WO2001012649A1 - Aga - Google Patents

Abstract

The invention provides aga promoter polynucleotides and methods for producing and using such polynucleotides, as well as their variants, agonists and antagonists, and their uses.

Description

What is claimed is:

1 An isolated polynucleotide selected from the group consisting of

(1) an isolated polynucleotide comprising a nucleotide sequence which has at least

(a) 70% identity,

(b) 80% identity,

(c) 90% identity; or

(d) 95% identity, to that of SEQ ID NO 1 over the entire length of SEQ ID NO 1,

(ύ) an isolated polynucleotide which is the polynucleotide of SEQ ID NO 1,

(lii) an isolated polynucleotide obtainable by screening an appropπate library under stringent hybridization conditions with a probe having the sequence of SEQ ID NO 1 or a fragment thereof,

(IV) an isolated polynucleotide comprising a promoter expressibly linked to the aga gene contained in the Streptococcus pneumomae, and

(v) a polynucleotide sequence complementary to said isolated polynucleotide of (1), (ii),

(iii) or (iv)

2. A method for the treatment of an individual

(I) in need of enhanced activity of the polynucleotide of claim 1 compπsmg the step of

(a) administering to the individual a therapeuticallv efFective amount of an agonist to said polynucleotide. or

(ii) having need to inhibit activity or expression of the polynucleotide of claim 1 comprising

(a) administering to the individual a therapeuticallv effective amount of an antagonist to said polynucleotide, or

(b) administering to the individual a therapeuticallv effective amount of a compound that competes with said polynucleotide for its hgand or binding factor

3 A method for screening to identify compounds that activate or that inhibit the function of the polynucleotide of claim 1 which comprises a method selected from the group consisting of

26 (a) mixing a candidate compound with a solution containing a polynucleotide of claim 1 to form a mixture, measuring activity of the pohmicleotide m the mixture, and comparing the

of the mixture to a standard, or

(b) detecting the effect of a candidate compound on the production of mRN A or pohpeptide promoter activity of the pohnucleotide in cells, using for instance, an ELISA assav. or

(c) ( 1 ) contacting a composition comprising the pohnucleotide with the compound to be screened under conditions to permit interaction between the compound and the pohnucleotide to assess the interaction of a compound, such interaction being associated with a second component capable of pro\ iding a detectable signal in response to the interaction of the polynucleotide ith the compound, and

(2) determining whether the compound interacts with and activates or inhibits an activity of the polynucleotide by detecting the presence or absence of a signal generated from the interaction of the compound with the polynucleotide

4 An agonist or an antagonist of the activity of or expression from polynucleotide of claim 1

5 A host cell comprising the polynucleotide of claim 1

6 A polynucleotide consisting of a polynucleotide of the formula

X-(R₁)_m-(R₂)-(R₃)_n-Y wherein, at the 5' end of the molecule. X is hydrogen, a metal or a modified nucleotide residue, or together with Y defines a covalent bond, and at the 3' end of the molecule, Y is hydrogen, a metal, or a modified nucleotide residue, or together with X defines the covalent bond, each occurrence of R_j and R3 is independently any nucleic acid residue or modified nucleic acid residue, m is an integer between 1 and 3000 or zero , n is an integer between 1 and 3000 or zero, and R₂ is a nucleic acid sequence or modified nucleic acid sequence set forth in SEQ ID NO 1

7 The method of ascertaining the functionality or essentiality of the target gene (gene- of-mterest) in a cell comprising the transcription or expression of gene-of-mterest by expressing an anti-sense sequence to the gene-of-interest under the transcπptional control of the promoter polynucleotides of claim 1 8 The method of ascertainmg the functionality or essentiality of the target gene (gene-of interest), m a cell, compπsmg

(a) disabling ("knockrng-out") the gene-of-mterest,

(b) re-introducrng, at the target gene locus, the gene-of-mterest now v der the operational control of the inducible promoter polynucleotides of claim 1, and

(c) add g the mducer thereby providing information to the essentiality or functionality of the gene of interest

9 A method of determining a functionality or essentiality of the target gene (gene-of interest), in a cell, compπsmg

(a) replacing the promoter sequence of the gene-of-mterest by the promoter polynucleotide of claim 1. and

(b) addmg the repressor thereby providing information to the essentiality or functionality of the gene-of-mterest

10 A method of determining a protem target of a antibacteπal compound comprising-

(a) placing the protem target under the control of the promoter polynucleotide of claim 1 , and

(b) addmg the mducer or repressor thereby changmg accordmgly the susceptibility of the bacteπa to the compound

28 aga

RELATED APPLICATIONS

This Application claims the benefit to U S Provisional Patent Application Number 60/149,575, filed August 18. 1999 FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides, and their production and uses, as well as their variants, agonists and antagonists, and their uses In particular, the invention relates to promoter polynucleotides, as well as their variants, hereinafter referred to as "aga," "aga promoter polynucleotιde(s)," and "aga polynucleotιde(s)" as the case may be BACKGROUND OF THE INVENTION

The Streptococci make up a medically important genera of microbes known to cause several types of disease in humans, including, for example, outis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid Smce its isolation more than 100 years ago, Streptococcus pneumoniae has been one of the more intensively studied microbes For example, much of our early understanding that DNA is, in fact, the genetic mateπal was predicated on the work of Griffith and of Avery, Macleod and McCarty using this microbe Despite the vast amount of research with S pneumoniae, many questions concerning the virulence of this microbe remain It is particularly preferred to employ Streptococcal genes and gene products as targets for the development of antibiotics The frequency of Streptococcus pneumoniae infections has πsen dramatically in the past few decades This has been attributed to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems It is no longer uncommon to isolate Streptococcus pneumoniae strains that are resistant to some or all of the standard antibiotics This phenomenon has created an unmet medical need and demand for new anti-microbial agents, vaccines, drug screening methods, and diagnostic tests for this organism

Moreover, the drug discovery process is currently undergoing a fundamental revolution as it embraces "functional genomics." that is. high throughput genome- or gene-based biology This approach is rapidly superseding earlier approaches based on "positional cloning" and other methods Functional genomics relies heavih on the vaπous tools of bioinformatics to identify gene sequences of potential interest from the many molecular biology databases now available as well as from other sources There is a continuing and significant need to identify and characterize further genes and other pohnucleotides sequences and their related polypeptides. as targets for drug discovery

Clearly, there exists a need for polynucleotides. such as the aga pohnucleotide embodiments of the invention, that have a present benefit of among other things being useful to screen compounds for antimicrobial activity Such factors are also useful to determine their role in gene regulation, pathogenesis of infection, dysfunction and disease T ere is also a need for identification and characterization of such factors and their antagonists and agonists to find ways to prevent, ameliorate or correct such infection, dysfunction and disease SUMMARY OF THE INVENTION

The present mvention relates to aga, m particular aga promoter polynucleotides, recombinant mateπals and methods for their production In another aspect, the invention relates to methods for using such polynucleotides, including treatment of microbial diseases, amongst others In a further aspect, the mvention relates to methods for identifying agonists and antagonists usmg the mateπals provided by the mvention. and for treatmg microbial infections and conditions associated with such infections with the identified agonist or antagonist compounds In a still further aspect, the mvention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting aga promoter dπven expression or activity

Vaπous changes and modifications within the spiπt and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following descπptions and from reading the other parts of the present disclosure

DESCRIPTION OF THE INVENTION The mvention relates to aga polynucleotides as descπbed in greater detail below In particular, the mvention relates to polynucleotides of a aga of Streptococcus pneumoniae. which is related by sequence homologv to no homolog polynucleotide or by the presence of characteπstic motifs The mvention relates especially to aga promoter polynucleotides having the nucleotides sequences set out m Table 1 Note that sequences recited m the Sequence Listing below as "DNA^" represent an exemplification of the mvention. smce those of ordinary skill will recognize that such sequences can be usefully employed m polynucleotides in general, including πbopolynucleotides

TABLE 1 aga Polynucleotide Promoter Sequences

(A) Streptococcus pneumoniae aga polynucleotide sequence [SEQ ID NO 1] 5'-

CATGTGCTACCTCCTACCTAACATTTTACCATATTCTTGAGACATTTTTCTATTTTAGAAA GCGATTTCAGGTGATAAAATATAATCAATAAAGTGATGAGGTGAAGTAAATG-3^,

- 2 - Methods to identify promoters mclude techniques known in the art as well as those provided herein. Art techniques mclude. but are not limited to. the following RT-PCR

RT-PCR analysis of total RNA isolated from infected tissue or in vitro grown cells Using genome databases, primer pairs are designed to predict transcπpts of the selected pathogen and arrayed m microtiter dish format Total RNA is isolated from an in vitro grown pathogen and RT- PCR performed with all the primer pairs Similarly RT-PCR is performed with total RNA isolated at varying times from infections of the selected pathogen m a vaπety of appropπate animal models Companson of the PCR profiles which reflect the ratio of a given mRNA to internal standards such as rRNA or housekeepmg genes provides identification of those transcπpts which are essentially absent in vitro, but are on throughout, or duπng. vaπous phases of infection

Putative promoters are characteπzed using TaqMan quantitative RT-PCR, or expression of reporter genes

Specific sequence detection occurs by amplification of target sequences m the PE Applied Biosystems 7700 Sequence Detection System m the presence of an ohgonucleotide probe labeled at the 5' and 3' ends with a reporter and quencher fluorescent dye, respectively (TaqMan FQ probe), which anneals between the two PCR primers Only specific product will be detected when the probe is bound between the pπmers As PCR amplification proceeds, the 5'-nuclease activity of Taq polymerase initially cleaves the reporter dye from the probe The signal generated when the reporter dye is physically separated from the quencher dye is measured with an attached CCD camera Each signal generated equals one probe cleaved which coπesponds to amplification of one target strand RT PCR controls may mclude +/- reverse transcπptase reactions, amplification along side genes known to be transcπbed under the conditions of study and amplification of serial dilutions of genomic DNA The level of transcription under in vivo and in vitro conditions is quantified b\ companson of signal generated from these samples to that of a standard curve generated from signal resulting from amplification of the genomic DNA

FAM and TAMRA labeling of pn ers and the uses of such primers has been reported

(Lee. LG, Connell. CR. and Bloch. W 1993 Allehc discrimination by nick-translation PCR with fluorogenic probes Nucleic Acids Research 21 3761-3766. Livak. KJ. Flood. SJA. Marmaro, J . Giusti. W. and Deetz. K 1995 Oligonucleotides with fluorescent dves at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybndization PCR

Methods and Applications 4 357-362 ) And/or the promoter region can be cloned upstream of a reporter gene m a vector appropπate for the selected pathogen By "appropnate" it is meant a vector capable of replicating stably m a selected pathogen

Potential reporter genes mclude, but are not limited to. beta-galactosidase, beta-lactamase, bactenal luciferase, firefly luciferase, beta-glucuronidase Many other reporter genes known in the art may be used m the methods and compositions of matter of the mvention

Primer extension

A pπmer especific for the gene which transcnption to be analyse is designed at the appropiate distance from the ATG start codon Total RNA is isolated from an in vitro grown pathogen and a reverse transcπptase reaction is earned out Primers are fluorescently labelled at the 5 'end usmg FAM, HEX or NED dyes Primer extension products can be separated on a polyacrylamide sequencmg gel and detected usmg a suitable gel scanning system (e g ABI Pπsm™ 377 Sequencer usmg GeneScan™ software as supplied by Perkin Elmer)

Deposited materials A deposit containing a Streptococcus pneumoniae 0100993 strain has been deposited with the

National Collections of Industπal and Marine Bacteπa Ltd (herein "NCIMB"), 23 St Machar Dπve, Aberdeen AB2 IRY, Scotland on 11 Apπl 1996 and assigned deposit number 40794 The deposit was descπbed as Streptococcus pneumoniae 0100993 on deposit

On 17 Apπl 1996 a Streptococcus pneumoniae 0100993 DNA library in E coli was similarly deposited with the NCIMB and assigned deposit number 40800 The Streptococcus pneumoniae strain deposit is refeπed to herein as "the deposited strain" or as "the DNA of the deposited strain "

The deposited strain contains the full length aga gene compπsmg the promoter polynucleotide of the invention The sequence of the promoter polynucleotides contained in the deposited strain, are controlling in the event of any conflict with anv descnption of sequences herein The deposit of the deposited strain has been made under the terms of the Budapest Treaty on the

International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure The deposited strain will be lπevocably and without restπction or condition released to the public upon the issuance of a patent The deposited strain is provided merely as convenience to those of skill in the art and is not an admission that a deposit is required for enablement such as that required under 35 U S C §112 A license ma\ be required to make, use or sell the deposited strain, and compounds deπved therefrom, and no such license is hereby granted

In one aspect of the mvention there is provided an isolated nucleic acid molecule functional m the Streptococcus pneumoniae 0100993 strain, which pohnucleotide is contained in the deposited strain Further provided b\ the invention are aga pohnucleotide sequences in the deposited strain, such as DNA and RNA

- 4 - and ammo acid sequences encoded thereby Also provided by the mvention are aga polynucleotide sequences isolated from the deposited strain Polynucleotides

It is an object of the mvention to provide promoter polynucleotides from a aga gene In a particularly prefeπed embodiment of the mvention the polynucleotide compπses a promoter region from aga gene cσmpnsing a sequence set out m Table 1 [SEQ ID NO 1], or a vaπant thereof

As a further aspect of the mvention there are provided isolated promoter nucleic aαd molecules from a aga gene, including, for example, polynucleotides deπved from such molecules, such as, unprocessed RNAs, πbozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs Further embodiments of the mvention mclude biologically, diagnostically, prophylactically. clinically or therapeutically useful polynucleotides, and vanants thereof, and compositions compπsing the same

Another aspect of the mvention relates to isolated polynucleotides, including, for example polynucleotides closely related to a aga promoter having a polynucleotide sequence of Table 1 [SEQ ID NO l] In another particularly prefeπed embodiment of the mvention there is a aga polynucleotide from

Streptococcus pneumoniae compπsmg or consisting of an nucleotide sequence of Table 1 [SEQ ID NO 1], or a vanant thereof

Using the information provided herein, such as a promoter polynucleotide sequence set out in Table 1 [SEQ ID NO 1], a polynucleotide of the mvention may be obtained usmg standard cloning and screening methods, such as those for cloning and sequencmg chromosomal (genomic) DNA fragments from bacteπa using Streptococcus pneumoniae 0100993 cells as starting mateπal, followed by obtaining a related or equivalent sequence For example, to obtain a polynucleotide sequence of the mvention, such as a polynucleotide sequence given m Table 1 [SEQ ID NO 1], typically a library of clones of chromosomal DNA of Streptococcus pneumoniae 0100993 in E coli or some other suitable host is probed with a radiolabeled oligonucleotide, preferably a 17-mer or longer, denved from a partial sequence Clones carrying DNA identical to that of the probe can then be distinguished using stringent hybndization conditions By sequencing the individual clones thus identified by hybridization with sequencmg primers designed from the original polynucleotide sequence it is then possible to extend the polynucleotide sequence m both directions to determine a functional promoter region sequence or full length gene sequence Conveniently, such sequencmg is performed, for example, usmg denatured double stranded DNA prepared from a plasmid clone Suitable techniques are descnbed by Mamatis, T . Fπtsch. E F and Sambrook et al . MOLECULAR CLONING, A LABORATORY MANUAL. 2nd Ed Cold Spring Harbor Laboratory Press. Cold Spring Harbor. New York (1989) (see m particular Screening By Hvbndization 1 90 and Sequencmg Denatured Double-Stranded DNA Templates 13 70) Direct genomic DNA sequencmg may also be performed to obtain a promoter sequence of expressibly linked full length gene sequence Illustrative of the mvention. each polynucleotide set out m Table 1 [SEQ ID NO 1] was discovered m a DNA hbrary deπved from Streptococcus pneumoniae 0100993

In a further aspect, the present mvention provides for an isolated polynucleotide compπsing or consisting of a polynucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NO 1 over the entire length of SEQ ID NO 1

A polynucleotide encoding a polypeptide of the present mvention. including homologs and orthologs from species other than Streptococcus pneumoniae, may be obtained by a process which comprises the steps of screening an appropnate hbrary under stπngent hybπdization conditions with a labeled or detectable probe consisting of or compπsmg the sequence of SEQ ID NO 1 or a fragment thereof, and isolating a promoter and/or full-length gene and/or genomic clones containing said polynucleotide sequence

The mvention also includes a polynucleotide consisting of or compπsmg a polynucleotide of the formula X-(R₁)_m-(R₂)-(R₃)_n-Y wherem, at the 5' end of the molecule, X is hydrogen, a metal or a modified nucleotide residue, or together with Y defines a covalent bond, and at the 3' end of the molecule, Y is hydrogen, a metal, or a modified nucleotide residue, or together with X defines the covalent bond, each occurrence of R\ and R3 is mdependently any nucleic acid residue or modified nucleic acid residue, m is an mteger between 1 and 3000 or zero . n is an mteger between 1 and 3000 or zero, and R₂ is a nucleic acid sequence or modified nucleic acid sequence of the mvention, particularly a nucleic acid sequence selected from Table 1 or a modified nucleic acid sequence thereof In the polynucleotide formula above, R₂ is onented so that its 5' end nucleic acid residue is at the left bound to R_] and its 3' end nucleic acid residue is at the πght. bound to R3 Any stretch of nucleic acid residues denoted by either R\ and/or R , where m and/or n is greater than 1. may be either a heteropolymer or a homopolymer, preferably a heteropolymer Where, m a preferred embodiment. X and Y together define a covalent bond, the polynucleotide of the above formula is a closed, circular polynucleotide. which can be a double-stranded polynucleotide wherein the formula shows a first strand to which the second strand is complementary In another prefeπed embodiment m and or n is an mteger between 1 and 1000. Other prefeπed embodiments of the mvention are provided where m is an mteger between 1 and 50, 100 or 500. and n is an mteger between 1 and 50 100. or 500

It is most prefeπed that a pohnucleotide of the mvention is deπved from Streptococcus pneumoniae however it ma\ preferably be obtained from other organisms of the same taxonomic genus A polynucleotide of the l ention ma\ also be obtained for example from organisms of the same taxonomic family or order Prefeπed embodiments are polynucleotides that retain substantially the same biological function or activity as the promoter region DNA of Table 1 [SEQ ED NO 1]

In accordance with certain prefeπed embodiments of this mvention there are provided polynucleotides that hybπdize, particularly under stπngent conditions, to aga polynucleotide sequences, such as those polynucleotides in Table 1 In this regard, the mvention especially relates to polynucleotides that hybπdize under stπngent conditions to the polynucleotides descπbed herein As herein used, the terms "stringent conditions" and "stringent hybπdization conditions" mean hybndization occurring only if there is at least 95% and preferably at least 97% identity between the sequences A specific example of stπngent hybndization conditions is overnight mcubation at 42°C m a solution compnsmg 50% formamide, 5x SSC (150mM NaCl, 15mM tnsodium citrate), 50 mM sodium phosphate (pH7 6), 5x Denhardt's solution, 10% dextran sulfate. and 20 micrograms/ml of denatured, sheared salmon sperm DNA, followed by washing the hybndization support m 0 lx SSC at about 65°C Hybndization and wash conditions are well known and exemplified m Sambrook, et al . Molecular Cloning A Laboratory Manual, Second Edition, Cold Spring Harbor, N Y , (1989), particularly Chapter 11 therein Solution hybndization may also be used with the polynucleotide sequences provided by the mvention Prefeπed polynucleotides that hybπdize under stringent conditions are polynucleotide sequences compnsmg of at least 50, 100, 500, 1000, or 3000 nucleotides

As discussed elsewhere herein regarding polynucleotide assays of the mvention, for instance, the polynucleotides of the mvention, may be used as a hybπdization probe genomic DNA to isolate genomic clones encoding aga and to isolate genomic clones of other genes that have a high identity, particularly high sequence identity, to the aga gene Such probes generally will compπse at least 15 nucleotide residues or base pairs Preferably, such probes will have at least 30 nucleotide residues or base parrs and may have at least 50 nucleotide residues or base pairs Particularly prefeπed probes will have at least 20 nucleotide residues or base pairs and will have lee than 30 nucleotide residues or base pairs The polynucleotides of the mvention may be employed, for example, as research reagents and mateπals for discovery of treatments of and diagnostics for diseases, particularly human diseases, as further discussed herem relating to polynucleotide assays

For each and every pohnucleotide of the mvention there is provided a polynucleotide complementary to it It is prefeπed that these complementary polynucleotides are fulh complementarv to each polynucleotide with which they are complementary

In addition to the standard A. G. C. T/U representations for nucleotides. the term "N" may also be used in describing certain polynucleotides of the invention "N" means that any of the four DNA or RNA nucleotides

appear at such a designated position in the DNA or RNA sequence, except it is prefeπed that N is not a nucleic acid that when taken in combination with adjacent nucleotide positions

- 7 - when read m the coπect reading frame, would have the effect of generating a premature termination codon m such reading frame

Vectors, Host Cells, Expression Systems

The mvention also relates to vectors that compnse a polynucleotide or polynucleotides of the mvention. host cells that are genetically engineered with vectors of the mvention and the production of polynucleotides of the mvention by recombinant techniques Further the promoter polynucleotides of the mvention can be used to dπve the expression of heterologous proteins or over-expression of naturally associated proteins Since the promoter polynucleotide of the mvention is inducible, such as by mannose or by any other appropnate rnducers as the case may be, in other aspect the further aspect relates to direct or indirect up- or down-regulation of expression or transcnption of a target gene Recombinant proteins (polypeptides) may be prepared by processes well known m the art from genetically engineered host cells compπsmg expression vectors

For recombinant production of the polynucleotides of the mvention, host cells can be genetically engineered to incorporate replication systems or portions thereof or polynucleotides of the mvention Introduction of a polynucleotide into the host cell can be effected by methods descnbed in many standard laboratory manuals, such as Davis, et al , BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al , MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed . Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N Y (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvecuon. micrornjection, catiomc lipid-mediated transfection. electroporation, transduction. scrape loading, balhstic mtroduction and infection Representative examples of appropnate hosts mclude bacteπal cells, such as cells of streptococci, staphylococci. enterococci. E coh. streptomyces cyanobacteπa. Bacillus subtihs. and Streptococcus pneumoniae. fungal cells, such as cells of a yeast Kluveromyces. Saccnaromyces. a basidiomycete. Candida albicans and Aspergillus. insect cells such as cells ϊDrosophila S2 and Spodoptera Sf9, animal cells such as CHO. COS. HeLa. C127. 3T3. BHK, 293. CV- 1 and Bowes melanoma cells, and plant cells, such as cells of a gymnosperm or angiosperm

A great variety of replication systems can be used to produce the polynucleotides of the mvention Such vectors mclude. among others, chromosomal-, episomal- and virus-deπved vectors, for example, vectors deπved from bacteπal plasmids. from bacteπophage, from transposons. from yeast episomes. from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses.

viruses, such as SV40, vaccinia viruses, adenoviruses. fowl pox viruses, pseudorabies viruses, picomaviruses and retroviruses. and vectors deπved from combinations thereof, such as those deπved from plasmid and bacteπophage genetic elements, such as cosmids and phagemids The replication system constructs may contain control regions other than the promoter of the mvention that regulate as well as engender expression, such as marker gene expression General!} any system or vector suitable to maintain, propagate or express polynucleotides m a host may be used for expression m this regard The appropnate DNA sequence may be inserted into the expression system by any of a vanety of well-known and routine techniques, such as, for example, those set forth m Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL, (supra) Assays, antagonists and Agonists

Characterization of Function: Assays of the mvention may be performed by determining the effect of transcnpt level on cell phenotype These assays will help to charactenze, among other things, temporal relevance of transcπption to phenotype

Protein Expression: Promoter polynucleotides of the mvention may be used for overproduction of heterologous proteins m bactena

Essentiality Testing: Promoter polynucleotides of the mvention may be used to assess gene essentiality m a bacteπa Example 2 provides one embodiment of this type of assay Skilled artisans can readily determine other ways to perform such analyses based on the present mvention and the teachings herem Polynucleotides of the mvention may also be used to assess the binding of small molecule substrates and hgands in, for example, cells, cell-free preparations, chemical hbranes. and natural product mixtures These substrates and hgands may be natural substrates and hgands or may be structural or functional mimetics See, e g , Cohgan et al , Current Protocols m Immunology 1(2) Chapter 5 (1991)

Polynucleotides of the present mvention are responsible for many biological functions, including many disease states, in particular the Diseases herembefore mentioned It is therefore desirable to devise screening methods to identify compounds which stimulate or which inhibit the function of the polynucleotide Accordmgly, rn a further aspect the present mvention provides for a method of screening compounds to identify those which stimulate or which inhibit the function of a polynucleotide of the mvention. as well as related polynucleotides In general, agonists or antagonists may be employed for therapeutic and prophylactic purposes for such Diseases as herembefore mentioned Compounds may be identified from a vanety of sources, for example, cells, cell-free preparations, chemical hbranes, and natural product mixtures Such agonists, antagonists or inhibitors so-identified may be natural or modified substrates, hgands, receptors, enzymes, etc . as the case may be. of aga polynucleotides. or may be structural or functional mimetics thereof (see Cohgan et al . Current Protocols in Immunology 1(2) Chapter 5 (1991)) The screening methods mav simply measure the binding of a candidate compound to the polynucleotide. or to cells or membranes bearing the polynucleotide Alternatively the screening method mav involve competition with a labeled competitor Further, these screening methods may test whether the candidate compound results a signal generated by activation or inhibition of the pohnucleotide. usmg detection systems appropriate to the cells comprising the pohnucleotide Inhibitors of activation are generally assayed m the presence of a known agonist and the effect on activation by the agomst by the presence of the candidate compound is observed Constitutively active promoter polynucleotides and/or constitutively expressed polynucleotides may be employed m screening methods for mverse agonists or inhibitors, m the absence of an agomst or inhibitor, by testing whether the candidate compound results m inhibition of activation of the polynucleotide, as the case may be Further, the screening methods may simply compπse the steps of mixing a candidate compound with a solution containing a polynucleotide of the present mvention. to form a mixture, measuring aga promoter polynucleotide activity m the mixture, and comparing the aga promoter polynucleotide activity of the mixture to a standard Fusion protems. such as those made from Fc portion and aga promoter polynucleotide, as herembefore descnbed, can also be used for high-throughput screening assays to identify antagonists of the polynucleotide of the present invention, as well as of phylogenetically and and/or functionally related promoters

The polynucleotides, polypeptides and antibodies that bmd to and/or interact with a polynucleotide of the present mvention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and/or promoter polynucleotide m cells

The mvention also provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) the action of aga polynucleotides, particularly those compounds that are bactenstatic and/or bactencidal The method of screening may mvolve high-throughput techniques For example, to screen for agonists or antagonists, a synthetic reaction mix. a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, compnsmg aga polynucleotide and a labeled substrate or gand of such polynucleotide is incubated m the absence or the presence of a candidate molecule that may be a aga agonist or antagonist The ability of the candidate molecule to agonize or antagonize the aga polynucleotide is reflected m decreased binding of the labeled gand or decreased production of product from such substrate Molecules that bmd gratuitously I e . without inducing the effects of aga pohnucleotide are most likely to be good antagorasts Molecules that bmd well and. as the case may be, increase the rate of product production from substrate, increase signal transduction. or increase chemical channel activity are agonists Detection of the rate or level of. as the case may be. production of product from substrate, signal transduction. or chemical channel activity mav be enhanced by usmg a reporter system Reporter systems that may be useful in this regard mclude but are not limited to colonmetnc. labeled substrate converted into product, a reporter gene that is responsive to changes in aga polynucleotide activity, and binding assays known in the art

Polynucleotides of the invention may be used to identify promoter binding protems. such as sigma factors, if any. for such pohnucleotide. through standard binding techniques known in the art for example, gel retardation assa\s Other of these techniques include, but are not limited to. hgand

- 10 - bmdmg and crosslrnkmg assays m which the polynucleotide is labeled with a radioactive isotope (for instance, ^P), chemically modified (for instance, biotinylated or fluorescent tagged), or fused to a polynucleotide sequence suitable for detection or punfication, and mcubated with a source of the putative bmdmg compound or gand (e g , cells, cell membranes, cell supernatants, tissue extracts, bodily matenals) Other methods mclude biophysical techmques such as surface plasmon resonance and spectroscopy These screening methods may also be used to identify agonists and antagonists of the polynucleotide which compete with the binding of the polynucleotide to its hgand(s), if any Standard methods for conducting such assays are well understood m the art

The fluorescence polanzation value for a fluorescently-tagged molecule depends on the rotational coπelation time or tumblmg rate Protem-polynucleotide complexes, such as formed by aga polynucleotide associating with polypeptide or other factor, labeled to compnse a fiuorescently-labeled molecule will have higher polanzation values than a fluorescently labeled monomenc polynucleotide It is prefeπed that this method be used to charactenze small molecules that disrupt polypeptide- polynucleotide complexes Fluorescence energy transfer may also be used to charactenze small molecules that interfere with the formation of aga polynucleotide-polypeptide dimers, tπmers, tetramers or higher order structures, or structures formed by aga polynucleotide and a polypeptide or polypeptides Aga polynucleotides can be labeled with both a donor and acceptor fluorophore Upon mixing of the two labeled species and excitation of the donor fluorophore. fluorescence energy transfer can be detected by observing fluorescence of the acceptor Compounds that block dimenzation w ll inhibit fluorescence energy transfer

In other embodiments of the mvention there are provided methods for identifying compounds which bmd to or otherwise interact with and inhibit or activate an activity or expression of a polynucleotide of the mvention compnsmg contacting a polynucleotide of the mvention with a compound to be screened under conditions to permit bmdmg to or other mteraction between the compoimd and the polynucleotide to assess the bmdmg to or other mteraction with the compound, such bmdmg or mteraction preferably bemg associated with a second component capable of providing a detectable signal m response to the bmdmg or mteraction of the polynucleotide with the compound, and determining whether the compound binds to or otherwise interacts with and activates or inhibits an activity or expression of the polynucleotide b\ detecting the presence or absence of a signal generated from the bmdmg or mteraction of the compound with the polynucleotide

Another example of an assav for aga agonists or antagonists is a competitive assay that combines aga and a potential agonist or antagonist with aga-binding molecules, recombinant aga bmdmg molecules, natural substrates or hgands or substrate or hgand mimetics under appropnate conditions for a competitive inhibition assa\ Aga can be labeled such as b\ radioactivits or a coloπmetπc compound, such that the

- 1 1 - number of aga molecules bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist or agomst

Numerous assays may be used with the prefeπed inducible promoters of the mvention, as provided herem an as known m the art These assays mclude, but are not limited to the following Antimicrobial Compound Testing

Promoter polynucleotides of the mvention may also be used to determine a target of unknown antibacteπal susceptibility

Promoter polynucleotides of the mvention may also be used to assess the effect of transcπpt level on antibacteπal susceptibility Still further, promoter polynucleotides of the mvention may also be used m whole cell screens

Inducible promoters can be used to up and down regulate expression of target gene directly, or indirectly by transcπption anti-sense RNA or nbozymes

Potential antagonists mclude, among others, small organic molecules, peptides, polypeptides that bmd to a polynucleotide of the mvention and thereby inhibit or extmgmsh its activity or expression Potential antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protem that binds the same sites on a binding molecule, such as a bmdmg molecule, without mducmg aga promoter- induced activities, thereby preventing the action of aga polynucleotides by excluding aga polynucleotides from binding Potential antagorasts mclude a small molecule that bmds to and occupies the bmdmg site of the polynucleotide thereby preventing bmdmg to cellular bmdmg molecules, such that normal biological activity is prevented Examples of small molecules mclude but are not limited to small organic molecules, peptides or peptide-like molecules Other potential antagonists mclude antisense molecules (see Okano. J Neurochem 56 560 (1991), OUGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press. Boca Raton. FL (1988), for a descπption of these molecules) Prefeπed potential antagonists mclude compounds related to and vaπants of aga

Other examples of potential polypeptide antagonists mclude oligonucleotides or proteins which are closely related to the hgands. substrates, receptors, enzymes, etc . as the case may be. of the polynucleotide. e g . a fragment of the hgands. substrates, receptors, enzymes etc . or small molecules which bmd to the polynucleotide of the present mvention but do not ehcit a response, so that the activity of the polynucleotide is prevented

Certain of the polynucleotides of the mvention are biomimetics. functional mimetics of the natural aga polynucleotide These functional mimetics may be used for among other tilings, antagonizing the activity of aga pohnucleotide Functional mimetics of the polynucleotides of the urv ention mclude but are not limited

- 12 - to truncated polynucleotides For example, prefened functional mimetics mclude, a polynucleotide compnsmg the polynucleotide sequence set forth m SEQ ED NO 1 lacking 5, 10, 20, 30, 40, 50, 60, 70 or 80 5' and/or 3' nucleotide residues, including fusion promoters comprising one or more of these truncated sequences Polynucleotides of these functional mimetics may be used to dnve the expression of expression cassettes and marker genes It is prefeπed that these cassettes compπse 5' and 3' restnction sites to allow for a convenient means to gate the cassettes together when desired It is further prefeπed that these cassettes compπse gene expression signals known m the art or descnbed elsewhere herein

Thus, m another aspect, the present mvention relates to a screening kit for identifying agonists, antagomsts. hgands, receptors, substrates, enzymes, etc for a polynucleotide of the present mvention; or compounds which decrease or enhance the production of such polynucleotides , which compnses (a) a polynucleotide of the present mvention, or (b) a recombinant cell expressmg a polynucleotide of the present mvention , which polynucleotide is preferably that of SEQ ID NO 1

It will be appreciated that m any such kit, (a) or (b) may compnse a substantial component

It will be readily appreciated by the skilled artisan that a polynucleotide of the present mvention may also be used m a method for the structure-based design of an agomst, antagomst or inhibitor of the polynucleotide, by (a) determining m the first instance the three-dimensional structure of the polynucleotide, or complexes thereof, (b) deducmg the three-dimensional structure for the likely reactive sιte(s), bmdmg sιte(s) or motif(s) of an agomst, antagomst or inhibitor, (c) synthesizing candidate compounds that are predicted to bmd to or react with the deduced bmdmg sιte(s), reactive sιte(s), and/or motif(s), and (d) testmg whether the candidate compounds are mdeed agorasts, antagomsts or inhibitors

It will be further appreciated that this will normally be an iterative process, and this iterative process may be performed usmg automated and computer-controlled steps

In a further aspect, the present mvention provides methods of treating abnormal conditions such as for instance, a Disease, related to either an excess of, an under-expression of. an elevated activity of. or a decreased activity of aga polynucleotide

If the expression and/or activity of the polynucleotide is in excess, several approaches are available One approach compnses administering to an individual in need thereof an inhibitor compound (antagomst) as herem descnbed, optionally m combination with a pharmaceutically acceptable earner, in an amount effective to inhibit the function and/or expression of the pohnucleotide. such as. for example, by blocking the bmdmg of hgands. substrates, receptors, enzymes, etc . or by inhibiting a second signal, and thereby alleviating the abnormal condition

In still another approach, promoter activity can be inhibited using expression blocking techniques This blocking is preferably targeted agamst transcription An examples of a known technique of this sort involve the use of antisense sequences, either internally generated or separateh

- 13 - administered (see, for example, O'Connor, J Neurochem (1991) 56 560 in Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton. FL (1988)) Alternatively, oligonucleotides which form tπple helices with the gene can be supplied (see, for example, Lee et al, Nucleic Acids Res (1979) 6 3073, Cooney et al , Science (1988) 241 456, Dervan et al , Science (1991) 2 1 1360) These ohgomers can be administered per se or the relevant o gomers can be expressed in vivo Thus promoter polynucleotides of the mvention are useful for ascertainmg the functionality or essentiality of the target gene (gene-of-mterest) m a cell through expression blocking techmques A method compnses "knocking-out" the transcnption or expression of gene-of-mterest by expressmg an anti-sense sequence to the gene-of-mterest under the transcnptional control of the promoter polynucleotides of the mvention, particularly those contamed m SEQ ID NO 1 In another embodiment, the method compnses, m a cell, (a) disabling ("knocking-out") the gene-of-mterest, (b) remtroducmg, at the target gene locus, the gene-of-mterest now under the operational control of the inducible promoter polynucleotides of the mvention (particularly those contamed m SEQ ID NO 1), and (c) adding the mducer thereby providing information to the essentiality or functionality of the gene of interest

Hehcobacter pylori (herein "H pylori") bactena infect the stomachs of over one-third of the world's population causmg stomach cancer, ulcers, and gastπtis (International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Hehcobacter Pylori (International Agency for Research on Cancer. Lyon, France, http //www uicc ch ecp/ecp2904 htm) Moreover, the International Agency for Research on Cancer recently recograzed a cause-and-effect relationship between H pylori and gastnc adenocarcinoma. classifying the bacteπum as a Group I (definite) carcinogen Prefeπed antimicrobial compounds of the mvention (agorasts and antagorasts of aga polynucleotides) found usmg screens provided by the mvention, or known m the art. particularlv naπow-spectrum antibiotics, should be useful in the treatment of H pylori infection Such treatment should decrease the advent of H /τy/σπ -induced cancers, such as gastrointestinal carcinoma Such treatment should also prevent, inhibit and/or cure gastnc ulcers and gastntis

GLOSSARY

The following definitions are provided to facilitate understanding of certain terms used frequent!} herein

"Bodily matenal(s) means any mateπal denved from an individual or from an organism infecting, infesting or inhabiting an individual, including but not limited to, cells, tissues and waste, such as. bone, blood, serum, cerebrospmal fluid, semen, saliva, muscle, cartilage, organ tissue, skin. unne. stool or autopsy matenals

- 14 - "Dιsease(s)" means any disease caused by or related to infection by a bacteπa. including , for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid "Host cell(s)" is a cell which has been transformed or transfected. or is capable of transformation or transfection by an exogenous polynucleotide sequence

"Identity," as known m the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be. as determined bv comparing the sequences In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be. as determined by the match between strings of such sequences "Identity" can be readily calculated by known methods, mcludmg but not limited to those descnbed m

(Computational Molecular Biology, Lesk, A M . ed , Oxford University Press. New York, 1988, Bwcomputing Informatics and Genome Projects. Smith. D W . ed , Academic Press, New York, 1993, Computer Analysis of Sequence Data. Part I, Gnffin, A M . and Gnffin. H G . eds . Humana Press. New Jersey, 1994. Sequence Analysis n Molecular Biology, von Hemje, G , Academic Press, 1987, and Sequence Analysis Primer, Gπbskov, M and Devereux, J , eds , M Stockton Press, New York, 1991, and Caπllo, H , and Lipman, D . SIAM J Applied Math , 48 1073 (1988) Methods to determme identity' are designed to give the largest match between the sequences tested Moreover, methods to determme identity are codified m publicly available computer programs Computer program methods to determme identity between two sequences mclude, but are not limited to. the GCG program package (Devereux. J . et al , Nucleic Acids Research 12(1) 387 (1984)), BLASTP. BLASTN and FASTA (Altschul S F et al . J o/ec Biol 215 403-410 (1990) The BLAST X program is pubhclv available from NCBI and other sources (BLAST Manual Altschul S et al NCBI NLM NIH Bethesda. MD 20894. Altschul. S et al J Mol Biol 215 403-410 (1990) The well known Smith Waterman algorithm ma} also be used to determme identity Parameters for polypeptide sequence comparison mclude the following Algorithm Needleman and Wunsch. J Mol Biol 48 443-453 (1970)

Companson matnx BLOSSUM62 from Hentikoff and Hentikoff Proc Natl Acad Sci USA 89 10915-10919 (1992) Gap Penalty 12 Gap Length Penalty 4

A program useful with these parameters is pubhch

as the "gap" program from Genetics Computer Group Madison Wl The aforementioned parameters are the default parameters for peptide compansons (along with no penalty for end gaps)

15 Parameters for polynucleotide companson mclude the following Algonthm- Needleman and Wunsch, J Mol Biol 48 443-453 (1970) Companson matnx matches = +10, mismatch = 0 Gap Penalty 50 Gap Length Penalty 3

Available as The "gap" program from Genetics Computer Group, Madison WI These are the default parameters for nucleic acid compaπsons

A prefeπed meaning for "identity" for polynucleotides and polypeptides, as the case may be, are provided m (1) and (2) below (1) Polynucleotide embodiments further mclude an isolated polynucleotide compπsmg a polynucleotide sequence having at least a 50. 60. 70. 80. 85. 90, 95. 97 or 100% identity to the reference sequence of SEQ ID NO 1. wherem said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO 1 or may mclude up to a certain mteger number of nucleotide alterations as compared to the reference sequence, wherem said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, mcludmg transition and transversion, or insertion, and wherem said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, mterspersed either individually among the nucleotides m the reference sequence or m one or more contiguous groups withm the reference sequence, and wherem said number of nucleotide alterations is determined by multiplying the total number of nucleotides m SEQ ID NO 1 by the mteger defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides m SEQ ID NO 1. or

ⁿn ≤ ^xn - (^xn ^• y)

wherein n_n is the number of nucleotide alterations. x_n is the total number of nucleotides m SEQ ID NO 1, y is 0 50 for 50%. 0 60 for 60%. 0 70 for 70%. 0 80 for 80%. 0 85 for 85%, 0 90 for 90%, 0 95 for 95%. 0 97 for 97% or 1 00 for 100%, and • is the symbol for the multiplication operator, and wherem anv non-mteger product of x_n and y is rounded down to the nearest integer pπor to subtractmg

By way of example, a pohnucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO 1. that is it may be 100% identical, or it may include up to a certain mteger number of nucleic acid alterations as compared to the reference sequence such that the percent ldentits is less than 100% identitΛ Such alterations are selected from the group consistmg of at least one nucleic acid deletion, substitution, including transition and trans\ersιon or insertion, and wherem

- 16 - said alterations may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleic acids in the reference sequence or m one or more contiguous groups within the reference sequence. The number of nucleic acid alterations for a given percent identity is determined by multiplying the total number of nucleic acids in SEQ ID NO:l by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleic acids in SEQ ID NO:l, or:

≤ ^xn " (^χn ' )>

wherein n_n is the number of nucleic acid alterations, x_n is the total number of nucleic acids in SEQ ID NO:l, y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc.. • is the symbol for the multiplication operator, and wherem any non-integer product of x_n and y is rounded down to the nearest integer pnor to subtracting it from x_n.

"Individual(s)" means a multicellular eukaryote. including, but not limited to a metazoan, a mammal, an ovid, a bovid, a simian, a primate, and a human.

"Isolated" means altered "by the hand of man" from its natural state, i.e., if it occurs in nature, it has been changed or removed from its oπginal environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein. Moreover, a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is "isolated" even if it is still present in said orgamsm. which organism may be living or non-living

"Orgamsm(s)" means a (i) prokaryote, including but not limited to. a member of the genus Streptococcus, Staphylococcus, Bordetella, Corynebactenum, Mycobactenum, Neissena, Haemophύus, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella, Moraxella, Acmetobacter, Erysipelothrix, Branhamella, Actinobacillus, Streptobacillus, Listena, Calymmatobactenum, Brucella, Bacillus. Clostndium, Treponema, Eschenchia, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia. Chlamydia, Borrelia and Mycoplasma. and further mcludmg. but not limited to. a member of the species or group. Group A Streptococcus, Group B Streptococcus, Group C Streptococcus, Group D Streptococcus. Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes. Streptococcus agalactiae, Streptococcus faecahs. Streptococcus faecium. Streptococcus durans, Neissena gonorrheae. Neissena menmgitidis, Staphylococcus aureus. Staphylococcus epidermidis, Corynebactenum dipthenae, Gardnerella vaginahs, Mycobactenum tuberculosis, Mycobactenum bovis

- 17 - Mycobactenum ulcerans, Mycobactenum leprae. Actinomyctes israehi, Listena monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella bronchiseptica, Eschenchia coll, Sh gella dysentenae, Haemophύus influenzae, Haemophilus aegyptius, Haemophilus paramfluenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacter freundii, Proteus mirabilis, Proteus vulgans, Yersinia pestis, Kle bsiella pneumoniae, Serratia marcessens, Serratia hquefaciens, Vibno cholera, Shigella dysenteni, Shigella flexneri, Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis, Bacillus anthracs, Bacillus cereus, Clostnάium perfnngens, Clostndium tetani, Clostndium botuhnum, Treponema pallidum, Rickettsia nclettsn and Chlamydta trachomitis, (n) an archaeon. mcludmg but not limited to Archaebacter, and (in) a unicellular or filamentous eukaryote. mcludmg but not limited to. a protozoan, a fungus, a member of the genus Saccharomyces, Kluveromyces, or Candida, and a member of the species Saccharomyces cenviseae, Kluveromyces lactis, or Candida albicans

"Polynucleotide(s)" generally refers to any polvnbonucleotide or polydeoxynbonucleσtide. which may be unmodified RNA or DNA or modified RNA or DNA "Polynucleotide(s)" mclude. without limitation, smgle- and double-stranded DNA. DNA that is a mixture of smgle- and double-stranded regions or single-, double- and tnple-stranded regions, smgle- and double-stranded RNA, and RNA that is mixture of smgle- and double-stranded regions, hybπd molecules compnsmg DNA and RNA that may be single-stranded or, more typically, double-stranded, or tnple-stranded regions, or a mixture of smgle- and double-stranded regions. In addition, "polynucleotide" as used herem refers to tnple-stranded regions compnsmg RNA or DNA or both RNA and DNA The strands m such regions may be from the same molecule or from different molecules The regions may mclude all of one or more of the molecules, but more typically mvolve only a region of some of the molecules One of the molecules of a tπple-he cal region often is an oligonucleotide As used herein, the term "polynucleotιde(s)" also mcludes DNAs or RNAs as descnbed above that contain one or more modified bases Thus. DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotιde(s)" as that term is intended herem Moreover. DNAs or RNAs compnsmg unusual bases, such as mosme. or modified bases, such as tntylated bases, to name just two examples, are polynucleotides as the term is used herem It will be appreciated that a great vanety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art The term "polynucleotide(s)" as it is employed herem embraces such chemically, enzymatically or metabohcally modified forms of polynucleotides. as well as the chemical forms of DNA and RNA characteπstic of viruses and cells, mcludmg. for example, simple and complex cells "Polynucleotide(s)" also embraces short polynucleotides often refeπed to as ohgonucleotιde(s)

"Polypeptιde(s)" refers to any peptide or protem compnsmg two or more ammo acids jomed to each other b\ peptide bonds or modified peptide bonds "Polypeptιde(s)" refers to both short chains, commonly refeπed to as peptides. ohgopeptides and ohgomers and to longer chains generally refeπed to as proteins Polypeptides may contain ammo acids other than the 20 gene encoded ammo acids "Polypeptide(s)" mclude those modified either by natural processes, such as processing and other post-translatiσnal modifications, but also by chemical modification techniques Such modifications are well descnbed in basic texts and m more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill m the art It will be appreciated that the same type of modification may be present m the same or varying degree at several sites m a given polypeptide Also, a given polypeptide may contain many types of modifications Modifications can occur anywhere m a polypeptide, mcludmg the peptide backbone, the ammo acid side-chains, and the ammo or carboxyl termini Modifications mclude, for example, acetylatiαn, acylation. ADP-nbosylation. amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide deπvative. covalent attachment of a hpid or hpid denvative, covalent attachment of phosphotidyhnositol, cross-linking, cychzation. disulfide bond formation, demethylation. formation of covalent cross-links, formation of cysterne, formation of pyroglutamate. formylation. gamma-carboxylation. GPI anchor formation, hydroxylation, lodination, methylation. myπstoylation. oxidation, proteolytic processing, phosphorylation. prenylation, racemization, glycosylation, hpid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP- ribosylation. selenoylation, sulfation, transfer-RNA mediated addition of ammo acids to proteins, such as arginylation. and ubiquitination See. for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T E Creighton, W H Freeman and Company, New York (1993) and Wold, F , Posttranslational Protem Modifications Perspectives and Prospects, pgs 1-12 m POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B C Johnson, Ed , Academic Press, New York (1983), Softer et al . Meth Enzymol 182626-646 (1990) and Rattan et al , Protem Synthesis Posttranslational Modifications and Aging. Ann N Y Acad Sci 663 48-62 (1992) Polypeptides may be branched or cyclic. with or without branching Cychc. branched and branched circular polypeptides may result from posttranslational natural processes and may be made by entirely synthetic methods, as well "Recombinant expression system(s)" and "recombinant replication system(s)"refers to expression systems or portions thereof or polynucleotides of the mvention introduced oi transformed mto a host cell or host cell lvsate for the production of the polynucleotides and polypeptides of the mvention

'Nanant(s)" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference pohnucleotide or polypeptide respectively, but retams essential properties A typical vanant of a polynucleotide differs m nucleotide sequence from another, reference pohnucleotide Changes in the nucleotide sequence of the variant mav or may not alter the ammo acid sequence of a polypeptide encoded the reference pohnucleotide Nucleotide changes ma} result m ammo acid substitutions, additions, deletions, fusion protems and truncations m the polypeptide encoded by the reference sequence, as discussed below A typical variant of a pohpeptide differs m ammo acid sequence from

- 19 - another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the vanant are closely similar overall and. m many regions, identical A vanant and reference polypeptide may differ m ammo acid sequence by one or more substitutions, additions, deletions m any combmation A substituted or inserted a mo acid residue may or may not be one encoded by the genetic code The present mvention also mcludes mclude vanants of each of the polypeptides of the rnventioα that is polypeptides that vary from the referents by conservative ammo acid substitutions, whereby a residue is substituted by another with like characteπstics Typical such substitutions are among Ala, Val, Leu and lie. among Ser and Thr, among the acidic residues Asp and Glu, among Asn and Gin, and among the basic residues Lys and Arg, or aromatic residues Phe and Tyr Particularly prefeπed are vanants m which several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acids are substituted, deleted, or added in any combination A vanant of a polynucleotide or polypeptide may be a naturally occurring such as an allehc vanant, or it may be a vanant that is not known to occur naturally Non-naturally occurring vanants of polynucleotides and polypeptides may be made by mutagenesis techmques, by direct synthesis, and by other recombinant methods known to skilled artisans

EXAMPLES The examples below are earned out usmg standard techniques, which are well known and routine to those of skill m the art except where otherwise descnbed in detail The examples are illustrative, but do not limit the mvention Example 1 Strain selection, Library Production and Sequencing

The polynucleotide havmg a DNA sequence given in Table 1 [SEQ ID NO 1] was obtained from a hbrarv of clones of chromosomal DNA of Streptococcus pneumoniae m E coll The sequencmg data from two or more clones containing overlappmg Streptococcus pneumoniae DNAs was used to construct the contiguous DNA sequence m SEQ ID NO 1 Libraries may be prepared by routine methods, for example Methods 1 and 2 below

Total cellular DNA is isolated from Streptococcus pneumoniae 0100993 accordmg to standard procedures and size-fractionated by either of two methods Method 1 Total cellular DNA is mechanically sheared by passage through a needle m order to size- fractionate according to standard procedures DNA fragments of up to 1 lkbp in size are rendered blunt by treatment with exonuclease and DNA polymerase. and EcoRI linkers added Fragments are hgated into the vector Lambda ZapII that has been cut with EcoRI. the library packaged by standard

20 procedures and E coli infected with the packaged library The library is amplified by standard procedures

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combination of restnction enzymes appropnate to generate a senes of fragments for cloning mto library vectors (e g , Rsal, Pall, Alul, Bshl235I), and such fragments are size-fractionated accordmg to standard procedures EcoRI linkers are hgated to the DNA and the fragments then hgated mto the vector Lambda ZapII that have been cut with EcoRI. the library packaged by standard procedures, and E cob infected with the packaged hbrary The library is amplified by standard procedures Example 2 Gene Essentiality Assay

A promoter replacement cassette is generated usmg PCR technology The cassette consists of a pair of 500bp chromosomal DNA fragments flanking an erythromycm resistance gene divergently transcnbed with respect to the rnducible/repressible promoter The chromosomal DNA sequences are the 500bp precedmg and following the promoter region of the gene whose essentiality is going to be tested

The promoter replacement cassette is mtroduced mto S pneumoniae R6 by transformation Competent cells are prepared accordmg to published protocols DNA is introduced mto the cells by incubation of μg quantities of promoter replacement cassette with 10" cells at 30°C for 30 minutes The cells are transfeπed to 37°C for 90 minutes to allow expression of the erythromycm resistance gene Cells are plated m agar containmg 1 μg erythromycm per ml and the appropnate concentration of the mducer molecule Following incubation at 37°C for 36 hours, colonies are picked and grown overnight m Todd-Hewitt broth supplemented with 0 5% veast extract and the appropiate amount of mducer Gene essentiality is tested by decreasmg the amount of mducer and/or increasing the amount of repressor and monitoring cell viability If the promoter replacement has occuπed upstream of an essential gene, viability of the bacteria will be absolutely dependent on the presence of mducer or the absence of repressor Example 3 Identification of promoter sequences

Blast searches are run usmg protems involved in sugar metabolism m E coh and B subtihs against SmithKlme Beecham's propπeton S pneumoniae genome sequence database This allows the identification of genes belonging to sugar operons The location of groups of sugar metabolism genes is used to identify mtergenic (noncoding) regions hich are considered to contam promoters In addition, putative promoter sequences are identified b\ homology with a consensus sequence for bacterial promoters Such a sequence consists of a -10 region (TATAAT). a 17bp/19bp spacer and a -35 region (TTGACA) S pneumoniae promoters haλ e sometimes a characteπstic extended -10

- 21 - region (TNTGNTATAAT) [SEQ ID NO:2] and lack a -35 region. In Seq ID Nol a -10 ("TATAAT⁸⁶) and -35 (⁵⁶TAGAAA⁶¹) regions have been identified. This promoter controls the multiple sugar metabolism operon. The first gene in the operon (aga) encodes an alpha-galactosidase. Example 4 aga Characterization Expression of the aga promoter is regulated by sugars a) Preparation of pre-inoculum and growth conditions

S. pneumoniae R6 is grown statically at 37oC in the semi-defined AGCH media (Lacks S. (1968).

Genetics 60: 4685-706.), supplemented with 0.2% yeast extract (YE) and different test sugars added as the sole sugar source. In a preliminary' experiment, S. pneumoniae was shown to grow to an ODβso of 0.6 at approximately the same growth rate, when sucrose, glucose, trehalose or laαose were added to the medium at the point of inoculation as the sugar source at 1% (w/v) final concentration. Where S. pneumoniae was unable to utilize a sugar as the single carbon source (fucose and galactose). or showed a significantly reduced growth rate (rafϊinose), glucose was supplemented to the media at a concentration of 0.2% (w/v) to maintain growth

The sugar growth conditions tested are 1% glucose, 1% sucrose, 1% lactose, 1% trehalose, 1% fructose, 1% mannose, 0.2% glucose, 0.2% glucose + 1% fucose, 0.2% glucose+1% galactose, 0.2% glucose

+ 0.5% raffinose and 1% rafϊinose.

To adapt S. pneumoniae to growth in the test sugar(s), 1 OOμl of a glycerol stock of S. pneumoniae R6 is diluted in 4ml of AGCH +YE media supplemented with the test sugar(s) and grown to exponential phase until OD₆₅₀ reached 0.29 -0.31 Cultures (0.1ml) are rediluted in 4ml fresh AGCH +YE containing the same sugar(s) and re-mcubated at 37°C to exponential phase. This is repeated for a third time. Glycerol is added to 10% (v/v) final concentration, and cells are frozen in dry ice/ ethanol bath and stored at -70oC as stocks of "synchronised " S. pneumoniae cells adapted to different sugar(s ) as carbon source(s)

To isolate RNA. S. pneumoniae cells are grown under appropnate sugar conditions to an OD₆₅₀ of 0.6. b) Isolation of Streptococcus pneumoniae R6 RNA.

RNA is extracted using the FastR A Kit (Biol 01) according to the manufacturers instuctions with slight modifications. Briefly. 5ml of S. pneumoniae cells are centrifuged at 6000 rpm and 4oC. the supematanrt is removed and the pellet is resuspended m 200μl of AGCH and added to a FastPrep tube containing lysing matπx and 1 ml of extraction reagents (FastRNA) The tubes are shaken m a reciprocating shaker (FastPrep FP120. BIO101) at 6000 rpm for 45 sec The crude RNA preparation is extracted with chloroform/isoamy] alcohol (24.1). and precipitated with DEPC-treated lsopropanol Precipitation Solution (BIO 101) RNA preparations are stored m this isopropanol solution at -80°C if necessary The RNA is pelleted (12.000g for 10 mm.), washed with 75% ethanol (v/v in DEPC-treated

22 - water), air-dπed for 5-10 mm, and resuspended m 0 1 ml of DEPC-treated water, followed by 5-10 minutes at 55 oC Finally, after at least 1 minute on ice, 200 umts of Rnasin (Promega) are added

RNA preparations are stored at -80 oC for up to one month For longer term storage the RNA precipitate can be stored at the wash stage of the protocol in 75% ethanol for at least one year at -20 oC

Quality of the RNA isolated is assessed by running samples on 1% agarose gels 1 x TBE gels stained with ethidium bromide are used to visualise total RNA yields c) The removal of DNA from Streptococcus pneumoniae-dsnved RNA

DNA is removed from 50 micrograrn samples of RNA by a 30 mmute treatment at 37°C with 20 umts of RNAase-free DNAasel (GenHunter) m the buffer supplied m a final volume of 57 microhters

The DNAase is mactivated and removed by treatment with TRIzol LS Reagent (Gibco BRL, Life Technologies) accordmg to the manufacturers protocol

DNAase treated RNA is resuspended m 100 microhtres of DEPC treated water with the addition of Rnasin as descnbed before d) The preparation of cDNA from RNA samples

5 microgram samples of DNAase-treated RNA are reverse transcnbed usmg a SuperScπpt

Preamplification System for First Strand cDNA Synthesis kit (Gibco BRL, Life Technologies) accordmg to the manufacturers instructions 250 nanogram of random hexamers is used to prime each reaction Controls without the addition of SuperScnptll reverse transcnptase (RT) are also run Both

+/-RT samples are treated with RnaseH cDNA samples were stored at -20oC e) The use of PCR and SybrGreen dye accumulation to quantitate bactenal mRNA

Specific sequence quantification occurs by amplification of target sequences m the PE Applied Biosystems 7700 Sequence Detection System with template specific primers and m the presence of Sybr Green dye Amplification with annealmg temperatures that only allow specific product formation is monitored by measuring the signal generated by accumulation of Sybr Green dye with an attached CCD camera Standardization to chromosomal DNA allows coπelation between signal generated and product accumulated Relative quantitation is normalized to a housekeeping gene and a calibrator sample (umnduced control) PCR reactions are set up using the PE Applied Biosvstem S\brGreen PCR Core Reagent Kit according to the instructions supplied such that each reaction contains 5 microhters 1 OX SybrGreen buffer. 7 microhters 25 mM MgCl . 5 microhters 300 nM forward primer, 5 microhters reverse pnmer. 1 microhter each 10 mM dATP. 10 mM dCTP. 10 mM dGTP and 20 mM dUTP, 13 25

- 22 microhters distilled water, 0 5 microhters AmpErase UNG, and 0 25 microhters AmphTaq DNA polymerase to give a total volume of 45 microhters

Amplification proceeds under the following thermal cyclmg conditions 50°C hold for 2 minutes, 95°C hold for 10 minutes, 40 cycles of 95°C for 15 seconds and 60°C for 1 minute, followed by a 25 °C hold until sample is retneved Detection occurs real-time Data is collected at the end of the reaction

RT/PCR controls mav mclude no reverse transcnptase reactions to veπfy the absence of contaminating genomic DNA

Primers used for Example 4 are as follows GGGCTTACCTTCTCCACATAG [SEQ ID NO 3] fwd pπmer

CCAATAGCCACCCGAACTT [SEQ ID NO 4] rev pπmer

Sybr Green quantitation of accumulating PCR product and the uses of such a technique have reported

(Morπson T B, Weis J J and Wittwer C T 1998 Quantification of low-copy transcnpts by continuous SYBR green I momtoπng dunng amplification BioTechmques 24 954-962 Wittwer C T , Herrmann M G , Moss A A , Rasmussen R P 1997 Contmuous fluorescence monitoπng of rapid cycle DNA amplification Biotechmques 22 130-131, 134-1388)

Such an experiment showed that expression of the aga promoter is mduced by 1% raffinose + 0 2% glucose and is repressed by 0 2% glucose Example 5. Characterization of the aga promoter using luxAB as a reporter system.

A cassette consistmg of

• a promoterless luxAB reporter gene from Vibrio harvevi encoding a luciferase

• transcπptional terminators from 5 pneumoniae πbosomal RNA operons positioned at both sides of the luxAB gene • an erythromycm resistance marker and

• sequences from the S pneumoniae ami operon flanking all the above has been constructed m pBlueScnpt to analyse the effects of different sugars on the expression level of promoters mvolved in sugar metabolism m S pneumoniae Different promoter sequences are cloned upstream of the promoterless luxAB gene usmg the unique Spel BamHl and or Sma\ restnction sites The cassette containing the promoter-reporter fusion is then transformed mto S pneumoniae R6 usmg published protocols DNA is mtroduced into the competent cells by incubation of μg quantities of cassette DNA with 10^ cells at 30°C for 30 minutes The cells are then transferred to 37°C for 90 minutes to allow expression of the ervthromvcm resistance gene Cells are plated in agar containing I μg ervthrom\cm per ml Following incubation at 37°C for 36 hours

- 24 - colonies are picked and grown overnight m Todd-Hewitt broth supplemented with 0 5% yeast extract Genomic DNA is isolated, and mtegration of the promoter-/«x45 fusion confirmed by diagnostic PCR and Southern analysis

Inducibility and repressibihty of the promoter sequence are measured by momtoπng luciferase expression under different conditions Smce this enzyme catalyses the oxidation of a long- chain aliphatic aldehyde m a hght-emiting reaction, promoter activity may be determined by the luminescence of the cell following the addition of the substrate, n-decyl aldehyde

Transformants are grown m AGCH supplemented with 0 2% yeast extract and 0 3% sucrose, or the mducer sugar, at 37°C in a CO₂ mcubator until OD₆₅₀ reaches 0 3 Cultures (0 1ml) are rediluted in 4ml fresh AGCH +YE containmg the same sugar and re-mcubated at 37°C to exponential phase This is repeated for a third time Glycerol is added to 10% (v/v) final concentration, and cells are frozen in dry ice/ ethanol bath and stored at -70oC as stocks of "synchronised " S pneumoniae cells adapted to different sugars as carbon source Glycerol stocks (0 1 ml) are inoculated m 4 ml of media containmg the mducer or repressor sugar at the pomt of lnnoculation Cultures are then grown under the same conditions and samples are taken at different stages of growth to determme luciferase expression

Such an experiment confirmed that expression of the aga promoter is mduced by 1% raffinose + 0 2% glucose and is repressed by 0 2% glucose

All publications and references, mcludmg but not limited to patents and patent applications. cited m this specification are herem incorporated by reference m their entirety as if each mdividual publication or reference were specifically and individually mdicated to be incorporated by reference herem as bemg fully set forth Any patent application to which this application claims pπontv is also incorporated by reference herem m its entirety m the manner described above for publications and references

25 -