FIELD OF THE INVENTION This invention relates to newly identified polynucleotides and polypeptides, and their production and uses. as well as their variants, agonists and antagonists, and their uses. In particular. the invention relates to polynucleotides and polypeptides of the nrd as well as their variants, hereinafter referred to as"nrdG.""nrdG polazucleotide (s),"and"nrdG polvpeptide (s)." BACKGROUND OF THE INVENTION It is particularly preferred to employ Staphylococcal genes and gene products as targets for the development of antibiotics. The Staphvlococci make up a medicallv important genera of microbes.

They are known to produce two types of disease. invasive and toxigenic, Invasive infections are byabscessformationeffectingbothskinsurfacesanddeeptissues.S. aureuscharacterizedgenerally is the second leading cause of bacteremia in cancer patients. Osteomyelitis, septic arthntts. septic thrombophlebitis and acute bacterial endocarditis are also relatigvely common. There are at least three clinical conditions resulting from the toxigenic properties of Staphvlococci. The manifestation of these diseases result from the actions of exotoxins as opposed to tissue invasion and bacteremia. These conditions include: Staphylococcal food poisoning, scalded skin syndrome and toxic shock syndrome.

The frequency of Staphylococcu. # aureus 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 unconu » on to isolate Staphylocdoccus aureus 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. te drug discovery process is currently undergoing a fundamental revolution as it embraces"ftmctional genonics,"diat is, high throughput genome-or gene-based biology. This approach is rapidly superseding earlier approaches based on"positional cloning"and other methods.<BR> <P>Functional genomics relies heavily on the various tools of bioiWonnatics 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 identifv and characterize further genes and other polynucleotides sequences and their related polypeptides. as targets for drug discovery.

Clearlv. there exists a need for polynucleotides and polypeptides. such as the nrdG embodiments of the invention. that have a a present benefit of, among other things, being useful to screen

compounds for antibiotic active. 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 their antagonists and agonists to find wavs to prevent. ameliorate or correct such infection, dysfimction and disease.

Certain polypeptidesoftheinventionpossesssignificantaminoacidsequenc ehomologyte to a known Phage T4 anaerobic ribonucleoside-tripllosphate reductase protein1 see Swissprot accession number P07075.

SUMMARY OF THE INVENTION The present invention relates to nrdG. in palticulal-nrdG polypcptides and nrdG polynucleotides, recombinant materials and methods for their production. In another aspect, the invention relates to methods for using such polypeptides and poivnucleotides, including me 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 treating microbial infections and conditions associated with such infections with the identified compounds. In a still further aspect, me invention relates to diagnostic assavs for detecting diseases associatedinfectionsandconditionsassociatedwithsuchinfection s,suchasassaysmicrobial for detecting nrdG expression or activity.

Various changes and modifications within the spirit 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 nrdG polypeptides and polynucleotides as described in greater detail below. In particular, the invention relates to polypeptides aud polvnucleotides of a nrdG of Staphylococcn. s aureu. s, wluch is related by amino acid sequence homology to Phage T4 anaerobic ribonucleoside-triphosphate reductase polvpeptide. The invention relates especially to nrdG having the nucleotide and ammo acid sequences set out in Table I as SEQ ID NO: 1 and SEQ ID NO: 2 respectively.

TABLE 1 NrdG Polynucleotide and Polypeptide Sequences

(A) Staphylococcu, aureus nrdG polynucleotide sequence [SEQ ID NO: 1].

5'-ATGACACTTTTAGACATTAAACAAGGACAAGGTTATATTGCTAAAATAGAATCA AATAGC <BR> <BR> <BR> <BR> TTTGTTGACGGTGAAGGAGTAAGATGCAGTGTTTATGTATCAGGATGTCCATTTAATTGT <BR> <BR> <BR> <BR> GTTGGATGTTATAACAAAGCCTCACAAAAGTTCAGATATGGCGAGS TACACTG. nTGA.

ATATTAGCAGAAATATTAGATGATTGCGATCATGATTATATATCTGGGCTAAGTCTA TTA GGTGGCGAACCATTTTGTAATTTGGATATTACATTAAATCTTGTCAAAGCATTTCGAGCA CGTTTTGGAAATACAAAGACAATTTGGGTATGGACTGGATTTTTATATGAATATTTAGCA AATGATTGTACAGAACGTCGAGAGTTATTATCATACATTGACGTTTTAGTAGATGGTCTA TTTATACAACACTTATTCAAACCTGATTTACCATATAAAGGTTCTTTAAATCAACGCATT ATAGATGTACAACAATCACTCTCGCATGCGCGTATGATTGAATATATAGTTAGT-3' (B) Staphylococcus aureus nrdG polypeptide sequence deduced from a polwnucleotide sequence in this table [SEQ ID NO: 2]. <BR> <BR> <BR> <P>NH-MTLLDIKQGQGYIAKIESNSFVDGEGVRCSVYVSGCPFNCVGCYNKAS QKFRYGEKYTDE<BR> <BR> <BR> <BR> ILAEILDDCDHDYISGLSLLGGEPFCNLDITLNLVKAFRARFGNTKTIWVWTGFLYEYLA NDCTERRELLSYIDVLVDGLFIQHLFKPDLPYKGSLNQRIIDVQQSLSHARMIEYIVS-C OOH Deposited materials A deposit containing a Staphylococcu. ceux aureu, s WCUH 29 strain has been deposited with me National Collections of Industrial and Marine Bacteria Ltd. (herein"NCIMB") 23 St. Machar Drivê, Aberdeen AB2 1 RY, Scotland on 11 September 1995 and assigned NCIMB Deposit No. 40771, and referred to as Staphylococcus aureus WCUH29 on deposit. The, Staphylococcu. s aureus strain deposit is referred to herein as"doe deposited strain"or as"the DNA of the deposited train." The deposited strain contais the full length nrdG gene. The sequence of the plynucleotides contained in the deposited strain, as well as the ammo acid sequence of any polypeptide. encoded thereby, are controlling in the event of any conflit with any description of sequences herein.

The deposit of the deposited strain has been made under the tenns of the Budapest Treaty on the International Recognition of the Deposit of Micro-orgunisms for Purposes of Patent Procedure.

The strain will be irrevocably and without restriction or condition released to the public upon the issuance of a patent. The deposited strain is provided merely as convenience to those of skill in the art and is not an admission mat a deposit is required for enablement, such as that required under 35 U. S. C.

§112.

A license may be required to make, use or sell the deposited strain, and compounds derived therefrom, and no such license is hereby granted.

In one aspect of the invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Staphylococcus aureus WCUH 29 strain, which polypeptide is contained in the deposited strain. Further provided by the invention are nrdG polynucleotide sequences in the deposited strain. such as DNA and RNA, and anillio acid sequences encoded tereby. Also provided by the invention are nrdG polypeptide and polynucleotide sequences iso ! atcd from the deposited train.

Polypeptides NrdG polypeptide of the invention is structurally related to other proteins of the nrd family. <BR> <BR> <BR> <BR> <P> In one aspect of the invention there are provided polypeptides of Staphylococcie. Y aurelis<BR> <BR> <BR> <BR> <BR> <BR> referred to hereui as"nrdG"and"nrdG polypeptides"as well as biologically. diagmostically. prophylactically, clinically or therapeutically useful variants tereof, and compositions comprising me same.

Among the particularly preferred embodiments of me invention are variants of nrdG polypeptide encoded by naturally occurring alleles of the nrdG 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, vet more preferably at least 95% identity. most preferably at least 7-99% or exact identity, to that of SEQ ID NO : 2 over the entire length of SEQ ID NO : 2 ; (b) a polypeptide encoded by an isolated polynucleotide comprising or consisting of a polynucleotide sequence which has at least 70°S, 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'S, or exact identity to SEQ 1D NO: 1 over the entire length of SEQ ID NO: I.

(c) a polypeptide encoded by an isolated polynucleotide comprising or consisting of 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 exact identity, to the ammo 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 N0 : 2] (in particular the mature polypeptide) as well as polypeptides and fragments, particularly those which have the biological activity of nrdG, and also those which have at least 70% identity to a polypeptide of Table 1 SEQ ID NO: 1]ot the relevant portion. preferably at least 80% identity to a polypeptide of Table I [SEQ ID NO : 2and more preferably at least 90% identity to a polypeptide of Table I #SEQ ID <BR> <BR> <BR> NO : 2] and still more preferably at least 95% identity to a polypeptide of Table i [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 comprising a polypeptide of the formula: X-(R1)m-(R2)-(R3)n-Y wherein, at the amino terminus. X is hydrogel. a metal or anv other moiett described herein for <BR> <BR> <BR> <BR> modified polypeptides, and at the carboxyl terminus, Y is hydrogen, a meta) or any other moiety described herein for modified polypeptides, R1 and R3 are any amino acid residue residue modified ammo 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 fonns thereof. In the formula above, R2 is oriente so that its amino terminal amino acid residue is at the left, covalently bound to R1, and its carboxy terminal amino acid residue is at the right, covalently bound to R3. Any stretch of amino acid residues denoted by either R1 or R3. where m and/or n is greater man 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 Staphylococcus aureus, however, it may preferably be obtained from other organisms of the same taxonomic genus. A polypeptide of me invention may also be obtained for example, from organisms of the same taxonomic familv or order.

A fragment is a variant polypeptide having an amino acid sequence that is entirety the same as part but not all of any amino acid sequence of any polypeptide of the invention. As va ith nrdG polypeptides, fragments may be "free-standing," or comprised 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, troncation polypeptides having a portion of an amino acid sequence of Table 1 [SEQ ID NO : 2], or of variants thereof, such as a contuiuous series of residues that includes an amino-and/or carboxyl-terminal amino acid sequence. Degradation fonts of the polypeptides of the invention produced by or in a host cell, particularly a Staphylococcus aureus, are also preferred. Further preferred are fragments characterized by structural or fimctional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions. beta-sheet and beta- sheet-formulg 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.

Also preferred are biologically active fragments which are those fragments that mediate activities of nrdG, 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 fonction essential for viability of Staphylococcus aureus 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 polypeptidebycorrespondingfull-length peptide thesevariantsmaybecmployedtherefore, as intennediates 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 polvnucleotides that encode nrdG pol\pcptides. particularly polynucleotides that encode trie polyeptide hereun designated nrdG. hi a particularly preferred embodiment of the invention the polynucleotide comprises a region encoding nrdG polypeptides comprising a sequence set out in Table 1 [SEQ ID NO: 1] which includes a full length gene, or a variant thereof. The Applicants believe that mis full length gene is essential to the groin and/or survival of an organism which possesses it, such as Staplivlococcus aureus.

As a further aspect of the uivention there are provided isolated nucleic acid molecules encoding and/or expression nrdG polypeptides and polynucleotides, particularly Staphylococcus polypeptidesandplynucleotides,including,forexample,unprocess edRNAs.aureusnrdG mRNAs,cDNAs,genomicDNAs,B-andZ-DNAs.Furtherembodimentsofribo zymeRNAs, the invention include biologically, diagnostically, 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 nrdG polypeptide having a deduced ammo acid sequence of Table 1 [SEQ ID NO: 2] and polynucleotides closely related thereto and variants thereof.

In another particularly preferred embodiment of the invention there is a nrdG polypeptide from Staphylococcuzs aurez comprising or consisting of an amino acid sequence of Table I [SEQ ID NO : 2]. or a variant thereof.

Using the information provided herein, such as a polvnucleotide sequence set out in Table 1 [SEQ ID NO: 1], a polynucleotide of the invention encoding nrdG polypeptide may be obtained using standard cloning and screening methods, such as those for clou'zig and sequencing chromosomal DNA fragments from bacteria using Staphylococcus aureus WCUH 29 cells as starting material, followed by obtalling a full length clone. For example, to obtain a polynucleotide sequence of the invention. such as a polvnucleotide sequence given in Table 1 [SEQ ID NO : 1], typically a library of clones of chromosomal DNA of Staphylococcs aureus WCUH 29 in E. coli or some other suitable host <BR> <BR> <BR> <BR> is probed with a radiolabeled oligonucleotide. preferably a 17-mer or longer_ deriv ed from a partial sequence. Clones carrying DNA identical to that of the probe can then be distinguished using stringent hybridization conditions. By sequencing the individual clones thus identified bN hybridization with sequencing primers designed from the original polypeptide or polynucleotide sequence it is then possible to extend the polynucleotide sequence in both directions to determine a full length gene sequence. Conveniently, such sequencing is performed, for example. using denatured double stranded DNA prepared from a plasmid clone. Suitable techniques are described bv Maniatis, T., Fritsch, E. F. and Sambrook et al.. MOLECULAR CLONING. A LABORATORYMANUAL, 2nd Ed. ; Cold Spring Harbor Laboratory Press. Cold Spring Harbor. Ne\ York (1989). (see in particular Screening By Hybridization 1.90 and Sequencing Denatured Doubie-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 in Table I [SEQ ID NO: 1] was discovered in a DNA library derived from Staphylococcus aureus WCUH 29. <BR> <BR> <BR> <BR> <BR> <P> Moreover, each DNA sequence set out in Table 1) SEQ ID NO:) <BR> <BR> <BR> <BR> <BR> <BR> frame encoding a protein having about the number of ammo acid residues set forth in Tab c 1 NO : 2] with a deduced molecular weight that can be calculated using 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 535 of SEQ ID NO : 1, encodes the polypeptide of SEQ ID NO : 2.

In a further aspect, the present invention provides for an isolated polynueleotide 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 preferably at least 97-99% or exact identity to SEQ ID NO: I over the entire length of SEQ ID NO : 1;

(b) a polynucleotide sequence encoding a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, vet 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 invention, including homologs and orthologs from species other than Staphylococcu. s aurezzx, may be obtained by a process which comprises the steps of screening an appropriate library under stringent hybridization conditions with a labeled or detectable probe consisting of or comprising the sequence of SEQ ID NO: 1 or a fragment thereof: and isolating a full-length gene and/or genomic clones containing said polvnucleotide sequcnce.

The invention provides a polynucleotide sequence identical over its entire length to a coding sequence (open reading frame) in Table 1 [SEQ ID NO: 1]. 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-protem sequence. The polynucleotide of the invention may also contain at least one non-coing sequence. including for example, but not limited to at least one non-coding 5' and 3' sequence, such as the transcribed but non- translated signals(suchasrho-dependentandrho-independentterminationterm ination signals), ribosome binding sites, Kozak sequences, sequences that stabilize mRNA, cintrons, and polyadenylation signals. The polynucleotide sequence may also comprise additional coding sequence encoding additional amino acids. For example, a marker sequence that facilitates purification of the fused polypeptide can be encoded. hi certain embodiments of me invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen. lnc.) and described in Gentz cl al., Proc.USA86:821-824(1989),oranHApeptidetag(Wilsonetal..Cell3@ :767Acad., (1984), both of which may be useful in purifying polypeptide sequence fused to them. Polynucleotides of the invention also include, but are not limited to, polynucleotides comprising a structural gene and its naturally associated sequences that control gene expression.

A preferred embodiment of the invention is a polynucleotide of consisting of or comprising nucleotide 1 to the nucleotide immediately upstream of or including nucleotide 535 set forth in SEQ ID NO: 1 of Table 1, both of which encode the nrdG polypeptide.

The invention also includes a polvnucleotide consisting of or comprising a polynucleotide of the fonnula: X-(E1)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 detines a covalent bond, and at the 3'end of the molecule. Y is hydrogel, a metal.

or a modified nucleotide residue. or together with X defines the covalent bond. each occurrence of RI and R3 is independently any nucleic acid residue or modified nucleic acid residue m is an integer between 1 and 3000 or zero, n is an integer between ! and 3000 or zero. and R2 is a nucleic acid sequence or modified nucleic acid sequence of the invention, particularly a nucleic acid sequence selected from Table 1 or a modified nucleic acid sequence thereof. In the polynucleotide formula above, R2 is oriented so that its 5'end nucleic acid residue is at the left, bound to R1, and its 3'end nucleic acid residue is at the right, bound to R3. Any stretch of <BR> <BR> <BR> <BR> nucleic acid residues denoted by either R1 and/or R2, where m and/or n is greater than). may be either a heteropolymer or a homopolymer. preferably a heteropoiymer. Where, in a preferred <BR> <BR> <BR> <BR> embodiment, X and Y together define a covalent bond. the poiynucteotide of the above formuta 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 integer between 1 and 1000. Other preferred embodiments of the <BR> <BR> <BR> <BR> 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 polvnucleotide of the invention is derived from, Staphylococcus aureus, however, it may preferably be obtained from other organisms of me same taxonomic genus. A polynucleotide of me invention may also be obtained, for example, from organisms of the same taxonomic family or order.

The temn"polynucleotide encoding a polypeptide"as used herein encompasses pohnucteotides that include a sequence encoding a polypeptide of the invention, particularly a bacterial polnpeptide and more jparticularly a polypeptide of the Staphylococcus aureus nrdG having an amino acid sequence set out in Table 1 [SEQ ID NO : 2 j. The term also encompasses poivnucieotidcs that mctudc a single continuous region or discontinuous regions encoding the polypeptide (for example, polynucleotides interrupted by integrated phage, an integrated insertion sequence-an integrated vector sequence, an integrated transposon sequence, or due to RNA editing or genomic DNA reorganization) together with additional regions, that also may contain coding and/or non-coding sequences.

The invention further relates to variants of the @ polynucleotides described herein that encode variants of a polypeptide having a deduced ammo acid sequence of Table l [SEQ ID NO : 2l.

Fragments of a poiynucleotides of the invention ma be used. for example. to synthesize full-length theinvention.polynucleotidesof Further particularly preferred embodunents are polyncleotides encoding nrdG variants, that have the amino acid sequence of nrdG 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 preferred among these are silent substitutions. additions Sand deletions. that do not alter the properties and activities of nrdG pol5peptide.

Further preferred embodiments of the invention are polynucleotides that are at least 70% identical over their entire length to a polynucleotide encoding nrdG polypeptide having an amino acid sequence set out in Table I [SEQ ID NO : 2], and polynucleotides mat are complementaw to such polynucleotides. Alternatively, most highly preferred are polynucleotides that comprise a region that is at least 80% identical over its entire length to a polynucleotide encoding nrdG polvpeptide and polynucleotides Inthisregard,polynucleotidesatleast90%identicaloverthereto. their entire thesameareparticularlypreferred,andamongtheseparticularlypre ferredto polynucleotides, those with at least 95% are especially preferred. Furthermore, those with at least 97% are highly preferred among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99% being the more preferred.

Preferred embodiments are polynucleotides encoding polypeptides that retain substantially thc same biological function or activity as the mature polypeptide encoded by a DNA of Table 1 [SEQ ID NO: 1].

In accordance with certain preferred embodiments of this invention there are provided polynucleotides that hybridize, particularly under stringent conditions, to nrdG polynucleotide sequences, such as those polynucleotides in Table 1.

The invention further relates to polynucleotides that hybridize to the polynucleotide sequences provided herein. In this regard, the invention especially relates to polynucleotides that hybridize under stringent conditions to the polynucleotides described herein. As herein used, the tenns rlstringent "stringenthybridizationconditions"meanhybridizationoccurring onlyifthereisatcondition"and 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 (I50mM NaCI, 15mM trisodium citrate), 50 mM sodium phosphate <BR> <BR> <BR> <BR> (pH7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml of denatured, sheared salmon spernz 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 cvl..

Molecular Cloning: A Laborator Manual. Second Edition, Cold Spring Harbor. N. Y.. (1989), particularly Chapter 11 therein. Solution hybridization may also be used with the polynucleotide sequences provided by the invention.

The invention also provides a polynucleotide consisting of or comprising a polynucleotide sequence obtained by screening an appropriate library containing the complete gene for a polynucleotide sequence set forth in SEQ ID NO: 1 under stringent hybridization

conditions with a probe having the sequence of said polynucleotide sequence set forth in SEQ ID NO : I or a fragment thereof ; and isolating said polvnucleotide sequence. Fragments useful for obtaining such a polynucleotide include, for example, probes and primers fully described elsewhere herein.

As discussed elsewhere herein regarding polvnucieotide assays of me invention, for instance. the polynucleotides of the invention, may be used as a hybridization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding nrdG and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to the nrdG gene. Such probes generaiiv wiltcompriseat least 15 nucleotide residues or base pairs.

Preferably, such probes will have at least 30 nucleotide residues or base pairs and may have at least 50 <BR> <BR> <BR> <BR> nucleotide residues or base pairs. Particularly preferred probes will have at least 20 nucleotidc residues or base pairs and will have lee than 30 nucleotide residues or base pairs.

A coding region of a nrdG gene may be isolated by screening using a DNA sequence provided in Table 1 [SEQ ID NO: 1] to syndiesize an olignoucleotide probe. A labeled oligonucleotide having a sequence complementary to that of a gene of the invention is then used to screen a library of cDNA. genomic DNA or mRNA to determine which members of the library the probe hvbridizes to.

There are several methods available and well known to those skilled in 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 MarathonT teCllllology (Clontech Laboratories Inc.) for example, have significantly simplified the search for longer cDNAs. In the MarathonT" technology. cDNAs have been prepared from mRNA extracted from a chosen tissue and an'adaptor'sequence ligated onto each end. Nucleic acid amplification (PCR) is then carried out to amplify the"missing"5'end of the DNA using a combination of gene specific and adaptor specific oligonucleotide primers. The PCR reaction is then repeated using <BR> <BR> <BR> <BR> "nested"primers, that is, primers designed to ameal within the amplified product (typically an adaptor specific primer that anneals further 3'in the adaptor sequence and a gene specific primer that anneals further 5'in the known gene sequence). The products of this reaction can then be @ analyzed by DNA sequencing and a full-length DNA constnicted either by joining the product directly to the existing DNA to give a complete sequence, or carrying out a separate full-lengtl PCR using the new sequence information for the design of the 5'primer.

The polynucleotides and polypeptides of the invention may be employed, for example, as research reagents and materials for discovery of treatments of and diagnostics for diseases, particularly human diseases. as further discussed herein relating to poiynucieotide assays.

The polynucleotides of the invention that are oligonucleotides derived from a sequence of Table 1 [SEQ ID NOS: 1 or 2] may be used in the processes herein as described, but preferably for PCR. to determine whether or not the polynucleotides identified herein in whole or in part are transcribed in bacteria in infected tissue. It is recognized that such sequences will also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained.

The invention also provides polynucleotides that encode a polypeptide that is the mature protein plus additional ammo or carboxyl-tenninal amino acids, or amino acids interior to the mature polypeptide (when the mature fonn has more than one poiypeptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protein transport, orshortenproteinhalf-lifeormayfacilitatemanipulationofaprote inlengthen for production,amongotherthings.Asgenerallyisthecaseinvivo,thead ditionalaminoor acids may be processed away from the mature protein bv cellular enzymes.

For each and every polynucleotide of me invention mere is provided a polynucleotide complementary to it. It is preferred that these complementary polynucleotides are fullv complementary to each polynucleotide with which they are complementary.

A precursor protein, having a mature fonn of the polypeptide fused to one or more <BR> <BR> <BR> <BR> prosequences may be an inactive fonn of the polvpeptide. When prosequences are removed such inactive precursors generally are activated. Some or all of me 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 invention."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 in combination 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 invention may encode a mature protein, a mature protein plus a leader sequence (which may be referred to as a preprotein), a precursor of a mature protein having one or more prosequences that are not the leader sequences of a preprotelli. or a preproprotem. which is a precursor to a proproteui. having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature fonns of the polypeptide.

Vectors, Host Cells, Expression Systems The invention also relates to vectors that comprise a polynucleotide or polynucleotides of me invention, host cells that are genetically engineered with vectors of the invention and the production of

polypeptides of the invention by recombinant techniques. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from me DNA constructs of the invention.

Recombinant polypeptides of me present invention may! be prepared by processes well known in those skilled in the art from genetically engineered host cells comprising expression systems.

Accordingly, in a further aspect, the present invention relates to expression systems which comprise a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression systems, and to the production of polypeptides of the invention bs recombinant techniques.

For recombinant production of the potypeptides of the invention, host cells can be genetically engineered to incorporate expression systems or portions thereof or polvnucleotides of the invention.

Introduction of a polynucleotide into the host cell can be effected by methods described in many standard laboratorv manuals, such as Davis. et al., BASIC METHODS IN MOLECULAR BIOLOGY.

(1986) and Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection. microinjection, cationic lipid-mediated transfection. electroporation, transduction, scrape loading, ballistic introduction and infection.

Representative examples of appropriate hosts include bacteria) celis-such as cells of streptococci, E.coli,streptomyces,cyanobacteria,Bacillussubtilis,andentero cocci fungalcells,suchascellsofayeast,Kluveromyces,Saccharomyces,a Staphylococcusaureus; basidiomycete, Candida albicans s and Aspergillus; insect cells such as cells of Di-o, sophila 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 ofagvmnosperm or angiospenn.

A great variez of expression systems can be used to produce the polypeptides of the invention. Such vectors include, among others, chromosomal-, episomal-and virus-derived vectors, for example, vectors derived from bacterial plasmids, from bacteriophage. from transposons. from yeast episomes, from insertion elements, from yeast chromosomal elements, from viroses such as baculoviruses, papova viruses, such as SV40, vaccina viruses ; adenoviruses. fowl pox viruses. pseudorabies viruses, picomaviruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and expressionsystemconstructsmaycontaincontrolregionsthatregula teaswellasphagemides.The engender expression. Generally, any system or vector suitable to maintain. propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression in this regard.

The appropriate DNA sequence may be inserted into me expression svstem by an ! of

known and routine techniques, such as, for example, those set forth in Sambrook et al., MOLECULAR LABORATORYMANUAL,(supra).CLONING,A In recombinant expression systems in eukaryotes, for secretion of a translated protein into the lumen of the endoplasmic reticulum, into the periplasmin space or into the extracellular environment. appropriate secretion signals may be incorporated into the expressed polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.

Polypeptides of the invention can be recovered and purified from recombinant cell culturels b well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatograpliy, phosphocellulose chromatography, hydrophobic interaction chromatography. affinit) chromatography-hvdroxvlapatite chromatography. and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification Diagnostic, Prognostic, Serotyping and Mutation Assays This invention is also related to the use of nrdG polsnnucleotides and polypeptides of the invention for use as diagnostic reagents. Detection of nrdG polynucleotides and/or polypeptides in a amammal,andespeciallyahuman,willprovideadiagnosticmethodfor. eukaryote,particularly diagnosis of disease, staging of disease or response of an infectious organism to drugs. Eukaryotes. particularly mammals, and especially humans, particularly those infected or suspecte to be infected with an organism comprising the nrdG gene or protein, may be detected at the nucleic acid or amino acid level by a variety of well known techniques as well as by methods provided herein.

Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putatively infected md/or illfected individual's bodily materials. Polynucleotides from any of these sources. particularly DNA or RNA, may be used directly for detection or may be amplified enzymatically by using PCR or any other amplification technique prior to analysis. RNA, particularly mRNA, cDNA and genomic DNA may also be used in the same ways. Using amplicication. characterization of the species and strain of infectious or resident organism present in an individual, may be made by an analysis of the genotype of a selected polynucleotide of the organism. Deletions and insertions can be detected by a change in size of the amplified product in comparison to a genotype of a reference sequence selected from a related organism, preferably a different species of the same genus or a different strain of the same species. Point mutations can be identified by hybridizing amplified DNA to labeled nrdG polynucleotide sequences. Perfectly or significantly matched sequences can be distinguished from imperfectly or more significantly 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 in die <BR> <BR> <BR> <BR> electrophoretic mobility of polynucleotide fragments in gels as compared to a reference sequence. This may be carried out with or without denaturing agents. Polyllucleotide differences may also be detected by direct DNA or RNA sequencing. See, for example, Myers el al., Science, 230: 1242 (1985).

Sequence changes at specific locations also may be revealed by nuclease protection assays. such as Rnase, V1 and S1 protection assay or a chemical cleavage method. See. for example. Cotton e/al Proc. Sci.,USA,85:4397-4401(1985).Acad.

In another embodiment, an array of oligonucleotides probes comprising nrdG 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 applicabiliy and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see. for examplc. Chce e al., Science, 274: 610 (1996)).

Thus in another aspect, the present invention relates to a diagnostic kit which comprises: (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 antibody to a polypeptide 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 comprise a substantial component. Such a kit will be of use in diagnosing a disease or susceptibility to a Disease, among others.

This invention also relates to the use of polynucleotides of the present invention as diagnostic reagents. Detection of a mutated form of a polynucleotide of the invention, preferable. SEQ ID NO: 1, which is associated with a disease or pathogenicity will provide a diagnostic tool mat can add to. or define, a diagnosis of a disease, a prognosis of a course of disease, a deternuination of a stage of disease, or a susceptibility to a disease, which results from under-expression. over-expression or altered expression of the polynucleotide. Organisms, particularly infectious organisms, carrying mutations in such polynucleotide may be detected at the plynucleotide level by a variety of techniques, such as those described elsewhere herein.

The nucleotide sequences of the present invention are also valuable for organism chromosome identification. The sequence is specifically targeted to, and can hybridize with, a particular location on

an organism's chromosome, particularly to a Staphyiococcus aureus chromosome. The mapping of relevant sequences to chromosomes according to me present invention may be an important step in correlating those sequences with pathogenic potential and/or an ecological niche of an organism 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-line in a sequence database. The relationship between genes and diseases khat have been mapped to the same chromosomal region are then identified through known genetic methods, for example. through linkage analysis (coinheritance of physically adjacent genes) or mating studies.

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

Cells from an organism carrying mutations or potymorphisms (allelic variations) in a polynucleotide and/or polypeptide of the invention may also be detected at the polynucleotide or polypeptide level by a variety of techniques, to allow for serotxping. for example. For example. RT- PCR can be used to detect mutations in me RNA. It is particularly preferred to use RT-PCR in conjunction with automated detection systems, 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 plynucleotide encoding nrdG polypeptide can be used to identify and analyze mutations. These primers may be used for, among other drings. amplifying nrdG DNA and/or RNA isolated from a sample derived from an individual, such as a bodily material. The primers may be used to amplify a pol) nucleotide isolated from an infected individual. such that the polynucleotide may the be subject to various techniques for elucidation of the polynucleotide sequence. In mis way, mutations in 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 ivention further provides a process for diagnosing, disease. preferably bacteria) infections, more preferably infections caused by Staphylococcus aureus, comprising determining from a sample derived from an individual. such as a bodily material, an increased level of expression of polynucleotide having a sequence of Table 1 [SEQ ID NO: 1]. lncreased or decreased expression of a nrdG polynucleotide can be measured using any on of the methods well known in the art for the quantitation of polvnucleotides. such as. for example. amplification.

PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and other hybridization methods.

In addition. a diagnostic assay in accordance with the invention for detecting over-expression of nrdG polx peptide compared to normal control tissue samples may be used to detect the presence of an infection, for example. Assay techniques mat can be used to determine Icvcls of a nrciG polypeptide, in a sample derived from a host, such as a bodily material, are wen-known to those of skill in the art. Such assay methods include radiouiununoassays, competitive-binding assays. Western Blot analysis, antibody sandwich assays, antibody detection and ELISA assays.

Differential Expression The polynucleotides and poiynucleotides of me invention may be used as reagents for differential screening methods. There are mam-differential screening and differential displav methods known in the art in which the polynucleotides and polypeptides of me invention may be used. For <BR> <BR> <BR> <BR> example, the differential display technique is described by Chuang el cil., J. Bticteriol.

175 : 2026-2036 (1993). This method identifies those genes which are expressed in an organism by identifying mRNA present using randomly-primed RT-PCR. By comparing pre-infection 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 (TVET) is described by Camilli et al.. Proc. Nat'l. Acad.

Sci. USA. 91 : 2634-2638 (1994). IVET identifies genes up-regulated during infection when compared to laboratory cultivation, implying an important role in infection. ORFs identified by this technique are implied to have a significant role in infection establishment and/or maintenance. In this technique random chromosomal fragments of target organism are cloned apromoter-lessrecombinasegeneinaplasmidvector.Thisconstructi sintroducedupstreamof into the target organism which carries m antibiotic resistance gene flanked by reso) vase sites.

Growth in the presence of the antibiotic removes from the population those fragments cloned into the plasmid vector capable of supporting transcription of the recombinase gene and therefore have caused loss of antibiotic resistance. The resistant pool is introduced into a host and at various times after infection bacteria may be recovered and assessed for the presence of antibiotic resistance. The chromosomal fragment carried by each antibiotic sensitive bacterium should carry a promoter or portion of a gene normally uprcgu) ated 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 polynucleotides of the invention, messenger RNA is isolated from bacterial infected tissue, e. g..

48 hour murine lung infections, and the amount of each mRNA species assessed by reverse

transcription of the RNA sample primed with random hexanucleotides followed by PCR with gene specific primer pairs. The detennination of the presence and amount of a particular mRNA species by quantification of the resultant PCR product provides information on the bacterial genes which are transcribed in the infected tissue. Analysis of gene transcription can be carried out at different times of infection to gain a detailed knowledge of gene regulation in bacterial pathogenesis allowing for a clearer understanding of which gene products represent targets for screens for antibacterials. Because of the gene specific nature of the PCR primers employed it should be understood that the bacterial 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 in the bacterium (in the order of 2 minute halflives). Optimallv the bacterial mRNA is prepared from infected murine lung tissue by mechanical disruption in the presence of TRIzole (GIBCO-BRL) for verx short periods of time, subsequent processing z ording to the manufacturers of TRIzole reagent and DNAase treatment to remove contaminating DNA. Preferablv the process is optimized by finding those conditions which give a maximum amount of Staphylococcus aureus 16S ribosomal RNA as detected by probing Northerns with a suitably labeled sequence specific oligonucleotide probe.

Typically a 5'due labeled primer is used in 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 using GeneScanner (manufactured by ABI).

Antibodies The polypeptides and polynucleotides of the invention or variants thereof. or cells expressing the same can be used as immunogens to produce antibodies immunospecific for such polypeptides or polynucleotides respectively.

In certain preferred embodiments of the invention there are provided antibodies against nrdG polypeptides or poinlilucleotides.

Antibodies generated against the polypeptides or polynucleotides of the invention can be obtained by administering the polypeptides and/or polvnucleotides of the invention, or epitope-bearing fragments of either or both. analogues of either or both. or cells expressing either or both. to an animal, preferably a nonhumm. using routine protocols. For preparation of monoclonal antibodies. any technique known u1 the art that provides antibodies produced bv continuousceltlineculturescanbe used. Examples include various techniques, such as those in Kolller. G. and Milstein. C.. Nature 256: 495-497 (1975)-, Kozbor et al., Immunology Today 4: 72 (1983): Cole et al., pg. 77-96 in MONOCLONASL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, hic. (1985).

Techniques for the production of single chain antibodies (U. S. Patent No. 4. 946.778) can be adapted to produce single chain antibodies to polypeptides or polynucleotides of this invention. Also. transgenic mice, or other organisms such as other mammals. may be used to express humanized antibodies immunospecific to me polypeptides or polynucleotides of the invention.

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-nrdG or from naive libraries (McCafferty, etal. (1990). Nature 348,552-554 ; Marks, et al., (1992) Biotechnology 10, 779- 783). The affinity of these antibodies can also be improved bv. for example. chain shuffling (Clackson et al.. (1991) Nature 352: 628).

The above-described antibodies may be employed to isolate or to identify clones expressing the polpeptides or polynucleotides of the invention to purify me polypeptides or polwucleotides by. for example. affinity chromatography.

Thus. among others, antibodics against nrdG-polypeptide or nrdG-polvnucleotide may be employed to treat infections, particularly bacteria) infections.

Polypeptide variants include antigenically, epitopically or immunologically equivalent variants form a particular aspect of this invention.

A polypeptide or plynucleotide of the invention, such as an antigenically or immunologically equivalent derivative or a fusion protein 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 conjugatiol1 with an imrnunogenic carrier protein for example bovine serum albumin, keynote iimpet haemocyanin or tetanus toxoid. Alternatively, a multiple antigenic polypeptide comprising multiple copies of the polypeptide, or an antigenically or immunologically equivalent polypeptide thereof may be sufficiently antigenic to improve immunogenicity so as to obviate the use of a carrier.

Preferably. the antibody or variant thereof is modified to make it less immunogenic in the individual. For example, if the individual is human the antibody may most preferably be "humanized,"where the complimentarity determining region or regions of the hybridoma-derivcd antibody has been transplanted into a human monoclonal antibody, for example as described in Jones et al. (1986). Nature 321, 522-525 or Tempest et cll, (1991) Biotechnology 9, 266-173.

In accordance with an aspect of the invention, there is provided the use of a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic theparticularlypreferredembodimentsoftheinventionarenaturall yoccurringimmunizatyion.Among allelic variants of nrdG polynucleotides and polypeptides encoded thereby.

The use of a polynucleotide of the invention in genetic inununization will preferably employ a suitable delivery method such as direct injection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet (1992) 1: 363, Manthorpe et al., Hum. Gene Ther. (1983) 4: 419), deliverv of DNA complexe with specific protein carriers (Wu et al., J Biol Chem. (1989) 264 : 16985). coprecipitation of DNA with calcium phosphate (Benvenisty & Reshef, PNAS USA, (1986) 83: 9551), encapsulation of DNA in various forms of liposomes (Kaneda el al.. Science (1989) 243: 375), particle bombardment (Tang et al.. Nature (1992) 356:152. Eisenbraun et al., DNA Cell Biol (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 polynucleotidesoftheinventionmayalsobeusedtoassessthebinding ofPolypeptidesand small molecule substrates and ligands in, for example, cells. cell-free preparations. chemical libraries. <BR> <BR> <BR> <BR> and natural product mixtures. These substrates and ligands may be natural substrats and ligands or may be structural or functionalmimetics.See;.,Coligan/.,CM/'//7-'/'/c/.s';M//??/? </-?/(' 5(1991).1(2):Chapter Polypeptides and polynucleotides of the present invention are responsible for many biological functions, including many disease statues, in particular the Diseases hereinbefore mentioned. It is therefore desirable to devise screening methods to identify compounds which stimulante or which inhibait the f mction of the polypeptide or polynucieotide. Accordingly, in a further aspect, me present invention provides for a method of screening compounds to identify those which stimulate or which mhibit the function of a polypeptide or polynucleotide of the invention, as well as related poypeptides and polynuc) eotides. hi general, agonists or antagonists may be employed tor therapeutic and prophylactic purposes for such Diseases as ilereulbefore 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 nrdG polypeptides and polynucleotides ; or may be structural or functional mimetics thereof (see Coligan et al., Current Protocole in Immurroloy 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 direct 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 bv activation or inhibition of the polvpeptidc or

polynucleotide, using detection systems appropriate to the cells comprising the polypeptide or polynucleotide. IWibitors 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 iWibition 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 fonn a mixture, measuring nrdG polypeptide and/or polynucleotide activity in the mixture, and comparing the nrdG polypeptide and/or polynucleotide activity of the mixture to a standard. Fusion proteins_ such as those made from Fc portion and nrdG polypeptide. as hereinbefore described. can also bc used for high-throughput screening assays to identify antagonists of the poiypeptide of the present invention, as well as of phylogenetically and and/or functionally related polypeptides (see D.

Bennett et al, J Mol Recognition, 8: 52-58 (1995); and K. Johanson et al., J Biol Chem, 270 (16): 9459-9471 (1995)).

The polymucleotides, 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 polypeptidc in cells.

For example, an ELISA assay may be constructed for measuring secreted or cell associated levels of poiypeptide using monoclonal and polyclonal antibodies by standard methods known in 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 invention also provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) trie action of nrdG polypeptides or polvnucleotidcs. particularlv those compounds that are bacteriostatic and/or bacteriocidal. The method of screening may involve high- <BR> <BR> <BR> <BR> throughput techniques. For example, to screen for agonists or antagonists, a sniitlietic reaction mix. a cellular compartment. such as a membrane, cell envelope or cell wall, or a preparation of any thereof. comprising nurG polypeptide and a labeled substrate or ligand of such poiypeptide is incubated in the absence or the presence of a candidate molecule that may be a nrdG agonist or antagonist. The ability of the candidate molecule to agonize or antagonize the nrdG polypeptide is reflected in decreased binding of the labeled ligand or decreased production of product from such substrate Molecules that bind gratuitously, i. e.. without induculg the effects of nrdG polypeptide are most likely to be good antagonists. Molecules that bind well and, as the case may be, increase me rate of product production

from substrate. increase signal transduction, or increase chemical chamiel activity are agonists.

Detection of the rate or level of. as the case may be. production of product from substrate. signal transduction, or chemical channel activity mav be enhanced bv using a reporter system. Reporter systems that may be useful in this regard include but are not limited to colorimetric. labeled substrate converted into product, a reporter gene that is responsive to changes in nrdG polynucleotide or polypeptide activity, and binding assays known in the art.

Polypeptides of the invention may be used to identify membrane bound or soluble receptors, if any, for such polypeptide. through standard receptor binding techniques known in the art. These techniques include, but are not limited to. ligand binding and crosslinking assays in which the polypeptide is labeled with a radioactive isotope (for instance, 125I), 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 supematants. tissue extracts, bodily materials). Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy. These screening methods may also be used to identify agonists and antagonists of the polypeptide which compete with the binding of the polypeptide to its receptor (s), if any. Standard methods for conducting such assays are well understood in the art.

In oftheinventionthereareprovidedmethodsforidentifyingcompounds embodiments which bind to or othenvise interact wnith and inhibit or activate an activity or expression of a polypeptide and/or polynucleotide of the invention comprising : contactai a polypeptide md/or polynucleotide of the invention with a compound to be screened under conditions to permit binding to or other interaction between the compound and the polypeptide and/or polynucleotide to assess the binding to or other interaction with the compound, such binding or interaction preferably! beulg associated with a second component capable of providing a detectable signa) in response to the binding or interaction of me polypeptide and/or polynucleotide with the compound: and determining w hcther the compound binds 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 binding or interaction of the compound with the polypeptide and/or polynucleotide.

Another example of an assay for nrdG agonists is a competitive assai that combines nrdG and a potential agonist with nrdG-bindulg molecules, recombinant nrdG binding molecules, natural substrats or ligands, or substrate or ligand minetics, under appropriate conditions for a competitive inhibition assay. NrdG can be labeled. such as by radioactivity or a colorimetric compound, such that the number of nrdG molecules bound to a binding molecule or converted to product can be detennined accurately to assess me effectiveness of the potential antagonist.

Potential antagonists include, among otllers, small organic molecules. peptides. polypeptides and antibodies that bind to a polynucleotide and/or polypeptide of the invention and thereby inhibit or extinguish its activity or expression. Potential antagonists also may be small organic molecules. a peptide, a poiypeptide such as a closely related protein or antibody that binds the same sites on a binding molecule, such as a binding molecule, without uiducing nrdG-uiduced activities, tllereby preventing me action or expression of nrdG polnpeptides and/or polvnucleotides by excluding nrdG polypeptides and/or polynucleotides from binding. <BR> <BR> <BR> <BR> <P> Potentialantagonistsinc)udeasmal!moleculetliatbindstoandoccu piesthebindingsiteof<BR> <BR> <BR> <BR> <BR> <BR> the OlyeLlC TIeT'e17'7CeVeIltll7f.T blllCllll t0 C ; Ill111' blI7C117 1110C ; CLII ; S. SLICI L71C 17011111IOOTICI activity is prevented. Exemples of small molecules include but are not limited to smalt organic molecules, peptides or peptide-like molecules. Other potential antagonists include antisense molecules (see Neurochem.56:560(1991);OLIGODEOXYNUCLEOTIDESASANTISENSEJ.

INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton. FL (1988), for a description of Preferredpotentialantagonistsincludecompoundsrelatedtoandvar iantsofnrdG.thesemolecules).

Other examples of potential polypeptide antagonists include antibodies or. in some cases. <BR> <BR> <BR> <BR> oligonucleotides or proteins which are closely related to the ligands. substrates. receptors, enzymes.<BR> <BR> <BR> <BR> <BR> <BR> etc., as the case may be, of the polypeptide, e. g., a fragment of the ligands. substrates. receptors. enzymes, etc.; or small molecules which bind to the polypeptide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented.

Thus. in another aspect, the present invention relates to a screening kit for identifying agonists. antagonists. ligands, receptors, substrates, enzymes, etc. for a polvpeptide 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 invention: which polypeptide is preferablv 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 comprise a substantial component.

It will be readily appreciated bv the skilled artisan that a polypeptide and/or polynucleotide of the present invention mav also be used in a method for the structure-based design of an agonist. antagonist or inhibitor of the polypeptide and/or polynucleotide. by :

(a) determining in the first instance the three-dimensional structure of the polvpeptide andUor polynucleotide, or complexes thereof ; (b) deducing the three-dimensional structure for the likely reactive site (s), binding site (s) or motif (s) of an agonist, antagonist or inhibitor : (c) synthesizing candidate compounds that are predicted to bind to or react with the deduced binding site (s), reactive site (s), and/or motif (s): and (d) testing whether the candidate compounds are indeed agoniste antagonists or inhibitors.

It will be further appreciated that this will nonnally be an iterative process, and this iterative process may be perfonned using automated and computer-controlled steps.

In a furtlier aspect, the present invention provides methods of treating abnonnal conditions such as. for instance, a Disease, related to either an excess of. an under-expression of. an elevated activity of, or a decreased activity of nrdG polypeptide and/or polynucleotide.

If the expression and/or activity of the polvpeptide and/or pofynucleotidc is in excess, several approaches are available. One approach comprises administering to an individual in need thereof an mhibitor compound (antagonist) as herein described, optionally in combination with a pharmaceuticaliy acceptable carrier, in an amount effective to inhibait the fonction and/or expression of the polypeptide and/or polynucleotide. such as. for example, by blocking the baiduig of ligands, substrates. receptors. enzymes, etc., or by inhibiting a second signal, and thereby alleviatuig the abnormal condition. In another approach, soluble fonts of the polypeptides still capable of binding the ligand. substrate. enzymes, receptors, etc. in competition with endogenous polypeptide and/or poh-nucleotide may be administered. Typical examples of such competitors include fragments of the nrdG polypeptide and/or polypeptide.

In a further aspect, the present invention relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present invention, or a fragment thereof, and various portions of the constant regions of heavy or light chains ofimmunoglobulins of various subclasses (IgG, IgM. IgA, IgE). Preferred as an immunoglobulin is the constant part of the heavy chain of human IgG, particularly IgG I. where fusion takes place at the hinge region. In 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 screening, diagnosis and therapy. A further aspect of the invention also relates to polynucleotides encoding such fusion proteins. Examples effusion protein teclmology can be found in International Patent Application Nos. W094/29458 and W094/22914.

In still another approach, expression of the gene encoding endogenous nrdG poiypeptide can be inhibited using expression blocking techniques. This blocking may be targeted against any step in 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. 0'Comor.. l Neunochem (1991) 56: 560 in Oligodeoxynucleotides as Antisense Ilihibitors of Genc Expression. CRC Press.

Boca Raton, FL (1988)). Altematively. 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 crl..

Science (1988) 241: 456 ; Dervan et al.. Science (1991) 251: 1360). These oligomers can be administered per. su or the relevant oligomers can be expressed in vivo.

Each of the polynucleotide sequences provided herein may be used in the discovery and <BR> <BR> <BR> <BR> development of antibacterial compounds. The encoded protein, upon expression, can be used as a target for the screening of antibacterial dnigs. Additionallw tlle polvnucleotide sequences encoding the amino terminal regions of the encoded protein or Sine-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 phvsical interaction between a pathogen or pathogens and a eukaryotic, preferably mammalian, host responsible for sequetac of infection. ln particular, the molecules of the invention may be used: in the prevention of adhesion of bactcria. in particular gram positive and/or gram negative bacteria. to eukaryotic, preferablymammalian. extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in rounds ; to block nrdG protein-mediated mammalian cell invasion by, for example, initiating phosphoryiation of mammalian tyrosine kinases (Rosenshine et nul., Infect. Immun. 60 : 2211 (1992) ; to block bacterial adhesion between eukaryotic, preferab) y mammalian, extracellular matrix proteins and bacterial nrdG 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 nrdG agonists and antagonists, preferably bactenostatic or bacteriocidal agonists and antagonists.

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

Helicobacter pylori (herein"H. pyloni") bacteria infect the stomachs of over onc-third of the world's population causing stomach cancer. ulcers. and gastritis (Internationa ! Agency for

Research on Cancer (1994) Schistosome. s. Liver FZuke. s ancl Helicobacter Pylori (International Agency for Research on Cancer, Lyon, France, http://wwv. uicc. cl/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 nrdG polypeptides and/or polynucleotides) found using screens provided by the invention, or known in the art, particularly narrow-spectnum antibiotics, should be useful in the <BR> <BR> <BR> <BR> <BR> treatment of H. pyl"i infection. Such treatment should decrease the advent of H. pvlori-induced cancers, such as gastrointestinat carcinoma. Such treatment should also prevent, inhibit and/or cure gastric ulcers and gastritis.

Vaccines There are provided by the invention, products, compositions and methods for assessing nrdG expression, treating disease, assaying genetic variation. and istering a nrdG polypeptide and/or anorganismtoraiseanimmunologicalresponseagainstabacteria,esp eciallyajpolynucleotideto bacteria.Staphylococcusaureus Another aspect of the invention relates to a method for inducing an immunological response in an individual, particularly a mamma ! which comprises inoculating the individual with nrdG polynucleotide and/or polypeptide or a fragment or variant thereof adequate to produce antibody and/or T cell immune response to protect said individual from infection, particular (y bacterial infection and most particularly Staphylococcus aureu. s infection. Also provided are methods whereby such immunologicai response slows bacterial replication. Yet another aspect of the invention relates to a method of inducing immunological response in an individual which comprises delivering to such individual a nucleic acid vector, sequence or ribozyme to direct expression of nrdG polynucleotide and/or polypeptide. or a fragment or a variant thereof. for expressing nrdG polynucleotide and/or polypeptide. or a fragment or a variant thereof isq vive in order to induce an immunological response, such as. to produce antibody and/or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells. to protect said individual. preferablv a human, from disease, whether that disease is alreadv established within the individual or not. One example of administering the gene is by accelerating it into the desired cells as a coating on particles or otherwise. Such nucleic acid vector may comprise DNA. RNA. a ribozyme. a modified nucleic acid. a DNA/RNA hvbrid, a DNA-protein complex or an RNA- protein 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. induces an inununological response in such individual to a nrdG <BR> <BR> <BR> <BR> polvnucleotide and/or polypeptide encoded therefrom. wherein the composition comprises a recombinant nrdG polynucleotide and/or polypeptide encoded therefrom and/or comprises DNA and/or RNA which encodes and expresses an antigen of said nrdG polynucleotidc. polypeptide encoded therefrom, or other polypeptide of the invention. The immunological response may be used therapeutically or prophylactically and may take the fonn of antibody immunity and/or cellular immunity, such as cellular inumunity arising from CTL or CD4+ T cells.

A nrdG polypeptide or a fragment thereof may be fused with co-protein or chemical moiety which may or may not bv itself produce antibodies. but which is capable of stabilizing the first protein and producing a fused or modified protein which will hav e 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 w hich 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 attache to either the amino-or carboxy-termmus 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: 2 (1996).

Also, provided by this invention are methods using the described polynucteotide 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 Staphylococcu. @ aurells. 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 <BR> <BR> <BR> <BR> successfully resisting or clearing infection. for the development of prophylactic agents or therapeutic treatments of bacterial infection, particularlv, Staphylococcus aureus 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 damagedtissue.Examplesoftissuedamageincludewoundsinskinorto

comiective 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 pharmaceuticatiy @eptable carrier. Since the polypeptides and polynucleotides may be broken thestomach,eachispreferablyadministeredparenterally,ioncludi ng,forin example, administration that is subcutaneous. intramuscular. intravenous, or intradermal.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants ; buffers, bacteriostatic compounds and solutes which render the formulation isotonic with the bodily fluid, preferably the blood. of the aqueousandnon-aqueoussterilesuspensionswhichmayincludesuspen dingindividual;and agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity 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 vaccine and can be readily determined by routine experimentation.

While the invention has been described with reference to certain nrdG 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 furtlier aspect of the invention there are provided compositions comprising a nrdG polynucleotide and/or a nrdG polypeptide for administration to a cell or to a multicellular organism.

The invention also relates to compositions comprising a polvnucleotide and/or a polypeptides discussed herein or their agonists or antagonists The pol) peptides and polyllucleotides of the invention may be employed in combination with a non-sterile or sterile carrier or carriers for use with cells. tissues or organisms, such as a pharmaceutical carrier suitable for administration to an individual.

Such compositions comprise, for instance, a media additive or a tlierapeuticalln, effective amount of a polypeptide md/or polynucleotide of the invention and a pharmaceutically acceptable carrier or excipient. Such carriers may include, but are not limited to. saline. buffered saline. dextrose, water.

glycerol, ethanol and combinations thereof. The formulation should suit the mode of administration.

The invention further relates to diagnostic and phannaccutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.

Polypeptides, polynucleotides and other compounds of me invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.

The pharmaceutical compositions may be administered in any effective. convenient mazer including, for instance, administration by topical, oral, anal. vagulal, intravenous. intraperitoneal, intramuscular, subcutaneous, intranasal or intradennal routes among others.

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

Altemativcly the composition may be formulated for topical application for example in the fonn of ointments. creams. lotions. eye ointments, eye drops. ear drops. mouthwash, impregnated dressings and sutures and aerosols. and may contain appropriate conventional additives, including. for example, preservatives. solvents to assist drug penetration. and emollients in ointments and creams. Such topica ! formulations may also contain compatible conventional carriers. for example cream or ointment bases, and ethanol or oleyl alcohol for lotions. Such carriers may constitute from about I% to about 98% by weight of the formulation : more usually they will constitute up to about 80% by weight of the formulation.

In a further aspect, the present invention provides for pharmaceutical compositions comprising a therapeutically effective amount of a polypeptide and/or polynucleotide, such as the soluble form of a polypeptide and/or polynucleotide of the present invention, agonist or antagonist peptide or small molecule compound, in combination with a pharmaceutically acceptable carrier or excipaient Such carriers include, but are not limited to, saline. buffered saline, dextrose, water, glycerol. ethanol, and combinations inventionfurtherrelatestopharmaceuticalpacksandkitscomprisin goneThe or more withoneormoreoftheingredientsoftheaforementionedcompositions ofthefilled invention. Polypeptides, polynucleotides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.

The composition will be adapted to the route of administration, for instance by a systemic or an oral route. Preferred fonns of systemic administration include injection, typically by intravenous injection. Other injection routes, such as subcutaneous, intramuscular, or intraperitoneal. can be used.

Alternative meus for systemic administration include transmucosal and transdenmal administration using penetrants such as bile salts or fnsidic acids or other detergents. In addition if a polypeptide or other compounds of the present invention can be formulated in an enteric or an encapsulated

formulation oral administration may also be possible. Administration of these compounds may also be topical and/or localized, in the fonn of salves, pastes, gels, and the like. tomammals,andparticularlyhumans,itisexpectedthatthedailyFora dministration dosage level of the active agent will be from 0.01 mg/kg to 10 mg/kg. typicallv around I mg/kg.

The physician in any event will detennine the actual dosage which will be most suitable for an individual and will vary with the age, weight and response of the particular individual. The above dosages are exemplary 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, i. 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 ambulator peritoneal dialysis (CAPD) catheters.

The composition of the invention may be administered by injection to achieve a systemic effect against relevant bacteria shortly before insertion of an in-dwelling device. Treatment may be continued after surgery during the in-body time of the device. In addition, the composition could also be used to broaden perioperative cover for anv surgical technique to prevent bacterial wound infections, especially Slc7phylococcz s aureus wound infections.

Many orthopedic surgeons consider that humans with prosthetic joints shoutd 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 generally as a wound treatment agent to prevent adhesion of bacteria to matrix proteins exposed in wound tissue and for prophylactic use in dental treatment as an alternative to, or in conjunction with, antibiotic prophylaxis.

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

A vaccine composition is conveniently in injectable form. Conventiona) adjuvants may be employed to enfance the immune response. A suitable unit dose for vaccination is 0. >-n <BR> <BR> <BR> <BR> microgram/kg of antigen, and such dose is preferably administered 1-3 times and w ith an interval of 1-3 weeks. With the indicated dose range, no adverse toxicological effects will be observed

with the compounds of the invention which would preclude their administration to suitable individuals.

Sequence Databases, Sequences in a Tangible Medium, and Algorithms Polynucleotide and polypeptide sequences forez a valuable information resource with which to determine their 2-and 3-dimensional structures as well as to identify further sequences of similar approachesaremosteasilyfacilitatedbystoringthesequenceinacom puterhomology.These readable thenusingthestoreddatainaknownmacromolecularstructureprogram ortoand search a sequence database using well known searching tools, such as GCC.

The polynucleotide and polypeptide sequences of the invention are particularly usefu ! as components in databases useful for search analyses as well as in sequence ana) ysis algorithms As used in this section entitled"Sequence Databases, Sequences in a Tangible Medium. and <BR> <BR> <BR> <BR> 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 fonii. 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 Algorithmes and in claims related thereto. the <BR> <BR> <BR> <BR> 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 po) ypcptide <BR> <BR> <BR> <BR> 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 polvpeptide 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 ofpotvnucieotide 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 ofpotynucieotide sequences wheron at <BR> <BR> <BR> <BR> least one of the sequences comprises the sequence of a polynuclconde sequence of the mvention: 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 oftheinvention.Thecomputerreadablemediumcanbeanycompositionp olypeptidesequence of matterusedtostoreinformationordata.including,forexample,comm erciallyavailable 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. Preferred methods of sequence analvsis include, for example, methods of sequence homology analysis. such as identity and similarity analysis. RNA structure analysis, sequence assembly. cladistic analysis. sequence <BR> <BR> <BR> <BR> motif analn, sis. open reading frame determination, nucieic acid base calling, nucleic acid base trimming, and sequencing chromatogram peak analysis.

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

A computer based method is also provided for perfonning homology identification, said method comprising the steps of: providing a polypeptide sequence comprising the sequence of a polypeptide of the invention in a computer readable medium ; and comparing said poiypeptide sequence to at least one 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 polvnucleotide sequence and a second polynucleotide sequence.

A further embodiment of the invention provides a computer based method for perfonning homology identification, said method comprising the steps of providing a polynucleotide sequence comprising the sequence of a polvnucleotide of the invention in a computer readable @ medium: and comparing said polynucleotide sequence to at least one polynucleotide or polypeptide sequence to identify homology.

A further 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 a polypeptide of the invention 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 polvnucleotide 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 a second polynucleotide sequence.

In another preferred embodiment of the invention there is provided a computer readable medium having stored thereon a member selected from the group consisting of: a nucleotide comprising the sequence of SEQ ID NO. @ 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. 1; a data set representing a polynucleotide sequence <BR> <BR> <BR> <BR> encoding a polypeptide sequence comprising the sequence of SEQ ID NO. 2; a polynucteotidc<BR> <BR> <BR> <BR> <BR> <BR> comprising the sequence of SEQ ID NO. 1: 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 <BR> <BR> <BR> <BR> comprising the sequence of SEQ ID NO. 1. a data set representing a polynucleotide sequence encoding a polypeptide sequence comprising the sequence of SEQ ID NO. 2. A further preferred embodiment of the invention provides a computer based method for perfonning homology identification. said method comprising the steps of providing a polynucleotide sequence comprising the sequence of SEQ ID NO. 1 in a computer readable medium: and comparing said polynucleotide sequence to at least one polynucleotide or polypeptide sequence to identif homology.

A still further preferred embodiment of the invention provides a computer based method for perfonning homology identification, said method comprising the steps of providing a <BR> <BR> <BR> <BR> polypeptide sequence comprising the sequence of SEQ ID NO. 2 in a computer readable medium : and comparing said polypeptide sequence to at least onc polvnuclcotidc or polvpeptide sequence to identifv 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. 1 in a computer readable medium: and screening for at least one overlapping region between said first polynucleotide sequence and a second 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. I in a computer readable medium; and comparing said polynucleotide sequence to at least one polynucleotide or polypeptide sequence to identifv homology.

A further embodiment of the invention provides a computer based method for performing homology identification, said method comprising the steps of: providing a poivpepude 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. 1 in a computer readable medium : and screening for at least one overlapping region between said first polynucteotide sequence and a second polyucleotide sequence.

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 individuallv indicated to be incorporated by reference herein as being fully set forth. Anv patent application to which this application claims priority is also incorporated by reference herein in its entrety.

GLOSSARY The following definitions are provided to facilitate understanding of certain terms used frequently herein.

"Antibody (ies)" as used herein includes polyclonal and monoclonal antibodies. chimeric. single chain. and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library.

"Antigenically equivalent derivative (s)" as used herein 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 polvnucleotide according to the

withtheimmediatephysicalinteractionbetweenpathogenandmammali aninvention.interferes host.

"Bispecific antibody (ies)" means an antibody comprising at least two antigen binding domains, each domain directed against a different epitope.

"Bodily material (s) means any material derived from an individual or from an organism infecting, infesting or inllabiting an individual, 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 materials..

"Disease (s)" means any disease caused by or related to infection by a bacteria. includulg. for example, disease, such as, infections of the upper respiratory tract (e. g.. otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis) lower respiratory (e. g., empyema. lung absccss). cardiac (e. g.. infective endocarditis), gastrointestinal (e. g., secretory diarrhoea, splenic absces, retroperitoneal abscess), CNS abscess),eye(e.g.,blepharitis,conjunctivitis,keratitis,endop hthalmitis.cerebral preseptal and orbital celluiitis, darcryocystitis), kidney and urinary tract (e. g., epidiymitis, intrarenal and perinephric absces. toxic shock syndrome). skin (e. g., impetigo. folliculitis, cutaneous abscesses. cellulitis, wound infection, bacterial myositis) bone and joint (e. g., septic arthritis. osteomyelitis).

"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 ofimmunoglobuhn molecules together with another human protein or part thereof. In many cases, employing an in munoglobulin Fc region as a part of a fusion protein is advantageous for use in therapy and diagnosis resulting in, for example, improved pharmacokinetic properties EP-A0232262].Ontheotherhand,forsomeusesite.g., 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 transfonned or transfected-or is capable of transformation or transfection by an exogenous polynucleotide sequence.

"Identity,"as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as detennined 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 detennined by the match betw een strings of such sequences."Identity"can be readily calculated by known methods, including but not limitcd to those described in Biology,Lesk.A.M.,Ed.,OxfordUniversityMolecular Press, New York, 1988; Biocomputing : Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1. Griffin, A. M.,

and Griffu. H. G., eds.. Humana Press, New Jersey, 1994: Sequence Analysi. s in Muleculan Biology, von Heinje, G.. Academic Press. 1987: and Sequence Analysis Primer. gribskov. M. and Devereux, J., eds., M Stockton Press, New York, 1991 ; and Carillo. H.. and Lipman. D..

SIAM J. AppliedMath., 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 in 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 AcidsResearch12(1):387(1984)),BLASTP,BLASTN,andFASTA(Atschul .Nucleic <BR> <BR> <BR> <BR> S. F. et al., J. Molec. biol. 215 : 403-410 (1990). The BLAST X program is pLibliclv availablc from NCBI and other sources (BLAST Manual, Altschul, S.. et ciel.. NCBI NLM NIH Bcthesda.

MD 20894; Altschul, S., et al., J. Mol. biol. 215: 403-410 (1990). The well known Smith Watennan algorithm may also be used to determine identity.

Parameters for polypeptide sequence comparison include the following: 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff. Proc. Natal. 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: 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48 : 443-453 () 970) 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 preferred meaning for"identitv"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: I 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' terminai 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 NO : 1 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: <BR> <BR> <BR> <BR> <BR> <BR> xn-(xn#y),nn# <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> wherein nn is the number of nucleotide alterations, xn is the total number of nucleotides in SEQ ID NO :1, y is 0.50 for 50%, 0.60 for 60%, 0. 70 for 70%). 0. 80 for 80%. 0 85 for 85%,. 0. 90 for <BR> <BR> <BR> <BR> 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 xll and y is rounded down to the nearest integer prior to subtracting it from xn. 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 : 2. that is it may be! 00% identical, or it may include 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 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'tenninal positions of the reference polynucleotide sequence or anyvhere between those terminal positions, interspersed either individuallv among the nucleic acids in the reference séquence or in one or more contiguous groups within the reference sequence. The number of nucleic acid alterations for a given percent identity is detennined by multiplying the total number of amino acids in SEQ ID NO : 2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID Nô 2, or : xn-(xn#y),nn#

whereein nn is the number of amino acid alterations, xn is the total number of amino acids in SEQ <BR> <BR> <BR> <BR> ID NO : 2, y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc,. is the svmbol for the multiplication operator. and wherein any non-integer product of xn and y is rounded down to the nearest integer prior to subtracting it from x, l (2) furtherincludeanisolatedpolypeptidecomprisingaembodiments polypeptide having at least a 50,60,70 80,85,90,95,97 or 100% identity to a polypcptide reference sequence of SEQ ID NO : 2, wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO: 2 or may include up to a certain integer number of amino 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 condservative and non- conservative substitution. or insertion. and wherein said alterations may occur at the ammo-or carboxy-tenninal positions of the reference polypeptide sequence or amw-here bctwecn those terminal positions, interspersed either individually among the amino acids in the reference ? sequence or in one or more contiguous groups within the reference sequence, and wherein said number of amino acid alterations is determined bv multiplying the total number of amino acids in SEQ ID NO : 2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number acidsinSEQIDNO:2.or:amino y),na#xa-(xa# wherein na is the number of amino acid alterations, is the total number of amino acids in SEQ <BR> <BR> <BR> <BR> ID NO : 2, y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70'/0,0.80 for 80%. 0.85 for 85%. 0.90 for<BR> <BR> <BR> <BR> <BR> <BR> <BR> 90%. 0.95 for 95%, 0. 97 for 97% or 1.00 for 00%,, and'is the symbol for the multiplication operator, and wherein any non-integer product of xa and y is rounded down to the nearest integer prior to subtracting it from xa By way of example, a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO :2, that is it may be 100% identical, or it may include 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 atleastoneaminoaciddeletion.substitution,includingconservati veandnon=consistingof <BR> <BR> <BR> <BR> conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-tenninal 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 within the reference sequence. The number of amino acid alterations for a given % identity is detennined by mu) tip) ying the total number of amino acids in SEQ ID NO : 2 by the integer defining the percent identitv divided by I 00 and then subtracting that product from said total number of amino acids in SEQ ID NO : 2. or : na-(xa#y),xa wherein na is the number of amino acid alterations, xa is 1 1 1 ID NO:2, y is. for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., and # is the symbol <BR> <BR> <BR> <BR> for the multiplication operator, and wherein any non-integer product of xa and v is rounded down to the nearest integer prior to subtracting it from Xa.

"Immunologically equivalent derivative (s)" as used herein encompasses a polypeptide. polynuc) eotide. or the equivalent of either which when used in a suitable formulation to raise antibodies in a vertebrate, the antibodies act to interfere with the immediate pllysical interaction between pathogen and mammalian host.

"Immunospecific" means jthat characteristic of an antibody whereby it possesses substantiallv greater affinity for the polypeptides of me invention or due polynucleotides of the invention than its afSnity for other related polypeptides or polynucleotides respectively-particularly those poiypeptides and polynucleotides in the prior art.

"Individual (s)" means a multicellular eukaryote. including, but not limited to a metazoan, a manunal, an ovid, a bovid, a simian, a primate, and a human. altered"bythehandofman"fromitsnaturalstate,i.e.,ifitoccursin nature,"Isolated"means it has been changed or removed from its originalenvironment,orboth.Forexample,apo)ynuc]eotide or a polypeptide naturally present in a living organism is not"isolated."but the same polynucleotide or polypeptide separated from the coexisting materials of its naWural state is"isolated". as the term is employed herein. Moreover, a polynucleotide or polypeptide that is introduced into an organism by transfonnation, genetic manipulation or by any other recombinant method is"isolated"even if it is still present in said organism, which organism may be living or non-living. a(i)prokaryote,includingbutnotlimitedto,amemberofthegenus"Or ganism(s)"means Bordetella,Corynehacterium,Mycobacterium.Neisseria,Streptoco ccus.Staphylococcus, Streptomycetes,Nocardia,Enterobacter.Yersinia,Fancisella,Hae mophlus.Actinomycetes, Acinetobacter,Erysipelothrix.Branhamella,actinobacillus.Stre ptobacillus.Pasturella,Moraxella, brucella.bacillus,Clostridium,Treponema,Escherichia,Lsteria, Calymmatobacterium, Vibrio,Proteus,Erwinia,Borrelia,Leptospira,Spirillum.Campylo bacter,Salmonella,Kleibsiella,

Pseudomonas.Aeromonas.rickettsia,Chlamydia,BorreliaandMycopl asma,Shigella,Legionella, and furtlier including, but not limited to. a member of the species or group. Group A. Streptococcu. y.

Group b CStreptococcus.GroupDStreptococcus.GroupGStreptococcus.Group Streptococcuspyogenes,Streptococcusagalactiae,StreptococcusS treptococcuspeneumoniae, faecium,Streptococcusdurans,Neisseriagonorrheae,Neisseriafae calis,Streptococcus aureus,staphylococcusepidermidis,Corynebacteriumdiptheriae.m eningitidis,Staphylococcus Garct7nerella vaginale. y, Mycobacterium tuberculosis, Mycobacterium bovis. Mycobacterium leprae,Actinomyctesisraelii,Listeriamonocytogenes,Bordetella pertusis.ulcerans,Mycobacterium Bordetellabronchiseptica,Escherichiacoli,Shigelladysenteriae .Bordatellaparapertusis, Haemphilusaegyptius,Haemophilusparainfluenzae.HaemophilusHae mophilusinfluenzae, ducreyi, typhiCitrobacterfreudii,P)roteusmirabilis.Proteusvulgaris.Sa lmonella Kleibsiellapneumoniae,Serratiamarcessens,Serratialiquefacien s,Vibriocholera.Yersiniapestis, Shigellaflexneri,Pseudomonasaeruginosa,Franscisellatularensi s.BrucellaShigelladysenterii, abortis,abortis,Bacillus cereus,Clostridiumperfringens,Clostridiumtetani.Bacillus Treponemapallidum,RickettsiarickettsiiandChlamydiatrachomiti s.(ii)anClostridiumbotulinum. <BR> <BR> <BR> <BR> arcliaeon, including but not limited to Achaebacter. and (iii) a uaicellular or filamentous eucaryote. including but not limited to, a protozoan, a fullgus. a member of the genus Sacharomyces. <BR> <BR> <BR> <BR> <P>Kluveromycex, or Candicla, and a member of the species Saccharomyces ceriviseae, Kluveromyces<BR> <BR> <BR> <BR> <BR> <BR> lactis, or Candida albicans. referstoanypolyribnucleotideorpolydeoxribonucleotide,'Polynu cleotide(s)"generally which may be unmodified RNA or DNA or modified RNA or DNA."Polynucleotide (s)" include. without limitation, single-and double-stranded DNA. DNA that is a mixture of single-and double- stranded regions or andtriple-strandedregionssingle-anddouble-strandedRNA,and#do uble- RNA that is mixture of single-and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or triple-stranded regions, or a mixture of single-and double-stranded regions. In addition,"polynucleotide"as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from me same molecule or from different molecules. The regions include all of one or <BR> <BR> <BR> <BR> more of the molecules. but more typically mvo) ve only a region of some of the moleculcs. Onc of the molecules of a triple-helical region often is an oligonucleotidc. As used hercin. thc 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 arc "polynucleotide (s)" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases. such as inosille or modified bases, such as triylated bases, to name just fiso 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 known to those of skill in the art.

The tenn"polynucleotide (s)" as it is employed herein embraces such chemically. enzymatically or metabolically modified fonns of pohnucleotides, as well as the chemical fonns of DNA and RNA characteristic of viruses and cells, iicluding, for example. simple and complex cells. embracesshortpolynucleotidesoftenreferredtoasoligonucletide( s)."Polynucleotide(s)"also "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. "Polvpeptide (s)" refers to both short chains. commonly referred to as peptides, oligopeptides and oligomers and to ionger chains generally referred to as proteins. Po) ypeptides may contain amino acids other man me 20 gene encoded amillo acids "Polypeptide(s)" include those modified either bv natura) 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 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 severa) sites in a given polvpeptide. givenpolypeptidemaycontainmanytypesofmodifications.Modificat ionscanoccurAlso,a apolypeptide,includingthepeptidebackbone,theaminoacidside-ch ains,andtheaminoanywherein or carboxyi terminai. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachement of a hemme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative. covalent attachment of phosphotidyluiositol, cross-hnking, cyclization. disulfide bond formation. demethylation. formation of ormationofcysteine,formationofpyroglutamate,formylation.cros s-links, GPIanchorformation,hydroxylation,iodination,methylation,gamm a-carboxylation,glycosylation, proteolyticprocessing,phosphorylation,prenylation,racemizati on.myristoylation,oxidation, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues. hydroxylation and ADP-ribosvlation, selenoylation, sulfation transfer-RNA mediated addition of amino acids to proteins, such as arginylation, and ubiquitination. See, for instance, PROTEINS - S7'RUCTURE AND MOLECULAR PROPERTIES. 2nd Ed.. T. E. Creighton. W. H. Freeman and Company, New York (1993) and Wold, F., Posttranslational Protes Modifications: Perspectives and Prospects, pgs. POSTTRANSLATIONALCOVALENTMODIFICATIONOFPROTEINS,B.in C. Jolmson. Ed., Academic Press, New York (1983) : Seifter et al., Meth. L'nzvlnol. 182: 626-646 (1990) and Rattm et al., Protein Synthesis: Posttranslational Modifications and Aging, Ann. N. Y.

Acad. Sci. 663: 48-62 (1992). Polypeptides may be branched or cvclic. with or without branching.

Cyclic. branched and branched circular polypeptides may result from post-translational saturai processes and may be made by entirely synthetic methods, as well.

''Recombiin t expression system (s)"refers to expression systems or portions thereof or polynucleotides of the invention introduced or transfonned into a host cell or host cell lysate for the production of the polynucleotides and polypeptides of the invention.

"Variant (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 variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions. deletions. fusion proteins and truncations in the polypeptide encoded by the reference sequence. as discussed below A typical variant of a polypeptide differs in amino acid sequence from another, reference polvpcpidc. Gencrallv. differences are limited so that the sequences of the reference potypeptide and the vanant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. The present invention also includes include variants of each of the polypeptides of the invention that is polypeptides that vary from the referents bv conservative amino acid substitutions. whereby a residue is substituted by another with like characteristics. Tyical such substitutions arc among Ala, Val, Leu and Ile; among Ser and Thr ; among the acidic residues Asp and Glu ; among Asn and Glii; and among the basic residues Lyis « mld Arg ; or aromatic residues Phe and Tyr. Particularly preferred are variants in which several, 5-10,1-5, 1-3, 1-2 or 1 amino acids are substituted, deleted, or added in any combination. A variant of a polyncleoitde or polypeptide may be a naturally occurring such as an allelic 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 skillcd artisans.

EXAMPLES The examples below are carried out using standard techniques. which are well known and routine to those of skill in the art. except where otherwise described in detail. The examples are illustrative. but do not limit the invention.

Example 1 Strain selection, Library Production and Sequencing

The polynucleotide having a DNA sequence given in Table 1 [SEQ ID NO: 1] was obtained from a library of clones of chromosomal DNA of aureus in E. coli. The sequencing data from two or more clones containing overlapping Staphylococcus aureus DNAs was used to construct the contiguous DNA sequence in SEQ ID NO: #. Librarics may be prepared by routine methods, for example: Methods 1 and 2 below.

Total cellular DNA is isolated from Staphylococcu. s aurez. s WCUH 29 according to standard procedures and size-fractionated by either of two methods.

Method 1 Total cellular DNA is mechanically sheared by passage through a needle in order to size- fractionate according to standard procedures. DNA fragments of up to I lkbp in size are reiidered blunt by treatment with exonuclease and DNA polymerase. and EcoRI linkers added. Fragments are ligated into the vector Lambda ZapII that has been cut with EcoRI, the library packaged bN, standard procedures and E.coli infected with the packaged library. The library is amplified by standard procedures.

Method 2 Total cellular DNA is partially hydrolvzed with a one or a combination of restriction <BR> <BR> <BR> <BR> enzymes appropriate to generate a series of fragments for cloning into library X ectors (c. g. Rsal.

PalI, Alul, Bshl2351), and such fragments are size-fractionated according to standard procedures.

EcoRI linkers are ligated to the DNA and the fragments then ligated into the vector Lambda ZapII that have 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.

Example 2 NrdG Characterization Nucleoside reductases (ribonucleoside diphosphate reductases), products of the nrd genes, are responsible for catalysing the reduction of the 2'hydroxyl group of the ribose ring of ribonucleosides to the corresponding deoxyribonucleosides. This is the first step in the synthesis of a DNA from a corresponding RNA and the nrd gene products are therefore ubiquitous in nature. There are 3 classes of Nrd, one of which (clashs-1) is subdivided into la (NrdA/B) and Ib (NrdE/F). In classes I and III, the enzymes are of alpha2-beta2 quatemary structure. The redox state of the alpha-subunit thiols is modulated by the action of the b subunit whose role is generation radicals.NrdA/BenzymesarewidelydistributedinnaturebutNrdE/Fr edox-active and NrdD/G may be more closely associated with bacteria. Inhibition of cellular activity of the bacterial NrdD/G enzyme can compromise the viability of the bacterial ccl) and the enzyme therefore presents as a molecular target against which antibacterial compounds can be directed.