalicatum ATCC14020 as the parent strain
-204 strain (Feikoken Bokuyori 5948) and L-312 strain (Feikoken Joyori 528) derived from Brevibacterium lactofamentum ATCC13655 as the parent strain
can be given. These lysozyme supersensitive strains have been deposited at the National Institute of Microbial Technology and have been assigned the Microbiological Research Institute as described above. Also included in the American Type Culture Collection in the United States.
ATCC No.31833, 31834, 31866, 31867 and
31868, respectively. The strain 225-106, named Corynebacterium glutamicum, was isolated from soil, and its mycological properties are as follows. For examination of mycological properties, refer to “Manual of Microbiological Methods”
by the Society of American Bacteriologist
Comittee or Bacteriological Technique (1957)
This was done using the method described in. Cell morphology Usually 0.7-1.0 x 1.0-3.0 microns elliptical or rod-shaped, but depending on the culture conditions, it shows pleomorphism in which multiple cells are linked in a linear or V-shape. Gram-positive, non-motile and does not produce spores. Growth characteristics on Fuei culture medium A single colony on an agar medium is circular, with a glossy surface and a pale yellow color. Growth on slants is also pale yellow and opaque. When puncture culture is carried out on agar medium, it grows well at the top and grows slightly in the deeper parts. In liquid culture, it grows slightly and settles in a slightly flocculent manner. Physiological properties (1) Temperature: The optimum temperature is 25-37℃, but it grows faintly at 42℃. (2) PH: The optimum temperature is 7-8, but it can also grow at PH 6-9. (3) Non-thermoresistant (4) Aerobic (5) Does not liquefy gelatin, (6) Does not degrade or metabolize casein, (7) Non-indole production, (8) Catalase positive, (9) Non-assimilable starch, (10) glucose, fructose, mannose,
Generates acid from maltose but not from xylose, galactose, lactose, and glycerose, (11) Requires biotin, (12) Produces large amounts of glutamic acid in biotin-limited media, (13) High concentration of biotin The containing medium produces lactic acid and α-ketoglutaric acid. The above results are based on J.Gen.Appl.Microbiol. 73 279～
301 (1967), it closely matches Corynebacterium glutamicum. Therefore, strain 225-106 is a strain identified as Corynebacterium glutamicum. A specific example of the method for obtaining the lysozyme hypersensitive mutant strain of the present invention from lysozyme-insensitive Corynebacterium and Brevibacterium strains will be described below. Inoculate the parent strain into a nutrient medium and culture with shaking at 20-35°C. Culture was stopped in the middle of the logarithmic growth phase, the bacteria were collected, and after washing with physiological saline,
Appropriate PH buffer (PH5.5-8.0), e.g. M/20
Approximately 1×10 ⁷ to Tris-malate buffer (PH6.0)
Suspend at 1×10 ⁹ cells/ml. Nitrosoguanidine is added to this suspension to give a final concentration of 200 to 500 μg/ml, and the suspension is left at 20 to 35° C. for 30 minutes to 1 hour. Collect bacteria by centrifugation, wash the cells with the same buffer, suspend in physiological saline, dilute appropriately with physiological saline, and add a certain amount of agar 1.8% to a complete medium, such as a rich medium (NB). Spread it on the medium with added. Cultivate at 25-35° C. for 1-4 days, and replica plate the resulting colonies onto a nutrient agar medium and a nutrient agar medium containing 2.5-25 μg/ml lysozyme. After culturing the replica plate at 25-35°C for 1-4 days,
A bacterium that grows on a nutrient agar medium but does not grow on a lysozyme-containing agar medium is obtained as a lysozyme hypersensitive mutant. The above-mentioned derivative strains are all lysozyme hypersensitive mutant strains obtained in this way. The lysozyme sensitivity of the lysozyme hypersensitive mutant strain is determined by dropwise inoculating the equivalent of approximately 10 ⁴ cells in the logarithmic growth phase onto an NB agar medium containing multiple concentrations of lysozyme, and after culturing at 30°C for 2 days, the growth status of the bacteria was determined. It can be measured by observation. The minimum lysozyme concentration at which no bacterial growth is observed is defined as the bacterial lysozyme susceptibility value (minimum growth-inhibitory concentration), and the results are shown in Table 1 when compared with each parent strain. The lysozyme supersensitive mutant strain of the present invention can be obtained by selecting a strain that cannot grow on an NB agar medium containing 2.5 to 25 μg/ml of lysozyme as described above, but it is described in JP-A-54-122794. Lysozyme-sensitive mutant strains of Corynebacterium and Brevibacterium species were obtained by selecting strains that cannot grow on NB agar medium containing 200 to 400 μg/ml of lysozyme, and both are essentially May have different properties. In fact, the lysozyme-insensitive strain used as the parent strain for these mutations has a minimum growth inhibitory concentration of 400 to 800 μg/ml using the above lysozyme sensitivity measurement method.
Although the microorganism of the present invention has a lysozyme sensitivity value of 1.6 to 3.2 μg/ml, it is different from the lysozyme sensitivity value of 25 to 400 μg/ml of the lysozyme susceptible strain described in JP-A-54-122794. It's clearly different. Furthermore, the former has the usefulness described below based on the fact that the cell wall is easily dissolved and removed without pretreatment with penicillin, whereas the latter does not have these characteristics, so lysozyme hypersensitive strains are It is clear that this strain is different from the lysozyme-sensitive strain.

[Table] The usefulness of the microorganism of the present invention whose cell wall is easily dissolved and removed by lysozyme is based on (1) the excellent regeneration ability of protoplasts from which the cell wall has been removed;
Enabling efficient transformation with DNA,
(2) It facilitates the extraction and separation of DNA such as plasmids from cells. This will be explained in detail below. (1) Preparation of protoplasts from the microorganism of the present invention and transformation with DNA using the protoplasts Protoplasts are prepared simply by directly treating cells grown in culture with lysozyme. The medium may be any medium that allows the bacteria to grow; for example, nutrient medium NB is used. Bacteria are inoculated into a medium and cultured with shaking at 25 to 37°C until the middle or late logarithmic phase to collect the cells. Then, it is suspended in a suitable hypertonic medium containing lysozyme at a final concentration of 0.2 to 10 mg/ml. As a hypertonic medium, for example, SSM medium [glucose 20g,
(NH ₄ ) ₂ SO ₄ 10g, urea 3g, yeast extract 1g,
KH ₂ PO ₄ 1g, MgCl ₂・6H ₂ O 0.4g, FeSO ₄・
_7H2O10mg , MnSO4・₄ ～ _6H2O0.2mg ,
ZnSO ₄・7H ₂ O0.9mg, CuSO ₄・5H ₂ O0.4mg,
Na ₂ B ₄ O ₇・10H ₂ O 0.09 mg, (NH ₄ ) ₆ MO ₇ O ₂₄・
0.4M sucrose, 0.01M in a 2-fold dilution of a medium containing 0.04 mg of 4H ₂ O, 30 μg of biotin, and 1 mg of thiamine hydrochloride in 1 part of pure water and adjusted to pH 7.2.
A medium PFM containing MgCl ₂ .6H ₂ O and adjusted to pH 7.0 to 8.5 can be used. By reacting at 30-37°C, normal cells gradually become protoplasts, and the extent of this can be observed with a light microscope. The time required for most cells to become protoplasts varies depending on the strain, but is 0.5 to 8 hours under the above conditions. After the action of lysozyme, the number of normal cells that survive without bursting to death under the hypotonic conditions in this bacterial suspension can be reduced to 10 ^-4 or less of the initial normal cells subjected to lysozyme treatment. The protoplasts prepared in this manner have the ability to form colonies (regenerate) on a suitable hypertonic agar medium. As a hypertonic agar medium, for example,
RCGP medium [glucose 5g, casein hydrolysate 5g, yeast extract 2.5g,
K ₂ HPO ₄ 3.5g, KH ₂ PO ₄ 1.5g, MgCl ₂・
6H ₂ O 0.41g, FeSO ₄・7H ₂ O 10mg, MnSO ₄・
4～ _6H2O2mg , _ZnSO4・_7H2O0.9mg ,
( _NH4 ) _6MO7O24・_4H2O0.04mg , _{biotin 30μ}
g, thiamine hydrochloride 2 mg, disodium succinate 135 g, polyvinylpyrrolidone (molecular weight
10000) containing 30g in 1 part of pure water and adjusting the pH to 7.2] can be used. Protoplast regeneration in RCGP medium varies slightly depending on the bacterial strain, lysozyme treatment concentration, and treatment time, but approximately 20 protoplasts per lysozyme-treated initial normal cell.
~70% efficiency. In addition, the number of culture days required for the appearance of regenerated colonies varies depending on the strain, but the number of days required for the culture to grow to the point where colonies can be caught is 5 to 5 days.
It is the 7th. Transformation of protoplasts obtained from the microorganism with the above characteristics with DNA,
This is carried out by mixing the protoplasts and DNA under hypertonic conditions in the coexistence of polyethylene glycol (PEG) or polyvinyl alcohol (PVA) and divalent metal cations. The transformed strain is obtained as normal cells by regenerating protoplasts on a hypertonic agar medium such as RCGP medium in the same manner as the protoplast regeneration described above. Transformants can be selected based on the characteristic traits imparted to the bacterium by genes derived from the donor DNA. This selection may be performed simultaneously with regeneration on a hypertonic agar medium, or it may be performed once non-selectively regenerated and then the regenerated cells collected and performed on an ordinary hypotonic agar medium. Applying this method to normal plasmids
When transformed with DNA, transformed strains are obtained at a frequency of 10 ^-4 to 10 ^-6 per regenerating strain. Specific examples of transformation will be described in Examples below. (2) Isolation of plasmid DNA from transformed strain Culture in a medium in which bacteria can grow, such as NB medium, and after harvesting, lysozyme is applied to dissolve the cell wall. From lysates, e.g. Biochim
Plasmids can be easily concentrated and isolated by commonly used methods such as those described in Biophys. Acta, 383 457-463 (1975). The concentration of lysozyme used varies depending on the target bacterial strain, but is usually 0.1 to 10 mg/ml. Recombination to Corynebacterium and Brevibacterium species used for industrial fermentation production of many amino acids such as glutamic acid and lysine
The application of DNA technology involves cloning genes involved in the biosynthesis or regulation of useful substances such as amino acids from these bacterial species or other microorganisms, and strengthening the biosynthetic system based on the amplification of that genetic information. Increasing the production of plants, and even cloning the genes of animals and plants,
There is a possibility that useful peptides can be produced using these bacterial species by expressing the genetic information, and this is extremely useful industrially. The lysozyme supersensitive microorganism of the present invention is capable of absorbing plasmids, etc. from cells, which is a prerequisite for introducing recombinant DNA technology.
It enables efficient extraction and separation of DNA, transformation of cells with DNA, etc., and therefore facilitates the application of recombinant DNA techniques in the genus Corynebacterium and Brevibacterium. The present inventors have discovered that the microorganism of the present invention is further recombinant.
It has been found useful as a safe host microorganism in DNA techniques. The first requirement in recombinant DNA techniques is the establishment of a safe host-vector system.
Conditions for a safe host include that it has extremely low pathogenicity, does not parasitize the human body, and quickly dies when released into the natural environment. Escherichia coli, which was first used in recombinant DNA techniques, was originally parasitic to the human body, and its safety was controversial, but at least the K-12 strain, which had been subcultured in the laboratory for many years, was parasitic. It was found that the parasitism and pathogenicity were significantly reduced, and it was certified as a B1 host. This K-
When 12 strains are administered to humans, bacteria are excreted over several days, and the total recovery rate is reported to be around 1% (Science 209, pp. 391-394, 1980). When applying recombinant DNA techniques to microorganisms that produce amino acids, nucleic acids, antibiotics, etc., many of these microorganisms have been isolated from soil and are thought to have low parasitic potential to the human body; It is conceivable that even if they can withstand these conditions and do not proliferate, they may be recovered alive. In order to further increase the safety of the harvested bacterial strains, it is conceivable to artificially give them the property that they will die under conditions within the gastrointestinal tract. B2 hosts are given a fragile property that allows them to die even if left in the natural environment. When the lysozyme-sensitive microorganism of the present invention was subjected to a survival test in the gastrointestinal tract of animals, it was found that it was extremely easy to die in the gastrointestinal tract of animals. In other words, ddY male mice (body weight 19g ±
1) Orally administer approximately 3 to 5 x 10 ⁹ of the following microorganisms per animal to a group of two animals, and measure the number of bacteria remaining in the intestines and the number of bacteria excreted in feces by adding 10% homogenate of each. Appropriate dilutions were spread on NB plate medium and measured. As shown in Table 2, it can be seen that the lysozyme-sensitive bacterial strain of the present invention is significantly killed and eliminated compared to the parent strain, and the number of viable bacteria in the intestine is also significantly reduced.

[Table] Furthermore, when the properties of the lysozyme-sensitive bacterial strain of the present invention against various drugs were investigated, it was found that the strain was sensitive to various antibiotics. That is, the microorganism of the present invention was inoculated dropwise into an NB agar medium containing about 10 ⁴ cells in the logarithmic growth phase, and cultured at 30°C for 2 days, and the growth state of the microorganism was observed. As a result of examining the susceptibility, it was found to be extremely sensitive to penicillin G, rifampicillin, etc., as shown in Table 3. This means that various drugs can be used to sterilize the parent strain,
This suggests that it is easy to bacteriostatic, and recombinant DNA
This study also shows that the use of drug-resistant plasmids as vectors has the effect of alleviating the increase in drug resistance.

[Table] As described above, the lysozyme-sensitive microorganism of the present invention is extremely easy to die in the gastrointestinal tract of animals, and is also susceptible to various drugs. This indicates that it has favorable properties. Example 1 Production of lysozyme hypersensitive mutant Corynebacterium glutamicum 225-106,
Corynebacterium hercyulis ATCC13868,
Brevibacterium deivalikatum
ATCC14020 and Brevibacterium lactofamentum ATCC13655 in NB medium (20 g of powdered bouillon, 5 g of yeast extract in 1 part of pure water,
Inoculate the cells into a medium (adjusted to pH 7.2) and culture with shaking at 30°C. The culture was stopped in the middle of the logarithmic growth phase, the bacteria were collected, washed with physiological saline, and suspended in M/20 Tris-malate buffer (PH6.0) to a density of approximately 5 x 10 ⁸ cells/ml. do. Nitrosoguanidine was added to this suspension to give a final concentration of 400 μg/ml, and the suspension was left at 25° C. for 30 minutes. Collect bacteria by centrifugation, wash the cells with the same buffer, suspend in physiological saline, dilute appropriately with physiological saline, and spread a certain amount onto an NB agar medium. After culturing at 30° C. for 2 days, the resulting colonies are replica plated onto a nutrient agar medium and an NB agar medium containing 12.5 μg/ml egg white lysozyme (Seikagaku Corporation, 6-time reconsolidation). After culturing the replica plate at 30°C for 2 days, bacteria that grow on NB agar medium but do not grow on lysozyme-containing agar medium are obtained as lysozyme hypersensitive mutants.

【table】

[Table] Example 2 Cell wall lysis removal and regeneration of generated protoplasts by lysozyme of the microorganism of the present invention Corynebacterium glutamicum 225-106,
Corynebacterium hercyulis ATCC13868,
Brevibacterium deivalikatum
Seed cultures of ATCC14020, Brevibacterium lactofamentum ATCC13655, and the lysozyme hypersensitive mutant derived therefrom obtained in Example 1 are inoculated into NB medium and cultured with shaking at 30°C. Measure the absorbance (OD) at 660 nm with a Tokyo Photoden colorimeter, and when the OD reaches 0.6, dilute it appropriately with physiological saline and apply a certain amount to the NB agar medium to determine the initial normal cell count. At the same time as the measurement, cells were collected from the culture solution, suspended in RCGP medium containing 1 mg/ml lysozyme (PH7.6) to a concentration of approximately 10 ⁹ cells/ml, and subjected to the L-type test. Transfer to a tube and incubate with gentle shaking at 30°C. After 5 hours, the cells were collected by centrifugation at 2500 xg for 10 minutes, suspended in the PFM medium, washed twice by centrifugation, and resuspended in an equal volume of PFM medium to prepare a protoplast bacterial solution. Take a part of this, one is
Appropriately diluted with RCGP medium, a certain amount was inoculated onto RCGP agar medium, and the other was appropriately diluted with physiological saline, applied onto NB agar medium, and cultured at 30°C, each under hypertonic conditions. The number of bacteria capable of forming colonies (cfu/ml) under hypotonic conditions was determined. The number of colonies formed under hypotonic conditions was measured on the second day of culture (the number does not increase with further culture). The number of colonies formed under hypertonic conditions was measured on the 7th day when the number of regenerated colonies did not increase even after further culturing. These results are shown in Table 3.

[Table] Even when lysozyme is applied to both parent strains, only short rod-shaped normal cells are observed under a microscope, and no production of spherical protoplasts is observed. Furthermore, the number of colonies formed under hypotonic conditions was almost the same as that before lysozyme treatment. On the other hand, lysozyme supersensitive strains are converted into protoplasts by the cell wall lytic action of lysozyme, and the number of remaining bacteria that can grow in hypotonic conditions where the protoplasts rupture and die (they are considered to be non-protoplast cells resistant to osmotic shock).
was 10 ^-4 to 10 ^-6 per number of lysozyme-treated test bacteria. These protoplasts had colony forming ability under hypertonic conditions, and their regeneration efficiency was 20 to 70% of the number of lysozyme-treated test bacteria. Once regenerated colonies were normal cells that could grow on NB agar medium. Example 3 Transformation of protoplasts obtained from the microorganism of the present invention with plasmid pCG4DNA (1) Isolation of plasmid pCG4 Plasmid pCG4 used for transformation in this example was Corynebacterium glutamicum 225-250, which is a carrier of pCG4. (Feikoken Bacterial Serial No. 5939) was extracted and isolated from the cultured bacterial cells as follows. Corynebacterium glutamicum 225
-250 in NB medium, 5 ml of seed culture was added to 400
ml semi-synthetic medium SSM and culture with shaking at 30°C. Measure the absorbance (OD) at 660nm with a Tokyo Photoden colorimeter, and when the OD reaches 0.2,
Add penicillin to a concentration of 0.5 units/ml in the culture medium. Further culture is continued at 30°C until the OD reaches approximately 0.6. Collect bacterial cells from the culture solution and add them to TES buffer [0.03M tris(hydroxymethyl)aminomethane (Tris), 0.005M disodium ethylenediaminetetraacetate (EDTA), 0.05M NaCl:PH
8.0], then lysozyme solution (25% sucrose,
Suspend in 10 ml with 0.1 M NaCl, 0.05 M Tris, 0.8 mg/ml lysozyme, pH 8.0) and react at 37°C for 4 hours. To the reaction solution, 2.4ml of 5M NaCl, 0.5M
Add 0.6 ml of EDTA (PH 8.5), 4.4 ml of a solution consisting of 4% sodium lauryl sulfate and 0.7 M NaCl in sequence, mix gently, and then place in ice water for 15 hours. Transfer the entire amount of the lysate to a centrifuge tube, centrifuge at 69,400 xg for 60 minutes at 4°C, and collect the supernatant.
Add PEG6000 equivalent to 10% weight percentage to this,
Mix gently and place in ice water after dissolving. After 16 hours, collect the pellet by centrifugation at 1500 xg for 10 minutes. Add 5 ml of TES buffer, gently redissolve the pellet, then add 2 ml of 1.5 mg/ml ethidium bromide, add cesium chloride, gently dissolve, and adjust the density to 1.580. This solution is centrifuged at 105,000 xg and 18°C for 48 hours. Through this density gradient centrifugation, the covalently closed circular DNA is found as a dense band in the lower part of the centrifuge tube by irradiation with an ultraviolet lamp. Plasmid pCG4 is isolated by extracting this band from the side of the centrifuge tube with a syringe.
Next, the fractionated solution was treated five times with an equal volume of isopropyl alcohol solution [volume percentage 90% isopropyl alcohol, 10% TES buffer (this mixture contains a saturated amount of cesium chloride)] to extract and remove ethidium bromide. and then dialyzed against TES buffer. In this way, 15 μg of plasmid pCG4 is obtained. Plasmid pCG4 has a molecular weight of approximately 19 megadaltons, contains a gene showing resistance to streptomycin and/or spectinomycin,
and EcoR, BamH, Hind,
This is a new plasmid having 4, 7, 9, 6, and 6 cleavage sites for Pst and Sal, respectively. (2) Transformation of protoplasts Transformation was performed using lysozyme-treated cells prepared in the same manner as in Example 2. Transfer 0.5 ml of protoplast bacteria solution to a small test tube and test at 2500 x g for 5 minutes.
Centrifuge for 1 minute, then use TSMC buffer [10mM magnesium chloride, 30mM calcium chloride, 50mM
Resuspend in 1 ml of Tris(hydroxymethyl)aminomethane (Tris), 400 mM sucrose, pH 7.5 and wash by centrifugation. to the precipitated protoplasts.
Add 0.1 ml of TSMC buffer and resuspend by shaking gently. This is combined with twice the concentration above.
pCG4DNA mixed 1:1 with TSMC buffer
Add 0.1ml of solution (containing 0.2μg DNA) and mix.
Next, add 0.8 ml of a solution containing 20% PEG6000 in TSMC buffer and mix gently. After 3 minutes, 2 ml of RCGP medium was added, centrifuged at 2500 x g for 5 minutes to remove the supernatant, and the precipitated protoplasts were suspended in 1 ml of RCGP medium, diluted in several stages with RCGP medium, and aliquoted into a fixed amount. smear onto RCGP agar medium containing 400 μg/ml spectinomycin. In order to know the number of bacteria that can form colonies at the same time,
Spread a certain amount of the highly diluted solution onto the RCGP agar medium. Culture at 30°C for 6 days and measure the number of colonies that appear. Colonies generated on RCGP medium containing spectinomycin were taken, spread on NB agar medium containing 12.5 μg/ml streptomycin, and cultured at 30° C. for 2 days to examine simultaneous acquisition of streptomycin resistance. Strains that showed cross-resistance were randomly selected, and plasmids were isolated by the method shown in Example 4. These results are summarized in Table 4.

【table】

[Table] ** Number of test strains is in parentheses.
*** No spectinomycin-resistant bacteria were obtained with this strain.
The spectinomycin-resistant strain that emerged from the lysozyme-insensitive parent strain was treated with lysozyme.
It did not show streptomycin cross-resistance, and a plasmid was isolated in the same manner as for the 225-250 strain described above, but no pCG4 was detected. On the other hand, spectinomycin-resistant strains that emerge from cells treated with lysozyme from lysozyme-hypersensitive strains have streptomycin cross-resistance, and the plasmids isolated from these strains are digested with the restriction enzyme Hind and then subjected to agarose electrophoresis. The fragment was identical to pCG4. Table 7 shows the deposited accession numbers of the pCG4 transformants obtained as described above.

[Table] Example 4 Isolation of plasmids from microorganisms harboring plasmids Corynebacterium glutamicum L15/pCG4, a transformed strain of pCG4 obtained in Example 3,
Corynebacterium hercyulis L103/
pCG4, Brevibacterium deivaricatum
5 ml of seed culture of L204/pCG4 and Brevibacterium lactofamentum L312/pCG4 cultured in NB medium was inoculated into 400 ml of NB medium, and incubated at 30℃.
Incubate with shaking for 12 hours. Collect bacteria from the culture solution, wash with TES buffer, and add to lysozyme solution (25% sucrose, 0.1M NaCl,
0.05M Tris, 0.8mg/ml lysozyme: PH8) 10
ml and react at 37°C for 1 hour. to the reaction solution
5M NaCl2.4ml, 0.5M EDTA (PH8.5) 0.6ml, 4
4.4 ml of a solution consisting of % sodium lauryl sulfate and 0.7 M NaCl are sequentially added, mixed gently and placed in ice water for 15 hours. Transfer the entire amount of the lysate to a centrifuge tube, centrifuge at 69,400 xg for 60 minutes at 4°C, and collect the supernatant. This is equivalent to a weight percentage of 10%.
Add PEG6000, mix gently and place in ice water after dissolving. After 16 hours, collect the pellet by centrifugation at 1500 xg for 10 minutes. Redissolve the pellet by adding 5 ml of TES buffer and then add 2 ml of 1.5 mg/ml ethidium bromide. Add cesium chloride to this, dissolve it, and adjust the density to 1580. This solution is subjected to density gradient centrifugation under the same conditions as described in Example 3, and the plasmid is fractionated. After extracting and removing ethidium bromide by treating the fractionated solution with isopropyl alcohol solution in the same manner as in Example 3,
Dialyze against TES buffer. In this way, 15 to 30 μg of pCG4 DNA was collected from any lysozyme hypersensitive strain harboring pCG4.
was gotten.

Claims (1)

[Scope of Claims] 1. A novel lysozyme-sensitive microorganism that belongs to Corynebacterium herkyulis, Brevibacterium deivalicatum or Brevibacterium lactofamentum and is sensitive to lysozyme. 2. The microorganisms are Corynebacterium hercyulis L-103 (FEI 5947, ATCC 31866), Brevibacterium deivaricatum L-204 (FEI 5948, ATCC 31867), and Brevibacterium lactoformentum L −312 (Feikoken Joyori)
528, ATCC 31868).