RESPIRATORY NITRATE REDUCTASE ALPHA SUBUNIT

Abstract: The invention provides respiratory nitrate reductase alpha subunit polypeptides and polynucleotides encoding respiratory nitrate reductase alpha subunit polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing respiratory nitrate reductase alpha subunit polypeptides to screen for antibacterial compounds.

Description: Respiratory nitrate reductase alpha subunit RELATED APPLICATIONS This invention claims benefit to US Provisional Patent Application Number 60/086.579. filed Mav 22. 1998 FIELD OF THE INVENTION This invention relates to newly identified polynucleotides and polypeptides. and their production and uses, as well as their vanants. agonists and antagonists, and their uses In particular, the invention relates to polynucleotides and polypeptides of the reductases family, as well as their vaπants. herein referred to as "respiratory nitrate reductase alpha subunit" "respiratory nitrate reductase alpha subunit poιvnucleotιde(s)," and "respiratory nitrate reductase alpha subunit polypeptιde(s)" as the case may be BACKGROUND OF THE INVENTION It is particularly preferred to employ Staphylococcal genes and gene products as targets for the development of antibiotics The Staphylococci make up a medically important genera of microbes They are known to produce two types of disease, invasive and toxigenic Invasive infections are characterized generally by abscess formation effecting both skin surfaces and deep tissues S aureus is the second leading cause of bacteremia in cancer patients Osteomyelitis, sepuc arthnus. septic thrombophlebitis and acute bactenal endocarditis are also relatively common There are at least three clinical conditions resulting from the toxigenic properties of Staphylococci The manifestation of these diseases result from the actions of exotoxrns as opposed to tissue invasion and bacteremia These conditions include Staphylococcal food poisoning, scalded skin syndrome and toxic shock The frequency of Staphylococcus aureus infections has nsen dramatically in the past few decades This has been attπbuted to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems It is no longer uncommon to isolate Staphylococcus aureus strains that are resistant to some or all of the standard antibioucs This phenomenon has created an unmet medical need and demand for new anti-microbial agents. \ accrnes. drug screening methods, and diagnostic tests for this organism Moreover, the drug discovery process is currently undergoing a fundamental revolution as it embraces "functional genomics." that is. high throughput genome- or gene-based biology This approach is rapidly superseding earlier approaches based on "positional cloning" and other methods Functional genomics relies heavily on the vanous tools of bioinformatics to identify gene sequences of potential interest from the many molecular biology databases now available as well as from other sources There is a continuing and significant need to identify and characterize further genes and other polynucleotides sequences and their related polypeptides. as targets for drug discovery Clearly, there exists a need for polynucleotides and polypeptides. such as the respiratory nitrate reductase alpha subunit embodiments ofthe mvention. that have a present benefit of. among other things, being useful to screen compounds for antimicrobial activity Such factors are also useful to determine their role m 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 ways to prevent ameliorate or correct such infection, dysfunction and disease SUMMARY OF THE INVENTION The present mvention relates to respiratory nitrate reductase alpha subunit in particular respiratory nitrate reductase alpha subunit polypeptides and respiratory nitrate reductase alpha subunit polynucleotides, recombinant matenals and methods for their production In another aspect, the invention relates to methods for using such polypeptides and polynucleotides, including treatment of microbial diseases, amongst others. In a further aspect, the mvention relates to methods for identifying agomsts and antagonists usmg the matenals provided by the mvention, and for treating microbial infections and conditions associated with such infections with the identified agomst or antagonist compounds In a still further aspect, the invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting respiratory nitrate reductase alpha subumt expression or activity Vaπous changes and modifications within the spiπt and scope of the disclosed invention will become readily apparent to those skilled m the art from reading the following descnptions and from reading the other parts ofthe present disclosure DESCRIPTION OF THE INVENTION The mvention relates to respiratory nitrate reductase alpha subunit polypeptides and polynucleotides as descπbed m greater detail below In particular, the invention relates to polypeptides and polynucleotides of a respiratory nitrate reductase alpha subunit of Staphylococcus aureus. that is related by ammo acid sequence homology to NarG [Staphylococcus camosus] polypeptide The invention relates especially to respiratory nitrate reductase alpha subunit havmg a nucleotide and ammo acid sequences set out in Table 1 as SEQ ID NO 1 or 3 and SEQ ID NO 2 respectively Note that sequences recited m the Sequence Listing below as " DNA" represent an exemplification of the mvention. since those of ordinary skill will recognize that such sequences can be usefully employed in polynucleotides m general, including nbopolynucleotides TABLE 1 Respiratory nitrate reductase alpha subunit Polynucleotide and Polypeptide Sequences (A) Staphylococcus aureus respiratory mtrate reductase alpha subumt polynucleotide sequence [SEQ ID NO: 1] 5 ' -ctgcaggaattcccaaatgatacatcgaatggcacatatcattcgtcgcaatatgattac tatgatgcatttattaagcagcaagaaaatgtaacatatatttcaaccgatcgtgcagat gctaatacagtgttatgtcactaatttataaaaaataaatgaataagtaaggtttcaacc gagagaatatattcgtgttgaagccttatttgtcgttgtgccaaatttgaacgatttaga ttggcaataagcacgaccatacatgattgtgta tatttcaatgaaatcccgttattgat agggaaattccttaagtaattaggtgattccctaatattttatatttgtgaaaaaatgta caatctaattaaagcaatagtcttgggcattttaaagtatttaaaaacacgacatctaca catcgtatttttatacgtaggaggatataaatatgggaaaatttggattgaatttcttta agccaacagaaaaatttaatgggaattggtcgatcctagaaagtaaaagtagagaatggg aaaaaatgtacagagaacgttggagccacgataaagaagtaagaacaacacatggtgtta actgtacaggctcatgttcttggaaagtatttgtgaaaaatggtgtgattaactgggaaa atcaacaaactgactatccaagttgtggtccggatatgcctgaatatgaaccgagaggat gtccacgaggtgcgtcattctcttggtatgaatacagtccgcttcgaatcaaata ccat atattcgtggaaaactctgggatttatggactgaagcattagaagaaaactatggtaatc gcgttgctgcatgggcgtctattgttgaaaatgaagacaaagccaaacaatataagcaag cccgaggtatgggagggcacgtgcgttcaaattggaaagacgttacagagataatcgcag cacaattactgtatacaataaaaaaatatggtccagatcgaatcgcaggatttacaccta ttccagcgatgtcaatgattagttatgcagcaggtgctcgattcatcaatttgcttggtg gtgaaatgcttagtttttatgactggtatgcagatttaccacctgcctctccacaaattt ggggagagcaaacagatgtgcctgaatcaagtgactggtataacgcatcatacattatta tgtggggctctaatgtacctttaacacgtactccggatgcacattttatgacagaagtcc gctataaaggtacaaaagtcatttcagtagcaccagattacgcagaaaatgtgaaatttg cagataactggctagcaccgaatcctggttcagatgctgcaattgctcaagcaatgacac atgttattttacaagaacattatgttaatcaacctaatgaacgctttataaattacgcta aacaatatacagatatgccgtttcttatcatgc ggatgaagatgaaaatggatataaag cgggtcgatttttaagagcgagtgacttaggtcaaacaacagagcaaggcgaatggaagc cagttattcatgatgcaatcagcgatagtttagtagtacctaatggcacaatgggtcaac gttgggaagaaggtaagaagtggaacttaaaactagaaacagaagatggttctaaaatta accctacattatcaatggcagaaggtgga acgaattagaaacaattcaattcccatact ttgatagtgatggagatgggatattcaatcgtccaattccaactcgacaagtcact tag caaatggtgacaaagtccgtattgctacaatttttgacttaatggcaagtcaatatggcg tgcgtcgttttgatcataaattagaatcaaaaggatacgacgatgcagaatcaaaatata cacctgc tggcaagaagccatttcaggcgtaaaacaaagtgttgtcattcaagtagcga aagaatttgcgcaaaacgctatcgatactgaagggcgttcaatgattatcatgggtgcgg gtactaaccattggtttaactcagatacgatttatcgttcaatcttaaacttagttatgt ta-grggctgtcaaggtgtgaatggrggcggttgggcccaccatgtgggacaagaaaaat gtcgtccgattgaaggatggagtactgtcgcatttgcgaaagactggcaaggaccaccac gtttgcaaaatggaacaagttggttctattttgcaacagaccaatggaaatatgaagagt caaatgtagatcgtttaaaatctccattagctaaaacagaggatttaaagcatcaacacc cagctgattataatgttttagcagctagacttggttggttaccatcatatccacaattta ataaaaatagtttgttgtttgcagaagaagctaaagatgaaggcattgagtcgaatgagg caattttaaaacgagcgataaatgaagttaagtcaaaacaaacgcaatttgcgatagaag atccggatttgaaaaagaatcatccgaaatcactgtttatatggcgctcaaatctaatct caagttctgcaaaaggtcaagaatactttatgaagcatttacttggcacaaaatcagggt tattagctacaccaaatgaagatgaaaagccagaagaaattacgtggcgtgaggaaacaa cagggaaattagatttagtcgtttctttagatttcagaatgacagcaacacctttatatt ctgacattgttttgccagcagcgacttggtatgagaagcatgatttgtca ctacagata tgcatccatatgtacatccttttaatccagctattgatccattatgggaatcgcgttcag actgggatatttataaaacgttggcaaaagcattttcagaaatggcaaaagactatttac ctggaacgtttaaagatgttgtgacaactccacttagtcatgatacaaagcaagaaattt caacaccatacggcgtagtgaaagattggtcgaagggtgaaattgaagcggtacctggac gtacaatgcctaactttgcaattgtagaacgcgactacactaaaatttacgacaaatatg tcacgcttggtcctgtacttgaaaaagggaaagttggagcacatggtgtaagtttcggtg tcagtgaacaatatgaagaattaaaaagtatgttaggtacgtggagtgatacaaatgatg attctgtgagagcgaatcgtccgcgtattgatacagcacgtaatgtagcagatgcaatac taagtatttcatctgctacgaatggtaaattatcacaaaaatcatatgaagatcttgaag aacaaactggaatgccgttaaaagatatttctagcgaacgtgctgctgagaaaatttcgt ttttaaatataacttcacaaccacgagaagtaataccgacagcagtattcccaggttcaa ataaacaaggtcgacgatattcaccatttacaacgaatatagaacgtctagtacctttta gaacattaacaggacgtcaaagttattatgtggatcacgaagttttccaacaatttgggg agagcttaccagtatataaaccgacattgccgccaatggtatttgggaatagagataaga aaattaaaggtggtacagatgctttggtactgcgttatttaacgcctcatggaaaatgga atatacactcaatgtatcaagataataagcatatgttgacactatttagaggtggtccac cggtttggatatcaaatgaagatgctgaaaaacacgatatccaagataatgatcggctag aagtgtataaccgtaatggtgttgtaacggcaagagcagttatttcgcatcgtacgccta aaggtacaatgtttatgtatcatgcacaagataaacatattcaaacgcctgggtcagaaa ttacagatacacgtggtggttcacacaacgcgccgactagaatccatttgaaaccaacac aactagtcggaggatacgcacaaattagttatcactttaattattatggaccaattggga accaaagggatttatatgtagcagttagaaagatgaaggaggttaattggcttgaagatt aaagcgcaagttgcgatggtattaaatttagataaatgcataggatgccatacgtgtagt gtgacatgtaaaaacacttggacaaatcgtccaggtgctgagtacatgtggttcaataac gtagaaacgaagccaggtgtagggtatccgaaacgttgggaagaccaagaacactacaaa ggtggttggg-3 ' (B) Staphylococcus aureus respiratory mtrate reductase alpha subunit polypeptide sequence deduced from a polynucleotide sequence in this table [SEQ ID NO 2] NH- - MGKFGLNFFKPTEKFNGNWSILESKSRE EKMYRER SHDKEVRTTHGVNCTGSCS KVFVKNGV INWENQQTDYPSCGP DMPEYEPRGCPRGASFSWYEYSPLRIKYPYIRGKLWDL TEALEENYGNRVAAWASIVENEDKAK QYKQARGMGGHVRSN KDVTEIIAAQLLYTIKKYGPDRIAGFTPIPAMSMISYAAGARFINLLGGEMLSFYD YADLPPA SPQIWGEQTDVPESS D YNASYIIMWGSNVPLTRTPDAHFMTEVRYKGTKVISVAPDYAENVKFADN LAPNPGSDAAIA QAMTHVILQEHYVNQ PNERFINYAKQYTDMPFLIMLDEDENGYKAGRFLRASDLGQTTEQGE KPVIHDAIΞDSLWPNG TMGQR EEGKK NLK LETEDGSKINPTLSMAEGGYELETIQFPYFDSDGDGIFNRPIPTRQVTLANGDKVRIATIFDLMA SQYGVRRFDHKLESK GYDDAESKYTPAWQEAISGVKQSVVIQVAKEFAQNAIDTEGRSMIIMGAGINH FNSDTIYRSIL NLVMLCGCQGVNGGG AHYVGQEKCRPIEG STVAFAKDWQGPPRLQNGTSWFYFATDQWKYEESNVDRLKSPLAKTEDL KHQHPADYNVLAARL GWLPSYPQFNKNSLLFAEEAKDEGIESNEAILKRAINEVKSKQTQFAIEDPDLKKNHPKSLFIWR SNLISSSAKGQEYFM KHLLGTKSGLLATPNEDEKPEEITWREETTGKLDLVVSLDFRMTATPLYSDIVLPAAT YEKHDL SSTDMHPYVHPFNPA IDPL ESRSD DIYKTLABCAFSEMAKDYLPGTFKDWTTPLSHDTKQEISTPYGVVKD SKGEIE AVPGRTMPNFAIVER DYTKIYDKYVTLGPVLEKGKVGAHGVSFGVSEQYEELKSMLGT SDTNDDSVRANRPRIDTARNV ADAILSISSATNGKL SQKSYEDLEEQTGMPLKDISSERAAEKISFLNITSQPREVIPTAVFPGSNKQGRRYSPFTTNIER LVPFRTLTGRQSYYV DHEVFQQFGESLPVYKPTLPPMVFGNRDKKIKGGTDALVLRYLTPHGK NIHSMYQCNKHMLTLF RGGPPV ISNEDAEK HDIQDND LEVYNRNGVVTARAVISHRMPKGTMFMYHAQDKHIQTPGSEITDTRGGSKNAPTRIH LKPTQLVGGYAQISY HFNYYGPIGNQRDLYVAVRKMKEVN LED-COOH (C) Staphylococcus aureus respiratory mtrate reductase alpha subumt polynucleotide sequence [SEQ ID NO-.3]. 5 ' - atgggaaaatttggattgaatttcttta agccaacagaaaaatttaatgggaattggtcgatcctagaaagtaaaagtagagaatggg aaaaaatgtacagagaacgttggagccacgataaagaagtaagaacaacacatggtgtta actgtacaggctcatgttcttggaaagtatttgtgaaaaatggtgtgattaactgggaaa arcaacaaactgactatccaagttgtggtccggatatgcctgaatatgaaccgagaggat gtccacgaggtgcgtcattctcttggtatgaatacagrccgcttcgaatcaaatatccat a .. ' - - :ggaaaact:ccgggatttatggactgaagcartagaagaaaacrat;ggtaatc gcgttgctgcatgggcgtctattgttgaaaatgaagacaaagccaaacaatataagcaag cccgaggtatgggagggcacgtgcgttcaaattggaaagacgttacagagataatcgcag cacaattactgtatacaataaaaaaatatggtccagatcgaatcgcaggatttacaccta ttccagcgatgtcaatgattagttatgcagcaggtgctcgattcatcaatttgcttggtg gtgaaatgcttagtttttatgactggtatgcagatttaccacctgcctctccacaaattt ggggagagcaaacagatgtgcctgaatcaagtgactggtataacgcatcatacattatta tgtggggctctaatgtacctttaacacgtactccggatgcacattttatgacagaagtcc gctataaaggtacaaaagtcatttcagtagcaccagattacgcagaaaatgtgaaatttg cagataactggctagcaccgaatcctggttcagatgctgcaattgctcaagcaatgacac atgttattttacaagaacattatgttaatcaacctaatgaacgctttataaattacgcta aacaatatacagatatgccgtttcttatcatgctggatgaagatgaaaatgga ataaag cgggtcgatttttaagagcgagtgacttaggtcaaacaacagagcaaggcgaatggaagc cagttattcatgatgcaatcagcgatagtttagtagtacctaatggcacaatgggtcaac gttgggaagaaggtaagaagtggaacttaaaactagaaacagaagatggttctaaaatta accctacat atcaatggcagaaggtggatacgaattagaaacaattcaattccca act ttgacagtgatggagatgggatattcaatcgtccaattccaactcgacaagtcactttag caaatggtgacaaagtccgtattgctacaatttttgacttaatggcaagtcaatatggcg tgcgtcgttttgatcataaattagaatcaaaaggatacgacgatgcagaatcaaaatata cacctgcttggcaagaagccatttcaggcgtaaaacaaagtgttgtcattcaagtagcga aagaatttgcgcaaaacgctatcgatactgaagggcgttcaatgattatcatgggtgcgg gtattaaccattggtttaactcagatacgatttatcgttcaatcttaaacttagttatgt tatgtggctgtcaaggtgtgaatggtggcggttgggctcactatgtgggacaagaaaaat gtcgtccgattgaaggatggagtactgtcgcatttgcgaaagactggcaaggaccaccac gtttgcaaaatggaacaagttggttctattttgcaacagaccaatggaaatatgaagagt caaatgtagatcgtttaaaatctccattagctaaaacagaggatttaaagcatcaacacc cagctgattataatgttttagcagctagacttggttggttaccatcatatccacaattta ataaaaatagtttgttgtttgcagaagaagctaaagatgaaggcattgagtcgaatgagg caattttaaaacgagcgataaatgaagttaagtcaaaacaaacgcaatttgcgatagaag atccggatttgaaaaagaatcatccgaaatcactgtttatatggcgctcaaatctaatct caagttctgcaaaaggtcaagaatactttatgaagcatttacttggcacaaaatcagggt tattagctacaccaaatgaagatgaaaagccagaagaaattacgtggcgtgaggaaacaa cagggaaattagatttagtcgtttctttagatttcagaatgacagcaacacctttatatt ctgacattgttttgccagcagcgacttggtatgagaagcatgatttgtcatctacagata tgcatccatatgtacatccttttaatccagctattgatccattatgggaatcgcgttcag actgggatatttataaaacgttggcaaaagcattttcagaaatggcaaaagactatttac ctggaacgtttaaagatgttgtgacaactccacttagtcatgatacaaagcaagaaattt caacaccatacggcgtagtgaaagattggtcgaagggtgaaattgaagcggtacctggac gtacaatgcctaactttgcaattgtagaacgcgactacactaaaatttacgacaaatatg tcacgcttggtcctgtacttgaaaaagggaaagttggagcacatggtgtaagtttcggtg tcagcgaacaatatgaagaattaaaaagtatgttaggtacgtggagtgatacaaatgatg attc grgagagcgaatcgtccgcgtattgatacagcacgtaatgtagcagatgcaatac taagtatttcatctgctacgaatggtaaattatcacaaaaatcatatgaagatcttgaag aacaaactggaatgccgttaaaagatatttctagcgaacgtgctgctgagaaaatttcgt rtttaaatataacttcacaaccacgagaagtaataccgacagcagtattcccaggr caa ataaacaaggtcgacgatattcaccatttacaacgaatatagaacgtctagtaccttt a gaacattaacaggacgtcaaagttattatgtggatcacgaagttttccaacaatttgggg agagcttaccagtatataaaccgacattgccgccaatggtatttgggaatagagataaga aaattaaaggtggtacagatgctttggtactgcgttatttaacgcctcatggaaaatgga atatacactcaatgtatcaagataataagcatatgttgacactatttagaggtggtccac cggtttggatatcaaatgaagatgctgaaaaacacgatatccaagataatgattggctag aagtgtataaccgtaatggtgttgtaacggcaagagcagttatttcgcatcgtatgccta aaggtacaatgtttatgtatcatgcacaagataaacatattcaaacgcctgggtcagaaa ttacagatacacgtggtggttcacacaacgcgccgactagaatccatttgaaaccaacac aactagtcggaggatacgcacaaattagttatcactttaattattatggaccaattggga accaaagggatttatatgtagcagttagaaagatgaaggaggttaattggcttgaagatt aa-3 1 Deposited materials A deposit compnsing a Staphylococcus aureus WCUH 29 strain has been deposited with the National Collections of Industrial and Marine Bacteπa Ltd (herem "NCIMB"). 23 St Machar Dπve Aberdeen AB2 1RY, Scotland on 11 September 1995 and assigned NCEMB Deposit No 40771. and referred to as Staphylococcus aureus WCUH29 on deposit The Staphylococcus aureus strain deposit is referred to herem as "the deposited strain" or as "the DNA ofthe deposited strain " The deposited strain comprises a full length respiratory mtrate reductase alpha subumt gene The sequence ofthe polynucleotides compπsed in the deposited strain, as well as the ammo acid sequence of any polypeptide encoded thereby, are controlling m the event of any conflict with anv descπption of sequences herem The deposit ofthe deposited strain has been made under the terms ofthe Budapest Treatv on the International Recogmtion of the Deposit of Micro-organisms for Purposes of Patent Procedure The deposited strain will be irrevocably and without restπction or condition released to the public upon the issuance of a patent The deposited strain is provided merely as convenience to those of skill in the art and is not an admission that a deposit is required for enablement such as that required under 35 U S C § 1 12 A license may be required to make, use or sell the deposited strain, and compounds denved therefrom, and no such license is hereby granted In one aspect of the mvention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Staphylococcus aureus WCUH 29 strain, which polvpeptide is compπsed in the deposited strain Further provided by the mvention are respiratorv mtrate reductase alpha subumt polynucleotide sequences m the deposited strain, such as DNA and RNA. and ammo acid sequences encoded thereby Also provided by the mvention are respiratory mtrate reductase alpha subumt polvpeptide and polynucleotide sequences isolated from the deposited strain Polypeptides Respiratory mtrate reductase alpha subumt polypeptide of the mvention is substantial phylogenetically related to other proteins ofthe reductases family In one aspect ofthe mvention there are provided polypeptides of Staphylococcus aureus referred to herem as "respiratory mtrate reductase alpha subumt" and "respiratory mtrate reductase alpha subumt polypeptides" as well as biologically, diagnostically, prophylactically, clinically or therapeuαcallv useful vaπants thereof, and compositions compπsing the same Among the particularly preferred embodiments ofthe mvention are vaπants of respiratory mtrate reductase alpha subunit polypeptide encoded by naturally occurring alleles of a respiratory mtrate reductase alpha subumt gene The present mvention further provides for an isolated polypeptide that (a) compπses or consists of an ammo acid sequence that has at least 95% identity, most preferablv at least 97-99% or exact identity, to that of SEQ ID NO 2 over the entire length of SEQ ID NO 2. (b) a polypeptide encoded by an isolated polynucleotide compπsmg or consistmg of a polynucleotide sequence that has at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NO 1 over the entire length of SEQ ID NO 1 , or the entire length of that portion of SEQ ID NO 1 which encodes SEQ ID NO 2, (c) a polypeptide encoded by an isolated polynucleotide compπsmg or consistmg of a polynucleotide sequence encoding a polypeptide that has at least 95% identity, even more preferably at least 97-99% or exact identity, to the aπuno acid sequence of SEQ ID NO 2. over the entire length of SEQ ID NO 2 The polypeptides ofthe mvention mclude a polypeptide of Table 1 [SEQ ID NO 2] (m particular a mature polypeptide) as well as polypeptides and fragments, particularly those that has a biological activity of respiratory mtrate reductase alpha subumt. and also those that have at least 95% identitv to a polypeptide of Table 1 [SEQ ID NO 2] and also mclude portions of such polypeptides with such portion of the polypeptide generally compπsmg at least 30 ammo acids and more preferably at least 50 ammo acids The mvention also includes a polypeptide consisting of or compπsmg a polypeptide of the formula X-(Rι ) m -(R 2 )-(R 3 ) n -Y wherein, at the aπuno termmus. X is hydrogen, a metal or anv other moiety descπbed herem for modified polypeptides, and at the carboxyl termmus, Y is hydrogen, a metal or any other moietv descnbed herem for modified polypeptides. Ri and R3 are any ammo acid residue or modified aπuno acid residue, m is an integer between 1 and 1000 or zero, n is an integer between 1 and 1000 or zero, and R is an ammo acid sequence of the mvention. parϋcularlv an aπuno acid sequence selected from Table 1 or modified forms thereof In the formula above. R 2 is oπented so that its ammo terminal ammo acid residue is at the left, covalently bound to Ri and its carboxy terminal ammo acid residue is at the nght covalentlv bound to R3 Any stretch of ammo acid residues denoted by either Ri or R3. where m and or n is greater than 1. may be either a heteropolymer or a homopolymer, preferably a heteropolvmer Other preferred embodiments ofthe mvention are provided where m is an mteger 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 mvention is deπved from Staphylococcus aureus. however, it may preferably be obtained from other organisms of the same taxonomic genus A polypeptide of the mvention may also be obtained, for example, from organisms of the same taxonomic family or order A fragment is a vaπant polypeptide havmg an ammo acid sequence that is entirely the same as part but not all of any ammo acid sequence of any polypeptide ofthe mvention As with respiratorv mtrate reductase alpha subumt polypeptides, fragments may be "free-standing," or compπsed within a larger polypeptide of which they form a part or region, most preferably as a smgle continuous region in a smgle larger polypeptide. Preferred fragments mclude, for example, truncation polypeptides having a portion of an ammo acid sequence of Table 1 [SEQ ID NO.2], or of vaπants thereof, such as a continuous seπes of residues that mcludes an aπuno- and/or carboxyl-terminal aπuno acid sequence Degradation forms of the polypeptides ofthe mvention produced by or m a host cell, particularly a Staphylococcus aureus, are also preferred Further preferred are fragments characterized by structural or functional attributes such as fragments that compπse alpha-hehx and alpha-helix forming regions, beta-sheet and beta-sheet-forrmng regions, turn and turn-forming regions, coil and cou-formmg regions, hydrophihc regions, hvdrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-foπnmg regions, substrate binding region, and high antigemc mdex regions. Further preferred fragments mclude an isolated polypeptide comprising an amino acid sequence havmg at least 15, 20, 30, 40, 50 or 100 contiguous amino acids trom the amino acid sequence of SEQ ID NO:2, or an isolated polypeptide compπsmg an ammo acid sequence havmg at least 15, 20, 30, 40, 50 or 100 contiguous ammo acids truncated or deleted from the ammo acid sequence of SEQ ID NO:2. Fragments ofthe polypeptides ofthe mvention may be employed for producmg the coπesponding full-length polypeptide by peptide synthesis, therefore, these vaπants mav be employed as intermediates for producmg the full-length polypeptides ofthe mvention Polynucleotides It is an object ofthe mvention to provide polynucleotides that encode respiratorv mtrate reductase alpha subumt polypeptides. particularly polynucleotides that encode a polvpeptide herem designated respiratory mtrate reductase alpha subumt In a particularly preferred embodiment of the mvention the polynucleotide compπses a region encoding respiratory mtrate reductase alpha subumt polypeptides compπsmg a sequence set out in Table 1 [SEQ ID NO 1, 3] that mcludes a full length gene, or a vaπant thereof The Applicants believe that this full length gene is essential to the growth and/or survival of an organism that possesses it such as Staphylococcus aureus As a further aspect of the mvention there are provided isolated nucleic acid molecules encoding and/or expressing respiratory mtrate reductase alpha subumt polypeptides and polynucleotides, particularly Staphylococcus aureus respiratory mtrate reductase alpha subumt polypeptides and polynucleotides. including, for example, unprocessed RNAs. nbozvme RNAs. mRNAs. cDNAs, genomic DNAs, B- and Z-DNAs Further embodiments ofthe mvention mclude biologically, diagnostically, prophylactically, clinically or therapeutically useful polynucleotides and polypeptides, and vaπants thereof, and compositions compπsmg the same Another aspect of the mvention relates to isolated polynucleotides. including at least one full length gene, that encodes a respiratory mtrate reductase alpha subumt polypeptide havmg a deduced aπuno acid sequence of Table 1 [SEQ ED NO 2] and polynucleotides closely related thereto and vaπants thereof In another particularly preferred embodiment of the invention there is a respiratory mtrate reductase alpha subumt polypeptide from Staphylococcus aureus compπsmg or consistmg of an amino acid sequence of Table 1 [SEQ ID NO 2], or a vaπant thereof Usmg the information provided herein, such as a polynucleotide sequence set out m Table 1 [SEQ ID NO 1. 3]. a polynucleotide of the mvention encoding respiratory mtrate reductase alpha subumt polvpeptide may be obtained usmg standard cloning and screening methods, such as those for cloning and sequencmg chromosomal DNA fragments from bacteπa usmg Staphylococcus aureus WCUH 29 cells as starting mateπal, followed by obtaining a full length clone For example, to obtain a polvnucleotide sequence of the mvention. such as a polynucleotide sequence given in Table 1 [SEQ ID NO 1. 3]. typically a library of clones of chromosomal DNA of Staphylococcus aureus WCUH 29 in E colt or some other suitable host is probed with a radiolabeled oligonucleotide. preferablv a 17-mer or longer, denved from a partial sequence Clones carrving DNA identical to that ofthe probe can then be distinguished usmg stringent hybndization conditions By sequencmg the individual clones thus identified by hybndization with sequencmg primers designed from the oπgmal polvpeptide or polynucleotide sequence it is then possible to extend the polynucleotide sequence in both directions to determine a full length gene sequence Convementlv. such sequencmg is performed, for example. usmg denatured double stranded DNA prepared from a plasmid clone Suitable techniques are descπbed by Mamatis. T . Fntsch. E F and Sambrook et al . MOLECULAR CLONING A LABORATORY MANUAL. 2nd Ed . Cold Spring Harbor Laboratory Press. Cold Spring Harbor. New York (1989) (see m particular Screening By Hybndization 1 90 and Sequencmg Denatured Double- Stranded DNA Templates 13 70) Direct genomic DNA sequencmg may also be performed to obtain a full length gene sequence Illustrative ofthe mvention. each polynucleotide set out m Table 1 [SEQ ID NO 1, 3] was discovered m a DNA library denved from Staphylococcus aureus WCUH 29 Moreover, each DNA sequence set out m Table 1 [SEQ ED NO 1. 3] contains an open reading frame encoding a protein having about the number of a mo acid residues set forth m Table 1 [SEQ ID NO 2] with a deduced molecular weight that can be calculated usmg a mo acid residαe molecular weight values well known to those skilled in the art The polynucleotide of SEQ ID NO 1. between nucleotide number 453 (ATG )and the codon that begins at nucleotide number 4142 (TAA) of SEQ ID NO 1 encodes the polypeptide of SEQ ID NO 2 In a further aspect the present mvention provides for an isolated polynucleotide compπsing or consisting of (a) a polynucleotide sequence that has at least 95 % identity, even more preferably at least 97-99% or exact identity to SEQ ID NO 1 over the entire length of SEQ ID NO 1. or the entire length of that portion of SEQ ID NO 1 which encodes SEQ ID NO 2, (b) a polynucleotide sequence encoding a polypeptide that has at least 95% identity, even more preferably at least 97-99% or 100% exact, to the ammo acid sequence of SEQ ED NO 2, over the entire length of SEQ ID NO 2 A polynucleotide encoding a polypeptide of the present mvention. mcludmg homologs and orthologs from species other than Staphylococcus aureus, may be obtained bv a process that compnses the steps of screening an appropπate library under stringent hybndization conditions with a labeled or detectable probe consistmg of or compnsing the sequence of SEQ ID NO 1. 3 or a fragment thereof, and isolating a full-length gene and/or genomic clones compπsmg said polynucleotide sequence The mvention provides a polynucleotide sequence identical over its entire length to a coding sequence (open reading frame) m Table 1 [SEQ ID NO 1, 3] Also provided by the mvention 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 mvention may also compπse at least one non-codmg sequence, mcludmg for example, but not limited to at least one non-coding 5 " and 3' sequence, such as the transcnbed but non-translated sequences, termination signals (such as rho-dependent and rho-mdependent termmation signals), πbosome binding sites. Kozak sequences, sequences that stabilize mRNA. mtrons. and polvadenvlation signals The pol y nucleotide sequence ma y also compπse additional coding sequence encoding additional ammo acids For example, a marker sequence that facmtates purification of a fused polvpeptide can be encoded In certain embodiments ofthe mvention. the marker sequence is a hexa-histidine peptide. as provided m the pQE vector (Qiagen. Inc ) and descnbed m Gentz et al . Proc Natl Acad Sci USA 86 821-824 (1989). or an HA peptide tag (Wilson et al , Cell 37 767 (1984), both of that may be useful m puπfvmg polypeptide sequence fused to them Polynucleotides of the mvention also mclude. but are not limited to. polynucleotides compπsmg a structural gene and its naturally associated sequences that control gene expression A preferred embodiment of the mvention is a polynucleotide of consisting of or compπsmg nucleotide 459 (ATG) to the nucleotide immediately upstream of or mcludmg nucleotide 4146 (TAA) set forth m SEQ ID NO 1. 3 of Table 1, both of that encode a respiratory mtrate reductase alplia subumt polvpeptide The mvention also mcludes a polynucleotide consisting of or compπsmg a polynucleotide ofthe formula wherem. at the 5 ' end of the molecule, X is hydrogen, a metal or a modified nucleotide residue, or together with Y defines a covalent bond, and at the 3' end ofthe molecule. Y is hydrogen, a metal, or a modified nucleotide residue, or together with X defines the covalent bond, each occunence of Ri and R 3 is independently any nucleic acid residue or modified nucleic acid residue, m is an mteger between 1 and 3000 or zero , n is an integer between 1 and 3000 or zero, and R 2 is a nucleic acid sequence or modified nucleic acid sequence of the mvention, particularly a nucleic acid sequence selected from Table 1 or a modified nucleic acid sequence thereof In the polynucleotide formula above. R 2 is oriented so that its 5' end nucleic acid residue is at the left, bound to Ri and its 3' end nucleic acid residue is at the nght. bound to R 3 Any stretch of nucleic acid residues denoted by either Ri and/or R 2 . where m and or n is greater than 1. may be either a heteropolymer or a homopolymer. preferably a heteropolymer Where, m a prefened embodiment. X and Y together define a covalent bond, the polynucleotide ofthe above formula is a closed, circular polynucleotide that can be a double-stranded polynucleotide wherem the formula shows a first strand to which the second strand is complementary In another prefened embodiment m and/or n is an integer between 1 and 1000. Other prefened embodiments ofthe mvention are provided where m is an mteger between 1 and 50. 100 or 500. and n is an mteger between 1 and 50. 100. or 500 It is most prefened that a polynucleotide ofthe mvention is denved from Staphylococcus aureus. however, it may preferably be obtained from other organisms of the same taxonomic genus A polynucleotide ofthe mvention may also be obtained, for example, from organisms ofthe same taxonomic family or order The term "pol y nucleotide encoding a polvpeptide" as used herem encompasses polynucleotides that mclude a sequence encodmg a polypeptide of the mvention. particularly a bacteπal polvpeptide and more particularly a polvpeptide ofthe Staphylococcus aureus respiratory mtrate reductase alpha subumt having an ammo acid sequence set out m Table 1 [SEQ ID NO 2] The term also encompasses polynucleotides that mclude a single continuous region or discontinuous regions encodmg 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 compnse coding and/or non-codmg sequences The mvention further relates to vaπants of the polynucleotides descnbed herem that encode vanants of a polypeptide having a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] Fragments of pol y nucleotides ofthe mvention mav be used, for example, to synthesize full-length polynucleotides of the mvention Further particularly prefened embodiments are polynucleotides encodmg respiratorv mtrate reductase alpha subumt vaπants, that have the ammo acid sequence of respiratory mtrate reductase alpha subumt 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 ammo acid residues are substituted, modified, deleted and/or added, m any combination Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of respiratory mtrate reductase alpha subumt polypeptide Prefened isolated polynucleotide embodiments also mclude polynucleotide fragments, such as a polynucleotide comprising a nuchc acid sequence havmg at least 15, 20, 30, 40. 50 or 100 contiguous nucleic acids from the polynucleotide sequence ot SEQ ID NO 1. 3, or an polynucleotide comprising a nucleic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids truncated or deleted from the 5' and/or 3' end ot the polynucleotide sequence ot SEQ ID NO 1, 3 Further prefened embodiments ofthe mvention are polynucleotides that are at least 95% or 97% identical over their entire length to a polynucleotide encodmg respiratory mtrate reductase alpha subumt polvpeptide havmg an ammo acid sequence set out m Table 1 [SEQ ID NO 2], and polynucleotides that are complementary to such polynucleotides Most highly prefened are polynucleotides that compnse a region that is at least 95% are especially prefened Furthermore, those with at least 97% are highlv prefened among those with at least 95%. and among these those with at least 98% and at least 99% are particularly highly prefened. with at least 99% being the more prefened Prefened embodiments are polynucleotides encoding polypeptides that retain substantially the same biological function or activity as a mature polypeptide encoded bv a DNA of Table 1 [SEQ ID NO 1. 3] In accordance with certain prefened embodiments of this mvention there are provided polynucleotides that hybπdize, particularly under stringent conditions, to respiratory mtrate reductase alpha subumt polynucleotide sequences, such as those polynucleotides m Table 1 The mvention further relates to polynucleotides that hybndize to the polynucleotide sequences provided herem In this regard, the mvention especially relates to polynucleotides that hybπdize under stringent conditions to the polynucleotides descπbed herem As herem used , the terms "stringent conditions" and "stringent hybndization conditions" mean hybndization occurring only if there is at least 95% and preferably at least 97% identity between the sequences A specific example of stringent h y bndization conditions is overnight incubation at 42°C m a solution comprising 50% formamide. 5x SSC (150mM NaCl. 15mM tnsodium citrate), 50 πiM sodium phosphate (pH7 6). 5x Denhardt's solution. 10% dextran sulfate. and 20 micrograms/ml of denatured, sheared salmon sperm DNA. followed by washing the hybndization support m 0 lx SSC at about 65°C Hybndization and wash conditions are well known and exemplified m Sambrook, et al , Molecular Cloning A Laboratory Manual, Second Edition, Cold Spring Harbor, N Y , (1989), particularly Chapter 1 1 therem Solution hybndization may also be used with the polynucleotide sequences provided by the mvention The invention also provides a polynucleotide consistmg of or compnsmg a polynucleotide sequence obtained by screemng an appropπate library compnsmg a complete gene for a pol y nucleotide sequence set forth in SEQ ID NO 1. 3 under stnngent hybndization conditions with a probe havmg the sequence of said polynucleotide sequence set forth m SEQ ID NO 1. 3 or a fragment thereof, and isolating said polynucleotide sequence Fragments useful for obtaining such a polynucleotide include, for example, probes and primers fully descnbed elsewhere herem As d i scussed elsewhere herem regarding polynucleotide assays ofthe mvention. for instance, the pol y nucleotides of the mvention. may be used as a hybndization probe for RNA. cDNA and genomic DNA to i solate full-length cDNAs and genomic clones encodmg respiratorv mtrate reductase alpha subumt and to i solate cDNA and genomic clones of other genes that have a high identity , particularly high sequence identity , to a respiratory mtrate reductase alpha subumt gene Such probes generally will compπse at least 15 nucleotide residues or base pairs Preferably, such probes will have at least 30 nucleotide res i dues or base pairs and may have at least 50 nucleotide residues or base pairs Particularlv prefened probes will have at least 20 nucleotide residues or base parrs and will have lee than 30 nucleotide residues or base pairs A coding region of a respiratory mtrate reductase alpha subumt gene mav be isolated bv screening using a DNA sequence provided m Table 1 [SEQ ED NO 1, 3] to synthesize an oligonucleotide probe A labeled oligonucleotide havmg a sequence complementary to that of a gene ofthe mvention is then used to screen a library of cDNA. genomic DNA or mRNA to determine which members ofthe library the probe hybπdizes to There are several methods available and well known to those skilled m the art to obtain full- length DNAs, or extend short DNAs, for example those based on the method of Rapid Amplification of cDNA ends (RACE) (see, for example, Frohman, et al , PNAS USA 85 8998-9002. 1988) Recent modifications ofthe technique, exemplified by the Marathon™ technology (Clontech Laboratoπes Inc ) for example, have sigmficantly sunphfied the search for longer cDNAs In the Marathon™ technology, cDNAs have been prepared from mRNA extracted from a chosen tissue and an 'adaptor' sequence gated onto each end Nucleic acid amplification (PCR) is then earned out to amp fv the "missing" 5' end ofthe DNA usmg a combmation of gene specific and adaptor specific oligonucleotide primers The PCR reaction is then repeated usmg "nested" primers, that is. pnmers designed to anneal within the amplified product (typically an adaptor specific pnmer that anneals further 3' m the adaptor sequence and a gene specific pnmer that anneals further 5' m the selected gene sequence) The products of this reaction can then be analyzed by DNA sequencmg and a full- length DNA constructed either by joining the product directly to the existmg DNA to give a complete sequence, or carrying out a separate full-length PCR usmg the new sequence information for the design of the 5' pnmer The polynucleotides and polypeptides ofthe mvention may be employed, for example, as research reagents and matenals for discovery of treatments of and diagnostics for diseases, particularlv human diseases, as further discussed herem relating to polynucleotide assays The polynucleotides of the mvention that are oligonucleotides denved from a sequence of Table 1 [SEQ ID NOS 1 or 2] may be used m the processes herem as descπbed. but preferably for PCR. to determine whether or not the polynucleotides identified herem in whole or m part are transcπbed in bactena in infected tissue It is recognized that such sequences will also have utility in diagnosis ofthe stage of infection and type of infection the pathogen has attained The mvention also provides polynucleotides that encode a polypeptide that is a mamre protein plus additional aπuno or carboxyl-terminal ammo acids, or ammo acids mteπor to a mamre polvpeptide (when a mature form has more than one polypeptide chain, for instance) Such sequences mav plav a role in processing of a protein from precursor to a mature form, may allow protein transport, mav lengthen or shorten protein half-life or mav facilitate manipulation of a protein for assay or production, among other tlungs As generally is the case in vivo, the additional ammo acids may be processed away from a mature protein by cellular enzymes For each and every polynucleotide of the mvention there is provided a polynucleotide complementary to i t It is prefened that these complementary polynucleotides are fully complementary to each polynucleotide with which they are complementary A precursor protein , havmg a mature form ofthe polypeptide fused to one or more prosequences may be an i nact i ve form of the polypeptide When prosequences are removed such inactive precursors generally are activated Some or all of the prosequences may be removed before activation Generally , such precursors are called proproterns As w i ll be recognized, the entire polypeptide encoded by an open reading frame is often not requ i red for activity Accordingly, it has become routine m molecular biology to map the boundaπes of the pr i mary structure required for activity with N-terminal and C-terminal deletion expeπments These expeπments util i ze exonuclease digestion or convement restπction sites to cleave coding nucleic acid sequence For example. Promega (Madison. WI) sell an Erase-a-base™ system that uses Exonuclease HI designed to facilitate analysis of the deletion products (protocol available at www promega com) The d i gested endpomts can be repaired (e.g., by hgation to synthetic linkers) to the extent necessary to preserve an open read i ng frame. In this way, the nucleic acid of SEQ ID NO- 1, 3 readily provides contiguous fragments of SEQ ED NO.2 sufficient to provide an activity, such as an enzymatic , binding or antibody- inducing activity Nucleic acid sequences encodmg such fragments of SEQ ID NO 2 and vanants thereof as descπbed herem are within the mvention. as are polypeptides so encoded In sum. a polynucleotide ofthe mvention may encode a mature protein, a mature protein plus a leader sequence (which may be refened to as a preprotein), a precursor of a mamre protem havmg one or more prosequences that are not the leader sequences of a preprotein, or a preproprotein. that is a precursor to a proprote i n. havmg a leader sequence and one or more prosequences. that generaUy are removed during processing steps that produce active and mature forms ofthe polypeptide Vectors, Host Cells, Expression Systems The mvent i on also relates to vectors that compπse a polynucleotide or polynucleotides of the mvention , host cells that are genetically engineered with vectors of the mvention and the production of polypeptides of the mvention by recombinant techmques CeU-free translation systems can also be employed to produce such proteins usmg RNAs denved from the DNA constructs ofthe mvention Recomb i nant polypeptides ofthe present mvention may be prepared by processes weU known m those sk i Ued m the art from geneticaUy engineered host ceUs compnsmg expression systems Accordingly , m a further aspect the present mvention relates to expression systems that compπse a polynucleotide or polvnucleotides ofthe present mvention, to host ceUs that are geneticaUy engineered with such expression systems, and to the production of polypeptides ofthe mvention by recombinant techmques For recombinant production of the polypeptides of the mvention. host ceUs can be geneticaUy engineered to incorporate expression systems or portions thereof or polynucleotides of the mvention Introduction of a polynucleotide mto the host ceU can be effected by methods descnbed in many standard laboratory manuals, such as Davis, et al . BASIC METHODS IN MOLECULAR BIOLOGY. (1986) and Sambrook, et al . MOLECULAR CLONING A LABORATORY MANUAL. 2nd E . 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 appropπate hosts mclude bacteπal ceUs. such as ceUs of streptococci, staphylococci, enterococci E coli, streptomyces. cyanobactena. Bacillus subtihs. and Staphylococcus aureus. fungal cells, such as ceUs of a yeast Kluveromyces, Saccharomyces. a basidiomycete. Candida albicans and Aspergillus. insect ceUs such as ceUs of Drosophila S2 and Spodoptera Sf9. animal ceUs such as CHO, COS, HeLa. C127, 3T3. BHK, 293. CV-1 and Bowes melanoma ceUs. and plant ceUs. such as ceUs of a gymnosperm or anmosperm A great vaπety of expression systems can be used to produce the polypeptides of the mvention Such vectors mclude. among others, chromosomal-, episomal- and vrrus-denved vectors, for example, vectors denved from bacteπal plasπuds. from bactenophage, from transposons. from yeast episomes. from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses. papova viruses, such as SV40, vaccinia viruses, adenovrruses. fowl pox viruses, pseudorabies viruses, picomavrruses and retrov-iruses. and vectors denved from combinations thereof, such as those denved from plasmid and bactenophage genetic elements, such as cosmids and phagemids The expression system constructs may compnse control regions that regulate as weU as engender expression GeneraUy. any system or vector smtable to mamtam propagate or express polynucleotides and/or to express a polypeptide m a host mav be used for expression m this regard The appropnate DNA sequence may be inserted mto the expression system by any of a vaπety of weU-known and routine techmques. such as. for example, those set forth in Sambrook et al . MOLECULAR CLONING. A LABORATORY MANUAL, (supra) In recombinant expression systems m eukaryotes. for secretion of a translated protein mto the lumen of the endoplasmic reticulum. mto the penplas ic space or mto the extracellular environment, appropπate secretion signals may be incorporated mto the expressed polypeptide These signals may be endogenous to the polypeptide or thev may be heterologous signals Polypeptides of the mvention can be recovered and puπfied from recombinant ceU cultures by weU-known methods mcludmg ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphoceUulose chromatography. hydrophobic interaction chromatography. affinity chromatography, hydroxylapatite chromatography. and lectin chromatography Most preferably, high performance liquid chromatography is employed for purification WeU known techmques 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 mvention is also related to the use of respiratory mtrate reductase alpha subumt polynucleotides and polypeptides ofthe mvention for use as diagnostic reagents Detection of respiratory mtrate reductase alpha subumt polynucleotides and/or polypeptides m a eukaryote. particularly a mammal. and especiaUy a human, wdl provide a diagnostic method for diagnosis of disease, staging of disease or response of an infectious orgamsm to drugs Eukaryotes, particularly and especiaUv humans, particularly those infected or suspected to be infected with an organism compπsmg the respiratory mtrate reductase alpha subumt gene or protein, may be detected at the nucleic acid or ammo acid level by a vaπety of weU known techmques as weU as by methods provided herem Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and/or infected mdividual's bodily matenals Polynucleotides from any of these sources, particularly DNA or RNA, may be used directly for detection or may be amplified enzymaticaUy by using PCR or any other amplification technique pnor to analysis RNA, particularly mRNA, cDNA and genomic DNA may also be used m the same ways Usmg amplification, characterization of the species and stram of infectious or resident orgamsm present m an mdividual. may be made by an analysis ofthe genotype of a selected polynucleotide ofthe orgamsm Deletions and insertions can be detected by a change m size ofthe amplified product m compaπson to a genotype of a reference sequence selected from a related orgamsm. preferably a different species of the same genus or a different stram of the same species Pomt mutations can be identified by hybndizmg amplified DNA to labeled respiratory mtrate reductase alpha subumt 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 m melting temperatures or renaturauon kinetics Polynucleotide sequence differences may also be detected by alterations m the electrophoretic mobility of polynucleotide fragments m gels as compared to a reference sequence This may be earned out with or without denatunng agents Polynucleotide differences may also be detected by direct DNA or RNA sequencing See. for example. Mvers et al . Science. 230 1242 (1985) Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase. VI and S 1 protection assay or a chemical cleavage method See. for example. Cotton et al . Proc Natl Acad Sci USA 85 4397- 4401 (1985) In another embodiment an arrav of ohgonucleotides probes compnsmg respiratorv mtrate reductase alpha subumt nucleotide sequence or fragments thereof can be constructed to conduct efficient screen i ng of, for example, genetic mutations, serotype, taxonomic classification or identification Array technology methods are weU known and have general applicability and can be used to address a vanety of questions m molecular genetics mcludmg gene expression, genetic linkage, and genetic vaπabmty (see. for example. Chee et al . Science, 274 610 (1996)) Thus m another aspect , the present mvention relates to a diagnostic kit that compπses (a) a polynucleot i de ofthe present mvention, preferably the nucleotide sequence of SEQ ID NO 1, 3. or a fragment thereof , (b) a nucleotide sequence complementary to that of (a), (c) a polypeptide of the present mvention, preferably the polypeptide of SEQ ID NO 2 or a fragment thereof , or (d) an ant i body to a polypeptide ofthe present mvention, preferably to the polypeptide of SEQ ID NO 2 It w i ll be apprec i ated that m any such kit. (a), (b), (c) or (d) may compnse a substantial component Such a k i t w i ll be of use m diagnosing a disease or susceptibility to a Disease, among others Th i s mvention also relates to the use of polynucleotides ofthe present mvention as diagnostic reagents Detection of a mutated form of a polynucleotide ofthe mvention. preferable, SEQ ED NO 1 , 3, that i s assoc i ated w i th a disease or pathogemcity will provide a diagnostic tool that can add to. or define , a d i agnos i s of a d i sease, a prognosis of a course of disease, a determination of a stage of disease , or a susceptib i l i ty to a d i sease, that results from underexpression, over-expression or altered expression ofthe polynucleotide Organisms, particularly infectious organisms, carrying mutations m such polynucleotide may be detected at the polynucleotide level by a vanety of techmques, such as those descπbed elsewhere herem The d i fferences m a polynucleotide and/or polypeptide sequence between organisms possessmg a first phenotype and organisms possessmg a different, second different phenotype can also be determ i ned If a mutation is observed in some or all organisms possessing the first phenotvpe but not i n any organisms possessmg the second phenotype, then the mutation is likely to be the causative agent ofthe first phenotype CeUs from an orgamsm carrying mutations or polymorphisms (aUehc vaπations) in a polynucleotide and/or polypeptide of the mvention may also be detected at the polymicleotide or polvpeptide level by a vaπety of techmques. to aUow for serotypmg, for example For example. RT-PCR can be used to detect mutations m the RNA It is particularly prefened to use RT-PCR m con j unction w i th 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 complementar to a polynucleotide encodmg respiratory mtrate reductase alpha subumt polypeptide can be used to identify and analvze mutations The mvention further provides these pπmers with I. 2. 3 or 4 nucleotides removed from the 5' and/or the 3' end These pπmers may be used for. among other things, amplifying respiratorv mtrate reductase alpha subumt DNA and/or RNA isolated from a sample denved from an mdividual. such as a bodily matenal The pπmers may be used to amplify a polynucleotide isolated from an infected mdividual. such that the polynucleotide may then be subject to vaπous techmques for elucidation of the polynucleotide sequence In this way, mutations m the polynucleotide sequence mav be detected and used to diagnose and/or prognose the infection or its stage or course, or to serotype and/or classify the infectious agent The mvention further provides a process for diagnosing, disease, preferablv bactenal infections, more preferably infections caused by Staphylococcus aureus, compnsmg deteπnining from a sample denved from an mdividual, such as a bodily matenal, an mcreased level of expression of polynucleotide havmg a sequence of Table 1 [SEQ ID NO 1, 3] Increased or decreased expression of a respiratory mtrate reductase alpha subumt polynucleotide can be measured using anv on of the methods well known in the art for the quantitation of polynucleotides. such as. for example, amplification, PCR. RT-PCR. RNase protection. Northern blotting, spectrometry and other hybndization methods In addition, a diagnostic assay in accordance with the mvention for detecting over-expression of respiratory mtrate reductase alpha subumt polypeptide compared to normal control tissue samples may be used to detect the presence of an infection, for example Assay techmques that can be used to determine levels of a respiratory mtrate reductase alpha subumt polypeptide, m a sample denved from a host such as a bodily matenal, are well-known to those of skill m the art Such assay methods mclude radioimmunoassays. competitive-bmdmg assays, Western Blot analysis, antibody sandwich assavs. antibody detection and ELISA assays Antagonists and Agonists - Assays and Molecules Polypeptides and polynucleotides ofthe mvention may also be used to assess the binding of smaU molecule substrates and hgands in, for example, cells. ceU-free preparations, chemical libraπes. and natural product mixtures These substrates and hgands may be natural substrates and hgands or may be structural or functional mimetics See. e g , Coligan et al . Current Protocols m Immunology 1(2) Chapter 5 (1991) Polypeptides and polynucleotides ofthe present mvention are responsible for manv biological functions, mcludmg manv disease states, in particular the Diseases herem mentioned It is therefore desirable to devise screening methods to identify compounds that agonize (e g , stimulate) or that antagonize (e g .inhibit) the function ofthe polypeptide or polynucleotide Accordingly, m a further aspect the present mvention provides for a method of screening compounds to ldentifv those that agonize or that antagonize the function of a polypeptide or polvnucleotide ofthe invention, as well as related polypeptides and polvnucleotides In general, agomsts or antagonists (e g , inhibitors) mav be emploved for therapeutic and prophylactic purposes for such Diseases as herem mentioned Compounds mav be identified from a vaπety of sources, for example, ceUs, ceU-free preparations, chemical hbraπes. and natural product mixtures Such agonists and antagonists so-identified may be natural or modified substrates, hgands. receptors, enzymes, etc , as the case may be. of respiratory mtrate reductase alpha subumt polypeptides and polynucleotides, or may be structural or functional mimetics thereof (see Cohgan et al . Current Protocols in Immunology 1(2) Chapter 5 (1991)) The screemng methods may sunply 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 ofthe polypeptide by means of a label directly or indirectly associated with the candidate compound Alternatively, the screemng method may mvolve competition with a labeled competitor Further, these screemng methods may test whether the candidate compound results in a signal generated by activation or inhibition ofthe polypeptide or polynucleotide. usmg detection systems appropnate to the cells compnsmg the polypeptide or polynucleotide Inhibitors of activation are generally assayed in the presence of a known agomst and the effect on activation by the agomst by the presence ofthe candidate compound is observed Constitutively active polypeptide and/or constitutively expressed polypeptides and polynucleotides may be employed m screemng methods for mverse agomsts, in the absence of an agomst or antagonist, by testmg whether the candidate compound results m inhibition of activation ofthe polypeptide or polynucleotide. as the case may be Further, the screemng methods may sunply compnse the steps of mixing a candidate compound with a solution compnsmg a polypeptide or polynucleotide ofthe present invention, to form a mixture, measuring respiratory nitrate reductase alpha subunit polvpeptide and/or polynucleotide activitv in the mixture, and comparing the respiratory nitrate reductase alpha subumt polvpeptide and or polynucleotide activity ofthe mixture to a standard Fusion proteins, such as those made from Fc portion and respiratory nitrate reductase alpha subunit polypeptide. as herein descnbed. can also be used for high-throughput screemng assays to identify antagonists ofthe polypeptide ofthe present mvention. as well as of phylogenetically and and or functionallv related polypeptides (see D Bennett et al , J Mol Recogmtion. 8 52-58 (1995). and K Johanson et al . J Bi Chem. 270(16) 9459-9471 (1995)) The polynucleotides. polypeptides and antibodies that bind to and/or interact with a polvpeptide ofthe present mvention may also be used to configure screenmg methods for detecting the effect of added compounds on the production of mRNA and/or polypeptide in cells For example, an ELISA assav mav be constructed for measuring secreted or cell associated levels of polvpeptide usmg monoclonal and polvclonal antibodies bv standard methods known in the art This can be used to d i scover agents that may inhibit or enhance the production of polypeptide (also called antagonist or agomst. respectivel y ) from suitably manipulated cells or tissues The mvention also provides a method of screemng compounds to identify those that enhance (agomst) or block (antagonist) the action of respiratory mtrate reductase alpha subumt polypeptides or polynucleotides, particularly those compounds that are bacteπstatic and or bactencidal The method of screen i ng may mvolve h i gh-throughput techmques For example, to screen for agomsts or antagonists , a synthet i c react i on m i x, a ceUular compartment such as a membrane. ceU envelope or ceU wall , or a preparation of any thereof, compπsmg respiratory mtrate reduclase alpha subumt polypeptide and a labeled substrate or l i gand of such polypeptide is incubated m the absence or the presence of a candidate molecule that may be a respiratory mtrate reductase alpha subumt agomst or antagonist The ability ofthe cand i date molecule to agonize or antagonize the respiratory mtrate reductase alpha subumt polypeptide is reflected m decreased binding of the labeled ligand or decreased production of product from such substrate Molecules that bmd gratuitously, / . without inducing the effects of respiratory mtrate reductase alpha subumt polypeptide are most likely to be good antagonists Molecules that bmd weU and. as the case may be. mcrease the rate of product production from substrate, increase signal transduction. or mcrease chem i cal channel activity are agomsts Detection of the rate or level of, as the case may be. production of product from substrate, signal transduction, or chemical channel activity may be enhanced by usmg a reporter system Reporter systems that may be useful in this regard mclude but are not limited to coloπmetnc, labeled substrate converted mto product a reporter gene that is responsive to changes m resp i ratory mtrate reductase alpha subumt polynucleotide or polypeptide activity, and binding assays known m the art Polypept i des ofthe invention may be used to identify membrane bound or soluble receptors , i f any, for such polypeptide. through standard receptor bmdmg techniques known in the art These techmques include , but are not limited to. ligand bmdmg and crosslinkmg assays in wh i ch the polypeptide is labeled with a radioactive isotope (for instance, 1 5 I), chemically modified (for i nstance , b i otinylated), or fused to a peptide sequence suitable for detection or punfication. and i ncubated w i th a source ofthe putative receptor (e g , cells, cell membranes , cell supernatants. t i ssue extracts , bod i ly matenals) Other methods include biophysical techmques such as surface plasmon resonance and spectroscopy These screemng methods may also be used to identifλ agomsts and antagonists ofthe polypeptide that compete with the bmdmg ofthe polypeptide to its receptor(s), i f any Standard methods for conductmg such assays are well understood m the art The fluorescence polanzation value for a fluorescently-tagged molecule depends on the rotat i onal conelat i on tune or tumbling rate Protem complexes, such as formed by respiratorv mtrate reductase alpha subumt polypeptide associatmg with another respiratorv mtrate reductase alpha subumt polypeptide or other polypeptide. labeled to compnse a fluorescentlv-labeled molecule will have higher polanzation values than a fluorescently labeled monomenc protein It is prefened that this method be used to charactenze small molecules that disrupt polvpeptide complexes Fluorescence energy transfer may also be used charactenze small molecules that interfere with the formation of respiratory mtrate reductase alpha subumt polypeptide dimers. tnmers. tetramers or higher order structures, or structures formed by respiratory mtrate reductase alpha subumt polypeptide bound to another polypeptide Respiratory mtrate reductase alpha subunit polypeptide can be labeled with both a donor and acceptor fluorophore Upon mixing ofthe two labeled species and excitation ofthe donor fluorophore, fluorescence energy transfer can be detected by observing fluorescence ofthe acceptor Compounds that block duneπzation will inhibit fluorescence energy transfer Surface plasmon resonance can be used to momtor the effect of small molecules on respiratory mtrate reductase alpha subumt polypeptide self-association as well as an association of respiratory mtrate reductase alpha subumt polypeptide and another polypeptide or small molecule Respiratory mtrate reductase alpha subumt polypeptide can be coupled to a sensor chip at low site densitv such that covalently bound molecules will be monomenc Solution protein can then passed over the respiratory mtrate reductase alpha subumt polypeptide -coated surface and specific bmdmg can be detected in real-time by monitoring the change m resonance angle caused by a change m local refractive dex This techmque can be used to charactenze the effect of small molecules on kinetic rates and equihbnum bmdmg constants for respiratorv nitrate reductase alpha subumt polypeptide self-association as well as an association of respiratorv mtrate reductase alpha subumt polypeptide and another polypeptide or small molecule A scintillation proximity assay may be used to characterize the interaction between an association of respiratory mtrate reductase alpha subunit polypeptide with another respiratorv mtrate reductase alpha subumt polypeptide or a different polypeptide Respiratory mtrate reductase alpha subumt polypeptide can be coupled to a scintillation-filled bead Addition of radio-labeled respiratory mtrate reductase alpha subumt polypeptide results in bmdmg where the radioactive source molecule is m close proximity to the scintillation fluid Thus, signal is emitted upon respiratory mtrate reductase alpha subumt polypeptide binding and compounds that prevent respiratorv mtrate reductase alpha subumt polypeptide self-association or an association of respiratorv mtrate reductase alpha subumt polypeptide and another polypeptide or small molecule will dimmish signal In other embodiments ofthe mvention there are provided methods for identifying compounds that bmd to or otherwise interact with and inhibit or activate an activity or expression of a polvpeptide and/or polynucleotide ofthe mvention compnsmg contacting a polypeptide and/or polynucleotide ofthe mvention with a compound to be screened under conditions to permit binding to or other mteraction between the compound and the polypeptide and or polynucleotide to assess the binding to or other mteraction with the compound, such binding or interaction preferably bemg associated with a second component capable of providing a detectable signal in response to the binding or mteraction ofthe polypeptide and/or polynucleotide with the compound, and determining whether the compound binds to or otherwise interacts with and activates or inhibits an activity or expression ofthe polypeptide and/or polvnucleotide by detecting the presence or absence of a signal generated from the bmdmg or mteraction ofthe compound with the polypeptide and/or polynucleotide Another example of an assay for respiratory mtrate reductase alpha subumt agomsts is a competitive assay that combines respiratory mtrate reductase alpha subumt and a potential agomst with respiratory mtrate reductase alpha subumt-bmdrng molecules, recombinant respiratory mtrate reductase alpha subumt bmdmg molecules, natural substrates or hgands, or substrate or ligand mimetics. under appropπate conditions for a competitive inhibition assay Respiratory mtrate reductase alpha subumt can be labeled, such as by radioactivity or a colonmetnc compound, such that the number of respiratory mtrate reductase alpha subumt molecules bound to a bmdmg molecule or converted to product can be determined accurately to assess the effectiveness ofthe potential antagomst It will be readily appreciated by the skilled artisan that a polypeptide and/or polvnucleotide ofthe present mvention may also be used m a method for the structure-based design of an agomst or antagomst ofthe polypeptide and/or polynucleotide. by (a) determining m the first instance the three-dimensional structure ofthe polypeptide and/or polynucleotide, or complexes thereof (b) deducmg the three-dimensional structure for the likely reactive sιte(s). bmdmg sιte(s) or motιf(s) of an agomst or antagomst. (c) synthesizmg candidate compounds that are predicted to bmd to or react with the deduced bmdmg sιte(s), reactive sιte(s), and or motιf(s), and (d) testing whether the candidate compounds are mdeed agomsts or antagonists It will be further appreciated that this will normally be an iterative process, and this iterative process may be performed usmg automated and computer-controlled steps In a further aspect, the present mvention provides methods of treating abnormal conditions such as. for instance, a Disease, related to either an excess of. an under-expression of. an elevated activity of. or a decreased activity of respiratorv mtrate reductase alpha subumt polypeptide and/or polynucleotide If the expression and/or activity ofthe polypeptide and/or polynucleotide is in excess, several approaches are available One approach compnses administering to an mdividual in need thereof an i nh i b i tor compound (antagomst) as herem descπbed. optionaUy m combination with a pharmaceuticaUy acceptable earner , m an amount effective to inhibit the function and/or expression ofthe polypeptide and or polynucleot i de. such as. for example, by blocking the binding of hgands. substrates , recep t ors , enzymes , etc . or by i nh i biting a second signal, and thereby aUeviating the abnormal condition In another approach, soluble forms ofthe polypeptides still capable of bmdmg the ligand. substrate , enzymes , receptors , etc m compet i t i on with endogenous polypeptide and/or polynucleotide may be administered Typ i cal examples of such competitors mclude fragments ofthe respiratory mtrate reductase alpha subumt polypeptide and/or polypeptide In still another approach, expression ofthe gene encodmg endogenous respiratory mtrate reductase alpha subunit polypeptide can be inhibited usmg expression blocking techmques This block i ng may be targeted against any step m gene expression, but is preferably targeted against transcnpt i on and/or translation An examples of a known techmque of this sort mvolve the use of ant i sense sequences , either internally generated or separately administered (see. for example. O ' Connor, J Neuroche (1991) 56 560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Express i on. CRC Press, Boca Raton, FL ( 1988)) Alternatively, oligonucleotides that form tnple hel i ces w i th the gene can be supplied (see, for example, Lee et al , Nucleic Acids Res (1979) 6 3073. Cooney et al , Sc i ence (1988) 241 456, Dervan et al , Science (1991) 251 1360) These o gomers can be adm i n i stered per se or the relevant ohgomers can be expressed in vivo Each of the polynucleotide sequences provided herem may be used in the discovery and development of ant i bacteπal compounds The encoded protem, upon expression , can be used as a target for the screemng of antibactenal drugs Additionally, the polynucleotide sequences encoding the ammo term i nal reg i ons of the encoded protem or Shme-Delgarno or other translation facilitating sequences of the respect i ve mRNA can be used to construct antisense sequences to control the expression ofthe codmg sequence of mterest The i nvent i on also provides the use of the polypeptide. polynucleotide. agonist or antagonist ofthe i nvent i on to i nterfere with the initial physical interaction between a pathogen or pathogens and a eukaryot i c. preferabl y mammalian, host responsible for sequelae of infection In particular , the molecules of the mvent i on may be used in the prevention of adhesion of bacteπa. in particular gram pos i t i ve and or gram negative bactena. to eukaryotic. preferably mammalian , extracellular matrix protems on i n-dwell i ng devices or to extracellular matnx protems in wounds , to block bactenal adhes i on between eukaryotic, preferabl y mammalian, extracellular matnx protems and bacteπal resp i rator y mtrate reductase alpha subumt proteins that mediate tissue damage and/or. to block the normal progress i on of pathogenesis in infections initiated other than bv the implantation of in ¬ dwelling devices or by other surgical techniques In accordance with yet another aspect of the mvention. there are provided respiratorv mtrate reductase alpha subumt agomsts and antagonists, preferablv bacteπstatic or bactencidal agomsts and antagonists The antagonists and agomsts ofthe mvention may be employed, for instance, to prevent, inhibit and/or treat diseases Hehcobacter pylori (herem "H pylon") bactena infect the stomachs of over one-third of the world's population causing stomach cancer, ulcers, and gastntis (International Agencv for Research on Cancer ( 1994) Schistosomes, Liver Flukes and Hehcobacter Pylori (International Agency for Research on Cancer. Lyon, France, http //www uicc ch/ecp/ecp2904 htm) Moreover, the International Agency for Research on Cancer recently recogmzed a cause-and-effect relationship between H pylori and gastric adenocarcinoma, classifying the bactenum as a Group I (definite) carcinogen Prefened antimicrobial compounds of the mvention (agomsts and antagonists of respiratory mtrate reductase alpha subumt polypeptides and/or polynucleotides) found usmg screens provided by the mvention, or known m the art. particularly nanow-spectrum antibiotics, should be useful m the treatment of H pylori infection Such treatment should decrease the advent of H pylon-mά ceά cancers, such as gastrointestinal carcinoma Such treatment should also prevent, inhibit and/or cure gastπc ulcers and gastntis All publications and references, mcludmg but not limited to patents and patent applications, cited m this specification are herem incorporated by reference in their entirety as if each mdividual publication or reference were specifically and mdividually mdicated to be incorporated bv reference herem as bemg fully set forth Any patent application to which this application claims pnoπty is also incorporated by reference herem in its entirety in the manner descnbed above for publications and references GLOSSARY The foUowing definitions are provided to facilitate understanding of certain terms used frequently herem "Bodily mateπal(s) means any matenal denved from an mdividual or from an organism infecting, infesting or inhabiting an mdividual. mcludmg but not limited to. ceUs. tissues and waste, such as. bone, blood, serum, cerebrospinal fluid, semen, sahva. muscle, cartilage, organ tissue, skin. unne. stool or autopsy matenals "Dιsease(s)" means anv disease caused by or related to infection by a bactena. including , for example, disease, such as. infections ofthe upper respiratorv tract (e g , otitis media, bacteπal tracheitis. acute epiglottitis. thvroiditis). lower respiratorv (e g . empvema. lung abscess), cardiac (e g . infective endocarditis), gastrointestinal (e g , secretory dianhoea. splenic absces. retropeπtoneal abscess) CNS (e g . cerebral abscess), eye (e g , blephaπtis. conjunctivitis, keratitis. endophthalmitis. preseptal and orbital ceUuhtis, darcryocystitis). kidney and urinary tract (e g , epididvmitis. lntrarenal and peπnephnc absces. toxic shock syndrome), skm (e g , impetigo, foUicuhtis, cutaneous abscesses. ceUuhtis. wound infection, bacteπal myositis) bone and jomt (e g , septic arthntis. osteomyelitis) "Host cell(s)" is a cell that has been introduced (e g , transformed or transfected) or is capable of introduction (e g , transformation or transfection) by an exogenous polvnucleotide sequence "Identity," as known m the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by comparmg the sequences In the art "identity" also means the degree of sequence relatedness between polypeptide or polvnucleotide sequences, as the case may be, as determined bv the match between strings of such sequences "Identity" can be readily calculated by known methods, mcludmg but not limited to those descπbed m (Computational Molecular Biology, Lesk, A M , ed , Oxford University Press. New York. 1988, Biocomputing Informatics and Genome Projects, Smith. D W . ed , Academic Press. New York, 1993, Computer Analysis of Sequence Data, Part I. Gnffin, A M , and Gnffm. H G . eds , Humana Press, New Jersey, 1994, Sequence Analysis in Molecular Biology, von Hemje, G , Academic Press, 1987, and Sequence Analysis Primer, Gπbskov, M and Devereux. J . eds , M Stockton Press, New York, 1991, and Canllo, H , and Lipman, D , SLAM J Applied Math 48 1073 ( 1988) Methods to determine identity are designed to give the largest match between the sequences tested Moreover, methods to determine identity are codified m publicly available computer programs Computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux. J . et al . Nucleic Acids Research 12(1) 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul. S F et al . J Molec Biol 215 403-410 (1990) The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul. S , et al , NCBI NLM NIH Bethesda. MD 20894 Altschul. S . et al J Mol Biol 215 403-410 (1990) The well known Smith Waterman algonthm mav also be used to determine identity Parameters for polypeptide sequence companson include the following Algonthm Needleman and Wunsch. J Mol Biol 48 443-453 (1970) Companson matnx BLOSSUM62 from Hentikoff and Hentikoff. Proc Natl Acad Sci USA 89 10915-10919 (1992) Gap Penalty 12 Gap Length Penalty 4 A program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group. Madison WI The aforementioned parameters are the default parameters for peptide compansons (along with no penalty for end gaps) Parameters for polynucleotide companson mclude the following Algonthm Needleman and Wunsch, J Mol Biol 48 443-453 (1970) Companson matnx matches = +10, mismatch = 0 Gap Penalty 50 Gap Length Penalty 3 Available as The "gap" program from Genetics Computer Group, Madison WI These are the default parameters for nucleic acid compansons A prefened meaning for "identity" for polynucleotides and polypeptides. as the case may be. are provided m (1) and (2) below (1) Polynucleotide embodiments further mclude an isolated polvnucleotide compnsmg a polynucleotide sequence havmg at least a 95, 97 or 100% identity to the reference sequence of SEQ ID NO 1. wherem said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO 1 or may mclude up to a certain mteger number of nucleotide alterations as compared to the reference sequence, wherem said alterations are selected from the group consistmg of at least one nucleotide deletion, substitution, mcludmg transition and transversion, or insertion, and wherem said alterations may occur at the 5' or 3' terminal positions ofthe reference nucleotide sequence oi anywhere between those termmal positions, mterspersed either individually among the nucleotides in the reference sequence or m one or more contiguous groups within the reference sequence, and wherem said number of nucleotide alterations is determined bv multiplying the total number of nucleotides in SEQ ID NO 1 by the mteger defining the percent identity divided bv 100 and then subtractmg that product from said total number of nucleotides in SEQ ID NO 1 or n n ≤ x n ( χ n • y)> wherem n n is the number of nucleotide alterations. x n is the total number of nucleotides m SEQ ID NO 1 y is 0 95 for 95%. 0 97 for 97% or 1 00 for 100%. and • is the symbol for the multiplication operator, and wherem any non-integer product of x n and y is rounded down to the nearest mteger pπor to subtractmg it from x n Alterations of a polynucleotide sequence encodmg the polvpeptide of SEQ ID NO 2 may create nonsense, missense or frameshift mutations in this coding sequence and therebv alter the polypeptide encoded bv the polynucleotide following such alterations (2) Polypeptide embodiments further mclude an isolated polypeptide compπsmg a polypeptide having at least a 95. 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2. wherem said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2 or may mclude up to a certain mteger number of ammo acid alterations as compared to the reference sequence , wherem said alterations are selected from the group consistmg of at least one aπuno ac i d deletion , substitution, mcludmg conservative and non-conservative substitution, or insertion , and wherem said alterations may occur at the ammo- or carboxy-terminal positions ofthe reference polypeptide sequence or anywhere between those termmal positions, mterspersed either mdiv i dually among the ammo acids the reference sequence or m one or more contiguous groups within the reference sequence, and wherem said number of aπuno acid alterations is determined bv multiplying the total number of ammo acids m SEQ ID NO 2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of ammo acids in SEQ ID NO 2. or n a < x a - (x a • y), wherem n a i s the number of ammo acid alterations, x a is the total number of ammo acids m SEQ ID NO 2. y i s 0 95 for 95%, 0 97 for 97% or 1 00 for 100%, and • is the symbol for the multiplication operator , and wherem any non-mteger product of x a and y is rounded down to the nearest integer pπor to subtracting it from x a "Indιvιdual(s)" means a multiceUular eukaryote. mcludmg, but not limited to a metazoan. a mammal, an ovid. a bovid. a simian, a pπmate. and a human "Isola t ed" means altered "by the hand of man" from its natural state. / e , if it occurs in nature, it has been changed or removed from its oπginal environment, or both For example, a polynucleotide or a polypept i de naturally present in a living orgamsm is not "isolated." but the same polvnucleotide or polypept i de separated from the coexisting matenals of its natural state is "isolated" , as the term is employed herem Moreover, a polynucleotide or polypeptide that is introduced mto an orgamsm bv transformat i on , genetic manipulation or by any other recombinant method is "isolated" even if it is still present in said orgamsm which orgamsm may be living or non-living "Organιsm(s)" means a (l) prokaryote. mcludmg but not limited to. a member of the genus Strep t ococcus. Staphylococcus Bordetella. Corynebactenum. Mycobactenum. Neissena Haemophilus. Ac ti nomvce t es Strep t omycetes Nocardia Enterobacter. Yersinia Fancisella Pasturella Moraxella Ac i ne t obacter Erysipelothnx Branhamella Actinobacillus Streptobacύlus Listena Cahmma t obac t enum Brucella Bacillus Clostnώum Trepone a Eschenchia Salmonella Kleώsiella Vibrio. Proteus. Erwmia Borreha Leptospira Spirillum Campylobacter Shigella Legionella, Pseudomonas. Aeromonas. Rickettsia. Chlamydia Borreha and Mycoplasma. and further mcluding, but not limited to, a member of the species or group, Group A Streptococcus Group B Streptococcus. Group C Streptococcus, Group D Streptococcus, Group G Streptococcus Streptococcus pneumomae, Streptococcus pyogenes, Streptococcus agalactiae. Streptococcus faecahs Streptococcus faecium. Streptococcus durans, Neissena gonorrheae, Neissena mentngitidis. Staphylococcus aureus Staphylococcus epidermidis, Corynebactenum dipthenae, Gardnereϊla vagtnalis Mycobactenum tuberculosis, Mycobactenum bovs, Mycobactenum ulcerans, Mycobactenum leprae Actmomyctes israeln Listena monocytogenes. Bordetella pertusis, Bordatella parapertusis Bordetella bronchtseptica, Eschenchta coli, Shigella dysentenae, Haemophtlus influenzae. Haemophilus aegyptius, Haemophilus paramfluenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacter freunώi, Proteus mirabihs, Proteus vulgans. Yersima pestis Kleibsiella pneumomae Serrano marcessens. Serratia hquefaciens, Vibno cholera. Shigella dysenteni, Shigella flexnen Pseudomonas aeruginosa, Franscisella tularensis. Brucella abortis. Bacillus anthraas. Bacillus cereus. Clostndium perfringens, Clostndium tetani. Clostndium botulmum. Treponema pallidum Rickettsia nckettsii and Chlamydia trachomitis, (u) an archaeon, mcluding but not limited to Archaebacter, and (m) a umceUular or filamentous eukaryote. mcluding but not limited to, a protozoan, a fungus, a member ofthe genus Saccharomyces, Kluveromyces, or Candida, and a member of the species Saccharomyces cenviseae, Kluveromyces lactis, or Candida albicans "Polynucleotide(s)" generaUy refers to any polynbonucleotide or polydeoxynbonucleotide. that may be unmodified RNA or DNA or modified RNA or DNA "Polynucleotide(s)" mclude. without limitation, smgle- and double-stranded DNA. DNA that is a mixture of smgle- and double-stranded regions or smgle-, double- and tπple-stranded regions, smgle- and double-stranded RNA, and RNA that is mixture of smgle- and double-stranded regions, hybnd molecules compπsmg DNA and RNA that may be smgle-stranded or. more typically, double-stranded, or tnple-stranded regions, or a mixture of smgle- and double-stranded regions In addition, "polynucleotide" as used herem refers to tπple-stranded regions compπsmg RNA or DNA or both RNA and DNA The strands m such regions may be from the same molecule or from different molecules The regions may mclude aU of one or more of the molecules, but more typicaUy mvolve only a region of some of the molecules One of the molecules of a tπple-hehcal region often is an ohgonucleotide As used herein, the term "polynucleotιde(s)" also mcludes DNAs or RNAs as descnbed above that compπse one or more modified bases Thus. DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotide(s)" as that term is intended herem Moreover. DNAs or RNAs compπsmg unusual bases, such as mosme. or modified bases, such as tnt lated bases, to name just two examples, are polynucleotides as the term is used herem It will be appreciated that a great vaπety of modifications have been made to DNA and RNA that serve manv useful purposes known to those of skiU m the art The term "polynucleotide(s)" as it is employed herem embraces such chermcaUy. enzymaticaUy or metabohcaUy modified forms of polynucleotides. as weU as the chemical forms of DNA and RNA charactenstic of viruses and ceUs, mcluding. for example, simple and complex ceUs "Polynucleotide(s)" also embraces short polynucleotides often refened to as ohgonucleotide(s) "Polypeptide(s)" refers to any peptide or protem compπsmg two or more aπuno acids jomed to each other by peptide bonds or modified peptide bonds "Polypeptide(s)" refers to both short chains, commonly refened to as peptides, ohgopeptides and ohgomers and to longer chains generaUy refened to as proteins Polypeptides may compπse aπuno acids other than the 20 gene encoded aπuno acids "Polypeptide(s)" mclude those modified either by natural processes, such as processing and other post- translational modifications, but also by chemical modification techmques Such modifications are weU descπbed m basic texts and m more detailed monographs, as weU as in a voluminous research literature, and they are weU known to those of skiU in the art It wiU be appreciated that the same type of modification may be present in the same or varying degree at several sites m a given polypeptide Also, a given polypeptide may compπse many types of modifications Modifications can occur anywhere m a polypeptide, mcluding the peptide backbone, the ammo acid side-chains, and the ammo or carboxyl termini Modifications mclude, for example, acetylation, acylation. ADP-nbosylation. amidation. covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide denvative, covalent attachment of a hpid or hpid denvative. covalent attachment of phosphotidyunositol. cross-linking, cychzation, disulfide bond formation, demethylation. formation of covalent cross-links, formation of cysteine. formation of pyroglutamate. formvlatio gamma- carboxylation, GPI anchor formation, hydroxylation, lodination, methylation. myπstovlation. oxidation, proteolytic processing, phosphorylation. prenylation. racemization. glycosylation. hpid attachment. sulfation. gamma-carboxylation of glutamic acid residues, hvdroxylation and ADP-nbosylation. selenoyiation. sulfation. transfer-RNA mediated addition of aπuno acids to protems. such as argmylation. and ubiquitination See. for instance. PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES 2nd Ed . T E Creighton. W H Freeman and Company, New York (1993) and Wold. F . Posttranslational Protem Modifications Perspectives and Prospects. pgs 1-12 in POSπRANSLATIONAL COVALENT MODIHCATION OF PROTEINS. B C Johnson. Ed . Academic Press. New York (1983), Seifter et al . Meth Enzymol 182626-646 (1990) and Rattan et al Protein Synthesis Posttranslational Modifications and Aging, Ann N Y Acad Sci 663 48-62 ( 1992) Pol y peptides may be branched or cyclic, with or without branching Cvclic branched and branched c i rcular polypeptides mav result from post-translational natural processes and mav be made bv entirelv synthetic methods, as weU "Recomb i nant expression svstem(s)" refers to expression systems or portions thereof or polvnucleotides ofthe mvention introduced or transformed mto a host ceU or host ceU lysate for the production ofthe polynucleotides and polypeptides ofthe mvention • Nanant(s)" as the term is used herem. is a polynucleotide or polypeptide that differs from a reference polynucleotide or polvpeptide respectively, but retains essential properties A typical vanant of a polynucleotide differs m nucleotide sequence from another, reference polvnucleotide Changes m the nucleotide sequence ofthe vaπant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result m ammo acid substitut i ons, add i tions, deletions, fusion protems and truncations m the polypeptide encoded by the reference sequence , as discussed below A typical vanant of a polypeptide differs in aπuno acid sequence from another , reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the vanant are closely similar overall and. in many reg i ons , i dent i cal A vanant and reference polypeptide may differ m ammo acid sequence by one or more subst i tut i ons, additions, deletions m any combmation A substituted or mserted ammo acid res i due may or may not be one encoded by the genetic code The present mvention also mcludes mclude vaπants of each of the polypeptides of the mvention, that is polypeptides that vary from the referents by conservative ammo acid substitutions, whereby a residue is substituted by another with like characteπstics Typical such substitutions are among Ala. Val, Leu and He. among Ser and Thr. among the ac i d i c res i dues Asp and Glu. among Asn and Gin. and among the basic residues Lvs and Arg, or aromatic res i dues Phe and Tvr Particularl y prefened are vaπants m which several. 5-10 1-5 1-3 1-2 or 1 ammo ac i ds are substituted , deleted, or added m anv combination A vaπant of a polvnucleotide or polvpept i de may be a naturally occumng such as an allelic vanant. or it may be a vanant that is not known to occur naturally Non-naturally occumng vanants of polynucleotides and polypeptides may be made by mutagenesis techmques. by direct synthesis, and by other recombinant methods known to skilled artisans EXAMPLES The examples below are earned out using standard techmques. that are weU known and routme to those of sk i U i n the art except where otherwise descπbed in detaU The examples are illustrative , but do not limit the mvention Example 1 Strain selection, Library Production and Sequencing The polvnucleotide havmg a DNA sequence given m Table 1 [SEQ ID NO 1] was obtained from a library of clones of chromosomal DNA of Staphylococcus aureus m E coli The sequencmg data from two or more clones compnsmg overlapping Staphylococcus aureus DNAs was used to construct the contiguous DNA sequence m SEQ ID NO 1 Libranes may be prepared by routine methods, for example Methods 1 and 2 below Total cellular DNA is isolated from Staphylococcus aureus WCUH 29 accordmg to standard procedures and size-fractionated by either of two methods Method 1 Total cellular DNA is mechanically sheared by passage through a needle m order to size- fractionate accordmg to standard procedures DNA fragments of up to 1 lkbp m size are rendered blunt by treatment with exonuclease and DNA polymerase. and EcoRI Imkers added Fragments are ligated mto the vector Lambda ZapII that has been cut with EcoRI. the library packaged bv standard procedures and E co infected with the packaged library The library is amplified by standard procedures Method 2 Total cellular DNA is partially hydrolyzed with a one or a combination of restriction enzymes appropπate to generate a senes of fragments for cloning mto library vectors (e g , Rsal, Pall. Alul, Bshl235I), and such fragments are size-fractionated accordmg to standard procedures EcoRI Imkers are ligated to the DNA and the fragments then ligated mto the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E co infected with the packaged library The library is amplified by standard procedures

Claims: What is claimed is: 1 An isolated polypeptide selected from the group consistmg of (I) an isolated polypeptide compnsmg an aπuno acid havmg at least 95% identity to the aπuno acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2. (π) an isolated polypeptide compnsmg the aπuno acid sequence of SEQ ID NO 2. (in) an isolated polypeptide that is the ammo acid sequence of SEQ ID NO 2. and (IV) a polypeptide that is encoded by a recombinant polynucleotide compπsmg the polyncleotide sequence of SEQ ID NO 3 2 An isolated polynucleotide selected from the group consisting of (I) an isolated polynucleotide compπsmg a polynucleotide sequence encodmg a polypeptide that has at least 95 % identity to the ammo acid sequence of SEQ ID NO 2, over the entire length of SEQ ID NO 2. (u) an isolated polynucleotide compnsmg a polynucleotide sequence that has at least 95% identity over its entire length to a polynucleotide sequence encoding the polypeptide of SEQ ID NO 2, (m) an isolated polynucleotide compnsmg a nucleotide sequence that has at least 95% identity to that of SEQ ID NO 3 over the entire length of SEQ ID NO 3, (iv) an isolated polynucleotide compnsmg a nucleotide sequence encoding the polypeptide of SEQ ID NO 2. (v) an isolated polynucleotide that is the polynucleotide of SEQ ID NO 3. (vi) an isolated polynucleotide of at least 30 nucleotides m length obtainable by screemng an appropnate hbrary under strmgent hybndization conditions with a probe havmg the sequence of SEQ ID NO 3 or a fragment thereof of of at least 30 nucleotides m length, (vu) an isolated polynucleotide encodmg a mature polypeptide expressed by the respiratorv mtrate reductase alpha subumt gene compπsed m the Staphylococcus aureus. and (vui) a polynucleotide sequence complementary to said isolated polynucleotide of (l). (u), (ui). 3 A method for the treatment of an mdividual (I) in need of enhanced activity or expression of or lmmunological response to the polypeptide of claim 1 compnsmg the step of administering to the mdividual a therapeuticallv effective amount of an antagomst to said polypeptide. or (n) having need to inhibit activity or expression ofthe polypeptide of claim 1 compnsmg (a) admuusteπng to the mdividual a therapeuticallv effective amount of an antagomst to said polypeptide. or (b) administering to the mdividual a nucleic acid molecule that inhibits the expression of a polynucleotide sequence encodmg said polypeptide (c) admuusteπng to the mdividual a therapeutically effective amount of a polypeptide that competes with said polypeptide for its ligand, substrate, or receptor, or (d) administering to the mdividual an amount of a polypeptide that induces an lmmunological response to said polypeptide in said mdividual 4 A process for diagnosing or prognosing a disease or a susceptibility to a disease m an mdividual related to expression or activity ofthe polypeptide of claim 1 m an mdividual compπsmg the step of (a) determining the presence or absence of a mutation m the nucleotide sequence encoding said polypeptide m an orgamsm m said mdividual, or (b) analyzing for the presence or amount of said polypeptide expression m a sample denved from said mdividual 5 A process for producing a polypeptide selected from the group consistmg of (I) an isolated polypeptide compnsmg an aπuno acid sequence selected from the group havmg at least 95% identity to the ammo acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2. (π) an isolated polypeptide compnsmg the aπuno acid sequence of SEQ ID NO 2 (in) an isolated polypeptide that is the aπuno acid sequence of SEQ ID NO 2. and (iv) a polypeptide that is encoded by a recombinant polynucleotide compnsmg the polvnucleotide sequence of SEQ ID NO 3, compnsmg the step of cultunng a host cell under conditions sufficient for the production of the polvpeptide 6 A process for producing a host cell compnsmg an expression s stem or a membrane thereof expressing a polypeptide selected from the group consisting of (l) an isolated polypeptide compnsmg an aπuno acid sequence selected from the group having at least 95% identity to the aπuno acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2 (n) an isolated polvpeptide compnsmg the aπuno acid sequence of SEQ ID NO 2 (iii) an isolated polypeptide that is the ammo acid sequence of SEQ ID NO:2. and (iv) a polypeptide that is encoded by a recombinant polynucleotide compnsmg the polynucleotide sequence of SEQ ID NO:3, said process comprising the step of transforming or transfecting a cell with an expression system compnsmg a polynucleotide capable of producing said polypeptide of (i), (u), (iii) or (iv) when said expression system is present a compatible host cell such the host cell, under appropnate culture conditions, produces said polypeptide of (i), (ii), (iii) or (iv). 7. A host cell or a membrane expressing a polypeptide selected from the group consistmg of: (i) an isolated polypeptide compnsmg an amino acid sequence selected from the group havmg at least 95% identity to the ammo acid sequence of SEQ ID NO:2 over the entire length of SEQ ID NO:2. (ii) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO.2. (iii) an isolated polypeptide that is the aπuno acid sequence of SEQ ID NO.2. and (iv) a polypeptide that is encoded by a recombinant polynucleotide compnsmg the polynucleotide sequence of SEQ ID NO:3. 8. An antibody immunospecific for the polypeptide of claim 1. 9. A method for screemng to identify compounds that agonize or that inhibit the function ofthe polypeptide of claim 1 that compπses a method selected from the group consisting of (a) measuring the binding of a candidate compound to the polypeptide (or to the cells or membranes bearing the polypeptide) or a fusion protein thereof by means of a label directly or mdirectly associated with the candidate compound: (b) measurmg the binding of a candidate compound to the polypeptide (or to the cells or membranes bearing the polypeptide) or a fusion protem thereof in the presence of a labeled competitor; (c) testing whether the candidate compound results in a signal generated by activation or inhibition ofthe polypeptide. using detection systems appropnate to the cells or cell membranes bearing the polypeptide: (d) mixing a candidate compound with a solution compnsmg a polypeptide of claim 1. to foπn a mixture, measuring activity ofthe polypeptide m the mixture, and companng the activity of the mixture to a standard: or (e) detectmg the effect of a candidate compound on the production of mRNA encodmg said polypeptide and said polypeptide in cells, usmg for mstance. an ELISA assay 10 An agomst or antagomst to the polypeptide of claim 1

Inventors: MATHIE THOMAS B, TRAINI CHRISTOPHER M, VAN HORN STEPHANIE, WARREN RICHARD L, MATHIE, THOMAS, B., TRAINI, CHRISTOPHER, M., VAN HORN, STEPHANIE, WARREN, RICHARD, L.
Applicants: SMITHKLINE BEECHAM CORP, SMITHKLINE BEECHAM CORPORATION
Application year: 1999
Main classification: C07K14
IPC codes: A61K38, A61K39, A61K45, A61P31, C07K14, C07K16, C12N9, C12N15, C12P21, C12Q1