WO1999030735A1 - UGC (UPSTREAM GENE OF cdsA) - Google Patents

Abstract

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

Description

ugc (upstream gene of cdsA)

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their variants, agonists and antagonists, and their uses In particular, the invention relates to novel polynucleotides and polypeptides of the ugc family, hereinafter referred to as "ugc "

BACKGROUND OF THE INVENTION

The frequency of Pseudomonas aeruginosa infections has nsen dramatically in the past few decades This has been attributed to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems It is no longer uncommon to isolate Pseudomonas aeruginosa strains which are resistant to some or all of the standard antibiotics This phenomenon has created a demand for both new anti-microbial agents, vaccines, and diagnostic tests for this organism

Clearly, there exists a need for factors, such as the ugc (upstream gene of cdsA) embodiments of the invention, that have a present benefit of being useful to screen compounds for antibiotic activity Such factors are also useful to determine their role in pathogenesis of infection, dysfunction and disease There is also a need for identification and characterization of such factors and their antagonists and agonists to find ways to prevent, ameliorate or correct such infection, dysfunction and disease

SUMMARY OF THE INVENTION

It is an object of the invention to provide polypeptides that have been identified as novel ugc (upstream gene of cdsA) polypeptides

It is a further object of the invention to provide polynucleotides that encode ugc polypeptides, particularly polynucleotides that encode the polypeptide herein designated ugc In a particularly preferred embodiment of the invention the polynucleotide compnses a region encoding ugc polypeptides compnsing a sequence set out in Table 1 [SEQ ID NO 1] which includes a full length gene, or a variant thereof

In another particularly preferred embodiment of the invention there is a novel ugc protem from Pseudomonas aeruginosa compπsmg the ammo acid sequence of Table 1 [SEQ ID NO 2]. or a variant thereof

A further aspect of the invention there are provided isolated nucleic acid molecules encoding ugc, particularly Pseudomonas aeruginosa ugc. including mRNAs, cDNAs, genomic DNAs Further embodiments of the invention include biologicalh diagnostically, prophylactically, clinically or therapeutically useful vanants thereof, and compositions compnsing the same

In accordance with another aspect of the invention, there is provided the use of a polynucleotide of the mvention for therapeutic or prophylactic purposes, in particular genetic immunization Among the particularly preferred embodiments of the invention are naturally occurring allelic vanants of ugc and polypeptides encoded thereby Another aspect of the invention there are provided novel polypeptides of Pseudomonas aeruginosa referred to herein as ugc as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful vanants thereof, and compositions compnsing the same

Among the particularly preferred embodiments of the mvention are vanants of ugc polypeptide encoded by naturally occurring alleles of the ugc gene In a preferred embodiment of the mvention there are provided methods for producing the aforementioned ugc polypeptides

In accordance with yet another aspect of the mvention, there are provided inhibitors to such polypeptides, useful as antibacterial agents, including, for example, antibodies

In accordance with certain preferred embodiments of the mvention, there are provided products, compositions and methods for assessing ugc expression, treating disease, assaymg genetic vanation, and administering a ugc polypeptide or polynucleotide to an organism to raise an lmmunological response against a bactena, especially a Pseudomonas aeruginosa bactena

In accordance with certain preferred embodiments of this and other aspects of the mvention there are provided polynucleotides that hybndize to ugc polynucleotide sequences, particularly under stringent conditions

In certain preferred embodiments of the mvention there are provided antibodies against ugc polypeptides In other embodiments of the mvention there are provided methods for identifying compounds which bmd to or otherwise interact with and inhibit or activate an activity of a polypeptide or polynucleotide of the mvention compnsmg contacting a polypeptide or polynucleotide of the mvention with a compound to be screened under conditions to permit bmdmg to or other mteraction between the compound and the polypeptide or polynucleotide to assess the bmding to or other mteraction with the compound, such bmdmg or mteraction bemg associated with a second component capable of providing a detectable signal m response to the bmdmg or mteraction of the polypeptide or polynucleotide with the compound, and determining whether the compound bmds to or otherwise interacts with and activates or inhibits an activity of the polypeptide or polynucleotide by detecting the presence or absence of a signal generated from the bmdmg or mteraction of the compound with the polypeptide or polynucleotide

In accordance with yet another aspect of the mvention, there are provided ugc agonists and antagonists, preferably bactenostatic or bactenocidal agonists and antagonists

In a further aspect of the mvention there are provided compositions compnsing a ugc polynucleotide or a ugc polypeptide for administration to a cell or to a multicellular organism

Vanous changes and modifications within the sprnt and scope of the disclosed mvention will become readily apparent to those skilled in the art from reading the following descnptions and from reading the other parts of the present disclosure

DESCRIPTION OF THE INVENTION

The mvention relates to novel ugc polypeptides and polynucleotides, which are essential to

Pseudomonas aeruginosa, as descnbed m greater detail below In particular, the mvention relates to polypeptides and polynucleotides of a novel ugc of Pseudomonas aeruginosa The mvention relates especially to ugc having the nucleotide and ammo acid sequences set out m Table 1 as SEQ ID NO

1 and SEQ ID NO 2 respectively

TABLE 1 ugc Polynucleotide and Polypeptide Sequences

(A) Sequences from Pseudomonas aeruginosa ugc polynucleotide sequence [SEQ ID NO l] 5 ' -ATGGAAAAGACCCGGAAGGATGTGTGCGTGCCACGCCACGTGGCCATTATCATGGACGGT AACAATCGCTGGGCGAAGAAGCGTCTTCTGCCCGGCGTCGCCGGCCACAAGGCCGGTGTC GATGCCGTCAGGGCGGTGATCGAGGTCTGCGCCGAGGCAGGGGTCGAGGTCCTCACCCTG TTCGCGTTCTCCAGCGAGAACTGGCAGCGTCCGGCGGACGAAGTCAGCGCGCTGATGGAG CTGTTTCTCGTGGCCCTGCGCCGCGAGGTGCGCAAGCTCGACGAGAACGGCATCCGCCTG CGCATCATCGGCGATCGCACGCGTTTCCATCCGGAGTTGCAGGCGGCCATGCGCGAAGCG GAAGCCGCCACTGCCGGCAATACCCGTTTCCTCCTCCAGGTCGCCGCCAACTACGGCGGC CAGTGGGACATCGTCCAGGCCGCACAGCGCCTGGCGCGCGAGGTCCAGGGCGGGCACCTG GCGGCGGACGATATCTCCGCCGAGCTGCTCCAGGGCTGCCTGGTGACCGGCGACCAGCCG CTGCCCGACCTGTGCATCCGCACCGGCGGCGAGCATCGCATCAGCAATTTCCTTCTCTGG CAGCTGGCCTACGCCGAGCTGTATTTCTCCGACCTGTTCTGGCCCGACTTCAAGCACGCG GCGATGCGGGCTGCCCTGGCGGATTTCTCCAAGCGCCAGCGCCGCTTCGGCAAGACCAGC GAGCAAGTCGAGGCCGAAGCCCGTCCGTCATGCTGA-3'

(B) Pseudomonas aeruginosa ugc polypeptide sequence deduced from the polynucleotide sequence m this table [SEQ ID NO 2]

NH₂-MEKTRKDVCVPRHVAIIMDGNNR AKKRLLPGVAGHKAGVDAVRAVIEVCAEAGVEVLTL

FAFSSEN QRPADEVSALMELFLVALRREVRKLDENGIRLRIIGDRTRFHPELQAAMREA

EAATAGNTRFLLQVAANYGGQ DIVQAAQRLAREVQGGHLAADDISAELLQGCLλ/TGDQP

LPDLCIRTGGEHRISNFLLWQLAYAELYFSDLFWPDFKHAAMRAALADFSKRQRRFGKTS

EQVEAEARPSC-COOH

Polypeptides

The polypeptides of the mvention mclude a polypeptide of Table 1 [SEQ ID NO 2] (m particular the mature polypeptide) as well as polypeptides and fragments, particularly those which have the biological activity of ugc, and also those which have at least 70% identity to a polypeptide of Table 1 [SEQ ID NO l]or the relevant portion, preferably at least 80% identity to a polypeptide of Table 1 [SEQ ID NO 2and more preferably at least 90% similarity (more preferably at least 90% identity) to a polypeptide of Table 1 [SEQ ID NO 2] and still more preferably at least 95% similanty (still more preferably at least 95% identity) to a polypeptide of Table 1 [SEQ ID NO 2] and also mclude portions of such polypeptides with such portion of the polypeptide generally containing at least 30 ammo acids and more preferably at least 50 ammo acids The mvention also includes polypeptides of the formula

X-(R₁)_m-(R₂)-(R₃)_n-Y wherein, at the ammo terminus, X is hydrogen, and at the carboxyl terminus. Y is hydrogen or a metal, Ri and R3 are any ammo acid residue, m is an integer between 1 and 1000 or zero, n is an integer between 1 and 1000 or zero, and R₂ is an ammo acid sequence of the mvention, particularly an ammo acid sequence selected from Table 1 In the formula above R₂ is onented so that its ammo terminal residue is at the left, bound to R\ and its carboxy terminal residue is at the nght, bound to R3 Any stretch of ammo acid residues denoted by either R group, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer

A fragment is a variant polypeptide having an ammo acid sequence that entirely is the same as part but not all of the ammo acid sequence of the aforementioned polypeptides As with ugc polypeptides fragments may be "free-standing," or compnsed within a larger polypeptide of which they form a part or region, most preferably as a smgle continuous region, 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 vanants thereof, such as a continuous senes of residues that mcludes the ammo terminus, or a continuous senes of residues that mcludes the carboxyl terminus Degradation forms of the polypeptides of the mvention m a host cell, particularly a Pseudomonas aeruginosa, are also preferred Further preferred are fragments characterized by structural or functional attributes such as fragments that compnse alpha-helix and alpha-hehx forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-formmg regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate bmdmg region, and high antigenic dex regions

Also preferred are biologically active fragments which are those fragments that mediate activities of ugc, including those with a similar activity or an improved activity, or with a decreased undesirable activity Also included are those fragments that are antigenic or lmmunogenic m an animal, especially m a human Particularly preferred are fragments compnsmg receptors or domains of enzymes that confer a function essential for viabihty of Pseudomonas aeruginosa or the abihty to initiate, or maintain cause disease in an individual, particularly a human

Vanants that are fragments of the polypeptides of the mvention may be employed for producmg the corresponding full-lengtii polypeptide by peptide synthesis, therefore, these vanants may be employed as intermediates for producmg the full-length polypeptides of the mvention In addition to the standard smgle and triple letter representations for ammo acids, the term "X" or "Xaa" may also be used m descπbmg certain polypeptides of the invention "X" and "Xaa" mean that any of the twenty naturally occuπng ammo acids may appear at such a designated position m the polypeptide sequence Polynucleotides Another aspect of the mvention relates to isolated polynucleotides, including the full length gene, that encode the ugc polypeptide having a deduced ammo acid sequence of Table 1 [SEQ ID NO 2] and polynucleotides closely related thereto and vanants thereof

Usmg the information provided herein, such as a polynucleotide sequence set out in Table 1 [SEQ ID NO 1], a polynucleotide of the mvention encoding ugc polypeptide may be obtained usmg standard cloning and screening methods, such as those for clonmg and sequencmg chromosomal DNA fragments from bactena usmg Pseudomonas aeruginosa strain 4 cells as starting matenal, followed by obtaining a full length clone For example, to obtain a polynucleotide sequence of the mvention, such as a sequence given m Table 1 [SEQ ID NO 1], typically a library of clones of chromosomal DNA of Pseudomonas aeruginosa strain 4 m E cob or some other suitable host is probed with a radiolabeled ohgonucleotide, preferably a 17-mer or longer, denved from a partial sequence Clones carrying DNA identical to that of the probe can then be distinguished usmg stringent conditions By sequencmg the individual clones thus identified with sequencmg primers designed from the oπgmal sequence it is then possible to extend the sequence m both directions to determine the full gene sequence Convemently, such sequencmg is performed usmg denatured double stranded DNA prepared from a plasmid clone Suitable techniques are described by Maniatis, T , Fπtsch, E F and Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed , Cold Spring Harbor Laboratory Press, Cold Spring Harbor New York (1989) (see in particular Screening By Hybridization 1 90 and Sequencmg Denatured Double-Stranded DNA Templates 13 70) Illustrative of the mvention, the polynucleotide set out m Table 1 [SEQ ID NO 1] was discovered m a DNA library denved from Pseudomonas aeruginosa strain 4 The DNA sequence set out m Table 1 [SEQ ID NO 1] contains an open reading frame encoding a protem having about the number of ammo acid residues set forth m Table 1 [SEQ ID NO 2] with a deduced molecular weight that can be calculated usmg ammo acid residue molecular weight values well known m the art The polynucleotide of SEQ ID NO 1, between nucleotide number 1 and the stop codon which begins at nucleotide number 754 of SEQ ID NO 1, encodes the polypeptide of SEQ ID NO 2

The mvention provides a polynucleotide sequence identical over its entire length to a coding sequence in Table 1 [SEQ ID NO 1] Also provided b the mvention is the coding sequence for the mature polypeptide or a fragment thereof, by itself as well as the coding sequence for the mature polypeptide or a fragment m reading frame with other coding sequence, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protem sequence The polynucleotide may also contain non-coding sequences, including for example, but not limited to non-coding 5' and 3^" sequences, such as the transcnbed, non-translated sequences, termination signals, nbosome bmdmg sites, sequences that stabilize mRNA, introns, pohadenylation signals, and additional coding sequence which encode additional ammo acids For example, a marker sequence that facilitates purification of the fused polypeptide can be encoded In certam embodiments of the 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 Set , USA 86 821-824 (1989), or an HA tag (Wilson et al , Cell 37 767 (1984) Polynucleotides of the mvention also mclude, but are not limited to, polynucleotides compnsing a structural gene and its naturally associated sequences that control gene expression

A preferred embodiment of the mvention is a polynucleotide of compnsmg nucleotide 1 to the nucleotide immediately upstream of or mcludmg nucleotide 754 set forth in SEQ ED NO 1 of Table 1, both of which encode the ugc polypeptide

The mvention also mcludes polynucleotides of the formula

X-(R₁)_m-(R₂)-(R₃)_n-Y wherem, at the 5' end of the molecule, X is hydrogen or together with Y defines a covalent bond, and at the 3' end of the molecule, Y is hydrogen or a metal or together with X defines the covalent bond, each occurance of R_j and R3 is independently any nucleic acid residue, m is an mteger between 1 and 3000 or zero , n is an mteger between 1 and 3000 or zero, and R₂ is a nucleic acid sequence of the mvention, particularly a nucleic acid sequence selected from Table 1 In the polynucleotide formula above R is oriented so that its 5' end residue is at the left, bound to Ri and its 3' end residue is at the πght, bound to R3 Any stretch of nucleic acid residues denoted by either R group, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer Where, m a preferred embodiment, X and Y together define a covalent bond, the polynucleotide of the above formula is a closed, circular polynucleotide, which can be a double-stranded polynucleotide wherem the formula shows a first strand to which the second strand is complementary In another preferred embodiment m and/or n is an mteger between 1 and 1000.

It is most preferred that the polynucleotides of the inventions are denved from Pseudomonas aeruginosa, however, they may preferably be obtained from organisms of the same taxonomic genus They may also be obtained, for example, from organisims of the same taxonomic family or order

The term "polynucleotide encodmg a polypeptide" as used herem encompasses polynucleotides that mclude a sequence encodmg a polypeptide of the mvention, particularly a bactenal polypeptide and more particularly a polypeptide of the Pseudomonas aeruginosa ugc having an amino acid sequence set out in Table 1 [SEQ ID NO 2] The term also encompasses polynucleotides that mclude a smgle continuous region or discontmuous regions encodmg the polypeptide (for example, interrupted by integrated phage or an insertion sequence or editing) together with additional regions, that also may contain coding and/or non-coding sequences The invention further relates to variants of the polynucleotides described herein that encode for variants of the polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NO:2]. Variants that are fragments of the polynucleotides of the invention may be used to synthesize full- length polynucleotides of the invention. Further particularly preferred embodiments are polynucleotides encoding ugc variants, that have the amino acid sequence of ugc polypeptide of Table 1 [SEQ ID NO:2] in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deleted or added, in any combination. Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of ugc. Further preferred embodiments of the invention are polynucleotides that are at least 70% identical over their entire length to a polynucleotide encoding ugc polypeptide having an amino acid sequence set out in Table 1 [SEQ ED NO:2], and polynucleotides that are complementary to such polynucleotides. Alternatively, most highly preferred are polynucleotides that comprise a region that is at least 80% identical over its entire length to a polynucleotide encoding ugc polypeptide and polynucleotides complementary thereto. In this regard, polynucleotides at least 90% identical over their entire length to the same are particularly preferred, and among these particularly preferred polynucleotides, those with at least 95% are especially preferred. Furthermore, those with at least 97% are highly preferred among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99% being the more preferred. Preferred embodiments are polynucleotides that encode polypeptides that retain substantially the same biological function or activity as the mature polypeptide encoded by a DNA ofTable l [SEQ ID NO:l].

The invention further relates to polynucleotides that hybridize to the herein above-described sequences. In this regard, the invention especially relates to polynucleotides that hybridize under stringent conditions to the herein above-described polynucleotides. As herein used, the terms "stringent conditions" and "stringent hybridization conditions" mean hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. An example of stringent hybridization conditions is overnight incubation at 42°C in a solution comprising: 50%) formamide, 5x SSC (150mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml denatured, sheared salmon sperm DNA, followed by washing the hybridization support in 0. lx SSC at about 65°C. Hybridization and wash conditions are well known and exemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter 11 therein.

The invention also provides a polynucleotide consisting essentially of a polynucleotide sequence obtainable by screening an appropriate library containing the complete gene for a polynucleotide sequence set forth in SEQ ID NO: l under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth in SEQ ID NO:l or a fragment thereof; and isolating said DNA sequence. Fragments useful for obtaining such a polynucleotide include, for example, probes and primers described elsewhere herein.

As discussed additionally herein regarding polynucleotide assays of the invention, for instance, polynucleotides of the invention as discussed above, may be used as a hybridization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding ugc and to isolate cDNA and genomic clones of other genes that have a high sequence similarity to the ugc gene. Such probes generally will comprise at least 15 bases. Preferably, such probes will have at least 30 bases and may have at least 50 bases. Particularly preferred probes will have at least 30 bases and will have 50 bases or less .

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

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

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

The invention also provides polynucleotides that may encode a polypeptide that is the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protem transport, may lengthen or shorten protem half-life or may facilitate manipulation of a protem for assay or production, among other things As generally is the case in vivo, the additional ammo acids may be processed away from the mature protem by cellular enzymes

A precursor protein, having the mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide When prosequences are removed such inactive precursors generally are activated Some or all of the prosequences may be removed before activation Generally, such precursors are called proproteins

In addition to the standard A, G, C, T U representations for nucleic acid bases, the term "N" may also be used m descπbmg certam polynucleotides of the mvention "N" means that any of the four DNA or RNA bases may appear at such a designated position m the DNA or RNA sequence, except it is preferred that N is not a base that when taken in combmation with adjacent nucleotide positions, when read in the correct readmg frame, would have the effect of generatmg a premature termination codon in such readmg frame

In sum, a polynucleotide of the mvention may encode a mature protein, a mature protem plus a leader sequence (which may be referred to as a preprotem), a precursor of a mature protem having one or more prosequences that are not the leader sequences of a preprotem, or a preproprotein. which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature forms of the polypeptide Vectors, host cells, expression

The mvention also relates to vectors that compnse a polynucleotide or polynucleotides of the mvention, host cells that are genetically engineered with vectors of the mvention and the production of polypeptides of the mvention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins usmg RNAs denved from the DNA constructs of the mvention For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the mvention Introduction of a polynucleotide into the host cell can be effected by methods descnbed m many standard laboratory manuals, such as Davis et al , BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook et al , MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed , Cold Spring Harbor Laboratory Press, Cold Sprmg Harbor, NY (1989), such as, calcium phosphate transfection. DEAE-dextran mediated transfection. transvection, micromjection, canonic hpid- mediated transfection, electroporation, transduction, scrape loadmg, balhstic introduction and infection

Representative examples of appropnate hosts mclude bactenal cells, such as streptococci, staphylococci, enterococci E coli, streptomyces and Bacillus subtihs cells, fiingal cells, such as yeast cells and Aspergillus cells, insect cells such as Drosophila S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells, and plant cells

A great vanety of expression systems can be used to produce the polypeptides of the mvention Such vectors mclude, among others, chromosomal, episomal and virus-denved vectors, e g , vectors denved from bactenal plasmids, from bactenophage, from transposons from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, 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 contam control regions that regulate as well as engender expression Generally, any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression in this regard The appropnate DNA sequence may be inserted into the expression system by any of a vanety of well-known and routine techmques, such as, for example, those set forth m Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL, (supra)

For secretion of the translated protem mto the lumen of the endoplasmic reticulum, mto the penplasmic space or mto the extracellular environment, appropnate secretion signals may be incorporated mto the expressed polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals Polypeptides of the mvention can be recovered and purified from recombinant cell cultures by well-known methods mcludmg ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography. phosphocellulose chromatography, hydrophobic mteraction chromatography, affinity chromatography, hvdroxylapatite chromatography, and lectin chromatography Most preferably, high performance hquid chromatography is employed for purification Well known techmques for refoldmg protem may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification Diagnostic Assays This mvention is also related to the use of the ugc polynucleotides of the mvention for use as diagnostic reagents Detection of ugc in a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of a disease Eukaryotes (herein also "ιndιvιdual(s)"), particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism compnsmg the ugc gene may be detected at the nucleic acid level by a variety of techmques

Nucleic acids for diagnosis may be obtained from an infected individual's cells and tissues, such as bone, blood, muscle, cartilage, and skin Genomic DNA may be used directly for detection or may be amplified enzymatically by usmg PCR or other amplification technique pnor to analysis RNA, cDNA and genomic DNA may also be used in the same ways Usmg amplification, characterization of the species and strain of prokaryote present m an individual, may be made by an analysis of the genotype of the prokaryote gene Deletions and insertions can be detected by a change m size of the amplified product m companson to the genotype of a reference sequence Point mutations can be identified by hybndizing amplified DNA to labeled ugc polynucleotide sequences Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences m melting temperatures DNA sequence differences may also be detected by alterations m the electrophoretic mobility of the DNA fragments m gels, with or without denaturing agents, or by direct DNA sequencmg See, e g , Myers et al , Science, 230 1242 (1985) Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase and SI protection or a chemical cleavage method See, e g , Cotton et al , Proc Natl Acad Sci , USA, 85 4397-4401 (1985)

Cells carrying mutations or polymorphisms m the gene of the mvention may also be detected at the DNA level by a vanety of techmques, to allow for serotyping, for example For example, RT- PCR can be used to detect mutations It is particularly preferred to used RT-PCR in conjunction with automated detection systems, such as, for example, GeneScan RNA, cDNA or genomic DNA may also be used for the same purpose, PCR or RT-PCR As an example. PCR primers complementary to a nucleic acid encodmg ugc can be used to identify and analyze mutations The mvention also mcludes primers of the formula

X-(R₁)_m-(R₂)-(R₃)_n-Y wherein, at the 5' end of the molecule, X is hydrogen, and at the 3' end of the molecule, Y is hydrogen or a metal, R\ and R3 is any nucleic acid residue, m is an mteger between 1 and 20 or zero , n is an integer between 1 and 20 or zero, and R₂ is a primer sequence of the mvention, particularly a primer sequence selected from Table 2 In the polynucleotide formula above R₂ is onented so that its 5' end residue is at the left, bound to Ri and its 3' end residue is at the nght, bound to R3 Any stretch of nucleic acid residues denoted by either R group, where m and or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer bemg complementary to a region of a polynucleotide of Table 1 In a preferred embodiment m and/or n is an mteger between 1 and 10.

The mvention further provides these primers with 1, 2, 3 or 4 nucleotides removed from the 5' and/or the 3' end These primers may be used for, among other things, amplifying ugc DNA isolated from a sample denved from an individual The primers may be used to amplify the gene isolated from an infected individual such that the gene may then be subject to various techmques for elucidation of the DNA sequence In this way, mutations m the DNA sequence may be detected and used to diagnose infection and to serotype and/or classify the infectious agent

The mvention further provides a process for diagnosing, disease, preferably bactenal infections, more preferably infections by Pseudomonas aeruginosa. compπsmg determining from a sample denved from an individual a mcreased level of expression of polynucleotide having a sequence of Table 1 [SEQ ID NO 1] Increased or decreased expression of ugc polynucleotide can be measured usmg any on of the methods well known in the art for the quantation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting and other hybπdization methods

In addition, a diagnostic assay in accordance with the mvention for detecting over- expression of ugc protem 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 ugc protein, m a sample denved from a host are well-known to those of skill m the art Such assay methods mclude radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays Antibodies

The polypeptides of the mvention or vanants thereof, or cells expressmg them can be used as an immunogen to produce antibodies lmmunospecific for such polypeptides "Antibodies" as used herem mcludes monoclonal and polyclonal antibodies, chimenc, smgle chain, simianized antibodies and humanized antibodies, as well as Fab fragments, mcludmg the products of an Fab immunolglobuhn expression library

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

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

Alternatively phage display technology may be utilized to select antibody genes with binding activities towards the polypeptide either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing anti-ugc or from naive libraries (McCafferty, J. et al, (1990), Nature 348, 552-554; Marks, J. et al., (1992) Biotechnology 10, 779-783). The affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et al, (1991) Nature 352, 624-628). If two antigen binding domains are present each domain may be directed against a different epitope - termed 'bispecific' antibodies.

The above-described antibodies may be employed to isolate or to identify clones expressing the polypeptides to purify the polypeptides by affinity chromatography.

Thus, among others, antibodies against ugc- polypeptide may be employed to treat infections, particularly bacterial infections.

Polypeptide variants include antigenically, epitopically or immunologically equivalent variants that form a particular aspect of this invention. The term "antigenically equivalent derivative" as used herein encompasses a polypeptide or its equivalent which will be specifically recognized by certain antibodies which, when raised to the protein or polypeptide according to the invention, interfere with the immediate physical interaction between pathogen and mammalian host. The term "immunologically equivalent derivative" as used herein encompasses a peptide or its equivalent which when used in a suitable formulation to raise antibodies in a vertebrate, the antibodies act to interfere with the immediate physical interaction between pathogen and mammalian host. The polypeptide, such as an antigenically or immunologically equivalent derivative or a fusion protein thereof is used as an antigen to immunize a mouse or other animal such as a rat or chicken. The fusion protein may provide stability to the polypeptide. The antigen may be associated, for example by conjugation, with an lmmunogemc carrier protem for example bovme serum albumin (BSA) or keyhole limpet haemocyanin (KLH) Alternatively a multiple antigenic peptide compπsmg multiple copies of the protem or polypeptide, or an antigenically or immunologically equivalent polypeptide thereof may be sufficiently antigenic to improve immunogenicity so as to obviate the use of a carrier

Preferably, the antibody or vanant thereof is modified to make it less lmmunogemc m the individual For example, if the individual is human the antibody may most preferably be "humanized", where the complimentanty determining regιon(s) of the hybπdoma-deπved antibody has been transplanted mto a human monoclonal antibody , for example as descnbed m Jones, P et al (1986), Nature 321, 522-525 or Tempest et al , (1991) Biotechnology 9, 266-273

The use of a polynucleotide of the mvention m genetic immunization will preferably employ a suitable delivery method such as direct injection of plasmid DNA mto muscles (Wolff et al , Hum Mol Genet 1992, 1 363, Manthorpe et al . Hum Gene Ther 1963 4, 419), delivery of DNA complexed with specific protem earners (Wu et al , J Biol Chem 1989 264,16985), coprecipitation of DNA with calcium phosphate (Benvenisty & Reshef, PNAS USA, 1986 83,9551), encapsulation of DNA m vaπous forms of posomes (Kaneda et al , Science 1989 243,375), particle bombardment (Tang et al , Nature 1992, 356 152, Eisenbraun et al , DNA Cell Biol 1993, 12 791) and in vivo infection usmg cloned retroviral vectors (Seeger et al , PNAS USA 1984 81,5849)

Antagonists and agonists - assays and molecules

Polypeptides of the mvention may also be used to assess the bmdmg of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical hbranes, and natural product mixtures These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics See, e g , Cohgan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991)

The mvention also provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) the action of ugc polypeptides or polynucleotides, particularly those compounds that are bactenostatic and/or bactenocidal The method of screening may mvolve high-throughput techmques For example, to screen for agomsts or antagoists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, compnsmg ugc polypeptide and a labeled substrate or hgand of such polypeptide is incubated m the absence or the presence of a candidate molecule that may be a ugc agonist or antagonist The abihty of the candidate molecule to agonize or antagonize the ugc polypeptide is reflected m decreased bmdmg of the labeled ligand or decreased production of product from such substrate Molecules that b d gratuitously, i e . without mducmg the effects of ugc polypeptide are most likely to be good antagonists Molecules that bmd well and mcrease the rate of product production from substrate are agomsts Detection of the rate or level of production of product from substrate may be enhanced by usmg a reporter system Reporter systems that may be useful m this regard mclude but are not limited to colonmetnc labeled substrate converted mto product, a reporter gene that is responsive to changes in ugc polynucleotide or polypeptide activity, and bmding assays known m the art

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

Potential antagonists mclude small orgamc molecules, peptides, polypeptides and antibodies that bmd to a polynucleotide or polypeptide of the mvention and thereby inhibit or extinguish its activity Potential antagonists also may be small orgamc molecules, a peptide, a polypeptide such as a closely related protem or antibody that bmds the same sites on a bmdmg molecule, such as a bmdmg molecule, without mducmg ugc-mduced activities, thereby preventing the action of ugc by excluding ugc from bmdmg

Potential antagonists mclude a small molecule that bmds to and occupies the bmdmg site of the polypeptide thereby preventing bmding to cellular bmd g molecules, such that normal biological activity is prevented Examples of small molecules mclude but are not limited to small orgamc molecules, peptides or peptide-like molecules Other potential antagonists mclude antisense molecules (see Okano, J Neurochem 56 560 (1991), OLIGODEOXYNUCLEOTLDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL (1988), for a descπption of these molecules) Prefened potential antagonists mclude compounds related to and vanants of ugc

Each of the DNA sequences provided herem may be used m the discovery and development of antibacterial compounds The encoded protem, upon expression, can be used as a target for the screening of antibacterial drugs Additionally, the DNA sequences encodmg the ammo terminal regions of the encoded protem or Shme-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest The mvention also provides the use of the polypeptide, polynucleotide or inhibitor of the mvention to interfere with the initial physical mteraction between a pathogen and mammalian host responsible for sequelae of infection In particular the molecules of the mvention may be used m the prevention of adhesion of bactena, m particular gram positive bactena, to mammalian extracellular matrix protems on m-dwelhng devices or to extracellular matnx protems in wounds, to block ugc protein-mediated mammalian cell mvasion by, for example, initiating phosphorylation of mammalian tyrosme kmases (Rosenshine et al , Infect Immun 60 2211 (1992), to block bacterial adhesion between mammalian extracellular matrix protems and bactenal ugc protems that mediate tissue damage and, to block the normal progression of pathogenesis m infections initiated other than by the implantation of m-dwelhng devices or by other surgical techmques

The antagomsts and agomsts of the mvention may be employed, for instance, to inhibit and treat diseases

Hehcobacter pylori (herein H pylori) bacteria infect the stomachs of over one-third of the world's population causing stomach cancer, ulcers, and gastntis (International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Hehcobacter Pylori (International Agency for Research on Cancer, Lyon, France, http //www uicc ch/ecp/ecp2904 htm) Moreover, the international Agency for Research on Cancer recently recognized a cause-and- effect relationship between H pylori and gastric adenocarcmoma, classifying the bactenum as a Group I (definite) carcinogen Prefeπed antimicrobial compounds of the mvention (agomsts and antagomsts of ugc) found usmg screens provided by the mvention, particularly broad- spectrum antibiotics, should be useful m the treatment of H pylori mfection Such treatment should decrease the advent of H pylor -mduced cancers, such as gastrointestinal carcmoma Such treatment should also cure gastnc ulcers and gastntis Vaccines Another aspect of the mvention relates to a method for mducmg an lmmunological response m an individual, particularly a mammal which comprises moculatmg the individual with ugc, or a fragment or vaπant thereof, adequate to produce antibody and/ or T cell immune response to protect said individual from infection, particularly bacterial infection and most particularly Pseudomonas aeruginosa infection. Also provided are methods whereby such immunological response slows bacterial replication. Yet another aspect of the invention relates to a method of inducing immunological response in an individual which comprises delivering to such individual a nucleic acid vector to direct expression of ugc, or a fragment or a variant thereof, for expressing ugc, or a fragment or a variant thereof in vivo in order to induce an immunological response, such as, to produce antibody and/ or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said individual from disease, whether that disease is already established within the individual or not. One way of administering the gene is by accelerating it into the desired cells as a coating on particles or otherwise. Such nucleic acid vector may comprise DNA. RNA, a modified nucleic acid, or a DNA/RNA hybrid.

A further aspect of the invention relates to an immunological composition which, when introduced into an individual capable or having induced within it an immunological response, induces an immunological response in such individual to a ugc or protein coded therefrom, wherein the composition comprises a recombinant ugc or protein coded therefrom comprising DNA which codes for and expresses an antigen of said ugc or protein coded therefrom. The immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity or cellular immunity such as that arising from CTL or CD4+ T cells. A ugc polypeptide or a fragment thereof may be fused with co-protein which may not by itself produce antibodies, but is capable of stabilizing the first protein and producing a fused protein which will have immunogenic and protective properties. Thus fused recombinant protein, preferably further comprises an antigenic co-protein, such as lipoprotein D from Hemophilus influenzae, Glutathione-S-transferase (GST) or beta-galactosidase, relatively large co-proteins which solubilize the protein and facilitate production and purification thereof. Moreover, the co-protein may act as an adjuvant in the sense of providing a generalized stimulation of the immune system. The co-protein may be attached to either the amino or carboxy terminus of the first protein.

Provided by this invention are compositions, particularly vaccine compositions, and methods comprising the polypeptides or polynucleotides of the invention and immunostimulatory DNA sequences, such as those described in Sato, Y. et al. Science 273: 352 (1996). Also, provided by this mvention are methods usmg the descπbed polynucleotide or particular fragments thereof which have been shown to encode non-variable regions of bactenal cell surface protems m DNA constructs used m such genetic immunization experiments m animal models of mfection with Pseudomonas aeruginosa will be particularly useful for identifymg protem epitopes able to provoke a prophylactic or therapeutic immune response It is believed that this approach will allow for the subsequent preparation of monoclonal antibodies of particular value from the requisite organ of the animal successfully resistmg or clearing mfection for the development of prophylactic agents or therapeutic treatments of bacterial infection, particularly Pseudomonas aeruginosa mfection, m mammals, particularly humans

The polypeptide may be used as an antigen for vaccmation of a host to produce specific antibodies which protect against mvasion of bactena, for example by blocking adherence of bactena to damaged tissue Examples of tissue damage mclude wounds m skin or connective tissue caused, e g , by mechanical, chemical or thermal damage or by implantation of mdwellmg devices, or wounds m the mucous membranes, such as the mouth, mammary glands, urethra or vagma

The mvention also mcludes a vaccine formulation which compnses an immunogenic recombinant protem of the mvention together with a suitable earner Smce the protein may be broken down in the stomach, it is preferably administered parenterally, mcludmg, for example, administration that is subcutaneous, intramuscular, mtravenous, or intradermal Formulations suitable for parenteral administration mclude aqueous and non-aqueous sterile injection solutions which may contam anti-oxidants, buffers, bactenostats and solutes which render the formulation msotonic with the bodily fluid, preferably the blood, of the individual, and aqueous and non-aqueous sterile suspensions which may mclude suspendmg agents or thickening agents The formulations may be presented m umt-dose or multi-dose contamers, for example, sealed ampules and vials and may be stored m a freeze-dned condition requiring only the addition of the stenle liquid earner immediately pnor to use The vaccme formulation may also mclude adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known m the art The dosage will depend on the specific activity of the vaccme and can be readily determined by routine experimentation

While the mvention has been described with reference to certam ugc protein, it is to be understood that this covers fragments of the naturally occurring protein and similar protems with additions, deletions or substitutions which do not substantially affect the lmmunogemc properties of the recombinant protem

Compositions, kits and administration

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

Polypeptides and other compounds of the mvention may be employed alone or in conjunction with other compounds, such as therapeutic compounds

The pharmaceutical compositions may be administered m any effective, convenient manner mcludmg, for instance, administration by topical, oral, anal, vaginal, mtravenous, intrapentoneaL intramuscular, subcutaneous, lntranasal or tradermal routes among others

In therapy or as a prophylactic, the active agent may be administered to an individual as an lnjectable composition, for example as a stenle aqueous dispersion, preferably isotomc

Alternatively the composition may be formulated for topical application for example m the form of omtments, creams, lotions, eye omtments, eye drops, ear drops, mouthwash, impregnated dressings and sutures and aerosols, and may contam appropnate conventional additives, mcludmg, for example, preservatives, solvents to assist drug penetration, and emollients in omtments and creams Such topical formulations may also contam compatible conventional earners, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions Such earners may constitute from about 1% to about 98% by weight of the formulation, more usually they will constitute up to about 80% by weight of the formulation For administration to mammals, and particularly humans, it is expected that the daily dosage level of the active agent will be from 0 01 mg/kg to 10 mg/kg, typically around 1 mg/kg The physician in any event will determine the actual dosage which will be most suitable for an individual and will vary with the age, weight and response of the particular individual The above dosages are exemplary of the average case There can, of course, be individual instances where higher or lower dosage ranges are mented, and such are within the scope of this mvention In-dwellmg devices mclude surgical implants, prosthetic devices and catheters, 1 e , devices that are mtroduced to the body of an individual and remain m position for an extended time Such devices mclude, for example, artificial jomts, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters, contmuous ambulatory pentoneal d alysis (CAPD) catheters The composition of the mvention may be administered by injection to achieve a systemic effect agamst relevant bactena shortly before msertion of an m-dwelling device Treatment may be contmued after surgery during the m-body time of the device In addition, the composition could also be used to broaden penoperative cover for any surgical technique to prevent bactenal wound infections, especially Pseudomonas aeruginosa wound infections Many orthopaedic surgeons consider that humans with prosthetic jomts should be considered for antibiotic prophylaxis before dental treatment that could produce a bacteremia Late deep infection is a serious complication sometimes leadmg to loss of the prosthetic jomt and is accompanied by significant morbidity and mortality It may therefore be possible to extend the use of the active agent as a replacement for prophylactic antibiotics m this situation In addition to the therapy descnbed above, the compositions of this mvention may be used generally as a wound treatment agent to prevent adhesion of bactena to matnx protems exposed m wound tissue and for prophylactic use m dental treatment as an alternative to, or m conjunction with, antibiotic prophylaxis

Alternatively, the composition of the mvention may be used to bathe an mdwellmg device immediately before msertion The active agent will preferably be present at a concentration of lμg/ml to lOmg/ml for bathing of wounds or mdwellmg devices

A vaccme composition is convemently m lnjectable form Conventional adjuvants may be employed to enhance the immune response A suitable umt dose for vaccmation is 0 5-5 microgram/kg of antigen, and such dose is preferably administered 1-3 times and with an mterval of 1-3 weeks With the mdicated dose range, no adverse toxicological effects will be observed with the compounds of the mvention which would preclude their administration to suitable individuals Each reference disclosed herem is incorporated by reference herem in its entirety Any patent application to which this application claims priority is also incorporated by reference herem m its entirety GLOSSARY The following definitions are provided to facilitate understanding of certain terms used frequently herem

"Host cell" is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence

"Identity," as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparmg the sequences In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences "Identity" and "similaπty" can be readily calculated by known methods, mcludmg but not limited to those descnbed m (Computational Molecular Biology, Lesk, A M , ed , Oxford University Press, New York, 1988, Biocomputing Informatics and Genome Projects, Smith, D W , ed , Academic Press. New York, 1993, Computer Analysis of Sequence Data, Part I, Gnfϊin, A M , and Gnf in, 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 , SIAM J Applied Math , 48 1073 (1988) Preferred methods to determme identity are designed to give the largest match between the sequences tested Methods to determme identity and similarity are codified m publicly available computer programs Prefened computer program methods to determine identity and similanty between two sequences mclude, but are not limited to, the GCG program package (Devereux, J , et al , Nucleic Acids Research 12(1) 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, 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 B ol 215 403-410 (1990) As an illustration, by a polynucleotide havmg a nucleotide sequence havmg at least, for example, 95% "identity" to a reference nucleotide sequence of SEQ ID NO 1 it is mtended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may mclude up to five pomt mutations per each 100 nucleotides of the reference nucleotide sequence of SEQ ID NO 1 In other words, to obtain a polynucleotide havmg a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides m the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides m the reference sequence may be inserted mto the reference sequence These mutations of the reference sequence may occur at the 5 or 3 ' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, mterspersed either mdividually among nucleotides m the reference sequence or in one or more contiguous groups withm the reference sequence Analogously , by a polypeptide havmg an ammo acid sequence havmg at least, for example, 95% identity to a reference ammo acid sequence of SEQ ID NO 2 is mtended that the ammo acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may mclude up to five ammo acid alterations per each 100 ammo acids of the reference ammo acid of SEQ ID NO 2 In other words, to obtam a polypeptide havmg an ammo acid sequence at least 95% identical to a reference ammo acid sequence, up to 5% of the ammo acid residues m the reference sequence may be deleted or substituted with another ammo acid, or a number of ammo acids up to 5% of the total ammo acid residues m the reference sequence may be inserted mto the reference sequence These alterations of the reference sequence may occur at the ammo or carboxy terminal positions of the reference ammo acid sequence or anywhere between those terminal positions, mterspersed either mdividually among residues m the reference sequence or m one or more contiguous groups within the reference sequence

"Isolated" means altered "by the hand of man" from its natural state, i e , if it occurs in nature, it has been changed or removed from its ongrnal environment, or both For example, a polynucleotide or a polypeptide naturally present in a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting matenals of its natural state is "isolated", as the term is employed herem Moreover, a polynucleotide or polypeptide that is introduced mto an organism by transformation, genetic manipulation or by any other recombinant method is "isolated" even if it is still present m said organism, which organism may be living or nonliving "Polynucleotide(s)" generally refers to any polynbonucleotide or polydeoxnbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA "Polynucleotide(s)" mclude, without limitation, smgle- and double-stranded DNA, DNA that is a mixture of smgle- and double- stranded regions or smgle-, double- and tnple-stranded regions, smgle- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybnd molecules compnsing DNA and RNA that may be single-stranded or, more typically, double-stranded, or tnple-stranded regions, or a mixture of smgle- and double-stranded regions In addition, "polynucleotide" as used herem refers to tnple-stranded regions compnsmg RNA or DNA or both RNA and DNA The strands in such regions may be from the same molecule or from different molecules The regions may mclude all of one or more of the molecules, but more typically mvolve only a region of some of the molecules One of the molecules of a tnple-hehcal region often is an ohgonucleotide As used herein, the term "polynucleotide(s)" also mcludes DNAs or RNAs as descnbed above that contam 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 compnsing unusual bases, such as mosme, or modified bases, such as tntylated bases, to name just two examples, are polynucleotides as the term is used herem It will be appreciated that a great vanety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skdl m the art The term "polynucleotide(s)" as it is employed herem embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA charactenstic of viruses and cells, mcludmg, for example, simple and complex cells "Polynucleσtide(s)" also embraces short polynucleotides often refened to as ohgonucleotide(s) "Polypeptide(s)" refers to any peptide or protem compnsmg two or more am o acids jo ed to each other by peptide bonds or modified peptide bonds "Polypeptide(s)" refers to both short chains, commonly referred to as peptides, ohgopeptides and ohgomers and to longer chains generally referred to as proteins Polypeptides may contam ammo acids other than the 20 gene encoded ammo acids "Polypeptide(s)" mclude those modified either by natural processes, such as processmg and other post-translational modifications, but also by chemical modification techmques Such modifications are well descnbed in basic texts and m more detailed monographs, as well as m a voluminous research literature, and they are well known to those of skill m the art It will be appreciated that the same type of modification may be present m the same or varying degree at several sites m a given polypeptide Also, a given polypeptide may contain many types of modifications Modifications can occur anywhere m a polypeptide, mcludmg the peptide backbone, the ammo acid side-chains, and the ammo or carboxyl termini Modifications mclude, for example, acetylation, acylation, ADP-nbosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation. gamma- carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins, such as arginylation, and ubiquitination. See, for instance, PROTEENS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed, T. E. Creighton, W. H. Freeman and Company, New York (1993) and Wold, F, Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed, Academic Press, New York (1983); Seifter et al, Meth. Enzymol. 182:626-646 (1990) and Rattan et al, Protein Synthesis: Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663: 48- 62 (1992). Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well.

'Nariant(s)" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non- naturally occurring vanants of polynucleotides and polypeptides may be made by mutagenesis techmques, by direct synthesis, and by other recombmant methods known to skilled artisans EXAMPLES

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

The polynucleotide havmg a DNA sequence given in Table 1 [SEQ ID NO 1] was obtamed from a library of clones of chromosomal DNA of Pseudomonas aeruginosa m E cob The sequencmg data from two or more clones contammg overlappmg Pseudomonas aeruginosa DNAs was used to construct the contiguous DNA sequence in SEQ ID NO 1 Libranes may be prepared by routme methods, for example Methods 1 and 2 below

Total cellular DNA is isolated from Pseudomonas aeruginosa strain 4 accordmg to standard procedures and size-fractionated by either of two methods

Method 1

Total cellular DNA is mechanically sheared by passage through a needle in 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 linkers added Fragments are ligated mto the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E cob 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 combmation of restnction enzymes appropπate to generate a senes of fragments for clonmg mto library vectors (e g , Rsal, Pall, Alul, Bshl235I), and such fragments are size-fractionated accordmg to standard procedures EcoRI linkers are ligated to the DNA and the fragments then ligated mto the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E cob infected with the packaged library The library is amplified by standard procedures

Example 2 ugc Characterization We have used chemical mutagenesis to isolate Ts mutants m an attempt to identify essential P aeruginosa gene products Over 100 mutants, which show Ts growth on complex medium at 44°C, have been isolated A genomic library contammg 5 to 6 kb DNA fragments of P aeruginosa was constructed to complement these Ts mutants Nucleotide sequence analysis of plasmids complementmg the Ts mutants revealed many known essential genes as well as genes with unknown functions One of the novel essential genes, encodmg a gene product with an unknown biochemical function, is called ugc Nucleotide sequence analysis of the Ts ugc allele revealed a C- T transition mutation at nucleotide position 404 m Table 1 [SEQ ID NO 1], which caused an ammo acid substitution resulting m the change of alanine at position 135 m Table 1 [SEQ ID NO 2] to valme m the Ugc ORF Flow cytometry studies of the ugc Ts mutant revealed that, following a shift to the non-permissive temperature, both cellular membrane potential and integrity were disrupted and cell lysis rapidly followed Viable cell count data supported this rapid loss of viability In contrast, there was no obvious effects on the complemented mutant at the non-permissive temperature This data and the fact that the gene is clustered with the cdsA gene encodmg the CDP-diglycende synthetase suggest that ugc could be mvolved m phosphohpid biosynthesis

Claims

What is claimed is:

1 An isolated polynucleotide compnsmg a polynucleotide havmg at least a 70% identity to a polynucleotide encodmg a polypeptide compnsmg the ammo acid sequence of SEQ ID NO 2

2 An isolated polynucleotide compnsmg a polynucleotide having at least a 70% identity to a polynucleotide encodmg the same mature polypeptide expressed by the ugc gene contained m the Pseudomonas aeruginosa of the deposited strain

3 An isolated polynucleotide compnsmg a polynucleotide encodmg a polypeptide compnsmg an am o acid sequence which is at least 70% identical to the ammo acid sequence of SEQ ID NO 2

4 An isolated polynucleotide that is complementary to the polynucleotide of claim 1

5 The polynucleotide of Claim 1 wherem the polynucleotide is DNA or RNA

6 The polynucleotide of Claim 1 compnsmg the nucleic acid sequence set forth m SEQ ID NO 1

7 The polynucleotide of Claim 1 compnsmg nucleotide 1 to the stop codon which begins at nucleotide number 754 set forth m SEQ ID NO 1

8 The polynucleotide of Claim 1 which encodes a polypeptide compnsmg the ammo acid sequence of SEQ ID NO 2

9 A vector compnsmg the polynucleotide of Claim 1

10 A host cell compπsmg the vector of Claim 9

11 A process for producmg a polypeptide compnsmg expressmg from the host cell of Claim 10 a polypeptide encoded by said DNA

12 A process for producmg a ugc polypeptide or fragment compnsmg cultuπng a host of claim 10 under conditions sufficient for the production of said polypeptide or fragment

13 A polypeptide compnsmg an ammo acid sequence which is at least 70% identical to the ammo acid sequence of SEQ ID NO 2

14 A polypeptide compπsmg an ammo acid sequence as set forth m SEQ ID NO 2

15 An antibody against the polypeptide of claim 14

16 An antagonist which inhibits the activity or expression of the polypeptide of claim 14

17 A method for the treatment of an individual in need of ugc polypeptide compπsmg administering to the individual a therapeutically effective amount of the polypeptide of claim 14 18 A method for the treatment of an mdividual havmg need to inhibit ugc polypeptide compπsmg administering to the mdividual a therapeutically effective amount of the antagomst of Claim 14

19 A process for diagnosing a disease related to expression or activity of the polypeptide of claim 14 m an mdividual compnsmg

(a) determining a nucleic acid sequence encodmg said polypeptide, and/or

(b) analyzing for the presence or amount of said polypeptide m a sample denved from the mdividual

20 A method for identifying compounds which mteract with and inhibit or activate an activity of the polypeptide of claim 14 compnsmg contacting a composition compnsmg the polypeptide with the compound to be screened under conditions to permit mteraction between the compound and the polypeptide to assess the mteraction of a compound, such mteraction bemg associated with a second component capable of providing a detectable signal m response to the mteraction of the polypeptide with the compound, and determining whether the compound interacts with and activates or inhibits an activity of the polypeptide by detectmg the presence or absence of a signal generated from the mteraction of the compound with the polypepti.de

21 A method for mducmg an immunological response m a mammal which compnses inoculating the mammal with ugc polypeptide of claim 14, or a fragment or vaπant thereof, adequate to produce antibody and/or T cell immune response to protect said animal from disease

22 A method of mducmg immunological response m a mammal which compnses delivering a nucleic acid vector to direct expression of ugc polypeptide of claim 14, or fragment or a vaπant thereof, for expressmg said ugc polypeptide, or a fragment or a vaπant thereof in vivo m order to induce an immunological response to produce antibody and/ or T cell immune response to protect said animal from disease