WO2000023611A1 - FabG - Google Patents

Abstract

The invention provides fabG polypeptides and polynucleotides encoding fabG polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing fabG polypeptides to screen for antibacterial compounds.

Description

FabG

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their vanants, agonists and antagonists, and their uses In particular, the invention relates to polynucleotides and polypeptides of the fabG (3-ketoacyl-ACP reductase) family, as well as their v.anants, hereinafter referred to as "fabG," "fabG polynucleotιde(s)," and "fabG polypeptιde(s)" as the case may be

BACKGROUND OF THE INVENTION

The frequency of Pseudomonas aerugmosa infections has risen 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 Pseudomonas aerugmosa strains which 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 "fiinctional 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 heavily 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 polynucleotides sequences and their related polypeptides, as targets for drug discovery

Clearly, there exists a need for polynucleotides and polypeptides, such as the fabG embodiments of the invention, that have a present benefit of, among other things, being useful to screen compounds for antimicrobial acUvity Such factors are also useful to determine their role in pathogenesis of infection, dysfunction and disease There is also a need for identification and characterization of such factors and then- antagonists and agonists to find ways to prevent, ameliorate or correct such infection, dysfunction and disease SUMMARY OF THE INVENTION

The present invention relates to fabG, in particular fabG polypeptides and fabG polynucleotides, recombinant matenals and methods for their production In another aspect, the invention relates to methods for using such polypeptides and polynucleotides, including treatment of microbial diseases, amongst others In a further aspect, the invention relates to methods for identifying agonists and antagonists using the materials provided by the invention, and for treatmg microbial infections and conditions associated with such infections with the identified agonist or antagonist compounds In a still further aspect, the invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting fabG expression or activity

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

DESCRIPTION OF THE INVENTION

The invention relates to fabG polypeptides and polynucleotides as described in greater detail below In particular the invention relates to polypeptides and polynucleotides of a fabG of Pseudomonas aerugxnosa, which is related by amino acid sequence homology to E coli FabG polypeptide The invention relates especially to fabG having the nucleotide and amino acid sequences set out in Table 1 as SEQ ID NO 1 and SEQ ID NO 2 respectively Note that sequences recited in 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 m general including πbopolynucleotides

TABLE 1 fabG Polynucleotide and Polypeptide Sequences

(A) Pseudomonas aeruginosa fabG polynucleotide sequence [SEQ ID NO 1]

5 ' -ATGAGTCTGCAAGGTAAGGTCGCATTGGTAACCGGCGCCAGCCGTGGCATCGGCCAGGCG ATTGCGCTGGAACTGGGGCGCCTGGGTGCCGTGGTCATCGGCACCGCGACCAGCGCGTCC GGCGCCGAGAAGATCGCCGAAACCCTCAAGGCCAATGGCGTCGAGGGTGCGGGCCTGGTC CTGGACGTTTCCAGCGACGAATCCGTAGCCGCGACCCTGGAGCACATCCAGCAGCATCTC GGCCAACCGCTGATCGTGGTCAATAACGCCGGCATCACCCGCGATAATCTGCTGGTGCGC ATGAAAGACGACGAGTGGTTCGATGTGGTCAACACCAACCTGAACAGTCTCTACCGTCTG TCGAAAGCCGTTCTGCGCGGTATGACCAAGGCCCGCTGGGGGCGCATCATCAACATCGGT TCCGTGGTCGGCGCCATGGGCAATGCCGGGCAAACCAACTATGCCGCGGCGAAGGCCGGC CTGGAGGGCTTCACCCGTGCGCTGGCCCGGGAAGTCGGTTCGCGTGCCATTACCGTGAAT GCGGTGGCGCCGGGCTTCATCGACACCGACATGACCCGCGAGCTGCCGGAAGCCCAGCGC GAAGCGCTGCTGGGCCAGATTCCGCTGGGTCGCCTGGGGCAGGCGGAAGAGATCGCCAAG GTGGTCGGCTTCCTCGCTTCGGACGGCGCAGCCTATGTGACCGGGGCCACCGTGCCGGTC AATGGTGGGATGTACATGAGCTGA-3" (B) Pseudomonas aerugino a fabG polypeptide sequence deduced from a polynucleotide sequence in this table [SEQ ID NO 2]

NH -MSLQGKVALVTGASRGIGQAIALELGRLGAVVIGTATSASGAEKIAETLKANGVEGAGLV LDVSSDESVAATLEHIQQHLGQPLIVVNNAGITRDNLLVRMKDDEWFDVVNTNLNSLYRL SKAVLRGMTKARWGRIINIGSVVGAMGNAGQTNYAAAKAGLEGFTRALAREVGSRAITVN AVAPGFIDTDMTRELPEAQREALLGQIPLGRLGQAEEIAKVVGFLASDGAAYVTGATVPV NGGMYMS-COOH

Polypeptides

FabG polypeptide of the invention is substantially phylogenetically related to other proteins of the fabG (3-ketoacyl-ACP reductase) family

In one aspect of the invention there are provided polypeptides of Pseudomonas aerugtnosa referred to herein as "fabG" and "fabG polypeptides" as well as biologically, diagnosticaUy, prophvlactically clinically or therapeutically useful variants thereof, and compositions compnsing the same Among the particularly preferred embodiments of the invention are variants of fabG polypeptide encoded by naturally occurring alleles of the fabG gene The present invention further provides for an isolated polypeptide which (a) comprises or consists of an amino acid sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferabl> at least 95% identity, most preferably at least 97-99% or exact identity to that of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (b) a polypeptide encoded bv an isolated polynucleotide compπsmg 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, (c) a polypeptide encoded by an isolated poh nucleotide comprising or consistmg of a polynucleotide sequence encoding a polypeptide which has at least 70% identity preferably at least 80%) identity, more preferabh at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% or exact identity, to the amino acid sequence of SEQ ID NO 2, over the entire length of SEQ ID NO 2

The polypeptides of the invention include a polypeptide of Table 1 [SEQ ID NO 2] (in particular the mature polypeptide) as well as polypeptides and fragments, particularly those which have the biological actn its of fabG and also those which have at least 70% identity to a polypeptide of Table 1 [SEQ ID NO 1 ]or the relev. nt portion preferabh at least 80% identity to a polypeptide of Table 1 [SEQ ID NO 2and more preferably at least 90% identity to a polypeptide of Table 1 [SEQ ID NO 2] and still more preferably at least 95% identity to a polypeptide of Table 1 [SEQ ID NO 2] and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids The invention also includes a polypeptide consisting of or compnsing a polypeptide of the formula

X-(Rι )_m-(R₂)-(R₃)_n-Y wherein, at the amino terminus, X is hydrogen, a metal or any other moiety descnbed herein for modified polypeptides .and at the carboxyl terminus, Y is hydrogen, a metal or any other moiety descnbed herein for modified polypeptides, R_j .and R3 are any amino acid residue or modified amino acid residue, m is an integer between 1 and 1000 or zero, n is an integer between 1 and 1000 or zero, and R₂ is an amino acid sequence of the invention, particularly an amino acid sequence selected from Table 1 or modified forms thereof In the formula above, R₂ is oriented so that its amino terminal amino acid residue is at the left, covalently bound to Rj and its carboxy terminal amino acid residue is at the right, covalently bound to R3 Am stretch of amino acid residues denoted by either Ri or R3, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer Other preferred embodiments of the invention are provided where m is an integer between 1 and 50, 100 or 500, and n is an integer between 1 and 50. 100, or 500

It is most preferred that a polypeptide of the invention is derived from Pseudomonas aerugtnosa, however, it may preferably be obtained from other organisms of the same taxonomic genus A polypeptide of the invention may also be obtained, for example, from organisms of the same taxonomic family or order A fragment is a vanant polypeptide having an amino acid sequence that is entirely the same as part but not all of any amino acid sequence of any polypeptide of the invention As with fabG polypeptides, fragments may be "free-standing," or compnsed within a larger polypeptide of which they form a part or region, most preferably as a single continuous region in a single larger polypeptide Preferred fragments include, for example, truncation polypeptides having a portion of an amino acid sequence of Table 1 [SEQ ID NO 2], or of vanants thereof, such as a continuous senes of residues that includes an amino- and/or carboxyl-termirial amino acid sequence Degradation forms of the polypeptides of the invention produced by or in a host cell, particularly a Pseudomonas aerugtnosa, are also preferred Further preferred are fragments characterized by structural or functional attributes such as fragments that compπse alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region and high antigenic index regions Further preferred fragments include an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids from the amino acid sequence of SEQ ID NO:2, or an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated or deleted from the amino acid sequence of SEQ ID NO:2.

Also preferred are biologically active fragments which are those fragments that mediate activities_^ of fabG, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those fragments that are antigenic or immunogenic in an animal, especially in a human. Particularly preferred are fragments comprising receptors or domains of enzymes that confer a function essential for viabihty of Pseudomonas aeruginosa or the ability to initiate, or maintain cause Disease in an individual, particularly a human.

Fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these variants may be employed as intermediates for producing the full-length polypeptides of the invention. In addition to the standard single and triple letter representations for amino acids, the term

"X" or "Xaa" may also be used in describing certain polypeptides of the invention. "X" and "Xaa" mean that any of the twenty naturally occurring amino acids may appear at such a designated position in the polypeptide sequence.

Polynucleotides It is an object of the invention to provide polynucleotides that encode fabG polypeptides, particularly polynucleotides that encode the polypeptide herein designated fabG.

In a particularly preferred embodiment of the invention the polynucleotide comprises a region encoding fabG polypeptides comprising a sequence set out in Table 1 [SEQ ID NO:l] which includes a full length gene, or a variant thereof. The AppUcants beheve that this full length gene is essential to the growth and/or survival of an organism which possesses it, such as Pseudomonas aeruginosa.

As a further aspect of the invention there are provided isolated nucleic acid molecules encoding and/or expressing fabG polypeptides and polynucleotides, particularly Pseudomonas aeruginosa fabG polypeptides and polynucleotides. including, for example, unprocessed RNAs, ribozyme RNAs, mRNAs, cDNAs. genomic DNAs. B- and Z-DNAs. Further embodiments of the invention include biologically. diagnosticaUy. prophylactically, clinically or therapeutically useful polynucleotides and polypeptides, and variants thereof, and compositions comprising the same. Another aspect of the invention relates to isolated polynucleotides, including at least one full length gene, that encodes a fabG polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NO 2] and polynucleotides closely related thereto and vaπants thereof

In another particularly preferred embodiment of the invention there is a fabG polypeptide from Pseudomonas aerugtnosa compπsing or consisting of an amino acid sequence of Table 1 [SEQ ID NO 2] or a variant thereof

Using the information provided herein, such as a polynucleotide sequence set out in Table 1 [SEQ ID NO 1 ] a polynucleotide of the invention encoding fabG polypeptide may be obtained usmg standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bacteπa using Pseudomonas aeruginosa strain 4 cells as starting matenal, followed by obtaining a full length clone For example, to obtain a polynucleotide sequence of the invention, such as a polynucleotide sequence given in Table 1 [SEQ ID NO 1], typically a library of clones of chromosomal DNA of Pseudomonas aeruginosa strain 4 m E coli or some other suitable host is probed with a radiolabeled o gonucleotide, preferably a 17-mer or longer, deπved from a partial sequence Clones carrying DNA identical to that of the probe can then be distinguished usmg stπngent hybridization conditions By sequencmg the individual clones thus identified by hybndization with sequencing primers designed from the oπgmal polypeptide or polynucleotide sequence it is then possible to extend the polynucleotide sequence m both directions to determine a 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 Maniatis, T , Fntsch, E F and Sambrook et al MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed , Cold Spring Harbor Laboratory Press, Cold Spnng Harbor, New York (1989) (see m particular Screening By Hybridization 1 90 and Sequencmg Denatured Double-Stranded DNA Templates 13 70) Direct genomic DNA sequencing may also be performed to obtain a full length gene sequence Illustrative of the invention, each polynucleotide set out m Table 1 [SEQ ID NO 1] was discovered m a DNA hbrary deπved from Pseudomonas aeruginosa strain 4

Moreover each DNA sequence set out in Table 1 [SEQ ID NO 1] contains an open reading frame encoding a protein having about the number of amino acid residues set forth in Table 1 [SEQ ID NO 2] a deduced molecular weight that can be calculated usmg amino acid residue molecular weight values well known to those skilled in the art The polynucleotide of SEQ ID NO 1. between nucleotide number 1 and the stop codon which begins at nucleotide number 742 of SEQ ED NO 1 encodes the polypeptide of SEQ ID NO 2 In a further aspect, the present mvention provides for an isolated polynucleotide comprising or consisting of (a) 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 preferabh at least 97-99% or exact identity to SEQ ID NO. l over the entire length of SEQ ID NO:l; (b) a polynucleotide sequence encoding a polypeptide 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 100% exact, to the amino acid .sequence of SEQ ID NO:2, over the entire length of SEQ ID NO.2

A polynucleotide encoding a polypeptide of the present inventioa including homologs and orthologs from species other than Pseudomonas aeruginosa, may be obtained by a process which comprises the steps of screening an appropπate library under stπngent hybndization conditions with a labeled or detectable probe consisting of or compnsmg the sequence of SEQ ED NO: 1 or a fragment thereof; and isolating a full- length gene and/or genomic clones containing said polynucleotide sequence

The mvention provides a polynucleotide sequence identical over its entire length to a coding sequence (open readmg frame) in Table 1 [SEQ ID NO:l] Also provided by the invention is a coding sequence for a mature polypeptide or a fragment thereof, by itself as well as a coding sequence for a mature polypeptide or a fragment in reading frame with another coding sequence, such as a sequence encoding a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence The polynucleotide of the invention may also contain at least one non-codmg sequence, including for example, but not limited to at least one non-codmg 5^" and 3^' sequence, such as the transcribed but non-translated sequences, termination signals (such as rho-dependent and rho-mdependent termination signals), ribosome binding sites, Kozak sequences, sequences that stabilize mRNA, introns. and polyadenylation signals The polynucleotide sequence may also compnse additional coding sequence encoding additional ammo acids For example, a marker sequence that facilitates purification of the fused polypeptide can be encoded In certain embodiments of the mvention, the marker sequence is a hexa-histidine peptide, as provided m the pQE vector (Qiagen, Inc.) and described in Gentz et al. , Proc. Natl. Acad. Sci., USA 86: 821-824 (1989), or an HA peptide tag (Wilson et al . Cell 37 767 (1984). both of which may be useful m purifying polypeptide sequence fused to them. Polynucleotides of the mvention also mclude. but are not limited to, polynucleotides comprising a structural gene and its naturally associated sequences that control gene expression A prefened embodiment of the mvention is a polynucleotide of consisting of or comprising nucleotide 1 to the nucleotide immediately upstream of or including nucleotide 742 set forth m SEQ ED NO 1 of Table 1, both of which encode the fabG polypeptide The mvention also mcludes a polynucleotide consisting of or compπsmg a polynucleotide of the formula

X-(Rι )_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 mteger between 1 and 3000 or zero , n is an mteger between 1 and 3000 or zero, and R 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, R is oriented so that its 5' end nucleic acid residue is at the left, bound to Ri and its 3' end nucleic acid residue is at the right, bound to R3 Any stretch of nucleic acid residues denoted by either Ri and/or R where m and/or n is greater than 1 may be either a heteropolymer or a homopolymer, preferably a heteropolymer Where, in 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 preferred embodiment m and/or n is an mteger between 1 and 1000. Other preferred 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 preferred that a polynucleotide of the mvention is denved from Pseudomonas aeruginosa however, it may preferably be obtained from other organisms of the s.ame taxonomic genus A pohnucleotide of the mvention may also be obtained, for example, from organisms of the same taxonomic famih or order

The term "polynucleotide encodmg a polypeptide" as used herem encompasses polynucleotides that mclude a sequence encodmg a polypeptide of the mvention, particularly a bactenal polypeptide and more particularly a polypeptide of the Pseudomonas aeruginosa fabG having an ammo acid sequence set out in Table 1 [SEQ ID NO 2] The term also encompasses polynucleotides that mclude a smgle continuous region or discontinuous regions encodmg the polypeptide (for example, polynucleotides interrupted by integrated phage an tegrated insertion sequence, an mtegrated vector sequence, an integrated transposon sequence or due to RNA editmg or genomic DNA reorgamzation) together with additional regions, that also ma\ contain codmg and/or non-codmg sequences

The mvention further relates to vanants of the polynucleotides descnbed herem that encode vanants of a polypeptide havmg a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] Fragments of a polynucleotides of the invention may be used, for example, to synthesize full-length polynucleotides of the invention.

Further particularly prefened embodiments are polynucleotides encoding fabG variants, that have the ammo acid sequence of fabG polypeptide of Table 1 [SEQ ID NO:2] in which several, a few, 5 to 10, 1 to 5. 1 to 3, 2, 1 or no amino acid residues are substituted, modified, deleted and/or added, in any combination. Especially prefened among these are silent substitutions, additions and deletions, that do not alter the properties and activities of fabG polypeptide.

Further prefened embodiments of the invention are polynucleotides that are at least 70% identical over their entire length to a polynucleotide encoding fabG polypeptide having an amino acid sequence set out in Table 1 [SEQ ID NO:2], and polynucleotides that are complementary to such polynucleotides.

Alternatively, most highly prefened are polynucleotides that comprise a region that is at least 80% identical over its entire length to a polynucleotide encoding fabG polypeptide and polynucleotides complementary thereto. In this regard, polynucleotides at least 90% identical over their entire length to the same are particularly prefened, and among these particularly prefened polynucleotides, those with at least 95% are especially prefened. Furthermore, those with at least 97% are highly prefened among those with at least

95%, and among these those with at least 98% and at least 99% are particularly highly prefened, with at least 99% bemg the more prefened.

Prefened embodiments are polynucleotides encoding polypeptides that retain substantially the same biological function or activity as the mature polypeptide encoded by a DNA of Table 1 [SEQ ED NO:l]. In accordance with certain prefened embodiments of this invention there are provided polynucleotides that hybridize, particularly under stringent conditions, to fabG polynucleotide sequences, such as those polynucleotides in Table 1.

The mvention further relates to polynucleotides that hybridize to the polynucleotide sequences provided herein. In this regard, the invention especially relates to polynucleotides that hybridize under strmgent conditions to the polynucleotides described herein. As herein used, the terms "stringent conditions" and "strmgent hybridization conditions" mean hybridization occurring only if there is at least 95% and preferably at least 97% identity between the sequences. A specific example of stringent hybridization conditions is overnight incubation at 42°C in a solution comprising: 50% formamide, 5x SSC

( 150mM NaCl, 15mM tπsodium 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 hybridization support in 0. lx SSC at about 65°C. Hybridization and wash conditions are well known and exemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N Y , (1989), particularly Chapter 11 therein Solution hybπdization may also be used with the polynucleotide sequences provided by the mvention

The invention also provides a polynucleotide consisting of or compnsing a polynucleotide sequence obtained by screening an appropnate library containing the complete gene for a poh nucleotide sequence set forth SEQ ID NO 1 under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth m SEQ ID NO 1 or a fragment thereof, and isolating said polynucleotide sequence Fragments useful for obtaining such a polvnucleotide include, for example, probes and pπmers fully descnbed elsewhere herem

As discussed elsewhere herem regarding polynucleotide assays of the mvention, for instance, the polynucleotides of the mvention, may be used as a hybπdization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encodmg fabG and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity to the fabG gene Such probes generally will compnse at least 15 nucleotide residues or base pairs Preferably, such probes will have at least 30 nucleotide residues or base pa rs and may have at least 50 nucleotide residues or base parrs Particularly prefened probes will have at least 20 nucleotide residues or base pairs and will have lee than 30 nucleotide residues or base pairs

A coding region of a fabG gene may be isolated by screening usmg a DNA sequence provided in Table 1 [SEQ ID NO I j to synthesize an ohgonucleotide probe A labeled oligonucleotide having a sequence complementary to that of a gene of the mvention is then used to screen a library of cDNA, genomic DNA or mRNA to deteimrne which members of the hbrary the probe hy bndrzes to

There are several methods available and well known to those skilled m the art to obtain full- length DNAs, or extend short DNAs for example those based on the method of Rapid Amplification of cDNA ends (RACE) (see for example Frohman, et al , PNAS USA 85 8998-9002, 1988) Recent modifications of the technique exemplified by the Marathon™ technology (Clontech Laboratoπes Inc ) for example, have significantly simplified the search for longer cDNAs In the Marathon™ technology, cDNAs have been prepared from mRNA extracted from a chosen tissue and an 'adaptor' sequence hgated onto each end Nucleic acid amplification (PCR) is then earned out to amplify the "missing" 5' end of the DNA using a combination of gene specific and adaptor specific ohgonucleotide primers The PCR reaction is then repeated using "nested" primers that is, primers designed to anneal w ithin the amplified product (tvpicalh an adaptor specific primer that anneals further 3' m the adaptor sequence and a gene specific primer that anneals further 5' in the selected gene sequence) The products of this reaction can then be analyzed by DNA sequencmg and a full-length DNA constructed either by joining the product directly to the existing DNA to give a complete sequence, or carrying out a separate full-length PCR usmg the new sequence information for the design of the 5' primer

The polynucleotides and polypeptides of the mvention may be employed, for example, as research reagents and matenals for discovery of treatments of and diagnostics for diseases, particularly human diseases as further discussed herem relating to polynucleotide assays

The poly nucleotides of the

ention that are ohgonucleotides deπved from a sequence of

Table 1 [SEQ ID NOS 1 or 2] may be used in the processes herem as descnbed, but preferably for

PCR, to determine whether or not the polynucleotides identified herem m whole or m part are transcribed in bacteπa m mfected tissue It is recogmzed that such sequences will also have utility m diagnosis of the stage of infection and type of infection the pathogen has attained

The mvention also provides pohnucleotides that encode a polypeptide that is the mature protem plus additional ammo or carboxyl-terminal ammo acids, or ammo acids mteπor to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance) Such sequences may play a role in processmg of a protem from precursor to a mature form, may allow protem transport, may lengthen or shorten protem half-lrfe or may facihtate mampulation of a protem for assay or production, among other things As generally is the case in v vo the additional am o acids may be processed away from the mature protem bv cellular enzymes

For each and every polynucleotide of the mvention there is provided a polynucleotide complementary to it It is prefened that these complementary polynucleotides are fully complementary to each polynucleotide with which they are complementary

A precursor protein, having a mature form of the polypeptide fused to one or more prosequences may be an mactive form of the polypeptide When prosequences are removed such inactive precursors generally are activated Some or all of the prosequences may be removed before activation Generally, such precursors are called proproteins 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 mvention "N" means that any of the four DNA or RNA nucleotides may appear at such a designated position in the DNA or RNA sequence, except it is preferred that N is not a nucleic acid that when taken m combmation with adjacent nucleotide positions when read in the correct reading frame, would have the effect of generating a premature termination codon in such reading frame

In sum, a polynucleotide of the mvention may encode a mature protein, a mature protem plus a leader sequence (which may be refened to as a preprotein), a precursor of a mature protem having one or more prosequences that are not the leader sequences of a preprotein, or a preproprotem, which is a precursor to a proprotem. havmg a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature forms of the polypeptide

Vectors, Host Cells, Expression Systems

The mvention also relates to vectors that compπse a polynucleotide or polynucleotides of the mvention host cells that are genetically engmeered with vectors of the mvention and the production of polypeptides of the mvention by recombinant techniques Cell-free translation systems can also be employed to produce such protems usmg RNAs deπved from the DNA constructs of the mvention

Recombinant polypeptides of the present mvention may be prepared by processes well known m those skilled in the art from genetically engmeered host cells compπsmg expression systems Accordingly, m a further aspect, the present mvention relates to expression systems which compnse a polynucleotide or polynucleotides of the present mvention. to host cells which are genetically engmeered with such expression systems, and to the production of polypeptides of the mvention by recombinant techniques

For recombinant production of the polypeptides of the mvention, host cells can be genetically engmeered to incorporate expression systems or portions thereof or polynucleotides of the mvention Introduction of a polynucleotide mto the host cell can be effected by methods descnbed m many standard laboratory manuals, such as Davis, et al , BASIC METHODS LN MOLECULAR BIOLOGY, (1986) and Sambrook et al . MOLECULAR CLON NG 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, transvection, microinjection, cationic pid-mediated transfection, electroporation transduction. scrape loading, ballistic mtroduction and infection

Representative examples of appropπate hosts mclude bactenal cells, such as cells of streptococci, staphylococci enterococci E coli, streptomyces, cyanobacteπa, Bacillus subtihs, and Pseudomonas aeruginosa fungal cells such as cells of a yeast. Kluveromyces, Saccharomyces. a basidiomycete, Candida albicans and Aspergillus, insect cells such as cells of Drosoph a 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 vaπety of expression systems can be used to produce the polypeptides of the mvention Such vectors mclude, among others, chromosomal-, episomal- and virus-denved vectors, for example, vectors denved from bactenal plasmids from bactenophage, from transposons, from yeast episomes, from insertion elements from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses, picornaviruses and retroviruses. and vectors denved from combinations thereof, such as those deπved from plasmid and bactenophage genetic elements, such as cosmids and phagemids The expression system constructs may contain control regions that regulate as well as engender expression Generally, any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide m a host may be used for expression m this regard The appropnate DNA sequence may be inserted mto the expression system by any of a vanety of well-lα own and routine techniques, such as, for example, those set forth in Sambrook el al MOLECULAR CLONING, A LABORATORY MANUAL, (supra)

In recombinant expression systems m eukaryotes, for secretion of a translated protem mto the lumen of the endoplasmic reticulum, mto the peπplasmic space or mto the extracellular environment, appropπate secretion signals may be incorporated mto the expressed polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals Polypeptides of the mvention can be recovered and purified from recombinant cell cultures by well- known methods including ammonium sulfate or ethanol precipitation acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography Most preferably, high performance liquid chromatography is employed for punfication Well known techniques for refolding protem may be employed to regenerate active conformation when the polypeptide is denatured durmg isolation and or punfication

Diagnostic, Prognostic, Serotypine .and Mutation Assays

This mvention is also related to the use of fabG polynucleotides and polypeptides of the mvention for use as diagnostic reagents Detection of fabG polynucleotides and/or polypeptides m a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of disease, staging of disease or response of an infectious organism to drugs Eukarvotes, particularly mammals, and especialh humans particularly those infected or suspected to be infected with an orgamsm compnsing the fabG gene or protem, may be detected at the nucleic acid or ammo acid level by a vanety of well known techniques as well as by methods provided herem Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and/or mfected individual's bodily mateπals Polynucleotides from any of these sources, particularly DNA or RNA, may be used directly for detection or may be amplified enzymatically by usmg PCR or any other amplification technique pnor to analysis RNA, particularly mRNA, cDNA and genomic D A may also be used m the same ways Usmg amplification, characterization of the species and strain of infectious or resident orgamsm present m an mdividual, may be made

an analysis of the genotype of a selected polynucleotide of the orgamsm Deletions and insertions can be detected by a change m size of the amplified product m compaπson to a genotype of a reference sequence selected from a related orgamsm, preferably a different species of the same genus or a different strain of the same species Point mutations can be identified by hybndizmg amplified DNA to labeled fabG polynucleotide sequences Perfectly or significantly matched sequences can be distinguished from imperfectly or more sigmficantly mismatched duplexes by DNase or RNase digestion, for DNA or RNA respectively, or by detecting differences in melting temperatures or renaturation kinetics Polynucleotide sequence differences may also be detected by alterations m the electrophoretic mobility of polynucleotide fragments m gels as compared to a reference sequence This may be earned out with or without denaturing agents Polynucleotide differences may also be detected b\ direct DNA or RNA sequencmg See for example, Myers et al , Science, 230 1242 (1985) Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase, VI and S I protection assay or a chemical cleavage method See, for example, Cotton et al , Proc Natl Acad Sci USA 85 4397-4401 (1985)

In another embodiment, an aπay of oligonucleotides probes compnsing fabG nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of for example, genetic mutations, serotype. taxonomic classification or identification Array technology methods are well known and have general applicability and can be used to address a vanety of questions in molecular genetics including gene expression genetic linkage, and genetic vaπabi ty (see, for example, Chee et al , Science, 274 610 ( 1996))

Thus in another aspect, the present invention relates to a diagnostic kit which compπses (a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO 1, or a fragment thereof , (b) a nucleotide sequence complementary to that of (a), (c) a polypeptide of the present invention, preferably the polypeptide of SEQ ID NO 2 or a fragment thereof, or (d) an antιbod\ to a poh peptide of the present invention preferably to the polypeptide of SEQ ID NO 2

It will be appreciated that in any such kit, (a), (b), (c) or (d) may compπse a substantial component Such a kit will be of use in diagnosing a disease or susceptibility to a Disease, among others This mvention also relates to the use of polynucleotides of the present mvention as diagnostic reagents Detection of a mutated form of a polynucleotide of the mvention, preferable, SEQ ED NO 1, which is associated with a disease or pathogenicity will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, a prognosis of a course of disease, a determination of a stage of disease, or a susceptibihtΛ to a disease, which results from under-expression, over-expression or altered expression of the polynucleotide Orgamsms, particularly infectious orgamsms, carrymg mutations m such polynucleotide may be detected at the polynucleotide level by a vanety of techniques, such as those descnbed elsewhere herem The nucleotide sequences of the present mvention are also valuable for orgamsm chromosome identification The sequence is specifically targeted to, and can hybndize with, a particular location on an organism's chromosome, particularly to a Pseudomonas aeruginosa chromosome The mapping of relevant s quences to chromosomes according to the present mvention may be an important step m conelating those sequences with pathogenic potential and/ or an ecological niche of an orgamsm and/or drug resistance of an organism as well as the essentiality of the gene to the organism Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data Such data may be found on-lme m a sequence database The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through known genetic methods for example through linkage analysis (coinhentance of physically adjacent genes) or matmg studies, such as by conjugation

The differences m a polynucleotide and/or polypeptide sequence between orgamsms possessmg a first phenotype and organisms possessmg a different, second different phenotype can also be determined If a mutation is observed m some or all orgamsms possessmg the first phenotype but not in any organisms possessmg the second phenotype, then the mutation is likely to be the causative agent of the first phenotype

Cells from an orgamsm carrymg mutations or polymorphisms (allelic vanations) m a pohmicleotide and/or polypeptide of the mvention may also be detected at the polynucleotide or polypeptide level bv a vanety of techniques, to allow for serotyping, foi example For example, RT-PCR can be used to detect mutations m the RNA It is particularly prefened to use RT-PCR m conjunction with automated detection svstems such as. for example GeneScan RNA. cDNA or genomic DNA may also be used for the same purpose PCR As an example PCR primers complementary to a polynucleotide encodmg fabG pohpeptide can be used to identify and analyze mutations These primers may be used for, among other things, amplifying fabG DNA and/or RNA isolated from a sample denved from an mdividual, such as a bodily matenal The primers may be used to amplify a polynucleotide isolated from an infected mdividual, such that the polynucleotide may then be subject to vanous techniques for elucidation of the polynucleotide sequence In this way, mutations m the polynucleotide sequence may be detected and used to diagnose and/or prognose the infection or its stage or course, or to serotype and/or classify the infectious agent

The mvention further provides a process for diagnosing, disease, preferably bacterial infections, more preferably infections caused by Pseudomonas aeruginosa, compnsmg determining from a sample derived from an individual, such as a bodily matenal, an mcreased level of expression of polynucleotide having a sequence of Table 1 [SEQ ID NO 1] Encreased or decreased expression of a fabG poh nucleotide can be measured using any on of the methods well known m the art for the quantitation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection Northern blotting spectrometry and other hybridization methods

In addition a diagnostic assay m accordance with the mvention for detecting over- expression of fabG polypeptide compared to normal control tissue samples may be used to detect the presence of an infection, for example Assay techniques that can be used to determine levels of a fabG pohpeptide in a sample deπved from a host such as a bodily matenal, are well-known to those of skill in the art Such assay methods mclude radioimmunoassays, competitive-binding assays, Western Blot analy sis antibody sandwich assays antibody detection and ELISA assays OiHei ential Expression The polvnucleotides and polynucleotides of the mvention may be used as reagents for differential screening methods There are many differential screening and differential display methods known m the art in which the polvnucleotides and polypeptides of the mvention may be used For example, the differential display techmque is descnbed by Chuang et al , J Bacte iol 175 2026-2036 (1993) This method identifies those genes which are expressed in an orgamsm by identifying mRNA present using randomh -primed RT-PCR By companng pre- fection and post infection profiles, genes up and down regulated during infection can be identified and the RT-PCR product sequenced and matched to ORF "unknowns "

In Vivo Expression Technology (IVET) is described by Camilh et al , Proc Nat'l Acad Sci USA 91 2634-2638 (1994) IVET identifies genes up-regulated duπng infection when compared to laboratory cultivation, implying an important role in infection ORFs identified by this techmque are implied to

a significant role in infection establishment and/or maintenance In this techmque random chromosomal fragments of target organism are cloned upstream of a promoter-less recombinase gene in a plasmid vector This construct is introduced into the target organism which canies an antibiotic resistance gene flanked by resolvase sites Growth in the presence of the antibiotic removes from the population those fragments cloned mto the plasmid vector capable of supporting transcription of the recombmase gene and therefore have caused loss of antibiotic resistance The resistant pool is introduced into a host and at vaπous times after infection bacteπa mav be recovered and assessed for the presence of antibiotic resistance The chromosomal fragment carried b\ each antibiotic sensitn'e bacterium should carry a promoter or portion of a gene normally upregulated during infection Sequencing upstream of the recombinase gene allows identification of the up regulated gene

RT-PCR may also be used to analyze gene expression patterns For RT PCR using the polvnucleotides of the invention, messenger RNA is isolated from bacterial mfected tissue, e g , 48 hour muπne lung infections, and the amount of each mRNA species assessed by reverse transcπption of the RNA sample primed with random hexanucleotides followed by PCR with gene specific primer pairs The determination of the presence and amount of a particular mRNA species by quantification of the resultant PCR product provides information on the bactenal genes which are transcπbed m the infected tissue Analysis of gene transcπption can be earned out at different tunes of infection to gam a detailed knowledge of gene regulation in bacterial pathogenesis allowing for a clearer understanding of which gene products represent targets for screens for antibactenals Because of the gene specific nature of the PCR primers employed it should be understood that the bactenal mRNA preparation need not be free of mammalian RNA This allows the investigator to carry out a simple and quick RNA preparation from infected tissue to obtain bacterial mRNA species which are very short lived m the bacterium (in the order of 2 minute halflives) Optimally the bacterial mRNA is prepared from infected murine lung tissue by mechanical disruption in the presence of TREzole (GIBCO-BRL) for veiΛ short periods of time, subsequent processing according to the manufacturers of TRIzole reagent and DNAase treatment to remove contaminating DNA Preferably the process is optimized by finding those conditions which give a maximum amount of Pseudomonas aeruginosa 16S πbosomal RNA as detected by probmg Northerns with a suitably labeled sequence specific ohgonucleotide probe Typically a 5' dye labeled primer is used m each PCR primer pair in a PCR reaction which is terminated optimally between 8 and 25 cycles The PCR products are separated on 6% polyacrylamide gels with detection and quantification usmg GeneScanner (manufactured by ABI) Gridding and Polynucleotide Subtraction

Methods have been described for obtaining information about gene expression and identity using so called "high density DNA aπays" or gπds See, e g , M Chee et al , Science, 274 610-614 ( 1996) and other references cited therein Such gπdding assays have been employed to identify certain novel gene sequences, referred to as Expressed Sequence Tags (EST) (Adams et a , Science, 252 1651-1656 (1991)) A variety of techniques have also been descnbed for identifying particular gene sequences on the basis of their gene products For example, see International Patent Application No W091/07087, published May 30, 1991 In addition, methods have been described for the amplification of desired sequences For example, see International Patent Application No W091/17271, published November 14, 1991 The polynucleotides of the mvention may be used as components of polynucleotide arrays, preferably high density arrays or gπds These high density arrays are particularly useful for diagnostic and prognostic purposes For example, a set of spots each compnsmg a different gene, and further comprising a polynucleotide or polynucleotides of the mvention, may be used for probing such as using hybridization or nucleic acid amplification, usmg a probes obtained or derived from a bodily sample to determine the presence of a particular polynucleotide sequence or related sequence in an individual Such a presence may indicate the presence of a pathogen, particularh Pseudomonas aeruginosa and may be useful in diagnosing and/or prognosing disease or a course of disease A grid comprising a number of variants of the polynucleotide sequence of SEQ ID NO 1 are preferred Also preferred is a comprising a number of vanants of a polynucleotide sequence encoding the polypeptide sequence of SEQ ID NO 2

Antibodies

The polypeptides and polynucleotides of the mvention or vanants thereof, or cells expressing the same can be used as lmmunogens to produce antibodies lmmunospecific for such polypeptides or polynucleotides respectively

In certain prefened embodiments of the mvention there are provided antibodies against fabG polypeptides or polynucleotides

Antibodies generated against the polypeptides or polynucleotides of the mvention can be obtained administering the polypeptides and/or polynucleotides of the mvention, or epitope-beaπng fragments of either or both, analogues of either or both, or cells expressing either or both, to an animal, preferably a nonhuman usmg routme protocols For preparation of monoclonal antibodies, any techmque known in the art that provides antibodies produced by continuous cell line cultures can be used Examples mclude vanous techniques such as those m Kohler, G and Milstein, C , Nature 256 495-497 (1975), Kozbor et al , Immunology Today 4 72 (1983), Cole et al , pg 77-96 m MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R Liss, Inc (1985)

Techniques for the production of smgle cham antibodies (U S Patent No 4,946,778) can be adapted to produce smgle cham antibodies to polypeptides or polynucleotides of this mvention Also, transgenic mice or other organisms such as other mammals, may be used to express humanized antibodies lmmunospecific to the polypeptides or polynucleotides of the mvention

Alternatively, phage display technology may be utilized to select antibody genes with binding activities towards a polypeptide of the invention either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing anti-fabG or from naive hbraπes (McCafferty, et al (1990) Nature 348 552-554 Marks et al , (1992) Biotechnology 10, 779-783) The affinity of these antibodies can also be improved by, for example, cham shufϊlmg (Clackson et al , (1991) Nature 352 628) The above-descπbed antibodies may be employed to isolate or to identify clones expressing the polypeptides or polynucleotides of the mvention to purify the polypeptides or polynucleotides by, for example, affinity chromatography

Thus, among others, antibodies against fabG-polypeptide or fabG-polynucleotide may be employed to treat infections, particularly bactenal infections

Poh peptide variants include antigenically, epitopically or lmmunologically equivalent vanants form a particular aspect of this invention

A polypeptide or polynucleotide of the invention, such as an antigenically or lmmunologically equivalent derivative or a fusion protem of the polypeptide is used as an antigen to immunize a mouse or other animal such as a rat or chicken The fusion protein may provide stability to the polypeptide The antigen may be associated, for example by conjugation, with an lmmunogenic carrier protem for example bovine serum albumin, keyhole limpet haemocyanin or tetanus toxoid Alternatively, a multiple antigemc polypeptide compnsmg multiple copies of the polypeptide, or an antigenically or lmmunologically equivalent polypeptide thereof may be sufficiently antigemc to improve lmmunogenicity so as to obviate the use of a earner

Preferably the antibody or vanant thereof is modified to make it less rmmunogenic in the idual For example if the individual is human the antibody may most preferably be "humanized," where the comphmentaπty determining region or regions of the hybπdoma-deπved antibody has been transplanted into a human monoclonal antibody, for example as descnbed m Jones et al (1986), Nature 321. 522-525 or Tempest et al . (1991) Biotechnology 9, 266-273

In accordance with an aspect of the mvention, there is provided the use of a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization Among the particularh prefened embodiments of the mvention are naturally occurring allehc vanants of fabG pohτιucleotιdes and polypeptides encoded thereby The use of a polynucleotide of the invention in genetic immunization will preferably employ a suitable delivery method such as direct injection of plasmid DNA mto muscles (Wolff et al , Hum Mol Genet (X992) 1 363, Manthorpe et al , Hum Gene Ther (1983) 4 419), delivery of DNA complexed with specific protem earners (Wu et al , J Biol Chem (1989) 264 16985), coprecipitation of DNA with calcium phosphate (Benvemsty & Reshef, PNAS USA, (1986) 83 9551), encapsulation of DNA in various forms of liposomes (Kaneda et al , Science (1989) 243 375), particle bombardment (Tang et al Nature ( 1992) 356 152, Eisenbraun et al , DNA Cell B ol (1993) 12 791) and in vivo infection using cloned retroviral vectors (Seeger et al , PNAS USA (1984) 81 5849)

Antagonists and Agonists - Assays and Molecules Polypeptides and polynucleotides of the invention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics. See, e.g., Coligan et al., Current Protocols in Immunology 1(2): Chapter 5 (1991). Polypeptides and polynucleotides of the present invention are responsible for many biological functions, including many disease states, in particular the Diseases hereinbefore mentioned. It is therefore desirable to devise screening methods to identify compounds which stimulate or which inhibit the function of the polypeptide or polynucleotide. Accordingly, in a further aspect, the present invention provides for a method of screening compounds to identify those which stimulate or which inhibit the function of a polypeptide or polynucleotide of the invention, as well as related polypeptides and polynucleotides. In general, agonists or antagonists may be employed for therapeutic and prophylactic purposes for such Diseases as hereinbefore mentioned. Compounds may be identified from a variety of sources, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. Such agonists, antagonists or inhibitors so-identified may be natural or modified substrates, ligands, receptors, enzymes, etc., as the case may be. of fabG polypeptides and polynucleotides; or may be structural or functional mimetics thereof (see Coligan et al., Current Protocols in Immunology 1 (2) : Chapter 5 (1991)).

The screening methods may simply measure the binding of a candidate compound to the polypeptide or polynucleotide, or to cells or membranes bearing the polypeptide or polynucleotide, or a fusion protein of the polypeptide by means of a label directly or indirectly associated with the candidate compound. Alternatively, the screening method may involve competition with a labeled competitor. Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide or polynucleotide, using detection systems appropriate to the cells comprising the polypeptide or polynucleotide. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed. Constitutively active polypeptide and/or constitutively expressed polypeptides and polynucleotides may be employed in screening methods for inverse agonists or inhibitors, in the absence of an agonist or inhibitor, by testing whether the candidate compound results in inhibition of activation of the polypeptide or polynucleotide, as the case may be. Further, the screening methods may simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide or polynucleotide of the present invention, to form a mixture, measuring fabG polypeptide and/or polynucleotide activity in the mixture, and comparing the fabG polypeptide and/or polynucleotide activity of the mixture to a standard. Fusion proteins, such as those made from Fc portion and fabG polypeptide, as hereinbefore described, can also be used for high-throughput screemng assays to identify antagonists of the polypeptide of the present invention, as well as of phylogenetically and and/or functionally related polypeptides (see D Bennett el al J Mol Recognition, 8 52-58 (1995), and K Johanson et al , J Biol Chem, 270(16) 9459-9471 ( 1995)) The pohτiucleotides polypeptides and antibodies that bind to and/or interact with a polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and/or polypeptide in cells For example, an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide usmg monoclonal and polyclonal antibodies by standard methods known m the art This can be used to discover agents which may inhibit or enhance the production of polypeptide (also called antagonist or agonist respectively) from suitably manipulated cells or tissues

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

Polypeptides of the invention may be used to identify membrane bound or soluble receptors, it am for such polypeptide through standard receptor binding techmques known in the art These techniques include but are not limited to ligand binding and crosslrnkrng assays m which the polypeptide is labeled with a radioactive isotope (for instance, 25τ) chemically modified (for instance, biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (e g , cells, cell membranes, cell supernatants, tissue extracts bodily materials) Other methods include biophysical techmques such as surface plasmon resonance and spectroscopy These screemng methods may also be used to identify agomsts and antagomsts of the polypeptide which compete with the bmdιng.of the polypeptide to its receptor(s), if any Standard methods for conducting such assays are well understood in the art The fluorescence polanzation value for a fluorescently-tagged molecule depends on the rotational correlation time or tumbling rate Protem complexes, such as formed by fabG poh peptide associating with another fabG polypeptide or other polypeptide, labeled to compπse a fluorescentlv-labeled molecule will have higher polarization values than a fluorescently labeled monomeπc protem It is preferred that this method be used to charactenze small molecules that disrupt polypeptide complexes

Fluorescence energy transfer may also be used characterize small molecules that interfere with the foπnation of fabG polypeptide dimers tπmers, tetramers or higher order structures, or structures formed by fabG polypeptide bound to another polypeptide FabG polypeptide 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 dimeπzation will inhibit fluorescence energy transfer

Surface plasmon resonance can be used to monitor the effect of small molecules on fabG poh peptide self-association as well as an association of fabG polypeptide and another polypeptide or small molecule fabG polypeptide can be coupled to a sensor chip at low site density such that covalently bound molecules will be monomeπc Solution protem can then passed over the fabG polypeptide -coated surface and specific bmdmg can be detected m real-time by monitoring the ch.ange in resonance angle caused by a change m local refractive mdex This techmque can be used to characterize the effect of small molecules on kinetic rates and equihbπum binding constants for fabG poh peptide self-association as well as an association of fabG polypeptide and another pohpeptide or small molecule

A scintillation proximity assay may be used to charactenze the interaction between an association of fabG polypeptide with another fabG polypeptide or a different polypeptide fabG polypeptide can be coupled to a scrntillation-fϊlled bead Addition of radio-labeled fabG polypeptide results in binding where the radioactive source molecule is m close proximity to the scmtillation fluid Thus signal is emitted upon fabG polypeptide binding and compounds that prevent fabG pohpeptide self-association or an association of fabG polypeptide and another polypeptide or small molecule will diminish signal ICS biosensors have been descnbed by AMBRI (Australian Membrane Biotechnology Research Institute) Thev couple the self-association of macromolecules to the closmg of gramacidin-facilitated ion channels in suspended membrane bilayers and hence to a measurable change in the admittance (similar to rmpedence) of the biosensor This approach is lmear over six decades of admittance change and is ideally suited for large scale, high through-put screemng of small molecule combmatonal hbraπes

In other embodiments of the

are provided methods for identifying compounds which bmd to or otherwise interact with and inhibit or activate an activity or expression of a polypeptide and/or polynucleotide of the mvention comprising contacting a polypeptide and/or polynucleotide of the mvention with a compound to be screened under conditions to permit binding to or other mteraction between the compound and the polypeptide and/or 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 polypeptide and/or pohnucleotide with the compound and determinmg whether the compound bmds to or otherwise interacts with and activates or inhibits an activity or expression of the polypeptide and/or polynucleotide by detecting the presence or absence of a signal generated from the bmdmg or mteraction of the compound with the polypeptide and/or polynucleotide

Another example of an assay for fabG agomsts is a competitive assay that combines fabG and a potential agonist with fabG-binding molecules, recombinant fabG binding molecules, natural substrates or ligands or substrate or ligand mimetics under appropπate conditions for a competitive inhibition assay fabG can be labeled, such as by radioactivity or a coloπmetnc compound, such that the number of fabG molecules bound to a bmdmg molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist

Potential antagomsts mclude. among others, small organic molecules, peptides, polypeptides and antibodies that bmd to a polynucleotide and/or polypeptide of the mvention and thereby inhibit or extinguish its activity or expression Potential antagomsts also may be small organic molecules, a peptide, a poh peptide such as a closely related protem or antibody that bmds the same sites on a bmdmg molecule, such as a bmdmg molecule without inducing fabG-induced activities, thereby preventing the action or expression of fabG polypeptides and/or polynucleotides by excluding fabG polypeptides and/or polynucleotides from bmdmg

Potential antagomsts mclude a small molecule that bmds to and occupies the bmdmg site of the polypeptide 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 antagomsts 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 descnption of these molecules) Preferred potential antagonists mclude compounds related to and vanants of fabG Other examples of potential polypeptide antagomsts mclude antibodies or, m some cases, oligonucleotides or proteins which are closely related to the ligands, substrates, receptors, enzymes, etc , as the case may be. of the polypeptide, e g . a fragment of the ligands, substrates, receptors, enzymes, etc , or small molecules which bmd to the polypeptide of the present mvention but do not elicit a response, so that the activity of the polypeptide is prevented Certain of the polypeptides of the mvention are biomimetics, functional mimetics of the natural fabG polypeptide These functional mimetics may be used for, among other things, antagonizing the activity of fabG polypeptide or as a antigen or immunogen m a manner descnbed elsewhere herem Functional mimetics of the polypeptides of the mvention mclude but are not limited to truncated polypeptides For example, prefened functional mimetics mclude, a polypeptide compnsmg the polypeptide sequence set forth m SEQ ID NO 2 lackmg 20, 30, 40, 50, 60, 70 or 80 ammo- or carboxy-terminal ammo acid residues, including fusion protems compnsmg one or more of these truncated sequences Polynucleotides encodmg each of these functional mimetics may be used as expression cassettes to express each mimetic polypeptide It is prefened that these cassettes compnse 5' and 3' restnction sites to allow for a convement means to ligate the cassettes together when desired It is further prefened that these cassettes compnse gene expression signals known the art or descnbed elsewhere herem

Thus, in another aspect, the present invention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc for a polypeptide and/or polynucleotide of the present invention or compounds which decrease or enhance the production of such polypeptides and/or polynucleotides , which comprises (a) a polypeptide and/or a polynucleotide of the present invention, (b) a recombinant cell expressing a polypeptide and/or polynucleotide of the present invention, (c) a cell membrane expressing a polypeptide and/or polynucleotide of the present invention, or (d) antibody to a polypeptide and/or polynucleotide of the present mvention, which poly peptide is preferably that of SEQ ID NO 2, and which polynucleotide is preferably that of SEQ ID NO 1 It will be appreciated that in any such kit, (a), (b), (c) or (d) may compπse a substantial component

It will be readily appreciated by the skilled artisan that a polypeptide and/or polynucleotide of the present invention may also be used m a method for the structure-based design of an agomst, antagonist or inhibitor of the polypeptide and/or polynucleotide, by (a) determining in the first instance the three-dimensional structure of the polypeptide and/or polynucleotide, or complexes thereof, (b) deducing the three-dimensional structure for the likely reactive sιte(s), bmdmg sιte(s) or motιf(s) of an agonist, antagonist or inhibitor, (c) synthesizing candidate compounds that are predicted to bind to or react with the deduced binding sιte(s), reactive sιte(s). and/or motιf(s), and (d) testing whether the candidate compounds are mdeed agomsts, antagomsts or inhibitors It will be further appreciated that this will normally be an iterative process, and this iterative process may be performed using automated and computer-controlled steps

In a further aspect, the present mvention provides methods of treating abnormal conditions such as, for mstance a Disease, related to either an excess of an under-expression of, an elevated activity of, or a decreased activity of fabG polypeptide and/or polynucleotide

If the expression and/or activity of the polypeptide and/or polynucleotide is m excess, several approaches are available One approach compnses administering to an mdividual m need thereof an inhibitor compound (antagonist) as herem descnbed, optionally m combination with a pharmaceutically acceptable earner in an amount effective to inhibit the function and/or expression of the polypeptide and/or polynucleotide, such as, for example, by blocking the bmdmg of ligands. substrates, receptors, enzymes, etc or by inhibiting a second signal, and thereby alleviating the abnormal condition In another approach, soluble forms of the polypeptides still capable of bmdmg the ligand substrate, enzymes, receptors, etc in competition with endogenous polypeptide and/or polynucleotide may be administered Typical examples of such competitors include fragments of the fabG polypeptide and/or polypeptide

In a further aspect, the present invention relates to geneticalh engineered soluble fusion proteins comprising a polypeptide of the present mvention, or a fragment thereof, and vaπous portions of the constant regions of heavy or light chains of immunoglobulms of various subclasses (IgG. IgM IgA. IgE) Preferred as an lmmunoglobuhn is the constant part of the heavy chain of human IgG, particularly IgG 1 where fusion takes place at the hinge region En a particular embodiment, the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa Furthermore, this invention relates to processes for the preparation of these fusion proteins by genetic engineering, and to the use thereof for drug screemng, diagnosis and therapy A further aspect of the invention also relates to polynucleotides encodmg such fusion proteins Examples of fusion protein technology can be found in International Patent Application Nos W094/29458 and W094/22914

In still another approach, expression of the gene encoding endogenous fabG polypeptide can be inhibited using expression blocking techniques This blocking may be targeted agamst any step m gene expression, but is preferably targeted against transcription and/or translation. An examples of a known technique of this sort involve the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor, J Neurochem (1991) 56:560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). Alternatively, oligonucleotides which form triple 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) 251 : 1360). These oligomers can be administered per se or the relevant oligomers can be expressed in vivo.

Each of the polynucleotide sequences provided herein may be used in the discovery and development of antibacterial compounds. The encoded protein, upon expression, can be used as a target for the screening of antibacterial drugs. Additionally, the polynucleotide sequences encoding the amino terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest. The invention also provides the use of the polypeptide, polynucleotide, agonist or antagonist of the invention to interfere with the initial physical interaction between a pathogen or pathogens and a eukaryotic. preferably mammalian, host responsible for sequelae of infection. In particular, the molecules of the invention may be used: in the prevention of adhesion of bacteria, in particular gram positive and/or gram negative bacteria, to eukaryotic, preferably mammalian, extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in wounds; to block bacterial adhesion between eukaryotic, preferably mammalian, extracellular matrix proteins and bacterial fabG proteins that mediate tissue damage and/or; to block the normal progression of pathogenesis in infections initiated other than by the implantation of in-dwelling devices or by other surgical techniques. In accordance with yet another aspect of the invention, there are provided fabG agonists and antagonists, preferably bacteristatic or bactericidal agonists and antagonists.

The antagonists and agonists of the invention may be employed, for instance, to prevent, inhibit and/or treat diseases.

Hehcobacter pylori (herein "H. pylori") bacteria infect the stomachs of over one-third of the world's population causing stomach cancer, ulcers, and gastritis (International Agency for Research on Cancer ( 1994) Schistosomes. Liver Flukes and Hehcobacter Pylori (Enternational Agency for Research on Cancer, Lyon, France, http://www.uicc.ch/ecp/ecp2904.htm). Moreover, the International Agency for Research on Cancer recently recognized a cause-and-effect relationship between H pylori and gastric adenocarcinoma, classifying the bacterium as a Group I (definite) carcinogen Preferred antimicrobial compounds of the invention (agonists and antagonists of fabG polypeptides and/or polynucleotides) found using screens provided by the mvention, or known in the art, particularly narrow-spectrum antibiotics, should be useful in the treatment of H. pylori infection. Such treatment should decrease the advent of H />v/orz-induced cancers, such as gastrointestinal carcinoma Such treatment should also prevent, inhibit and/or cure gastric ulcers and gastritis Vaccines

There are provided by the mvention, products, compositions and methods for assessing fabG expression, treatmg disease, assaymg genetic vaπation, and administering a fabG polypeptide and/or polynucleotide to an orgamsm to raise an immunological response against a bacteria, especially a Pseudomonas aeruginosa bactena

Another aspect of the invention relates to a method for inducing an immunological response in an individual, particularly a mammal which compnses inoculating the mdividual with fabG polynucleotide and/or polypeptide, or a fragment or variant thereof, adequate to produce antibody and/ or T cell immune response to protect said mdividual from infection, particularly bacterial infection and most particularly Pseudomonas aeruginosa infection Also provided are methods whereby such immunological response slows bactenal replication. Yet another aspect of the invention relates to a method of inducing immunological response m an individual which compnses delivering to such individual a nucleic acid vector, sequence or πbozyme to direct expression of fabG polynucleotide and/or polypeptide, or a fragment or a variant thereof, for expressmg fabG polynucleotide and/or polypeptide, or a fragment or a vaπant thereof in vivo in order to induce an immunological response, such as. to produce antibody and/ or T cell immune response, mcludmg, for ex,ample, cytokine- producing T cells or cytotoxic T cells, to protect said individual, preferably a human, from disease, whether that disease is already established within the individual or not One example of administering the gene is by accelerating it mto the desired cells as a coating on particles or otherwise Such nucleic acid vector may compπse DNA, RNA. a ribozyme, a modified nucleic acid, a DNA/RNA hybrid, a DNA-protein complex or an RNA-protern complex.

A further aspect of the invention relates to an immunological composition that when introduced into an individual, preferably a human, capable of having induced within it an immunological response, mduces an immunological response m such mdividual to a fabG polynucleotide and/or polypeptide encoded therefrom, wherein the composition comprises a recombinant fabG polynucleotide and/or polypeptide encoded therefrom and/or compnses DNA and/or RNA which encodes and expresses an antigen of said fabG polynucleotide, polypeptide encoded therefrom, or other polypeptide of the invention. The immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity and/or cellular immunity, such as cellular immunity arising from CTL or CD4+ T cells.

A fabG polypeptide or a fragment thereof may be fused with co-protein or chemical moiety which may or may not by itself produce antibodies, but which is capable of stabilizing the first protein and producing a fused or modified protein which will have antigenic and/or immunogenic properties, and preferably protective properties. Thus fused recombinant protein, preferably further comprises an antigenic co-protein, such as lipoprotein D from Hemophilus influenzae, Glutathione-S-transferase (GST) or beta-galactosidase. or any other relatively large co-protein which solubilizes the protein and facilitates production and purification thereof. Moreover, the co-protein may act as an adjuvant in the sense of providing a generalized stimulation of the immune system of the organism receiving the protein. The co-protein may be attached to either the amino- or carboxy-terminus of the first protein.

Provided by this invention are compositions, particularly vaccine compositions, and methods comprising the polypeptides and/or polynucleotides of the invention and immunostimulatory DNA sequences, such as those described in Sato, Y. et al. Science 273: 352 (1996).

Also, provided by this invention are methods using the described polynucleotide or particular fragments thereof, which have been shown to encode non-variable regions of bacterial cell surface proteins, in polynucleotide constructs used in such genetic immunization experiments in animal models of infection with Pseudomonas aeruginosa. Such experiments will be particularly useful for identifying protein epitopes able to provoke a prophylactic or therapeutic immune response. It is believed that this approach will allow for the subsequent preparation of monoclonal antibodies of particular value, derived from the requisite organ of the animal successfully resisting or clearing infection, for the development of prophylactic agents or therapeutic treatments of bacterial infection, particularly Pseudomonas aeruginosa infection, in mammals, particularly humans. A polypeptide of the invention may be used as an antigen for vaccination of a host to produce specific antibodies which protect against invasion of bacteria, for example by blocking adherence of bacteria to damaged tissue. Examples of tissue damage include wounds in skin or connective tissue caused, for example, by mechanical, chemical, thermal or radiation damage or by implantation of indwelling devices, or wounds in the mucous membranes, such as the mouth, throat, mammary glands, urethra or vagina.

The invention also includes a vaccine formulation which comprises an immunogenic recombinant polypeptide and/or polynucleotide of the invention together with a suitable carrier, such as a pharmaceutically acceptable carrier. Since the polypeptides and polynucleotides may be broken down in the stomach, each is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or lntradermal Formulations suitable for parenteral administration mclude aqueous and oon-aqueous steπle injection solutions which may contain anti-oxidants, buffers, bacteπstatic compounds and solutes which render the formulation lsotomc with the bodily fluid, preferably the blood, of the mdividual, and aqueous and non-aqueous steπle suspensions which ma's include suspending agents or thickening agents The formulations may be presented in umt-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dned condition requiring only the addition of the steπle liquid carrier immediately prior to use The vaccine formulation may also include adjuvant systems for enhancing the immunogemcity of the formulation, such as oil-in water systems and other systems known in the art The dosage will depend on the specific activity of the vaccme and can be readily determined bv routine experimentation

While the mvention has been descnbed with reference to certain fabG polypeptides and polynucleotides, it is to be understood that this covers fragments of the naturally occurring polypeptides and polynucleotides, and similar polypeptides and polynucleotides with additions, deletions or substitutions which do not substantially affect the immunogenic properties of the recombinant polypeptides or polynucleotides Compositions, kits .and administration In a further aspect of the mvention there are provided compositions compnsmg a fabG polynucleotide and/or a fabG polypeptide for administration to a cell or to a multicellular orgamsm

The mvention also relates to compositions compnsmg a polynucleotide and/or a polypeptides discussed herem or their agomsts or antagomsts The polypeptides and polynucleotides of the mvention may be employed in combination with a non-sterile or sterile earner or earners for use with cells, tissues or orgamsms such as a pharmaceutical earner suitable for admmistration to an mdividual Such compositions compnse, for instance, a media additive or a therapeutically effective amount of a polypeptide and/or polynucleotide of the mvention and a pharmaceutically acceptable earner or excipient Such earners may mclude but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof The formulation should suit the mode of admmistration The invention further relates to diagnostic and pharmaceutical packs and kits compnsmg one or more containers filled with one or more of the ingredients of the aforementioned compositions of the mvention

Pohpeptides polynucleotides and other compounds of the mvention may be employed alone or m conjunction with other compounds, such as therapeutic compounds The pharmaceutical compositions may be administered m any effective, convenient manner including, for instance, admmistration by topical, oral, anal, vaginal, intravenous, mtrapeπtoneal, intramuscular, subcutaneous, mtranasal or lntradermal routes among others

In therapy or as a prophylactic, the active agent may be administered to an mdividual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotomc

Alternatively the composition may be formulated for topical application for example in the form of ointments creams, lotions, eye omtments, eye drops, ear drops. mouthwash impregnated dressings and sutures and aerosols, and may contam appropπate conventional additives including, for example, preservatives, solvents to assist drug penetration, and emollients in ointments and creams Such topical formulations may also contam compatible conv entional carriers for example cream or ointment bases, and ethanol or oleyl alcohol for lotions Such earners may constitute from about 1% to about 98% by weight of the formulation, more usually thev ill constitute up to about 80% bv weight of the formulation

In a further aspect, the present mvention provides for pharmaceutical compositions compnsmg a therapeutically effective amount of a polypeptide and/or polynucleotide, such as the soluble form of a polypeptide and/or polynucleotide of the present mvention, agonist or antagonist peptide or small molecule compound in combmation with a pharmaceutically acceptable earner or excipient Such earners mclude, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof The mvention further relates to pharmaceutical packs and kits compnsmg one or more containers filled with one or more of the mgredients of the aforementioned compositions of the mvention Polypeptides, polynucleotides and other compounds of the present mvention may be employed alone or in conjunction with other compounds, such as therapeutic compounds

The composition will be adapted to the route of admmistration, for instance by a systemic or an oral route Prefened forms of systemic administration mclude injection, typically by intravenous injection Other injection routes, such as subcutaneous, intramuscular, or lntrapentoneal, can be used Alternative means for s stemic administration mclude transmucosal and transdermal administration usmg penetraπts such as bile salts or fusidic acids or other detergents In addition, if a polypeptide or other compounds of the present mvention can be formulated m an entenc or an encapsulated formulation, oral administration may also be possible Admmistration of these compounds may also be topical and/or localized, m the form of salves, pastes gels, and the like

For administration to mammals, and particularly humans, it is expected that the daily dosage level of the active agent will be from 0 01 mg/kg to 10 mg/kg, typically around 1 mg/kg The phv sician in any event will determine the actual dosage which will be most suitable for an mdividual and will vary with the age weight and response of the particular individual The above dosages are exemplars of the average case There can of course, be individual instances where higher or lower dosage ranges are merited and such are within the scope of this invention

In-dwelling devices include surgical implants prosthetic devices and catheters, 1 e , devices that are introduced to the body of an individual and remain in position for an extended time Such devices include, for example, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters cerebrospinal fluid shunts urinary catheters, continuous ambulatory pentoneal dialysis

(CAPD) catheters

The composition of the invention may be administered by injection to achieve a systemic effect against relev ant bacteria shortly before insertion of an in-dwelling device Treatment may be continued after surgery durmg the -body time of the device In addition, the composition could also be used to broaden peπoperative cover for any surgical technique to prevent bactenal wound infections especially Pseudomonas aeruginosa wound infections

Manv orthopedic surgeons consider that humans with prosthetic joints should be considered for antibiotic prophylaxis before dental treatment that could produce a bacteremia Late deep infection is a serious complication sometimes leading to loss of the prosthetic joint and is accompanied by significant morbidity and mortality It may therefore be possible to extend the use of the active agent as a replacement for prophylactic antibiotics in this situation

In addition to the therapy described above, the compositions of this invention may be used generalh as a wound treatment agent to prevent adhesion of bactena to matnx proteins exposed m wound tissue and for prophylactic use m dental treatment as an alternative to, or m conjunction with, antibiotic prophylaxis

Alternatively, the composition of the invention may be used to bathe an indwelling device immediately before insertion The active agent will preferably be present at a concentration of 1 μg/ml to lOmg/ml for bathing of wounds or indwelling devices

A accine composition is convenienfh injectable form Conventional adjuvants may be employed to enhance the immune response A suitable umt dose for vaccmation is 0 5-5 microgram/kg of antigen and such dose is preferably administered 1-3 times and with an interval of 1- 3 weeks W ith the indicated dose range no adv erse toxicological effects will be observed with the compounds of the mvention which would preclude their administration to suitable individuals Sequence Databases, Sequences in a Tangible Medium, and Algorithms

Polynucleotide and polypeptide sequences form a valuable information resource with which to determine their 2- and 3 -dimensional structures as well as to identify further sequences of similar homology These approaches are most easily facilitated by storing the sequence in a computer readable medium and then using the stored data in a known macromolecular structure program or to search a sequence database using well known searching tools, such as GCC.

The polynucleotide and pol peptide sequences of the invention are particularly useful as components in databases useful for search analyses as well as in sequence analysis algorithms. As used in this section entitled "Sequence Databases. Sequences in a Tangible Medium, and Algorithms," and in claims related to this section, the terms "polynucleotide of the invention" and "polynucleotide sequence of the invention" mean any detectable chemical or physical characteristic of a polynucleotide of the invention that is or may be reduced to or stored in a tangible medium, preferably a computer readable form. For example, chromatographic scan data or peak data, photographic data or scan data therefrom, called bases, and mass spectrographic data. As used in this section entitled Databases and Algorithms and in claims related thereto, the terms "polypeptide of the invention" and "polypeptide sequence of the invention" mean any detectable chemical or physical characteristic of a polypeptide of the invention that is or may be reduced to or stored in a tangible medium, preferably a computer readable form. For example, chromatographic scan data or peak data, photographic data or scan data therefrom, and mass spectrographic data.

The invention provides a computer readable medium having stored thereon polypeptide sequences of the invention and/or polynucleotide sequences of the invention. For example, a computer readable medium is provided comprising and having stored thereon a member selected from the group consisting of: a polynucleotide comprising the sequence of a polynucleotide of the invention; a polypeptide comprising the sequence of a polypeptide sequence of the invention; a set of polynucleotide sequences wherein at least one of the sequences comprises the sequence of a polynucleotide sequence of the invention; a set of polypeptide sequences wherein at least one of the sequences comprises the sequence of a polypeptide sequence of the invention; a data set representing a polynucleotide sequence comprising the sequence of polynucleotide sequence of the invention; a data set representing a polynucleotide sequence encoding a polypeptide sequence comprising the sequence of a polypeptide sequence of the invention; a polynucleotide comprising the sequence of a polynucleotide sequence of the invention: a polypeptide comprising the sequence of a polypeptide sequence of the invention; a set of polynucleotide sequences wherein at least one of the sequences comprises the sequence of a polynucleotide sequence of the invention; a set of polypeptide sequences wherein at least one of said sequences comprises the sequence of a polypeptide sequence of the invention; a data set representing a polynucleotide sequence comprising the sequence of a polynucleotide sequence of the invention; a data set representing a polynucleotide sequence encoding a polypeptide sequence comprising the sequence of a polypeptide sequence of the invention. The computer readable medium can be any composition of matter used to store information or data, including, for example, commercially available floppy disks, tapes, chips, hard drives, compact disks, and video disks. Also provided by the invention are methods for the analysis of character sequences or strings, particularly genetic sequences or encoded genetic sequences. Prefened methods of sequence analysis include, for example, methods of sequence homology analysis, such as identity and similarity analysis, RNA structure analysis, sequence assembly, cladistic analysis, sequence motif analysis, open reading frame determination, nucleic acid base calling, nucleic acid base trimming, and sequencing chromatogram peak analysis.

A computer based method is provided for performing homology identification. This method comprises the steps of providing a first polynucleotide sequence comprising the sequence a polynucleotide of the invention in a computer readable medium; and comparing said first polynucleotide sequence to at least one second polynucleotide or polypeptide sequence to identify homology.

A computer based method is also provided for performing homology identification, said method comprising the steps of: providing a first polypeptide sequence comprising the sequence of a polypeptide of the invention in a computer readable medium; and comparing said first polypeptide sequence to at least one second polynucleotide or polypeptide sequence to identify homology. A computer based method is still further provided for polynucleotide assembly, said method comprising the steps of: providing a first polynucleotide sequence comprising the sequence of a polynucleotide of the invention in a computer readable medium; and screening for at least one overlapping region between said first polynucleotide sequence and at least one second polynucleotide or polypeptide sequence. A computer based method is still further provided for polynucleotide assembly, said method comprising the steps of: providing a first polypeptide sequence comprising a polypeptide of the invention in a computer readable medium; and screening for at least one overlapping region between said first polypeptide sequence and at least one second polynucleotide or polypeptide sequence.

In another prefened embodiment of the invention there is provided a computer readable medium having stored thereon a member selected from the group consisting of: a polynucleotide comprising the sequence of SEQ ID NO: l ; a polypeptide comprising the sequence of SEQ ID NO:2; a set of polynucleotide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO: 1 : a set of polypeptide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO:2; a data set representing a polynucleotide sequence comprising the sequence of SEQ ID NO: l; a data set representing a polynucleotide sequence encoding a polypeptide sequence comprising the sequence of SEQ ID NO:2; a polynucleotide comprising the sequence of SEQ ID NO:l; a polypeptide comprising the sequence of SEQ ID NO:2; a set of polynucleotide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO: l; a set of polypeptide sequences wherein at least one of said sequences comprises the sequence of SEQ ID NO:2; a data set representing a polynucleotide sequence comprising the sequence of SEQ ID NO: l : a data set representing a polynucleotide sequence encoding a polypeptide sequence comprising the sequence of SEQ ID NO:2. A further prefened embodiment of the invention provides a computer based method for performing homology identification, said method comprising the steps of providing a polynucleotide sequence comprising the sequence of SEQ ID NO:l in a computer readable medium; and comparing said polynucleotide sequence to at least one polynucleotide or polypeptide sequence to identify homology.

A still further preferred embodiment of the invention provides a computer based method for performing homology identification, said method comprising the steps of: providing a polypeptide sequence comprising the sequence of SEQ ID NO:2 in a computer readable medium; and comparing said polypeptide sequence to at least one polynucleotide or polypeptide sequence to identify homology.

A further embodiment of the invention provides a computer based method for polynucleotide assembly, said method comprising the steps of: providing a first polynucleotide sequence comprising the sequence of SEQ ID NO: l in a computer readable medium; and screening for at least one overlapping region between said first polynucleotide sequence and a second polynucleotide sequence.

A further embodiment of the invention provides a computer based method for performing homology identification, said method comprising the steps of: providing a polynucleotide sequence comprising the sequence of SEQ ID NO: l in a computer readable medium; and comparing said polynucleotide sequence to at least one polynucleotide or polypeptide sequence to identify homology.

All publications and references, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference in their entirety as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth. Any patent application to which this application claims priority is also incorporated by reference herein in its entirety in the manner described above for publications and references. GLOSSARY

The following definitions are provided to facilitate understanding of certain terms used frequently herem

"Antιbody(ιes)" as used herem includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, mcluding the products of an Fab or other immunoglobulin expression library.

"Antigenically equivalent denvative(s)" as used herem encompasses a polypeptide, polynucleotide, or the equivalent of either which will be specifically recognized by certain antibodies which, when raised to the protein, polypeptide or polynucleotide according to the invention, interferes with the immediate physical interaction between pathogen and mammalian host

"Bispecific antιbody(ιes)" means an antibody compnsmg at least two antigen bmdmg domains, each domain directed agamst a different epitope

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

"Dιsease(s)" means any disease caused by or related to infection by a bacteria, 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. "Fusion protein(s)" refers to a protein encoded by two, often unrelated, fused genes or fragments thereof In one example, EP-A-0464 discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protem or part thereof. In many cases, employing an immunoglobulin Fc region as a part of a fusion protem is advantageous for use in therapy and diagnosis resultmg m, for example, improved pharmacokmetic properties [see, e g , EP-A 0232262] On the other hand, for some uses it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and purified.

"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 by 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, including but not limited to those described in (Computational Molecular Biology. Lesk, A M . ed , Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith, D W., ed . Academic Press, New York, 1993, Computer Analysis of Sequence Data, Part I, Griffin, A.M , and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heiηje, G., Academic Press, 1987, and Sequence Analysis Primer. Gπbskov, M and Devereux, J . eds . M Stockton Press. New York, 1991; and Carillo, H , and Lipman, D., SLAM J Applied Math , 48: 1073 (1988). Methods to determine identity are designed to give the largest match between the sequences tested Moreover, methods to determine identity are codified m publicly available computer programs. Computer program methods to determine identity between two sequences include, 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 Molec Biol 215 403-410 (1990). The BLAST X program is publicly 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 algonthm may also be used to determine identity Parameters for polypeptide sequence comparison include the following Algonthm-

Needleman and Wunsch, J. Mol Biol 48 443-453 (1970)

Companson matrix 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 publicly available as the "gap" program from Genetics Computer Group, Madison WI The aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps)

Parameters for polynucleotide comparison include the following Algonthm- Needleman and Wunsch, J Mol Biol 48 443-453 (1970)

Comparison matrix' 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 comparisons

A prefened meaning for "identity" for polynucleotides and polypeptides, as the case may be, are provided in (1) and (2) below ( 1 ) Polynucleotide embodiments further include an isolated polynucleotide comprising 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 , wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO: 1 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NOT by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides in SEQ ID NO: 1, or:

"n ≤ ^xn " (^xn • y)>

wherein n_n is the number of nucleotide alterations, x_n is the total number of nucleotides in SEQ ID NO: l , 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 wherein any non-integer product of x_n and y is rounded down to the nearest integer prior to subtracting it from x_n. Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO: 2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.

By way of example, a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO: l, that is it may be 100% identical, or it may include up to a certain integer number of nucleic acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity. Such alterations are selected from the group consisting of at least one nucleic acid deletion, substitution, including transition and transversion, or insertion, and wherein 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 in 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: 1 by the mteger 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

n_n < x_n - (x_n • y),

wherein n_n is the number of nucleic acid alterations, x_n is the total number of nucleic acids m SEQ ID NO 1, y is, for mstance 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 prior to subtracting it from x_n (2) Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 50,60, 70 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2 wherem said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2 or may mclude up to a certain mteger number of ammo acid alterations as compared to the reference sequence wherein said alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherem said alterations may occur at the ammo- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the ammo acids m the reference sequence or m one or more contiguous groups within the reference sequence, and wherem said number of ammo acid alterations is determined by multiplying the total number of amino acids in SEQ ID NO 2 by the mteger definmg the percent identity divided by 100 and then subtracting that product from said total number of ammo acids m SEQ ID NO 2, or

n_a < x_a (*a • y)»

wherein n_a is the number of amino acid alterations, x_a is the total number of ammo acids in SEQ ID NO 2 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 any non-mteger product of x_a and y is rounded down to the nearest mteger pπor to subtractmg it from x_a

By way of example, a polypeptide sequence of the present mvention may be identical to the reference sequence of SEQ ID NO 2. that is it may be 100% identical, or it may mclude up to a certain integer number of amino acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity Such alterations are selected from the group consistmg of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherem said alterations may occur at the ammo- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups withm the reference sequence The number of ammo acid alterations for a given % identity is determined by multiplying the total number of ammo acids in SEQ ID NO 2 by the mteger definmg the percent identity divided by 100 and then subtractmg that product from said total number of amino acids in SEQ ID NO 2, or

ⁿa ≤ ^xa ^" (^xa * )»

wherein n_a is the number of amino acid alterations x_a is the total number of amino acids in SEQ ID NO 2 y is, for instance 0 70 for 70%, 0 80 for 80%, 0 85 for 85% etc , and • is the symbol for the multiplication operator, and wherein any non-mteger product of x_a and y is rounded down to the nearest integer prior to subtractmg it from x_a

"lmmunologically equivalent deπvatιve(s)" as used herein encompasses a polypeptide, polynucleotide, or the equivalent of either which when used in a suitable formulation to raise antibodies m a vertebrate, the antibodies act to interfere with the immediate physical mteraction between pathogen and mammalian host

"lmmunospecific' means that charactenstic of an antibody whereby it possesses substantially greater affinity for the polypeptides of the mvention or the polynucleotides of the mvention than its affinity for other related polypeptides or polynucleotides respectively, particularly those polypeptides and polynucleotides m the pnor art

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

"Isolated" means altered "by the hand of man" from its natural state, / e , if it occurs m nature it has been changed or removed from its ongmal environment or both For example, a polynucleotide or a polypeptide naturally present m a living orgamsm is not "isolated," but the same polynucleotide or polypeptide separated from the coexistmg matenals of its natural state is "isolated" as the term is employed herem Moreover, a polynucleotide or polypeptide that is mtroduced mto an orgamsm by transformation, genetic mampulation or by any other recombinant method is "isolated" even if it is still present m said orgamsm which orgamsm may be living or non-hving

"Organιsm(s)" means a (1) prokaryote, including but not limited to, a member of the genus Streptococcus Staphylococcus, Bordetella, Corynebacterium, Mycobactenum, Neissena, Haemophύus, Actinomycetes, Streptomycetes, Nocardia Enterobacter, Yersinia Fancisella, Pasturella, Moraxella, Acinetobacter Erysipelothnx Branhamella Actinobacillus Streptobacillus, Listena, Calymmatobactenum Brucella Bacillus Clostridium Treponema Escherichia, Salmonella Kleώsiella, Vibrio Proteus Erwinia, Borreha, Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas Aeromonas, Rickettsia Chlamydia, Borreha and Mycoplasma, and further including, 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 meningitidis Staphylococcus aureus, Staphylococcus epidermidis, Corynebactenum dipthenae, Gardnerella vagina s, Mycobactenum tuberculosis Mycobactenum bovis, Mycobactenum ulcerans, Mycobactenum leprae, Actinomyctes israelu, Listena monocytogenes Bordetella pertusis, Bordatella parapertusis, Bordetella bronchiseptica, Eschenchia coli, Shigella dysentenae, Haemophilus influenzae, Haemophilus aegyptius, Haemophilus parainfluenzae Haemophilus ducreyi Bordetella, Salmonella typhi Citrobacter freundu, Proteus mirabihs Proteus vulgans Yersinia pestis Kleibsiella pneumoniae Serratia marcessens, Serratia liquefaaens Vibrio cholera Shigella dysenteni Shigellaflexnen, Pseudomonas aeruginosa, Franscisella tularensis Brucella abortis Bacillus anthracis, Bacillus cereus Clostndium perfnngens, Clostridium tetani, Clostndium botuhnum, Treponema palhdum, Rickettsia nckettsii and Chlamydia trachomitis, ( ) an archaeon, including but not limited to Archaebacter, and (m) a unicellular or filamentous eukaryote, including 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

"Polynucleotιde(s)" generally refers to any polyπbonucleotide or polydeoxynbonucleotide, 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 smgle-, double- and tnple-stranded regions, smgle- and double-stranded RNA, and RNA that is mixture of smgle- and double-stranded regions, hybnd 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 in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an ohgonucleotide. As used herein, the term "polynucleotide(s)" also includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotide(s)" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes lαiown to those of skill in the art. The term "polynucleotide(s)" as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells. "Polynucleotide(s)" also embraces short polynucleotides often refened to as oligonucleotid ).

"Polypeptide(s)" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds. "Polypeptide(s)" refers to both short chains, commonly referred to as peptides, oligopeptides and oligomers and to longer chains generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene encoded amino acids. "Polypeptide(s)" include those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art. It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the ammo acid side-chains, and the amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP- ribosylation, selenoylation, sulfation, transfer-RNA mediated addition of amino 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), Seifter et al Meth Enzymol 182626-646 (1990) and Rattan et al Protein Synthesis Posttranslational Modifications and Aging Ann N Y Acad Sci 663 48-62 (1992) Polypeptides may be branched or cyclic with or without branching Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well

"Recombinant expression system(s)" refers to expression systems or portions thereof or polynucleotides of the mvention introduced or transformed into a host cell or host cell lysate for the production of the polynucleotides and polypeptides of the mvention

"Subtraction set" is one or more, but preferably less than 100, polynucleotides compnsmg at least one polynucleotide of the invention

Vaπant(s) as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties A typical vaπant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes in the nucleotide sequence of the vanant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may 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 polypeptide differs m ammo acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the vaπant are closely similar overall and, in many regions, identical A vaπant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, additions, deletions in any combmation A substituted or inserted ammo acid residue may or may not be one encoded by the genetic code The present mvention also mcludes mclude vanants of e^ch of the polypeptides of the mvention, that is polypeptides that vary from the referents by conservative ammo acid substitutions whereby a residue is substituted by another with like charactenstics 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 prefened are vanants m which several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acids are substituted, deleted, or added m any combination A vaπant of a polynucleotide or polypeptide may be a naturally occurring such as an allehc variant, or it may be a variant that is not known to occur naturally Non- naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques by direct synthesis, and by other recombinant methods known to skilled artisans EXAMPLES

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

The polynucleotide having a DNA sequence given m Table 1 [SEQ ID NO 1] was obtained from a library of clones of chromosomal DNA of Pseudomonas aeruginosa in E coli The sequencmg data from two or more clones containing overlapping Pseudomonas aeruginosa DNAs was used to construct the contiguous DNA sequence in SEQ ID NO 1 Libraπes may be prepared by routine methods for example Methods 1 and 2 below

Total cellular DNA is isolated from Pseudomonas aeruginosa strain 4 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 m size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added Fragments are ligated mto the vector Lambda ZapII that has been cut with EcoRI the library packaged by standard 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 restπction enzymes appropriate to generate a series of fragments for cloning into library vectors (e g , Rsal, Pall, Alul, Bshl235I), and such fragments are size-fractionated accordmg to standard procedures EcoRI linkers are ligated to the DNA and the fragments then ligated mto the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E coh mfected with the packaged library The library is amplified by standard procedures Example 2 FabG Characterization

We have used chemical mutagenesis to isolate temperature-sensitive (ts) mutants m an attempt to identify essential P aerugmosa gene products Over 100 mutants, which show ts growth on complex medium at 44oC, have been isolated A genomic library containing 5 to 6 kb DNA fragments of P aeruginosa was constructed to complement these ts mutants Nucleotide sequence analysis of plasmids complementing the ts mutants revealed many known essential genes as well as genes with unknown functions. One of the ts mutants, ts-67, was shown to contain a C->T transition mutation at nucleotide position 394 in Table 1 [SEQ ID NO: l], which caused an amino acid substitution resulting in the change of arginine at position 135 in Table 1 [SEQ ID NO: 2] to cysteine in the FabG ORF. Furthermore, a revertant (i.e., restore growth at 44oC) of the fabG ts mutant was isolated and found to have the original mutation (C to T) reverted back to wild type sequence (T to C). Therefore, these results demonstrate that the fabG gene is essential for cell growth and can be used as an antimicrobial target.

Claims

What is claimed is:

1 An isolated polypeptide selected from the group consisting of

(0 an isolated polypeptide compnsmg an ammo acid having at least

(a) 70% identity,

(b) 80% identity.

(c) 90% identity, or

(d) 95% identity to the ammo acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (n) an isolated polypeptide compnsmg the ammo acid sequence of SEQ ID NO 2, (in) an isolated polypeptide which is the ammo acid sequence of SEQ ID NO 2, and (iv) a polypeptide which is encoded by a recombmant polynucleotide compnsmg the poh nucleotide sequence of SEQ ID NO 1

2 An isolated polynucleotide selected from the group consisting of

(0 an isolated polynucleotide compnsmg a polynucleotide sequence encodmg a polypeptide that has at least

(a) 70% ιdeπtm

(b) 80% identity.

(c) 90% identity, or

(d) 95% identity, to the ammo acid sequence of SEQ ED NO 2, over the entire length of SEQ ED NO 2, (u) an isolated polynucleotide compnsmg a polynucleotide sequence that has at least

(a) 70% identity

(b) 80% identity,

(c) 90% identity, or

(d) 95% identity, over its entire length to a polynucleotide sequence encodmg the polypeptide of SEQ ED

NO 2,

(in) an isolated polynucleotide compnsmg a nucleotide sequence which has at least

(a) 70% identity.

(b) 80% identity

(c) 90% identity, or