Description

DETECTION OF CONVERSION TO MUCOIDY IN PSEUDOMONAS AERUGINOSA

INFECTING CYSTIC FIBROSIS PATIENTS

The present application is a continuation-in-part of co-pending US Patent Application Serial No. 08/017,114, filed February 12, 1993. The entire text of the above- referenced disclosures is specifically incorporated by reference herein without disclaimer. The government owns rights in the present invention pursuant to grant number AI31139 from the National Institutes of Health) .

BACKGROUND OF THE INVENTION

Cystic Fibrosis (CF) is the most common inheritable lethal disease among Caucasians. There are approximately 25,000 CF patients in the U.S.A. The frequency of CF in several other countries (e.g., Canada, United Kingdom, Denmark) is high (ranging from 1 in 400 to 1 in 1,600 live births) . There are numerous CF centers in the U.S.A. and Europe - specialized clinical facilities for diagnosing and treating children and adolescents with CF.

Chronic respiratory infections caused by mucoid Pseudomonas aeruginosa are the leading cause of high morbidity and mortality in CF. The initially colonizing P . aeruginosa strains are nonmucoid but in the CF lung they inevitably convert into the mucoid form. The mucoid coating composed of the exopolysaccharide alginate leads to the inability of patients to clear the infection, even under aggressive antibiotic therapies. The emergence of the mucoid form of P. aeruginosa is associated with further disease deterioration and poor prognosis.

The microcolony mode of growth of P. aeruginosa , embedded in exopolysaccharide biofilms in the lungs of CF patients (Costerton et al., 1983), among other functions, plays a role in hindering effective opsonization and phagocytosis of P . aeruginosa cells (Pier et al., 1987; Pier 1992) . Although CF patients can produce opsonic antibodies against P. aeruginosa antigens, in most cases phagocytic cells cannot effectively interact with such opsonins (Pressler et al., 1992; Pier et al., 1990; Pier 1992) . Physical hindrance caused by the exopolysaccharide alginate and a functionally important receptor-opsonin mismatch caused by chronic inflammation and proteolysis are contributing factors to these processes (Pedersen et al., 1990; Tosi et al. , 1990; Pier, 1992) . Under such circumstances, the ability of P. aeruginosa to produce alginate becomes a critical persistence factor in CF; consequently, selection for alginate overproducing (mucoid) strains predominates in the CF lung.

Synthesis of alginate and its regulation has been the object of numerous studies (Govan, 1988; Ohman et al., 1990; Deretic et al., 1991; May et al., 1991). It has been shown that several alginate biosynthetic genes form a cluster at 34 min of the chromosome (Darzins et al., 1985), and that the algD gene, encoding GDPmannose dehydrogenase, undergoes strong transcriptional activation in mucoid cells (Deretic et al., 1987; 1991). GDP mannose dehydrogenase catalyzes double oxidation of GDP mannose into its uronic acid, a reaction that channels sugar intermediates into alginate production. The transcriptional activation of algD has become a benchmark for measuring molecular events controlling mucoidy (Deretic et al., 1991; Ohman et al., 1990; May et al., 1991). Studies of these processes have lead to the uncovering of several ciε- and trans-acting elements controlling algD promoter activity: (i) The algD

promoter has been shown to consist of sequences unusually far upstream of the mRNA start site (Mohr et al., 1990). These sequences (termed RBI and RB2) , as well as a sequence closer to the mRNA start site (RB3) are needed for the full activation of algD (Mohr et al., 1990; 1991; 1992) . (ii) AlgR, a response regulator from the superfamily of bacterial signal transduction systems (Deretic et al., 1989), binds to RBI, RB2, and RB3, and is absolutely required for high levels of algD transcription (Mohr et al., 1990; 1991; 1992). (iii) Another signal transduction factor, AlgB, also contributes to the expression of genes required for alginate synthesis ( ozniak and Ohman, 1991) . (iv) The peculiar spatial organization of AlgR binding sites imposes steric requirements for the activation process. The conformation of the algD promoter appears to be affected by histone like proteins [e.g. Alg (ILl) (Deretic et al., 1992) and possibly IHF (Mohr and Deretic, 1992)], and perhaps by other elements controlling nucleoid structure and DNA topology. (v) The algD promoter does not have a typical -35/-10 canonical sequence (Deretic et al., 1989) . It has been proposed that RpoN may be the sigma factor transcribing this promoter; however, several independent studies have clearly ruled out its direct involvement (Mohr et al., 1990; Totten et al., 1990).

The present inventors have cloned and characterized a new gene, algU, which plays a critical role in algrD expression (Martin et al., 1993). The algU gene encodes a polypeptide product that shows sequence and domainal similarities to the alternative sigma factor SpoOH from Bacillus spp. (Dubnau et al., 1988). SpoOH, although dispensable for vegetative growth, is responsible for the initial events in the triggering of the major developmental processes in Bacillus subtiliε, viz. sporulation and competence (Dubnau et al., 1988; Dubnau, 1991) . These findings suggest that activation of alginate synthesis may represent a cell differentiation

process participating in interconversions between planktonic organisms and biofilm embedded forms in natural environments (Martin et al., 1993; Costerton et al., 1987).

Inactivation of algU abrogates algD transcription and renders cells nonmucoid, further strengthening the notion that algU plays an essential role in the initiation of mRNA synthesis at algD (Martin et al., 1993) . algU maps in the close vicinity of roue markers that have been demonstrated in the classical genetic studies by Fyfe and Govan (1980) to cause the emergence of mucoid strains constitutively overproducing alginate. The mucoidy-causing property of muc mutations has been based on the ability of different roue alleles (e.g. muc- 2, muc-22, and muc-25) to confer mucoidy in genetic crosses (Fyfe and Govan, 1980; 1983) . The present application describes the presence of additional genes immediately downstream of algU, termed mucA and roucB, which also play a role in the regulation of mucoidy.

Detection of mucoid P. aeruginosa is a standard practice, however, due to the variability in expression of mucoidy on standard clinical media, more objective detection methods are needed. An early detection of conversion to mucoidy will be possible by using the present invention.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for the early detection and diagnosis of the conversion to mucoidy of Pseudomonas aeruginosa . The present invention also provides a molecular mechanism for the conversion from the nonmucoid to the mucoid state, including specific sequence alterations that occur in the

mucA gene that cause the conversion and molecular probes for the early detection of this disease state.

"Recombinant," as used herein, means that a protein is derived from recombinant (e.g., microbial) expression systems. "Microbial" refers to recombinant proteins made in bacterial or fungal (e.g., yeast) expression systems. As a product, "recombinant microbial" defines a protein produced in a microbial expression system which is essentially free of native endogenous substances.

Protein expressed in most bacterial cultures, e.g., E . coli , will be free of glycan.

"Biologically active," as used throughout the specification means that a particular molecule shares sufficient amino acid sequence similarity with the embodiments of the present invention disclosed herein to be capable of forming a algU-mucA-mucB complex, thereby repressing gene transcription from the algD promoter.

"DNA sequence" refers to a DNA polymer, in the form of a separate fragment or as a component of a larger DNA construct. Preferably, the DNA sequences are in a quantity or concentration enabling identification, manipulation, and recovery of the sequence and its component nucleotide sequences by standard biochemical methods, for example, using a cloning vector. Such sequences are preferably provided in the form of an open reading frame uninterrupted by internal nontranslated sequences. Genomic DNA containing the relevant sequences could also be used. Sequences of non-translated DNA may be present 5' or 3' from the open reading frame, where the same do not interfere with manipulation or expression of the coding regions.

"Nucleotide sequence" refers to a heteropolymer of deoxyribonucleotides. DNA sequences encoding the

proteins provided of this invention can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, to provide a synthetic gene which is capable of being expressed in a recombinant transciptional unit.

"Recombinant expression vector" refers to a replicable DNA construct used either to amplify or to express DNA which encodes the fusion proteins of the present invention and which includes a transciptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structure or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences. Structural elements intended for use in yeast expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, where recombinant protein is expressed without a leader or transport sequence, it may include an N-terminal methionine residue. This residue may optionally by subsequently cleaved from the expressed recombinant protein to provide a final product.

"Recombinant microbial expression system" means a substantially homogeneous monoculture of suitable host microorganisms, for example, bacteria such as E. coli or yeast such as S. cerevisiae, which have stably integrated a recombinant transcriptional unit into chromosomal DNA or carry the recombinant transcriptional unit as a component of a resident plasmid. Generally, cells constituting the system are the progeny of a single ancestral transformant. Recombinant expression systems as defined herein will express heterologous protein upon induction of the regulatory elements linked to the DNA sequence or synthetic gene to be expressed.

The present invention provides a composition of matter comprising a first polynucleotide having the sequence of SEQ ID NO:10, a second polynucleotide complementary to the first polynucleotide or a polynucleotide differing from the first or second polynucleotide by codon degeneracy. Also claimed is a polynucleotide which hybridizes with the first or second polynucleotide, or an oligonucleotide probe for the first or second polynucleotide which hybridizes with said polynucleotide.

A further composition of matter of the present invention is a Pseudomonas aeruginosa mucA or mucB gene in substantially pure form. The mucA gene is defined as substantially comprising the sequence of Figure 2 (SEQ ID NO:10) defined further as excluding the mucB coding region. Also claimed is a mucA gene defined further as differing from having an altered sequence. The alteration may be an insertion or deletion of at least one nucleotide, it may be a frameshift mutation, or a nonsense mutation. Alteration of the gene results in an inactive roucA gene product. In a preferred embodiment of the invention, the altered gene sequence has a deletion of nucleotide "A" from position 371 of the sequence of Figure 2. In an alternatively preferred embodiment of the invention, the altered gene sequence has a deletion of nucleotide "G" from position 440 of the sequence of Figure 2. In also a preferred embodiment of the invention, the altered gene sequence has an alteration of nucleotide "C" from position 362 of the sequence of Figure 2.

The polynucleotide may be a polydeoxyribonucleotide or a polyribonucleotide. The oligonucleotide may be an oligodeoxyribonucleotide or an oligoribonucleotide. A further composition of matter is an oligonucleotide useful as a probe for and which hybridizes with the

polynucleotide sequence of Figure 2 or sequence alterations thereof. A preferred embodiment of the invention is an oligonucleotide comprising a sequence complementary to a region spanning the deletion at position 371. In particular, the oligonucleotide comprises the sequence 5'-GGGACCCCCCGCA-3' (SEQ ID NO:2). The altered sequence of the mucA gene may have a deletion of the nucleotide "G" from position 439 or 440. One skilled in the art would see that since there is a "G" in both positions 439 and 440 it is not possible to know which "G" is deleted in this altered sequence.

A further embodiment of the present invention is an oligonucleotide comprising a sequence complementary to a region spanning the deletion at position 439 or 440. In particular, this oligonucleotide comprises the sequence 5'-GAGCAGGGGCGCC-3' (SEQ ID NO:3) .

A further composition of matter of the present invention is a mucA gene having an altered sequence wherein the altered sequence is an insertion of nucleotides 5'-CAGGGGGC-3' between positions 433 and 434. Also claimed is an oligonucleotide comprising a sequence complementary to a region spanning the insertion of the nucleotides 5'-CAGGGGGC-3 ' . A preferred embodiment is an oligonucleotide comprising 5'-CAGGGGGCCAGGGGGC-3' (SEQ ID NO:4) .

A further embodiment of the present invention is the use of these compositions of matter for a method of detecting conversion to mucoidy in Pseudomonas aeruginosa comprising detecting a loss of roucA or mucB function. In particular, a method of detecting conversion to mucoidy in Pseudomonas aeruginosa having an inactive mucA gene product comprising the detection of an altered sequence in the mucA gene is claimed. A preferred embodiment is a method of detecting conversion to mucoidy in Pseudomonas

aeruginoεa having an inactive mucA gene product comprising the detection of an altered sequence in the mucA gene. In this case, the altered sequence encodes an inactive product and the altered sequence is detected by hybridization with a complementary oligonucleotide. The complementary oligonucleotide may be 5'-GGGACCCCCCGCA-3' , 5 ^/ -GAGCAGGGGCGCC-3 , or 5'-CAGGGGGCCAGGGGGC-3' (SEQ ID NOS:2-4). One skilled in the art could see that an altered sequence may comprise nucleotide changes, insertions, deletions or changes in the sequence leading to a nonsense or stop codon anywhere within this locus between about positions 300 and 500 of the sequence of Figure 2.

A further embodiment of the present invention is a method of detecting conversion to mucoidy in Pεeudomonaε aeruginoεa having an inactive mucA gene comprising the steps of: 1) obtaining Pεeudomonaε aeruginosa suspected of conversion to mucoidy to provide a test sample, and 2) hybridizing the test sample with an oligonucleotide 5'- GGGACCCCCCGCA-3' , 5'-GAGCAGGGGCGCC-3 ' , or 5'- CAGGGGGCCAGGGGGC-3' (SEQ ID NOS:2-4). Positive hybridization indicates conversion to mucoidy in Pseudomonas aeruginoεa .

A further embodiment of the present invention is a Pεeudomonaε aeruginoεa algU gene in substantially pure form. The algU gene comprises the sequence of Figure 1.

The DNA sequences disclosed herein will also find utility as probes or primers in nucleic acid hybridization embodiments. As such, it is contemplated that oligonucleotide fragments corresponding to the sequence(s) of seq id no:1-14 and 20-23. For stretches of between about 10 nucleotides to about 20 or to about 30 nucleotides will find particular utility, with even longer sequences, e.g., 40, 50, 100, even up to full

length, being more preferred for certain embodiments. The ability of such nucleic acid probes to specifically hybridize to algU, mucA and roucB-encoding sequences will enable them to be of use in a variety of embodiments. For example, the probes can be used in a variety of assays for detecting the presence of complementary sequences in a given sample. However, other uses are envisioned, including the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions.

Nucleic acid molecules having stretches of 10, 20, 30, 50, or even of 100 nucleotides or so, complementary to seq id no:1-14, 20-23, will have utility as hybridization probes. These probes will be useful in a variety of hybridization embodiments, such as Southern and Northern blotting in connection with analysing the complex interaction of structural and regulatory genes in diverse microorganisms and in clinical isoltaes from CF patients. The total size of fragment, as well as the size of the complementary stretch(es) , will ultimately depend on the intended use or application of the particular nucleic acid segment. Smaller fragments will generally find use in hybridization embodiments, wherein the length of the complementary region may be varied, such as between about 10 and about 100 nucleotides, or even up to full length DNA insert of SEQ ID NOS:1-14, 20- 23, according to the complementary sequences one wishes to detect.

The use of a hybridization probe of about 10 nucleotides in length allows the formation of a duplex molecule that is both stable and selective. Molecules having complementary sequences over stretches greater than 10 bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of

specific hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having gene- complementary stretches of 15 to 20 nucleotides, or even longer where desired. Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCR technology of U.S. Patent 4,603,102 (herein incorporated by reference) or by introducing selected sequences into recombinant vectors for recombinant production.

Accordingly, the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of homologous, or heterologous genes or cDNAs. Depending on the application envisioned, one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence. For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, e.g., one will select relatively low salt and\or high temperature conditions, such as provided by 0.02M-0.15M NaCI at temperatures of 50°C to 70°C. Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating functionally related genes.

Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template or where one seeks to isolate specific mutant algU , mucA or roucB- encoding sequences from related species, functional equivalents, or the like, less stringent hybridization conditions will typically be needed in order to allow formation of the heteroduplex. In these circumstances, one may desire to employ conditions such as 0.15M-0.9M

salt, at temperatures ranging from 20°C to 55°C. Cross- hybridizing species can thereby be readily identified as positively hybridizing signals with respect to control hybridizations. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.

In certain embodiments, it will be advantageous to employ nucleic acid sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator means are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal. In preferred embodiments, one will likely desire to employ a fluorescent label or an enzyme tag, such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmental undesirable reagents. In the case of enzyme tags, colorimetric indicator substrates are known which can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.

In general, it is envisioned that the hybridization probes described herein will be useful both as reagents in solution hybridization as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes under desired

conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C contents, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Following washing of the hybridized surface so as to remove nonspecifically bound probe molecules, specific hybridization is detected, or even quantified, by means of the label.

Longer DNA segments will often find particular utility in the recombinant production of peptides or proteins. DNA segments which encode peptide antigens from about 15 to about 50 amino acids in length, or more preferably, from about 15 to about 30 amino acids in length are contemplated to be particularly useful, as are DNA segments encoding entire algU, mucA or roucB proteins. DNA segments encoding peptides will generally have a minimum coding length in the order of about 45 to about 150, or to about 90 nucleotides.

The nucleic acid segments of the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. For example, nucleic acid fragments may be prepared in accordance with the present invention which are up to 10,000 base pairs in length, with segments of 5,000 or 3,000 being preferred and segments of about 1,000 base pairs in length being particularly preferred.

It will be understood that this invention is not limited to the particular nucleic acid and amino acid sequences of seq id nos:1-23. Therefore, DNA segments prepared in accordance with the present invention may also encode biologically functional equivalent proteins or peptides which have variant amino acids sequences. Such sequences may arise as a consequence of codon redundancy and functional equivalency which are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged.

DNA segments encoding a algU , mucA or roucB gene may be introduced into recombinant host cells and employed for expressing a algϋ , mucA or mucB structural or functionally related protein. Alternatively, through the application of genetic engineering techniques, subportions or derivatives of selected algϋ, mucA or mucB genes may be employed. Equally, through the application of site-directed mutagenesis techniques, one may re- engineer DNA segments of the present invention to alter the coding sequence, e . g. , to introduce improvements to the antigenicity of the protein or to test algU, mucA or roucB mutants in order to examine transcription from the algD or related promoter activity at the molecular level. Where desired, one may also prepare fusion peptides, e . g. , where the algϋ, mucA or mucB coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for immunodetection purposes (e.g., enzyme label coding regions) .

As mentioned above, modification and changes may be made in the structure of algϋ, mucA or mucB coding regions and still obtain a molecule having like or otherwise desirable characteristics. As used herein, the term "biological functional equivalent" refers to such proteins. For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence (or, of course, its underlying DNA coding sequence) and nevertheless obtain a protein with like or even counterveiling properties (e.g., antagonistic v. agonistic) . It is thus contemplated by the inventors that various changes may be made in the sequence of algϋ, mucA or mucB proteins or peptides (or underlying DNA) without appreciable loss of their biological utility or activity.

In still further embodiments, the present invention concerns a method for identifying new compounds that inhibit transcription from the algD promoter, which may be termed as "candidate substances." Such coumpounds may include anti-sense oligonucleotides or molecule that encourage algϋ-mucA-mucB mediated repression of the algD promoter. The present invention provides for a method for screening a candidate substance for preventing P. aeruginosa conversion to mucoidy comprising: contacting the E. coli bacteria as described in Example 6 with an effective amount of a candidate substance; and assaying for reporter gene activity, wherein a decrease in the expression of the reporter gene indicates inhibition of algD promoter activity.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

Figure 1. DNA sequence of algϋ. Bent arrows denote the endpoints of deletions: ΔU4/76 suppresses mucoidy and produces P27 (+) ; U4/33 has no effect on mucoidy and is not capable of producing P27 (-) . EcoRV, a site used for insertional inactivation of algϋ on the chromosome is shown.

Figure 2. The complete nucleotide sequence of mucA and mucB . The open reading frames compatible with the direction of transcription, suppression activity of deletions, and the observed K. of the roucA gene product [20 kDa (Martin et al., 1992)] and that of the roucB gene product.

Figure 3A Mutations causing mucoidy in P. aeruginoεa in the region of mucA containing mutations causing conversion to mucoidy is shown [dots indicate omitted sequence. Highlighted are nucleotides missing or substituted in mucoid strains (CFl, CF8, CF14, CF23, and PA0578) . Arrows indicate duplicated sequence (eight nucleotides) in PA0568 (mucA2 ) . Boxed are termination codons (TGA or TAG) placed in frame with the roucA sequence as a result of muc mutations.

Figure 3B DNA sequence (from position 423 to 442) of the wild-type mucA ⁺ , mucA2 , and mucA22 alleles. The

location of the duplicated sequence in mucA2 (+ 8 bp) is indicated by arrows; triangle and AC denote a missing nucleotide (-1 bp) in mucA22.

Figure 3C detection of roue mutations by hybridization with ASOs. PCR-amplified sequences containing mucA from PA0568 (roucA2) and PA0381 (mucA ⁺ ) were blotted onto nitrocellulose membrane and hybridized with ASO 381 or ASO 568. Under the conditions used, ASO 568 hybridizes to mucA2 only while ASO 381 hybridizes to most mucA alleles.

Figure 3D the sequence of mucA mutations in CFl (deletion of a nucleotide at 371) (Center) and CF8 (C-362 — T-362 transition) (Right) are compared to the wild- type sequence of the nonmucoid strain PAOl (Left) . The antisense strand is shown (nucleotides in parentheses correspond to the residues in A) .

Figure 4A DNA sequence analysis of gene replacement from the mucoid mucA2 mutation in PA0381a2-3.

Figure 4B DNA sequence analysis of gene replacement from the parental non-mucoid strain.

Figure 4C DNA sequence analysis of gene replacement from the mucoid mucA22 mutation in PA0381a22-l.

Figure 5A. Regulatory interactions of algU, mucA, mucB, and algR in the control of the algD promoter (PalgD) .

Figure 5B. Partial nucleotide sequence and organization of the algD promoter. T

Figure 5C. Diagram depicting the inserts of plasmids pDM76, pDM76UA, pDM76A, and pDM76B, used in

this study. Balloons indicate the locations of the Tcr cassette used for insertional inactivation of genes on the plasmid pDM76. Jagged edges of the boxes corresponding to algU (stippled) and mucA (open) indicate that the 3' end of algU and the 5' end of mucA have been deleted in the construct pDM76UA.

Figure 6. Stimulation of algD::lacZ expression by algU. LacZ (β-galactosidase) activity (expressed in mU per mg of total cell protein) was determined in sonic extracts of the algD::lacZ lysogen strain VD1870 harboring one of the following plasmids: pUC12 (vector) ; pDM76 carrying the wild type algU mucA mucB cluster from PAOl (U+A+B+) ; pDM76A carrying the same cluster except that the mucA gene was insertionally inactivated

(U+A-B+) ; pDM76B (U+A+B-) carrying the cluster with insertionally inactivated mucB; pDM76UA, with a deletion of the 3' end of algU and the 5' end of mucA (U-A-B+) . T bars, standard error.

Figure 7. Suppression of AlgU-dependent algD transcription by in trans complementation of mucA and mucB mutations. The E. coli algD::lacZ transcriptional fusion strain VD1870 harbored plasmids pDM76A (U+A-B+) , or pDM76B (U+A+B-) . All strains harbored a second plasmid, pMJStac84AB, containing functional but promoterless mucA mucB genes cloned behind the tac promoter on pMJStac84. This plasmid also contains the laclq gene. Open bars, no IPTG. Filled bars, 1 mM IPTG added to the medium.

Figure 8. AlgR stimulates algD expression in E. coli. VD1870 (algD::lacZ lysogen E. coli) harbored plasmids pDM76 (U+A+B+) or pDM76UA (U-A-B+) . Stippled bars, no algR; open bars, algR present on the second plasmid pMJStac84R.

Figure 9A. Second site supressor mutations in algU and pseudoreversion to nonmucoidy. Partial nucleotide sequence of the algU gene from PA0578 (Accession number L02119) . Dots, sequences not shown. Highlighted nucleotides: deleted residue (ΔT) or substitution (A below the sequence) . Boxed triplets represent stop codons generated or brought in frame with the coding sequence of algU.

Figure 9B Illustration of the sequence alterations within algU in spontaneous nonmucoid derivatives (type III) of the mucoid strain PA0578 (mucA22) . Gel sections show the corresponding regions of algU from strains PA0578 (algU+ mucA22, mucoid), PA057874 (algU74 mucA22, nonmucoid) and PAO578160 (algU160 mucA22, nonmucoid). Bars show positions of deleted or substituted nucleotides. The sequences in B are that of the anti-sense strand; nucleotides in parenthesis indicate corresponding residues in the sense strand.

Figure 9C Illustration of the sequence alterations within algU in spontaneous nonmucoid derivatives (type III) of the mucoid strain PA0578 (mucA22) . Gel sections show the corresponding regions of algU from strains PA0578 (algUt mucA22, mucoid), PA057874 (algU74 mucA22, nonmucoid) and PAO578160 (algU160 mucA22, nonmucoid) .

Figure 10A shows regulatory circuits controlling mucoidy in P. aeruginoεa and effects of roue mutations. Under conditions when all genes are complete, MucA and MucB negatively regulate algϋ. One possibility shown here is that MucA and MucB are complexed with algϋ. This renders algϋ inactive or unavailable for transcription of algD

the sequestering or inactivity of the sigma factor-like element algϋ there is no transcription of algD.

Figure 10B when mucA is inactivated by a fra eshift mutation, algϋ becomes active and allows initiation of transcription at algD.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Mucoidy in Pεeudomonaε aeruginoεa is a critical virulence factor associated with chronic infections in cystic fibrosis (CF) . The initially colonizing strains are nonmucoid but once in the CF lung, they almost inevitably convert into mucoid phenotype. Three tightly linked genes algϋ, mucA, and mucB have been identified with a chromosomal region shown by genetic means to represent the site where mutations cause conversion to mucoidy. Mutations causing mucoidy occur in mucA. The complete nucleotide sequence of the roucA gene is shown in Fig. 2. The positions of mutations in PA0568 (muc-2) , PA0578 (muc-22) , and CF isolates (CFl, CF14, and CF23) are also indicated in Fig. 2. The oligonucleotides designed to detect such mutations by hybridization.

The algϋ gene plays a positive regulatory role in the transcription of algD, a gene encoding GDPmannose dehydrogenase. The algD gene must be expressed at high levels in order for cells to attain mucoid phenotype (Fig. 17) . mucA and mucB play a negative regulatory role, and, when active, these genes suppress mucoidy.

When either mucA or mucB are inactivated, this results in derepression of algD transcription and conversion to mucoidy (Fig. 17) . The present inventors have isolated, sequenced, and characterized the entire region containing algϋ, mucA, and roucB. When a clone of algϋ, mucA and roucB, isolated from nonmucoid cells, is placed into mucoid derivatives of the standard genetic strain PAO and

in CF isolates, it can cause suppression of mucoidy, viz . the cells become phenotypically nonmucoid and the algD promoter is silenced. Using gene replacements on the chromosome and phage-mediated generalized transduction, the present inventors have shown that algϋ and the downstream genes described here as mucA and roucB map at about 67.5 minutes on the P. aeruginoεa chromosome. These genetic markers represent a site where mutations causing conversion from nonmucoid to mucoid phenotype occur, and have not been previously isolated or characterized. Mutations (deletions and insertions) causing frameshift mutations and premature termination of the mucA open reading frame have been identified through the work described herein.

Even though the invention has been described with a certain degree of particularity, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing disclosure. Accordingly, it is intended that all such alternatives, modifications, and variations which fall within the spirit and the scope of the invention be embraced by the defined claims.

Even though the invention has been described with a certain degree of particularity, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing disclosure. Accordingly, it is intended that all such alternatives, modifications, and variations which fall within the spirit and the scope of the invention be embraced by the defined claims.

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow

represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1 Characterization of a Locus Determining the Mucoid Status of Pseudomonas aeruginosa: algϋ Shows Sequence Similarities with a Sigma Factor from Bacillus

Several genetic studies have indicated that muc loci have the property to affect mucoidy when present in trans. For example, it has been observed that R' derivatives of R68.45, which carry pruAB ⁺ and an adjacent muc locus from a nonmucoid PAO strain, are capable of switching off (suppressing) alginate production in mucoid strains PA0568, PA0578, and PA0581 (Fyfe, 1985). This effect appeared to be specific since another mucoid PAO derivative, strain PA0579, was not affected (Fyfe, 1985). This suggested to the present inventors that changes in mucoidy could be used as a screening tool to clone and isolate additional regulatory genes. A generation of a comprehensive genomic library from P. aeruginosa has been reported previously (Ratnaningsih et al . , 1990). Several cosmids from this library have been successfully used for construction of a combined physical and genetic map of P. aeruginosa PAO (Ratnaningsih et al . , 1990). This cosmid library was constructed in pLA2917 (which can replicate in P. aeruginosa) using DNA from a derivative of the strain PA01 (nonmucoid) (Holloway, 1955; Ratnaningsih et al . , 1990). The library was introduced into several

mucoid strains by conjugation and ten independent and nonoverlapping clones capable of altering the mucoid character were isolated: pMO010533, pMO010921, pM0011021, pMO011537, pMO011644, pMO011744, pM0011801, pMO011809, pMO011920, and pMO012046. Two of the clones had previously been described as carrying other genetic markers (Ratnaningsih et al . , 1990). pMO011809 contains hisl and has been used to demonstrate that this locus resides on the Spel fragment E in the late region of the chromosome (Ratnaningsih et al . , 1990). In the same study, pMO011644 was shown to carry the oruJ gene, also mapping in the late region of the chromosome, but hybridizing to a different Spel fragment. One of the clones, pMO012046, rendered a significant number of strains completely nonmucoid, and was chosen for further study. The locus affecting alginate production on this chromosomal fragment was designated algU.

Media and bacterial growth. E. coli was grown on LB supplemented with 10 μg/ml tetracycline (Tc) , 40 μg/ml ampicillin (Ap) , and 25 μg/ml kanamycin (Km) when required. P. aeruginoεa was grown on LB, minimal media (Deretic et al . , 1990; Meile et al . , 1982), and Pεeudomonaε isolation agar (PIA) (DIFCO) . The nitrogen free medium (P) , used to test the ability to utilize proline (supplemented at the concentration of 20 mM) as the sole carbon and nitrogen source, has been previously described (Meile et al . , 1982). Other amino acids were supplied as 1 mM when necessary. Media for environmental modulation by different nitrogen sources (nitrate or ammonia) have been described previously (Deretic et al . , 1990; Mohr et al . , 1990). 300 mM NaCI was added to LB when required (Deretic et al . , 1990). Antibiotics supplements for P. aeruginosa were: 300 μg/ml Tc for PIA, 50 μg/ml Tc for LB and minimal media, and 300 μg/ml carbenicillin (Cb) for all media.

-24-

Plasmids and bacterial strains . Strains of P. aeruginoεa and plasmids used in this study are shown in Table 1.

Table 1. Bacterial strains, plasmids, and bacteriophages.

Species, strain, plasmid, or phage Relevant properties ⁸

P. aeruginoεa

PAOl prototroph Alg ^"

PA01293 prototroph Alg ^"

PA0568 FP2 ⁺ rouc-2 (Alg ⁺ⁱ ) leu-38

PA0578 FP2 ⁺ rouσ-22 (Alg ⁺ ) leu-38

PA0579 FP2 ⁺ rouc-23 (Alg ⁺ ) leu-38

PA0581 FP2 ⁺ rouc-25 (Alg ⁺ ) leu-38

PAO540 cys-5605 his-5075 argA171 Alg ^"

PA0669 FP2 ⁺ muc-2 (Alg ⁺ⁱ ) leu-38 Cb ^r algD ⁺ algD'. '.xylE (Derived from PA0568)

PA0670 FP2 ⁺ algϋ'. :Tc ^r (Alg ^" ) (Derived from PA0568)

PA0964 pru-354 aroi-151 hut C107 Alg ^"

PAM425 muc-3739 (Alg ⁺ ) lys-13

Plasmids pLA2917 JncPl moJ ⁺ tra cos ⁺ Tc ^r Km ^r pCMob ColEl mob ⁺ (RK2) tra coε ⁺ Ap ^r (Cb ^r ) Tc ^r pSF4 Ori (pl5A) mojb ⁺ (RK2) coε ⁺ Tc ^r pRK2013 ColEl roo ⁺ tra ⁺ (RK2) Km ^r pT7-5 ColEl Ap ^r φl0 pro oter-EcoRI-polylinker- Hindlll

Table 1. Cont.

Species, strain, plasmid, or phage Relevant properties ⁸

pT7-6 ColEl Ap ^r øl0 promoter-Hindlll- polylinker-

EcoRI pGPl-2 Ori (pl5A) P _L T7 gene 1 (T7 RNA polymerase)

P _lac -cl857 Km ^r pVDZ'2 IncPl mob ⁺ tra lacZ' (lacZ_) Tc ^r pCMR7 algR as 827 bp Hindlll-BamHI in pT7-6

PPA0M3 pVDXlδ IngQ/P4 al gD : :xylE Ap ^r (Cb ^r ) pMO011809 hisl ⁺ (cosmid clone in pLA2917) pMO012046 algU ⁺ (cosmid clone in pLA2917) pDMUl algU ⁺ (a 6 kb Hindlll-EcoRI fragment from pMO012046 subcloned on pVDZ'2)

PDMU4/76 algϋ - ^' as Δ4/76 subcloned on pVDZ'2 pRC l a 6 kb Hindlll-Nsil subclone from the cosmid pMO011809 pDMUlOO pUC12 mob ⁺ algU: :Tc ^r Ap ^r (Cb ^r ) pDMDX pCMobB algD: zxylE mob ⁺ Ap ^r (Cb ^r )

Phaqes

F116L Generalized transduction phage

Alg ⁺¹ , inducible production of alginate resulting in mucoid phenotype (Deretic et al . , 1990). Alg ⁺ , mucoid phenotype, Alg ^" , nonmucoid phenotype.

Strains PA0669 and PAO670 were derivatives of P. aeruginoεa PA0568 (muc- 2) . The strain PA0669 was generated by integration of a nonreplicative plasmid carrying an algD: : xylE fusion on the chromosome of PA0568. An 11.5 kb Hindlll fragment carrying algD with

xylE inserted in the Xhol site of algD, was cloned in the Hindlll site of pCMobB (Mohr and Deretic, 1990) , and the resulting plasmid pDMDX conjugated into PA0568. pCMobB and its derivative pDMDX cannot replicate in Pεeudomonaε but can be effectively mobilized into this bacterium

(Mohr and Deretic, 1990). Cb ^r exconjugants were obtained and tested for the presence of other plasmid markers [development of a yellow color when sprayed with a solution of catechol (Konyecsni and Deretic, 1989) ] and insertions on the chromosome verified by Southern blot analysis. The strain PA0669 was mucoid and produced alginate on inducing media. PAO670, a strain used to determine effects of the inactivation of algϋ on the chromosome, was constructed by gene replacement of the chromosomal algϋ with an insertionally inactivated algϋ (algϋ : :Tc ^r ) . This was accomplished as follows: A 2.4 kb Hindlll-EcoRI fragment from ΔU4/76 was inserted into pUC12. The resulting construct was digested with EcoRV, and NotI linkers were added. A NotI modified Tc ^r cassette (Ishimoto and Lory, 1989) was inserted, and the resulting plasmid digested with EcoRI. Into this site an 1.4 kb EcoRI fragment with mob from pCMobA (originating from pSF4) (Mohr and Deretic, 1990; Selvaraj et al . ) was inserted to produce pDMUlOO. This plasmid was transferred into P. aeruginoεa PA0568 by conjugation and exconjugants selected on PIA supplemented with Tc. Since pUC12 and its derivative pDMUlOO cannot replicate in Pεeudomonaε , Tc ^r strains had this plasmid integrated on the chromosome via homologous recombination. Double cross-over events were identified as Tc ^r Cb ^s strains, chromosomal DNA extracted, digested with appropriate enzymes, and gene replacements verified by Southern blot analysis. CF strains were from a combined collection of mucoid isolates from CF patients in Edinburgh, Scotland, and San Antonio, Texas. Cosmid clones not shown in Table 1 are described in Results. The source of regA was a 1.9 kb Pstl-Xhol subclone in mpl8 (Hindahl et al . , 1988).

The use of E. coli strains for subcloning in pVDZ2 (JM83) , triparental conjugations (HB101 harboring pRK2013) , and deletion subcloning (WB373) has been described elsewhere (Deretic and Konyecsni, 1989; Konyecsni and Deretic, 1989) .

Nucleic acids manipulations and recombinant DNA methods. All DNA manipulations and Southern blot analyses were according to the previously published methods (Deretic and Konyecsni, 1989; Konyecsni and Deretic, 1990; Mohr et al . , 1990; Ratnaningsih et al . , 1990) or standard recombinant DNA procedures (Ausubel et al . , 1989). Radiolabeled probes (Ausubel et al . , 1989) were generated using random priming labeling method and [α- ³² P]dCTP (3,000 Ci/mmol; DuPont NEN) . RNA extraction and SI nuclease analysis have been previously published (Deretic and Konyecsni, 1989; Konyecsni and Deretic, 1990) . The construction of the cosmid clone library has been reported (Ratnaningsih et al . , 1990). Overlapping deletions of the clones in M13 were generated as previously described (Deretic and Koonyecsni, 1989) . DNA was sequenced by a modification of the chain termination method with the substitution of dGTP by its analog 7- deaza-dGTP to avoid compressions as previously described (Konyecsni and Deretic, 1990) , and using 17 bp or custom made primers when needed. Similarity searches were performed using FASTA program (Pearson and Lipman, 1988) and GenBank databases, as well as through NBRF-PIR protein identification resource network server.

Genetic methods. Clones made in broad host-range plasmids (pVDXlδ and pVDZ'2) were transferred into P. aeruginoεa by triparental filter matings as described previously (Konyecsni and Deretic, 1989) , using E. coli harboring pRK2013 as the helper. Cosmid clones were mobilized into P . aeruginoεa from E. coli S17-1 (Simon et al . , 1983) as previously reported (Ratnaningsih et al . ,

1990) . Generalized transduction using F116L (Krishnapillai, 1971) was performed as follows: Serially diluted (to achieve near confluency) single plaque preparations of F116L were grown mixed with the donor strain in top agar for 17 h at 37°C. The top agar was scraped and phage eluted in equal volume of TNM (10 mM Tris-HCl pK 7.4, 150 mM NaCI, 10 mM MgS0 ₄ ) , centrifuged at 9000 rpm in SM24 rotor, and supernatant filtered through a 0.45 μm membrane to generate transducing phage stock (used within one month) . 500 μl of freshly grown overnight recipient cells was incubated with 500 μl of transducing phage stock (diluted to 5 x 10 ⁹ ; multiplicity of infection 5:1) for 20 min at 37 ^* C. Cells were centrifuged for 1 min in a microcentrifuge and resuspended in 1 ml of TNM. Aliquots were plated on selective media and incubated for 1 to 2 days, strains purified on selective media, and then spot tested for coinheritance of unselected markers.

Enzyme and alginate assays and scoring; of suppression of mucoidy. Catechol 2,3-dioxygenase (CDO) , the gene product of xylE, was assayed in cell-free sonic extracts as previously described (Konyecsni and Deretic, 1989) . The activity was monitored in 50 mM phosphate buffer (pH 7.5)-0.33 mM catechol by following the increase of A ₃₇₅ in a Shimadzu UV160 spectrophotometer. The molar extinction coefficient of the reaction product, 2-hydroxymuconic semialdehyde, is 4.4xl0 ⁴ at 375nm. Suppression of mucoidy by plasmid borne genes was monitored on PIA plates unless specified otherwise, and the phenotypic appearance of the colonies scored as mucoid or nonmucoid. A control strain harboring the vector without an insert was always used for comparison. Alginate was assayed by a colorimetric method (Knutson and Jeaneε, 1976) .

Visualization of gene products using T7 RNA polvmerase/promoter system. Polypeptides encoded by cloned genes were visualized by expression in E. coli using a temperature-inducible T7 expression system (plasmid vectors pT7-5 and pT7-6 and T7 RNA polymerase encoded by pGPl-2) (Tabor and Richardson) and protein labeling with [ ³⁵ S]methionine and [ ³⁵ S]cysteine (Expre ³⁵ S ³⁵ S protein labeling mix; 1000 Ci/mmol; DuPont NEN) with previously described modifications (Konyecsni and Deretic, 1990; Mohr and Deretic, 1990). Proteins were separated on 12% sodium dodecyl sulfate-polyacrylamide gels. ¹⁴ C-labeled methylated proteins (.Amersham) were used as molecular weight standards. The gels were fixed in 10% acetic acid, washed with H ₂ 0, impregnated with IM salicylic acid, and bands representing radiolabeled polypeptides detected by autofluorography at -70 ^* C.

Pulsed-field gel electrophoresis and Southern blot analysis. Localization of genes on the Spel map of P. aeruginoεa PAO was performed by previously published methods (Ratnaningsih et al . , 1990; Shortridge et al . , 1991) . Identification of Spel fragments was done by comparison to the lambda phage concatameric ladder ranging in size from 48.5 to 582 kb (Ratnaningsih et al . , 1990) as well as based on the hybridization to the previously mapped genes (Ratnaningsih et al . , 1990; Shortridge et al . , 1991).

Nucleotide seguence accession number. The sequence reported here has been deposited in GenBank (accession number L02119) .

RESULTS Isolation of cosmid clones affecting mucoidy in trans.

Deletion mapping of the alσϋ locus. In order to facilitate molecular characterization of algϋ , this locus

was examined by deletion mapping. The subcloning of the ability of algϋ to suppress alginate production and mucoid phenotype was done using the broad host range subcloning vector pVDZ ^, 2 (Deretic et al . , 1987). Initially, a 6 kb Hindlll-EcoRI fragment from pMO012046 was found to carry the suppressing activity, and was subjected to further deletion mapping. Two series of consecutive overlapping deletions were produced from each end of the 6 kb fragment, using the previously described deletion-subcloning strategy (Deretic and Konyecsni,

1989) . Subclones of these deletion products in pVDZ'2 were transferred by conjugation into PA0568, a mucoid derivative of the standard genetic strain PAO (Fyfe and Govan, 1980) . The exconjugants were screened for the loss of mucoid character. All deletion clones which retained the suppressing activity caused phenotypically indistinguishable effect; all negative deletions completely lost the ability to affect mucoidy. The activity was delimited to a region demarcated by the endpoints of deletions ΔU4/76 and ΔUM9.

alσU has a strain-specific effect on suppression of mucoidy. It has been shown that different mucoid PAO derivatives and clinical CF isolates display significant differences in algD promoter activity and alginate production in response to modulation by environmental stimuli, such as the salt concentration in the medium or growth on nitrate (Deretic et al . , 1990). For example, the algD promoter in strains PA0568 and PA0578 is induced by salt or growth on nitrate (Deretic et al . , 1990), although the effects differ in magnitude. PA0568 and PA0578 carry muc determinants designated muc-2 and muc-22 (Fyfe and Govan, 1980) , respectively, which map close to each other and to pruAB (Fyfe, 1985; Fyfe and Govan, 1983) . PA0579 has a different muc locus (designated muc- 23) which maps between hisl and proB and displays a completely opposite response to increased salt

concentration in the medium when compared to PA0568 and PA0578 (Deretic et al . , 1990).

Genetic map of the late region of the P. aeruginosa chromosome indicates that the muc loci is linked to the genetic markers pru-70, pruAB, hisl, and proB. muc-2 , muc-22 , and algU are cotransducible with pruAB (indicated by arcs) . muc-25 and muc-3739 map between hisl and pur- 70 ; it is not known whether they are cotransducible with pruAB (indicated by asterisks) . The muc-23 marker maps between hisl and proB . The positions of several genetic markers, alg genes and probes used in this study on a physical map (Spel) of P. aeruginosa PAO. The algD gene hybridizes to two Spel fragments. The genetic map of the late region and the corresponding Spel fragments are aligned to permit overlaps of markers known to hybridize to a given fragment, but precise relative positions are not known. Probes known to hybridize, or have been shown to hybridize, to a given Spel fragment.

Another possibly different muc locus is represented by muσ-3739 (strain PAM425) (MacGeorge et al . ) . When the plasmid pDMUl, containing an active algU locus on the 6 kb Hindlll-EcoRI insert in pVDZ ^, 2 was introduced into a panel of strains representative of different mucoid PAO derivatives and CF clinical isolates, a specific pattern of suppression of mucoidy was observed (Table 2) .

Table 2. Strain specific suppression of mucoidy by algU.

Plasmid ^b

Straii.na' pVDZ'2 pDMUl pRCWl Suppression of mucoidy ⁰

PA0568 (muc-2) + PA0578 (muc-22) + PA0581 (muc-25) + PA0579 (muc-23) PAM425 (muc-3739) + CF strains - (18/18)' + (7/18)' + (3/8)f

10 I u> ^a PAO strains are isogenic mucoid derivatives of P. aeruginosa PA0381 carrying to I different mapped muc markers (Fyfe and Govan, 1980) . P.AM425 is a cross between PAO and a mucoid clinical P. aeruginosa isolate, Ps3739 (MacGeorge et al . , 1986); the corresponding muc-3739 locus

15 has been mapped (MacGeorge et al . , 1986). CF strains were mucoid P. aeruginosa isolates from different cystic fibrosis patients. ^b pDMUl is algϋ from PAOl cloned as a 6 kb Hindlll-EcoRI fragment on the broad host range vector pVDZ ^, 2 (Deretic et al . , 1987). pRCWl is a subclone of a 6 kb Hindlll-Nsil fragment

(see Results) from pMO011809 in pVDZ ^, 2.

20

Table 2. Cont. ^c Suppression was scored on PIA supplemented with Tc as + (transition from mucoid to nonmucoid status when harboring the plasmid) or - (the strain remained mucoid when harboring the plasmid) . ^d Of 18 strains tested none were affected by the vector pVDZ'2. ^e ' ^f Of 18 strains tested (denominator) , 7 lost mucoidy when harboring pDMUl; of 8 strains

(denominator) in which pRCWl was introduced, 3 lost mucoidy. The strains affected by pDMUl were different from those affected by pRCWl, except in one case with variable results. Not all strains tested with pRCWl were tested with pDMUl and vice versa.

10

UJ UJ I

pDMUl rendered muc-2, muc-22 and muc-25 strains (PA0568, PA0578, and PA0581) nonmucoid. In contrast, it had no detectable effect on the muc-23 strain PA0579 and a muc-3739 strain (PAM425) . It also affected a substantial number of mucoid clinical isolates (7 out of 18 tested) . Congruent with these results was the finding that the mucoid phenotype of some of the strains not affected by algU were affected by a different clone. For example, the strain PAM425 which was not affected by pDMUl lost its mucoid character when pRCWl, containing a 6 kb Hindlll-Nsil subclone from the cosmid pMO011809 (Ratnaningsih et al . , 1990), was introduced (Table 2). pRCWl affected 3 out of 8 CF isolates tested. Thus, the CF strains fell into three categories: (i) Those affected by pDMUl, (ii) those affected by pRCWl, and (iii) those not affected by either of the plasmids.

The results presented in this section indicated that: (i) The suppression of mucoidy in trans was strain dependent; (ii) algU affected a significant number of CF isolates; and (iii) there was a correlation between different muc linkage groups and different clones exerting effects.

Two polypeptides, P27 and P20. are encoded by the region affecting mucoidy in muc-2 . muc-22 . and muc-25 strains. Since deletion inactivation of the algU locus from either end had similar effects, suppression of mucoidy was unlikely to be due to the titration of a diffusable factor (e.g. AlgR) by its binding to DNA. Whether this locus had a coding capacity for a possible trans-acting factor was tested by analysis of [ ³⁵ S] methionine and [ ³⁵ S] cysteine labeled polypeptides encoded by the insert in a T7 expression system. [ ³⁵ S] methionine and [ ³⁵ S] cysteine labeled polypeptides encoded by different deletion derivatives of the algϋ region were separated by SDS-

polyacrylamide gel electrophoresis and visualized by autofluorography. Two polypeptides, with apparent M-. of 27.5 kDa (P27) and 20 kDa (P20) were observed as encoded by the algU containing DNA fragment. The consecutive deletions were then used to establish the order of genes and their importance for the suppressing activity. Deletions extending from the Hindlll end abolished P27 synthesis while not affecting P20, thus establishing the order of genes as P27 followed by P20. The gene encoding P27 was designated algU. Deletion ΔU4/33, which lost the ability to produce P27, but still directed the synthesis of P20, was no longer capable of suppressing mucoidy. Thus, algU was necessary for the activity of this region.

Suppression of mucoidy by algU is exerted at the level of algD transcription. Both algD and algJ? undergo transcriptional activation in mucoid cells (Deretic and Konyecsni, 1989) . The difference in transcription is very profound at the algD promoter, which remains silent in nonmucoid cells and is highly active in mucoid strains (Deretic et al . , 1987; Deretic et al . , 1990; Deretic and Konyecsni, 1989) . algJ? is transcribed from two promoters, one distal and constitutive (Mohr and Deretic, 1990; Mohr et al . , 1990), and the other proximal and induced in mucoid cells (Deretic and Konyecsni, 1989) . The present inventors have investigated whether the presence of algϋ affected transcription of algD and algR. To assay algD transcription under different conditions in the presence of algϋ on a plasmid, first a transcriptional fusion of algD and xylE was constructed [used as a reporter gene (Konyecsni and Deretic, 1989) ] on the chromosome of PA0568. The strain was constructed as a merodiploid for algD, with one intact copy of algD while the other was inactivated due to the fusion with xylE (strain PA0669; for construction details see Materials and Methods) .

The parental strain PA0568 (Fyfe and Govan, 1980) has a remarkable feature in that it displays a broad dynamic range of algD expression (Deretic et al . , 1990). Both algD transcription and colony morphology (changing from nonmucoid to mucoid) respond dramatically to inducing conditions (high salt concentration in the medium or growth on nitrate) (Deretic et al . , 1990). The strain PA0669 retained these properties (since PA0669 was merodiploid for algD it could synthesize alginate) . The induction of algD on the chromosome of PA0669 was analyzed to verify the previously established parameters of algD response to environmental conditions (Deretic et al. , 1990; Konyecsni and Deretic, 1989; Mohr et al . , 1990) . The results of xylE fusion assays and phenotypic induction of mucoidy indicated that the chromosomal fusion reacted to environmental modulation in the same manner previously reported for algD-xylE fusions on plasmids (Table 3) .

Table 3. Effects of plasmid borne algϋ from PAOl on algD transcription in the muc-2 backgroun .

CDO (U/mg) ^d

Growth conditions ⁰

Strain and plasmids ⁸ Phenotype* LB LB+NaCl NH ₄ NO3

CDO (ϋ/mg) ^d

10 PA0669 [None] M 0.43 (ND) 2.84 (ND) 0.22 5.69 (±0.02) (±1.19)

UJ

PA0669 M 0.76 4.61 0.59 3.25 I

[pVDZ'2] (±0.14) (±1.19) (±0.10) (±0.47)

PA0669 NM 0.39 0.40 0.20 0.20

[pDMU4/76] (±0.08) (±0.08) (±0.03) (±0.02)

15 ^a PA0669 is a derivative of PA0568 (muc-2) in which an algD-xylE fusion has been placed on the chromosome. The plasmid pDMU4/76 was constructed by cloning the deletion product

ΔU4/76 into pVDZ'2. This plasmid suppresses mucoidy in muc-2, muc-22, and muc-25 PAO derivatives.

20 ^b Phenotype was scored on inducing media (PIA, LB+NaCl, and N0 ₃ ) . M, mucoid; NM, nonmucoid.

^c Growth conditions and media were as previously reported (Deretic et al., 1990). LB+NaCl, LB supplemented with 300 mM NaCI. NH ₄ and N0 ₃ , minimal media with ammonia or nitrate as the

nitrogen source, respectively. The composition and the use of these media for algD induction have been previously described (Detetic et al . , 1990; Mohr et al . , 1990). Table 3. Cont. ^d Activity of catechol 2,3 dioxygenase (CDO), the xylE gene product, was determined in cell free extracts as previously described (Konyecsni and Deretic, 1989) . One unit of CDO is defined as the amount of enzyme that oxidizes 1 μ ol of catechol per min at 24°C. ±, standard error; ND, not determined.

I

00

When plasmid pDMU4/76, carrying algU and capable of suppressing mucoidy, was introduced into PA0669, this resulted in a complete loss of alginate synthesis and algD transcription. No induction was observed in response to environmental stimuli known to induce algD in PA0568 (Deretic et al . , 1990) (Table 3). When PA0669 harboring pDMU4/76, which displayed nonmucoid colony morphology, was transferred to a medium that no longer supplied selective pressure for plasmid maintenance, colonies segregated into outgrowing mucoid and nonmucoid sectors (data not shown) . This was accompanied by a loss of the plasmid in mucoid segregants, as evidenced by the loss of Tc ^r in such cells. The Tc ^s bacteria (devoid of pDMU4/76) had algD activity restored, as indicated by activities of the chromosomal algD- xylE fusion in strains purified from the corresponding sectors. The mucoid segregants grown on PIA showed CDO (the xylE gene product) activities ranging from 1.76-2.01 U/mg, while the nonmucoid strains originating from the same colonies had CDO activities ranging from 0.401-0.445 U/mg of protein in crude cell extracts. The effects of algU on algD was confirmed by SI nuclease protection analysis of algD mRNA levels (data not shown) . The SI nuclease protection studies also indicated that neither of the algR promoters were affected in PA0568 harboring algU on a plasmid (not shown) . These results strongly suggested that the effect of algU on mucoidy was at the level of algD transcription.

Insertional inactivation of the alσU locus on the chromosome of PA0568 renders cells nonmucoid and abrogates algD transcription. The studies presented in the previous sections were not sufficient to conclude that algU participates in algD promoter regulation under normal circumstances. In order to investigate this possibility, and to explore whether algU is a positive or

a negative regulator of algD transcription, this locus on the chromosome was insertionally inactivated. Transposon mutagenesis of algU on a plasmid using Tn5 proved to be uninterpretable, possibly due to the reported instability of Tn5 in Pεeudomonaε (Goldberg et al . , 1990) and was not pursued further. Instead, a Tc ^r cassette was inserted into a conveniently located restriction site within the algU region.

These studies were performed as follows: (i) The presence of two closely spaced EcoRV sites was noted in the region where the gene encoding P27 (algU) resided. This was based on the estimated size of the gene needed to encode a 27.5 kDa polypeptide, the detailed mapping of the coding region of algU using T7 expression studies, and was further confirmed by DNA sequence analysis. Since the endpoint of the last positive deletion still producing P27 was located 540 bp upstream from the first EcoRV site, it was concluded that this site must be within the algU coding region. (ii) A suicide plasmid

(pDMUlOO) was constructed (see Materials and Methods) in which the 2.4 kb Hindlll-EcoRI fragment from ΔU4/76 was placed on pUC12 which cannot replicate in P. aeruginosa . EcoRV sites within the algU insert were converted into NotI specificity, and a Tc ^r cassette (Ishimoto and Lory, 1989) , modified as a NotI fragment, was inserted. After addition of a DNA fragment with the mob functions to facilitate plasmid mobilization into Pseudomonas (Selvaraj et al . ) , the final construct (pDMUlOO) was conjugated into PA0568 and Tc ^r exconjugants were selected.

These strains were expected to have the plasmid with algU::Tc ^r integrated on the chromosome via homologous recombination. Two possible types of recombinants were anticipated: (i) Merodiploids for algU, retaining an active algU copy, which would have an insertion of the

entire plasmid as the result of a single cross-over event; and (ii) true gene replacements, products of double cross-overs, in which case the plasmid moiety and the associated markers would be lost. The present inventors have observed in other gene replacement studies using this procedure that double cross-over events on the P. aeruginosa chromosome are frequent and that they range from 10% to 70% for various genes studied (unpublished results) , obviating in all cases examined the need for a positive selection against markers encoded by the plasmid moiety. In 9 independent samples with algϋ::Tc ^r , 1663 Tc ^r exconjugants were examined. Of these 29% lost Cb ^r encoded by the plasmid moiety, indicative of double cross-over events. All such Tc ^r Cb ^s strains were nonmucoid and did not produce alginate under any of the conditions tested. Most of the colonies with Tc ^r and Cb ^r markers (results of single cross-over events and thus expected to have a functional copy of algU) were mucoid, while a portion of such strains showed a delayed mucoid phenotype (mucoidy was developing after 3-4 days as compared with 48 hours needed for the parental strain PA0568) . Further studies with Tc ^r Cb ^s recombinants using Southern blotting analysis confirmed that these nonmucoid strains had a true gene replacement with the chromosomal copy of algU disrupted by the Tc ^r cassette. Moreover, when the mutation in such strains was purified by transduction (using the generalized transducing phage F116L) into the parental strain PA0568, all Tc ^r transductants displayed nonmucoid phenotype. One of the algU::Tc ^r derivatives characterized in these studies (strain PA0670) was used to investigate algD transcription. This time, the previously characterized al D-xylE fusion plasmid pPAOM3 (Konyecsni and Deretic, 1989) was introduced into PAO670, and algD promoter activity assayed.

Table 4. Analysis of algD transcription in PAO670 (algϋ: :Tc ^r ) .

Growth conditions ^b

Strain and plasmid ⁸ PIA LB+NaCl N0 ₃

CDO (ϋ/mg) ^c

PAO568 [pPA0M3] 12.10 11.54 10.95 PA0670 [pPA0M3] 1.02 1.85 1.40

10 ^a PA0568 (muc-2) is the mucoid parental strain of PAO670. PAO670 has algϋ I insertionally inactivated on the chromsome. Both strains harbored the to I algD-xylE transcriptional fusion plasmid pPA0M3. ^b PIA is a rich medium on which all mucoid strains, including PA0568, present their mucoid phenotype. Other media induce mucoidy and algD transcription in PA0568

15 (Deretic et al . , 1990) and are defined in Table 3. ^c CDO, catechol 2,3 dioxygenase. Relative error did not exceed 20%.

These results (Table 4) indicated that inactivation of the algϋ locus on the chromosome resulted in a loss of algD transcription, and strongly suggested a positive role for algϋ in algD expression.

Genetic and physical mapping of algU indicates its close linkage or identity with a subset of muc loci. Plasmid borne algϋ showed specific suppression of mucoidy in strains containing muc- 2 and muc-22. These and other muc loci have been suggested to participate in the emergence of mucoid strains (Fyfe and Govan, 1980; MacGeorge et al . , 1986), although their nature and the mechanism of action have not been studied. Extensive information is available on the linkage of muc to genetic markers within the late region of the PAO chromosome

(Fyfe, 1985; Fyfe and Govan, 1980; Fyfe and Govan, 1983; MacGeorge et al . , 1986).

Of particular significance is the cotransducibility of muc-2 and muc-22 with the pru-354 marker [a mutation in pruAB, genes required for the utilization of proline as the sole carbon and nitrogen source (Meile et al . , 1982)] demonstrated by F116L bacteriophage mediated genetic exchange (Fyfe, 1985; Fyfe and Govan, 1983). This indicates that these muc loci and the pruAB genes are very close, since F116L can transduce regions in the range of one min of the chromosome.

The present inventors proceeded to localize algU on the chromosome: The first approach was based on the recently determined physical map of P. aeruginoεa PAO (Ratnaningsih et al . , 1990); in these studies algU was used as a probe for Southern hybridization analysis of Spel fragments separated by pulsed field gel electrophoresis. The second approach was to map algU via F116L transduction; in this case the inventors took

advantage of having a strain (PAO670) with the algU gene on the chromosome tagged with the Tc ^r cassette and monitored the coinheritance of pruAB with Tc ^r .

The results of the Southern blot analyses with Spel digested PAO chromosome subjected to separation by pulse field gel electrophoresis. Physical mapping of algϋ on the chromosome of P. aeruginoεa was performed by Southern blot hybridization of various probes with PAOl DNA digested with Spel. Fragments were separated on agarose gel by pulsed-field electrophoresis, and blotted onto a membrane. The radiolabeled probes were hybridized, autoradiograms obtained, probes stripped of the filter and checked for completeness of the process, and the blot reprobed with a different gene. Probes: algϋ; regA, a gene that regulates toxin A synthesis; algR, a response regulator controlling algD transcription; algW, a 6 kb Hindlll-Nsil fragment from pMO011809, that also affects mucoidy.

Several consecutively applied probes were used to confirm identification of the Spel fragments. The algU gene hybridized to the 330 kb Spel fragment (#6, F) known to carry two genetic markers linked to muc-2 and muc-22, viz. pur-70 at 66 min, and pruAB at 67.5 min

(Ratnaningsih et al . , 1990). This indicated that algU may be close to the muc-2 and muc-22 markers. To explore this possibility, cotransducibility of pruAB with algU::Tc ^r was tested. The results of transductional crosses between PAO670 [algU::Tc ^r on the chromosome of PA0568 (muc-2)] and PA0964 (pru-354), a mutant in pruAB which cannot grow on proline as the sole carbon and nitrogen source, revealed a high degree of coinheritance of pruAB with algU::Tc ^r (Table 5).

Table 5. Co ransduc ion of algϋ and pruAB ^a .

Donor x Recipient Selected marker ^b % coinheritance of the unselected marker ⁰

Tc ^r Mucoidy

PA0670 X PA0964 pru-354 ⁺ 20.3 0 (<0.3%) PA0670 X PA0540 hisl ⁺ 0 (9.25%) 0 (<0.25%)

10 ^a F116L transduction was performed using an algC7::Tc ^r derivative of PA0568 (.muc-2) (strain PAO670) as the donor, and PA0964 (pru- 354) or PAO540 (cys-5605 his-5075 argA171) as recipients. PAO670 is nonmucoid due to the inactivation of algC7 by the insertion of Tc ^r cassette. PA0964 and PAO540 are nonmucoid. ^b pru-354 is a mutant allele of pruAB (Meile et al . , 1982). PA0964 (pru-354) cannot grow on

15 proline as the sole carbon and nitrogen source. The selection was performed for pruAB ⁺ or hisJ as described in Materials and Methods. ^c pruAB ⁺ transductants (300 colonies) and hisJ transductants (400 colonies) were tested for coinheritance of Tc ^r . Tc ^r in transduction crosses originates from algϋ: :Tc ^r on the

20 PAO670 chromosome. No strain displayed mucoid character in at least two independent transduction determinations. In a reciprocal study, in which Tc ^r was the selected marker, a 50% coinheritance of pruAB ⁺ with Tc ^r was observed (not shown) .

The % coinheritance of Tc ^r with pruAB corresponded closely to the values previously reported for JΠUC-2 and muc-22 (20-49%) (Fyfe, 1985; Fyfe and Govan, 1983). In a control determination, no coinheritance of hisl and Tc ^r was observed using the same transducing phage lysates (Table 5) . Significantly, no mucoid transductants (expected from the transfer of muc-2) among over 700 colonies examined were observed in these crosses regardless whether the selection was for pru ⁺ or Tc ^r . This was in sharp contrast with the results obtained with the recipient strain PA0964 and the donor strain PA0568 (muc-2; the strain parental to PAO670) . Normally, 49% of the pru ⁺ colonies are mucoid in transductions involving PA0568 and PA0964 (Fyfe, 1985; Fyfe and Govan, 1983). Although PA0568 had the capacity to transfer the muc-2 marker conferring mucoidy upon the recipient cells, its algU: :Tc ^r derivative PAO670 completely lost this ability. This effect could be attributed to the insertional inactivation of algU in PAO670. These results indicate that algU is in the close vicinity of the muc loci represented by muc-2 and muc-22 and may even be allelic with these determinants.

Alaϋ shows sequence similarity with SPOOH. a sigma factor reguired for developmental processes in Bacilluε subtilis. In order to gain information about the nature and possible function of genetic elements within the algU region, the nucleotide sequence of the DNA region from the endpoint of the deletion ΔU4/76 (the last 5' deletion positive for suppression of mucoidy and synthesis of P27) and extending through one of the EcoRV sites used for insertional inactivation of algU was determined (Figure 1) . An open reading frame was identified within the region defined as algU by deletion and functional mapping. This sequence contained translational initiation signals, conformed with Pseudomonaε codon

usage (West and Iglewski, 1988) , and was in the direction of transcription determined in T7 expression studies.

When a global homology search was performed using the translated sequence of algU with GenBank and NBRF databases, two known proteins showed statistically significant similarity with AlgU: SpoOH from B. licheniformis and B. subtilis. SpoOH is dispensable for growth, and is primarily required for initiation of sporulation and other developmental processes

(competence) in B. subtilis (Dubnau et al . ; Tatti et al . , 1989). The sequence similarity observed (24.9% identity over the entire length of both sequences, and the optimized score of 155) , although limited, was equivalent to the extent of similarity of sigma ¹¹ to other known sigma factors (ranging between 22% and 31% identity with optimized scores between 113 and 145) (Dubnau et al . ) .

All regions noted in several sequence compilations and alignments of sigma factors (Helmann and Chamberlin, 1988; Lonetto et al . , 1992) were represented in the regions of homology between SpoOH and AlgU. The predicted pi of AlgU was 5.315, similar to the pi of SpoOH (5.052-5.146). A relatively low pi is characteristic of sigma factors (Merrick and Gibbins,

1985) and is known to cause anomalous mobility of several members of this class of proteins during SDS-PAGE (Merrick and Gibbins, 1985) . This may help explain a discrepancy in the observed electrophoretic mobility corresponding to 27.5 kDa and predicted M-. of AlgU from the sequence (22,194 Da) which is in the range of discrepancies reported for several sigma factors (Merrick and Gibbins, 1985) . B . εubtiliε sigma ¹¹ shows electrophoretic mobility corresponding to 30 kDa, while its predicted M,. is 25,331 (Carter and Moran, 1986).

EXAMPLE 2

Differentiation of Pseudomonas aeruginosa into the

Alginate Producing Form: Inactivation of mucB causes

Conversion to Mucoidy

This example further characterizes genes within the chromosomal region at 67.5 min which play a critical role in determining the mucoid status of P. aeruginoεa . Two new genes within this locus, mucA and mucB , have been identified, characterized, and demonstrated to participate in the control of mucoidy.

PROCEDURES

References cited herein are listed at the end of the specification.

Bacterial strains, plasmids and growth conditions. All strains of Pεeudomonaε aeruginosa used in this study are derivatives of the standard genetic strain PAOl. PA0671 was generated by the insertional inactivation of mucB (mucB: :Tc ^r ) on the chromosome of the nonmucoid parental strain PA0381 (FP2 ⁺ leu-38 mucA ⁺ mucB ⁺ ; Fyfe and Govan, 1980) . This was accomplished using a previously described procedure for allele replacement (Martin et al., 1993). A 2.4 Hindlll-EcoRI fragment (U4/76) was inserted into pUC12. A Bglll fragment containing the Tc ^r cassette was cloned into the unique Bglll site within the mucB open reading frame. Next a l.4-kb EcoRI fragment with mob from pCMobA (Mohr and Deretic 1990; Selvaray et al., 1984) was inserted into a unique EcoRI site resulting in pDMBlOO. This plasmid was transferred by triparental conjugations into PA0381 to generate PA0671, and additionally into three other nonmucoid PAO strains. Exconjugants were selected on PIA supplemented with tetracycline and double crossovers were identified as Tc ^r

and Cb ^s . In all cases, Tc ^r Cb ^s exconjugants were mucoid, while Tc ^r Cb ^r (single crossovers) were nonmucoid. Gene replacements in Tc ^r Cb ^s strains (all mucoid) were verified by Southern blot analysis.

P. aeruginoεa was grown on LB and Pεeudomonaε Isolation agar (PIA, Difco) . Antibiotic supplements for P. aeruginoεa were 300 μg/ml tetracycline for PIA, 50 μg/ml of tetracycline for LB and 300 μg/ml carbenicillin for all media. Eεcherichia coli was grown on LB supplemented with tetracycline (10 μg/ml) , ampicillin (40 μg/ml) and kanamycin (25 μg/ml) when required. All incubation were at 37'C.

Nucleic acid manipulations and recombinant DNA techniques. All recombinant DNA manipulations and Southern blot analysis were carried out using standard procedures (Ausubel et al., 1989; Martin et al., 1993). DNA sequencing was carried out using the United States Biochemical Sequenase kit with 7-deaza GTP.

Labeling and detection of the mucB gene product. The gene product of mucB was specifically labeled and expressed in E. coli using a temperature-inducible T7 RNA polymerase/promoter expression system (plasmid vectors pT7-6 or pT7-5 and T7 RNA polymerase encoded by pGPl-2) (Tabor and Richardson 1985) . Nascent polypeptides were labeled with [ ³⁵ S]methionine and [ ³⁵ S]cysteine (Expre ³⁵ S ³⁵ S protein labeling mix; 1,000 Ci/mmol; DuPont NEN) . Proteins were separated on a sodium dodecyl sulfate (SDS)-12% polyacrylamide gel. ¹⁴ C-labeled methylated proteins (Amersham) were used as molecular weight standards. Gels were fixed in 10% acetic acid, washed with H ₂ 0, impregnated with IM salicylic acid, and bands representing radiolabeled peptides were detected by autofluorography at - 70°C.

Phenotvpic scoring, enzyme assays and alginate measurements. Suppression of mucoidy by plasmid-borne genes was monitored on PIA plates, and the phenotypic appearance of the colonies was scored as mucoid or nonmucoid. Alginate was assayed as previously described (Knutson and Jeanes, 1976) . Various deletion products of the region containing the genes algϋ , mucA, and mucB were placed in the broad host range vector pVDZ'2 (Martin et al., 1993) and introduced into PA0568 and PA0581 to test their ability to suppress mucoidy. The plasmid pPA0M3 (Cb ^r ; Konyecsni and Deretic 1988) , containing an algD: :xylE transcriptional fusion, was introduced into PA0671 carrying muc.B::Tc ^r and the parental strain PA0381 (Table 6) by triparental conjugation (Konyecsni and Deretic, 1988) . Cell- free sonic extracts were assayed for catechol 2,3-dioxygenase (CDO) activity using previously described methods (Konyecsni and Deretic 1988) . The activity was monitored in 50 mM phosphate buffer (pH 7.5)-0.33 mM catechol by following the increase of A ₃₇₅ in a Shi adzu UV160 spectrophotometer.

The molar extinction coefficient of the reaction product, 2-hydroxymuconic semialdehyde, is 4.4 X IO ⁴ at 375 nm. 1 unit of CDO is defined as the amount of enzyme that oxidizes l μmol of catechol per min at 24 ^* C.

RESULTS

Complementation of muc-25 reguires two genes downstream of alαϋ . The present inventors have discovered that the chromosomal muc mutations can be suppressed to nonmucoidy (Martin et al., 1993). This can be accomplished by in trans complementation with a cosmid clone and its derivatives carrying DNA from a nonmucoid PAO strain (Martin et al., 1993). It has also been shown that this suppression activity was at the level of reducing algD transcription (Martin et al., 1993). The region needed

for complementation includes algϋ, but this process also requires additional sequences downstream of algU (Martin et al., 1993). These studies have also indicated the presence of at least one additional gene, termed mucA, immediately following algϋ , which encodes a polypeptide (P20) with an apparent M,. of 20 kDa (Martin et al., 1993). algϋ and mucA are necessary to exert detectable suppression of mucoidy in the PA0568 (muc-2) strain (Fyfe and Govan, 1980) . Finer analyses indicated that a region further downstream of mucA was also needed to completely abrogate mucoidy in this strain. Moreover, another isogenic mucoid strain, PA0581, carrying a different muc mutation (muc-25) known to map close to muc-2, was not affected by the DNA fragment containing only algϋ and mucA unless downstream sequences were included. The present inventors further defined this additional region. The results of these studies are shown in Fig. 7. Based on the size of additional DNA required for the suppression activity, it seemed likely that there was another gene, located downstream of the mucA gene, that was needed for suppression of mucoidy in PA0581. To test this hypothesis it was determined whether a polypeptide product encoded by this DNA region could be detected. T7 expression analysis of mucB expression was studies by separated ³⁵ S labeled polypeptides through SDS- polyacrylamide gel electrophoresis and visualized by autoradiography. A polypeptide with an apparent M,. of 32.8 kDa (P33) was encoded by the insert required for the suppression of the muc-25 mutation. No polypeptide product was observed when the same inserts were expressed in the opposite direction. However, P33 was expressed relatively poorly when compared to algϋ and mucA (not shown) (Martin et al., 1993). The gene encoding P33 was designated mucB .

Co plete nucleotide seguence of the mucA and mucB genes. In order to further characterize the mucA and mucB genes, the complete nucleotide sequence of this region from the prototype PAO strain PAOl (nonmucoid) , parental to PA0381 and its mucoid derivatives PA0568 and PA0581, was determined. The sequence of algϋ has been reported previously (Martin et al., 1993). The assignment of open reading frames for mucA and mucB in this region was facilitated by protein expression and other analyses. The only two open reading frames compatible with: (i) the order of genes (mucA followed by mucB) ; (ii) direction of transcription; (iii) apparent M-. of gene products (P20 and P33) ; (iv) the endpoints of deletions encroaching on the mucB open reading frame that abrogate the suppression activity in PA0581; and (v) conforming with the codon usage typical of Pεeudomonaε (West and Iglewski, 1988) are shown in Fig. 2. The end of the algϋ gene encoding a polypeptide homologous to sigma factor from Bacillus spp. (Martin et al., 1993) is shown. SD, putative ribosomal binding sites. Stars underneath the sequence, stop codons. The position of the Bglll site used to insertionally inactivate mucB on the P. aeruginoεa chromosome is shown. The end point of deletion UM9 that does not suppress mucoidy in PA0581 (muσ-25) is shown (bent arrow) . This deletion still partially suppresses mucoidy in PA0568 (muc-2) . These sequence data have been submitted to GenBank (accession numbers L04794) .

The mucA open reading frame, encodes a polypeptide with predicted M-. of 20,997, immediately follows algϋ. The mucB open reading frame, within the region necessary for suppression of mucoidy in PA0581 encodes a polypeptide with predicted M-. of 34,471 kDa. To further confirm the correct assignment of the genes, this same region was cloned using PCR from several different

strains, including PA0381, and in each case the complete nucleotide sequence was determined in multiple PCR clones confirming the one presented in Fig. 2.

Insertional inactivation of mucB on the chromosome of the nonmucoid strain PA0381 results in mucoid phenotype. Any explanation of the requirement for all three genes (algϋ, mucA, and mucB) for suppression of mucoidy must take into account that algϋ plays a positive regulatory role in algD expression, possibly as the sigma factor required for mRNA initiation at the algD promoter (Martin et al., 1993) . One of the models compatible with this function of algϋ in conjunction with the requirement for mucA and mucB (from a nonmucoid strain) to complement muc mutations and suppress mucoidy, is that mucA and mucB counteract the activity of algϋ and are needed for the maintenance of nonmucoid phenotype. If this is the case, then inactivation of mucB on the chromosome of P. aeruginoεa should result in the mucoid phenotype.

To test this hypothesis the mucB on the chromosome of the nonmucoid strain PA0381 was inactivated. This strain is parental to the mucoid derivatives PA0568 ( uc- 2) , and PA0581 (muc-25) that have muc mutations mapping in the same chromosomal region (67.5 min) as the algϋ- mucAB cluster (Fyfe and Govan, 1980; 1983; Martin et al., 1993). Thus, PA0381 is capable of conversion to mucoidy via mutations in the muc genes. To inactivate mucB , the algϋ- mucAB cluster was first cloned on pUC12. The conveniently located Bglll site (Fig. 2) within the coding region of mucB was used to insert a Tc ^r cassette (Totten, et al., 1990), resulting in the disruption of mucB , as described in Procedures. To this construct was added a fragment containing mob functions (to facilitate its mobilization into P. aeruginosa) , resulting in the plasmid pDMBlOO. Since pUC12 and its derivative pDMBlOO

cannot replicate in Pεeudomonaε , upon a transfer of this plasmid into P. aeruginoεa via triparental conjugation (see Procedures) , any Tc ^r exconjugants must carry this marker integrated on the chromosome. This can occur via homologous recombination involving the a gU-mucAB region through single crossover or double crossover events. In the case of single crossovers, the exconjugants are expected to be merodiploids, and should also display Cb ^r (Ap ^r ; encoded by the vector moiety) ; in the case of double crossovers, a true gene replacement is expected to take place with the vector moiety of the plasmid being lost, and thus the resulting strains should be sensitive to carbenicillin (Cb ^s ) . Of 129 Tc ^r P. aeruginoεa exconjugants obtained from 4 independent crosses between E. coli -JM83 harboring pDMBlOO and PA0381, 28% of exconjugants were Cb ^r , indicative of a single crossover event. As expected, all such strains were nonmucoid, since they were merodiploids, and contained a functional copy of mucB . These strains were indistinguishable from the parental strain PA0381. In contrast, all Tc ^r exconjugants that were Cb ^s (72% of total Tc ^r exconjugants) , thus indicative of double crossover events, became mucoid. Thus, a complete and stable conversion to mucoidy was achieved by inactivating mucB on the chromosome of a previously nonmucoid strain. A true gene replacement event of mucB with mucB : :Tc ^r on the chromosome of such strains was further confirmed by Southern blot hybridization.

To determine whether inactivation of mucB resulted in transcriptional activation of algD, one such muσB::Tc ^r strain (PA0671) was further examined. A plasmid containing algD-xylE transcriptional fusion was introduced into PA0671 and the levels of algD transcription in the parental strain PA0381 (mucB ⁺ ) and its mucoid derivative PA0671 (mucB : :Tc ^r ) were compared.

The results of these studies indicated a 26-fold activation of algD in PA0671 vs PA0381, under identical growth conditions (Table 6) . Thus, inactivation of mucB is an event that results in increased algD transcription, alginate overproduction, and the establishment of mucoid phenotype.

Table 6. Effects of mucB inactivation on algD promoter activity

Strain ⁸ Phenotype algD: :xylE activity ⁰ (U/mg of CDO)

PA0381 (mucB ⁺ ) NM 0.4

PA0671 (mucB : :Tc ^r ) M 10.5 a All strains harbored the algD: :xylE transcription fusion plasmid pPA0M3 (Konyecsni and Deretic, 1989; Mohr et al., 1990) . ^b Phenotype was scored as mucoid (alginate producing) or nonmucoid after growth for 24 h on PIA. ^c The activity was expressed as units of catechol 2,3 deoxygenase (CDO; the xylE gene product) per milligram of total protein in crude extracts. Standard error did not exceed 20%. Growth conditions, extract preparation, activity measurements, and unit definitions (see

Procedures) are as previously described (Martin et al., 1992; Konyecsni and Deretic, 1989).

The studies presented here demonstrate that inactivation of genes such as mucB can lead to a derepression of the algD promoter and conversion to mucoid (alginate overproducing) status. More importantly, using an isogenic series of strains, different frameshift mutations within the mucAB region

that were present in several mucoid strains including CF isolates and absent in the nonmucoid strains have been detected, see Example 3.

A model founded on recently reported evidence (Martin et al., 1993), the results presented in this work, and studies by others (Fyfe and Govan, 1980; 1983; Costerton et al., 1983), is based on the premise that the synthesis of alginate and the emergence of alginate overproducing strains may be a developmental or a cell- differentiation process. Signal transduction involving response regulators such as AlgR and AlgB (Deretic et al., 1989; 1991; Wozniak and Ohman, 1991), nucleoid structure (Deretic et al., 1992; Mohr and Deretic, 1992), and activation of the specific sigma factor(s) (Martin et al., 1993) are most likely different contributing mechanisms for activation of alginate synthesis in natural environments. In the CF lung, while this environment may also be conducive to the induction of the alginate system, due to strong selective pressures (e.g. increased resistance of mucoid forms to phagocytosis, physical clearance mechanisms, antibiotic treatments, etc.) mutants are being selected that overproduce alginate and render cells constitutively mucoid. Such mutants, once extracted from the CF lung, retain mucoid character (Govan, 1988; Martin et al., unpublished results) . Mutations in the a gU-mucAB region, e.g. inactivation of mucA by frameshift mutations (see Example 3) , or mutations affecting mucB activity, represent major pathways for conversion into the mucoid phenotype.

Understanding of the principles of signal transduction processes activating the alginate system at several levels, as well as the precise definition of the mutations causing mucoidy in CF strains which is currently in progress, will provide improved diagnostic

tools and present potential targets for therapeutic interventions.

EXAMPLE 3 Mechanism of Conversion to Mucoidy in P. aeruginosa

Infecting Cystic Fibrosis Patients: Frameshift Mutations of mucA Cause Conversion to Mucoidy

In the course of performing gene replacements with algϋ in the mucoid strain PA0568 (carrying the muc-2 mutation) , the present inventors discovered an informative class of recombinants. The gene replacements on the chromosome were carried out via homologous recombination with algϋ: :Tc ^r on a plasmid that cannot replicate in Pεeudomonaε . A set of studies was performed using a deletion that simultaneously removed the 3 ' end of algϋ and the 5' end of the downstream gene mucA. Two types of recombinants were expected: (i) Nonmucoid strains containing true gene replacements with inactivated algϋ (results of double crossovers) ; and (ii) mucoid strains that were merodiploids (results of single crossovers) . As expected, all double crossovers were nonmucoid since they lost a functional algϋ. The majority of merodiploids were mucoid, since they retained a functional copy of algϋ. However, a third class of recombinants was also observed which consisted of nonmucoid merodiploids. Since the plasmid DNA came from the nonmucoid strain PAOl, parental to the PA0568 lineage, a plausible explanation for the existence of nonmucoid merodiploids was that the crossover took place between the deletion in mucA on the plasmid and a putative mutation (muc- 2) in mucA on the chromosome of the mucoid strain PA0568. Only such a crossover could restore a wild type copy of mucA resulting in nonmucoid merodiploids. The mutation had to be located between the EcoRV site of mucA, where the 5' deletion in the plasmid

construct ended, and the 3' end of mucA. In order to test this hypothesis, the present inventors cloned the corresponding region from the strain PA0568 by PCR and determined its complete nucleotide sequence in multiple independent clones. A duplication of 8 nucleotides at position 433 was observed within mucA in all PCR clones from PA0568. The existence of this mutation was further confirmed by hybridization with allele specific oligonucleotides, oligo 381 and oligo 568, to PCR amplified chromosomal sequences from PA0568 (muc-2) and its direct nonmucoid parental strain PA0381 (Fig. 3C) . Next, the entire algϋ-mucAB region was cloned by PCR from PA0568 and its parental strain PA0381, and the complete nucleotide sequence of this region was determined in at least three independent clones. Figure 3A contains the sequence of the mucA gene and encoded protein. The only difference between PA0381 (muc ⁺ ) and PA0568 (muσ-2) was the octanucleotide duplication in mucA. The present inventors concluded that this was the muc-2 mutation and the mucA allele was designated mucA2. The muc-2 mutation results in a frameshift causing premature termination of mucA at a downstream TGA codon (see Fig. 3A, and Example 5) .

Amplification of alσU-mucA-mucB sequences. and hybridizations with allele specific oligonucleotides. The al U-mucA-mucB region was amplified using the following pairs of oligonucleotides: (i) UL5 GCCGCACGTCACGAGC and UR16 GAGTCCATCCGCTTCG for sequences containing mostly algϋ and a 5' portion of mucA; and (ii) UL3 CTGTCCGCTGTGATGG and UR12 CGCCCCTGCTCCTCGA for sequences containing most of mucA and the entire mucB gene. For amplification of genomic sequences, a loopful of bacteria from a P. aeruginoεa colony was washed in 0.85% saline, centrifuged, resuspended in 500 μl H ₂ 0, boiled for 10 min, and stored at -20° until use. One μl

of boiled preparations is sufficient to obtain necessary amounts of products for amplification by polymerase chain reaction (PCR) . PCR was carried out in 50 μl volumes using standard procedures. Amplification products were tested by electrophoresis on agarose gels. Equal amounts of amplification products were electrophoretically separated on 1% agarose gels and then blotted onto a nitrocellulose filter using standard methods. After the transfer, and crosslinking using UV light (254 nm) , blots were prehybridized in lOxSSC, δxDenhardt solution

(without BSA) for at least 30 min. Allele specific oligonucleotides were kinased with ³² P following standard methods, purified using chromatography on Sep-Pak C ₁₈ columns (Waters) and lyophilized by evaporation in a Savant SpeedVac apparatus. Hybridization with radiolabeled allele specific oligonucleotides was performed in 10 ml of lOxSSC, 5x Denhardt solution for 12 h at 42°C. Membranes were washed 3x for 10 min at 42°C and autoradiograms taken overnight at -70°C. The blots were boiled for 3 min between hybridizations with different probes.

A simplified version of differential hybridization was also carried out using dot blots. In this case, 5 μl taken directly from the PCR mixture was blotted onto a nitrocellulose or nylon membrane presoaked in lOxSSC, and after denaturation, neutralization, crosslinking (by standard methods or as described above) , hybridized and processed as explained for Southern blots.

RESULTS

The present inventors examined whether the allele specific oligonucleotides could be used to screen other mapped muc mutations in PAO and mucoid CF isolates.

Although the oligonucleotide probe 568 (specific for the mucA2 allele) did not hybridize with the PCR amplified sequences from several strains, the control oligonucleotide (381) did hybridize but with a reduced intensity relative to PA0381. This suggested that although the tested strains did not have the octanucleotide duplication observed in mucA2 , there were other alterations within the region complementary to the oligonucleotide probe. The corresponding regions from several strains hybridizing weakly with the oligonucleotide 381 were cloned and examined. The following strains were included: PA0578 (mucoid derivative of PA0381 with the mutation muc-22 mapping close to muc-2 as determined by transduction) and representative clinical mucoid isolates obtained from different cystic fibrosis patients. Following the procedure outlined for cloning and sequencing of the region encompassing the muc-2 mutation, the corresponding nucleotide sequences in the strain PA0578, and the clinical isolates tested were determined. Instead of the duplication of the octanucleotide sequence in PA0568, there was a deletion of a G residue within a string of 5 Gs within the same general region (see Fig. 3B) . Since this was a deletion of one nucleotide, the net result was a similar frameshift as in PA0568, placing the same TGA termination codon in frame with the mucA sequence. The results of these analyses were additionally confirmed by designing an allele specific oligonucleotide designed for this mutation (oligo 578) . The mutant allele in PA0578 was designated mucA22.

Next, the strains that were complemented by plasmids carrying the algU-mucAB region from PAOl but did not show reduced hybridization with the oligonucleotides were examined. One such cystic fibrosis isolate was subjected to the same cloning and sequencing procedure as outlined above. No changes were detected within the location of the mucA2 and mucA22 mutations. Instead, a deletion of a single nucleotide at the position 371 was detected (see Fig. 3D) . This deletion was confirmed by sequencing multiple clones and by hybridizations with an allele specific oligonucleotide CFl. This frameshift mutation also results in a premature termination of mucA although at an upstream termination codon (see Fig. 3A) . Another strain, PA0581, that did not show differential hybridization with the allele specific oligonucleotides was also examined. PA0581 carries a muc mutation (muc- 25) which has not be mapped by transduction. In this case the present inventors could not find any sequence differences between PA0581 and PA0381 within the region examined here. Similar to PA0581, several mucoid CF strains did not show detectable alterations in mucA.

The work described herein identifies a major site of mutations causing mucoidy in P. aeruginoεa . The mucA gene and a tightly linked downstream gene, mucB are both required for suppression of mucoidy. When these functions are lost by insertional inactivation on the chromosome of previously nonmucoid strains, provided that the first gene of the cluster (algϋ) is intact, this results in a strong activation of algD transcription and conversion to mucoidy.

Mucoidy in P. aeruginoεa has received attention mainly due to its association with CF. However, almost all strains of P. aeruginoεa have the genetic capacity to synthesize alginate suggesting that this system must play

a role in other ecological niches. The vast majority of P. aeruginoεa biomass in nature exists as the form embedded in the exopolysaccharide biofilm attached to surfaces. It has been shown that P. aeruginoεa undergoes interconversions between the free floating planktonic form and the sessile form in biofilms, a process which has been viewed as a developmental or cell differentiation phenomenon. Regulation of alginate production by a factor (algU) homologous to an alternative sigma factor SpoOH, controlling the initial stages of development in Bacillus spp. (e.g. sporulation and competence) , may reflect the nature of regulatory processes controlling development of biofilms. The genetic data indicate that mucA and mucB suppress the function of algϋ. There are now ample examples of accessory factors associated with or linked to alternative sigma factors in Bacilluε and other organisms that post-translationally modify (e.g. inhibit) their function. By analogy, MucA and MucB may play a similar role. This system, along with signal transduction regulators and histone-like elements, is likely designed to control development of biofilms in response to appropriate environmental cues. Mutations in mucA that lock the system in its constitutive state, which is favorable due to the antiphagocytic properties of the mucoid coating, are being selected in the course of chronic respiratory infection in CF.

In addition to the improved understanding of the molecular mechanisms controlling an important bacterial virulence factor, several aspects of the regulation of mucoidy presented here shed light on developmental processes in Gram negative organisms. The finding that algϋ shows similarities with a sigma factor specializing in developmental processes of a Gram positive sporulating organism, suggests that bacterial cell differentiation

phenomena (e.g. sporulation, biofilm development, and bacterial encystment) may share common regulatory mechanisms.

EXAMPLE 4

Complementation of a mucA Mutation by the Wild-Type mucA

Gene Results in Suppression of Alginate Production and

Nonmucoid Phenotype.

Frameshift mutations in mucA, which result in conversion to mucoidy and activation of the algD promoter, suggest a negative regulatory role for the mucA gene product. If this is correct, then it should be possible to complement a mutation in mucA to nonmucoidy by the plas id-borne wild-type mucA gene. To test this hypothesis, a 852-bp Bεt Yl fragment containing the mucA gene from the nonmucoid strain PAOl was cloned behind the tac promoter on the broad-host-range plasmid pVDtac24, resulting in the construct termed "ptac-mucA ⁺ ." This plasmid also carries the lacJ ^q gene, which renders tac transcription dependent on the presence of the inducer IPTG. The plasmid was transferred into the mucoid strain PA0568 (mucA2 ) by triparental conjugation, and exconjugants were examined for colony morphology and alginate production on plates supplemented with 1 mM IPTG or in the absence of the inducer. The colonies grown in the absence of IPTG showed a mucoid morphology identical to that of the parental strain PA0568.

Next the exchange or mucA ⁺ with mucA2 caused activation of algD was examined. Mucoidy is dependent on a strong transcription activation of the algD gene encoding a key biosynthetic enzyme for the mucoid exopolysaccharide alginate. The plasmid pPAOM3 containing an algD: zxylE transcriptional fusion was

-64- introduced into one such mucoid strain, and the reporter gene activity was determined (Table 7) . The results of such measurements indicated that the gene replacement with the mucA2 allele resulted in a 320-fold activation of the algD promoter, which correlated with the levels of alginate production.

Table 7. Conversion to mucoidy and activation of algD transcription as a result of gene replacement with the mucA2 allele

Alginate algD: :xylE

Strain Phenotype production ⁷ activity, units/mg of CDO* PA0381 NM <0.1 0.14±0.03 (mucA ⁺ )

PA0381a2-3 M 64.9 44.911.0 (mucA2 )

For transcriptional fusion measurements, all strains harbored pPAOM3 with an algD: :xylE transcriptional fusion. PA0381a2-3 was one of several mucoid isolates generated by exchange of the chromosomal mucA ⁺ in PA0381 with plasmid borne mucA2. ** Phenotype was scored as mucoid (M) or nonmucoid (NM) . ⁷ Alginate production was expressed in μg of alginate per mg of wet cell weight. ^§ Transcriptional activity was expressed as units of catechol 2,3-deoxygenase (CDO; the xylE gene product) per mg of crude protein extracts ± SEM.

When the same strain harboring ptac-mucA ⁺ was grown in the presence of 1 mM IPTG, it displayed a nonmucoid phenotype. This was accompanied by a decrease in

detectable alginate production by a factor of 15 (Table 8) .

Table 8. Complementation of the mucA2 mutation to nonmucoidy in PA0568 harboring ptac-mucA ⁺

Alginate production, IPTG Phenotype μg/mg of wet cell weight M 59.8 1 mM NM 3.6X

IPTG was used as inducer of tac transcription. M, mucoid; NM, nonmucoid.

These experiments showed that mucoid phenotype can be complemented by a functional mucA gene alone, thus additionally confirming that the observed muc mutations are responsible for mucoid phenotype and that mucA plays a negative regulatory role.

EXAMPLE 5 Detection of mucA Mutations in Mucoid P. aeruginosa Isolates from CF Patients.

To determine whether similar mutations in mucA occur in mucoid CF isolates, several strains from different patients were tested. Strains previously shown to be complemented with plasmids carrying the algU-mucAB region from PAOl were examined. Two randomly chosen strains that showed a reduced hybridization with ASO 381 were subjected to PCR and sequence analysis. These strains, CF14 and CF23, displayed a mutation identical to the mucA22 allele (a loss of one G within the string of five G residues in the wild-type mucA) , resulting in a premature termination of muc A at the same position as in PA0578.

Next, the strains that did not show reduced hybridization with ASO 381 were examined. One such cystic fibrosis isolate (CFl) was subjected to the same cloning and sequencing procedure as outlined above. No changes were detected within the location of the mucA2 and mucA22 mutations. Instead, a deletion of a single nucleotide at the position 371 was detected (Fig. 3A and 3D) . This deletion was confirmed by sequencing multiple cones. This frameshift mutation also results in a premature termination of mucA, although at an upstream termination codon (position 396) . Another CF strain from this category, CF8, was examined, and a different mutation was observed. There was a transition of C into a T (Fig. 3A and 3D) at position 362 of the mucA sequence. Interestingly, this mutation, although not causing a frameshift, generates a stop codon (TAG) , known as a nonsense mutation, at this position (Fig. 3A) . These results showed that mutations inactivating mucA, identical or similar to the mucA mutations responsible for conversion of PAO to mucoidy, can be observed in clinical CF isolates.

Strains that can be complemented with algϋ-mucAB from PAOl were also found in which no mutation in mucA could be detected in the two general areas where mucA could be detected in the areas where mucA2 (and mucA22) or mutations in CFl (and CF8) were observed. Similar to these strains, PA0581, a mucoid PAO derivative that carries a muc mutation (muc-25) which has not been mapped by transduction, did not have sequence differences relative to its nonmucoid parent PA0381 within the regions examined here. These results are consistent with the existence of additional types of mutations within the algU-mucAB region or participation of additional sites or processes affecting conversion to mucoidy.

EXAMPLE 6 Functional Analysis in a Heterologous Host and Role in the Instability of Mucoidy

In the preceeding examples the present inventors have demonstrated that the conversion to mucoidy is caused by the overproduction of the exopolysaccharide alginate in laboratory and cystic fibrosis strains of Pseudomonas aeruginosa that occur via frameshift or nonsense mutations in the second gene of the algU mucA mucB cluster. A model system was developed to characterize novel compounds for use in controlling conversion to mucoidy. To that end, the wild type algU mucA mucB cluster from the standard genetic strain PAOl was used to reconstitute algD transcription in

Escherichia coli. Transcription of a algD::lacZ chromosomal fusion in E. coli was detected upon introduction of plasmid borne algU mucA mucB. Moreover, when either mucA or mucB were insertionally inactivated, this resulted in further stimulation of transcriptional activity from the algD promoter.

This activation was dependent on algU, since a double algU mucA mutation abrogated transcription of algD. These studies suggest that the phenotypic manifestations of muc mutations, i.e. increased algD expression and mucoid phenotype, depend upon the presence of an active algU gene, and that algU and the downstream factors interact.

Bacterial strains, plasmids. and bacteroiophaαes

Strains, plasmids, and phages used in this study are shown in Table 1. P. aeruginosa strains PA057874, PA0578160 and PAO57800 were spontaneous nonmucoid derivatives of P. aeruginosa PA0578, obtained from

initially pure mucoid cultures of PA0578 by repeated passages on Pseudomonas isolation agar (PIA) (DIFCO) .

Plasmids pDM76, pDMU76A, pDMU76B, and pDM76UA represent a series of pUC12 derivatives (Fig. 5C) carrying the wild type algU mucA mucB cluster from PAOl, or its insertionally inactivated variants of mucA, mucB, or both algU and mucA, respectively. Plasmid pDMU76UA also has a deletion spanning the region beginning 105 bp upstream of the 3 ' end of algU, and ending 180 bp downstream of the 5' end of mucA. This deletion was generated by digestion with EcoRV, modification of the ends into the NotI specificity followed by insertion of a NotI linkered tetracycline resistance (Tcr) cassette in this position. Plasmid pDMU76A contained a Bglll Tcr cassette inserted into the unique Bell site of mucA. Plasmid pDMU76B contained the same Tcr cassette inserted into the unique Bglll site of mucB.

The plasmid vector pMJStac84 was constructed by cloning the 2.45 kb BamHI fragment (containing the laclq gene, the tac promoter, and the multiple cloning site from pUC12) from pVDtac24 into the BamHI site of pACYC184. This new vector is based on the pl5A replicon, which is fully compatibe with ColEl replicon-based plasmids, and can be used for carrying out studies that require two-plasmid systems. The useful cloning sites are Bglll, Smal, and SstI, in the order of their positions downstream of the tac promoter. The plasmid pMJStac84AB was constructed by cloning the 1.9 kb mucA-mucB containing fragment from PAOl as BstBI-EcoRI, which was made blunt by incubation with Klenow fragment of DNA polymerase I, into the Smal site of pMJStac84 in the direction of tac transcription. pMJStac84R was constructed by cloning the 826 bp BamHI algR containing fragment from pCMR7 into pMJStac84.

Media and Bacterial Growth

E. coli was grown on LB supplemented with 10 ?g/ml tetracycline (Tc) , 100 μg/ml ampicillin (Ap) , 25 μg/ml kanamycin (Km) and 0.7 mM isopropyl-D-thio galactopyranoside (IPTG) , and 40 μg/ml of

5-bromo-4-chloro-indolyl-β-D-galactopyramoside (XGal) when required. P. aeruginoεa was grown on LB or PIA. When necessary, PIA was supplemented with 300 μg/ml carbenicillin, or 300 μg/ml Tc. All incubation were at 37°C.

Construction of alqD::lacZ lysoα/ens in E. coli

Using the strategy of Simons, et al . (1987) single copy chromosomal fusions in E. coli were generated. A 1.2 kb Hindlll-EcoRl fragment carrying the algD promoter from P. aeruginoεa PAO was excised from pPAOM3, converted at the Hindlll end into the BamHI specificity, and cloned as a BamHI-EcoRI fragment into the BamHI and EcoRI digested transcriptional fusion plasmid pRS550. The resulting plasmid pBED, carrying the algD::lacZ fusion, was introduced in E. coli strain DR459, which was then infected with the bacteriophage λRS74. This λ phage derivative carries a 7.9 kb EcoRI insert that matches the genes flanking the lacZ fusion in pRS550, and thus permits genetic exchange between the plasmid and the λ phage. Such in vivo recombinational events can be selected based on the associated transfer of Kmr from the plasmid to the phage. E. coli DR459 was infected with λ phage lysates prepared on E. coli harboring pBED. λ phage lysogens carrying the algD::lacZ fusion integrated on the chromosome were selected as Kmr colonies. Several strains were obtained and the presence of single-copy intact algD::lacZ fusion was verified by Southern blot analysis. One such strain, designated VD1870, was used in further studies.

DNA amplification. Cloning and Sequencing

Oligonucleotides UL5 (5'-GCCGCACGTCACGAGC-3') (SEQ ID NO:20) and UR20 (5'-CGCGAACCGCACCATCGCTC-3') (SEQ ID NO:21) were used for PCR amplification of the sequences beginning 120-bp upstream of the algU initiation codon and ending 193-bp past the stop codon of mucA resulting in a 1.5-kb PCR product. Where indicated, direct cycle sequencing of PCR products was performed using AmpliTaq Cycle Sequencing kit (Perkin-Elmer) . Sequences illustrating algU mutations were generated with the sequencing primers UR14 (5'ATCGCTGTCCGGTCGGC3') (SEQ ID NO:22) and UR16 (5'GAGTTCATCCGCTTCG3') SEQ ID NO:23.

Transcriptional Fusion and Alginate Assays For xylE transcriptional fusion measurements, all strains harbored the algD::xylE transcriptional fusion plasmid pPA0M3 which was introduced into P. aeruginosa by triparental filter matings. Activity of the xylE gene product, catechol 2,3 dioxygenase (CDO), was assayed as previously described. Expression of the algD::lacZ fusion was measured by following 3-galactosidose activity in sonic extracts obtained from cells harboring various plasmids and grown for 48h on LB plates supplemented with Ap, Km, Tc, and IPTG. The specific activity of /3-galactosidase was determined by hydrolysis of o-nitrophenyl 0-D-galactopyranoside (ONPG) at 420 nm in a spectrophotometer and expressed in mU/mg. One unit is defined as the amount of enzyme hydrolyzing lnmole of ONPG in 1 min. Molar extinction coefficient of the reaction product, o-nitrophenol, is 4500. Alginate measurements were performed in triplicate by the method of Knutson and Jeanes (1976) .

Reconstitution of algϋ-dependent algD transcription in E. coli

The algD promoter is a critical site for the regulation of mucoidy. All known elements required for the full activation of this promoter are contained within the region beginning at -533 and extending to +112 relative to the algD mRNA start site. A 1.2 kb DNA fragment including this region (extending from the Hindlll site, located 1.1 kb upstream, to the EcoRI site located at +112 downstream of the algD mRNA start site) was modified and cloned as a BamHI-EcoRI fragment into pRS550. This way, the algD promoter was positioned upstream of the promoterless lacZYA operon. The lacZ transcriptional fusion vector pRS550 permits in vivo transfer of the in vitro generated fusions from the plasmid to a companion λ phage via genetic recombination of the regions flanking the fusion. After exchange of algD::lacZ carrying region with the phage λRS74, the algD: :lacZ lysogen E. coli strain VD1870 was constructed (Fig. 5B) . The nucleotide sequence of the region immediately upstream of the mRNA start site is shown; overlined bold letters indicate the conserved -35 and -10 motifs of promoters regulated by AlgU homologs. The three AlgR binding sites RBI, RB2, and RB3 have the highly conserved core sequence 5'ACCGTTGTC3'-3', which in the case of RB3 runs in the opposite direction of RBI and RB2, and due to two mismatches has a lower affinity for AlgR. Numbers underneath the sequence denote positions of the core sequences relative to the algD mRNA start site (+1). A 1.2 kb fragment containing the algD promoter was used to generate the transcriptional fusion with lacZ in the E. coli lysogen VD1870 carrying chromosomally integrated algD::lacZ (PalgD: :lacZ) . Other symbols: kan, kanamycin resistance; T14, four tandem copies of the strong tanscriptional terminator Tl of the E. coli rrnB operon. Wavy lines indicate the E . coli chromosome.

To assess whether algU directly activates algD transcription, the present inventors attempted to clone the algU gene alone on a plasmid and to introduce such a construct into VD1870. This presented difficulties, since transformants containing plasmid constructs with inserts carrying algU devoid of mucA and mucB quickly lost their viability. However, when algU was cloned together with mucA and mucB in pUC12, this permitted its stable maintenance in E. coli. When the resulting plasmid pDM76 was introduced into the algD::lacZ transcriptional fusion strain VD1870, the transformant colonies displayed a light blue color on plates supplemented with XGal. /3-galactosidase activity was detectable in sonic extracts prepared from VD1870 harboring pDM76 (Fig. 6) . These results indicated that algD transcription can be initiated, albeit at a low level, by algU in the presence of its presumptive negative regulators mucA and mucB. A very low 3-galactosidase activity was also detectable in sonic extracts of VD1870 carrying the pUC12 vector only, indicating that a background algD transcription was present in E.coli.

Inactivation of mucA or mucB results in stimulation of algD transcription

The inventors determined whether this phenomenon could be reconstructed in E. coli. When VD1870 was transformed with the variants of the plasmid pDM76 carrying insertionally inactivated mucA (pDM76A) or mucB (pDM76B) , this further increased the transcription of algD::lacZ on the chromosome as evidenced by a darker blue coloration of colonies on plates supplemented with XGal and accompanied increase of /3-galactosidase activity in sonic extracts (Fig. 6) . This activation was dependent on the presence of a functional algU gene, since the plasmid pDM76UA with a double mutation, which

simultaneously inactivated algU and mucA, did not stimulate algD transcription over the background level (Fig. 5) . Furthermore, it was possible to complement mucA and mucB mutations on pDM76A and pDM76B, respectively, by introducing a compatible second plasmid carrying mucA and mucB cloned behind the tac promoter. Introduction of such a construct, pMJStac84AB, which also carries the lacl ^q gene, into the strain VD1870 harboring plasmids with insertionally inactivated mucA or mucB (pDM76A or pDM76B) but carrying an active algU, did not significantly affect algD::lacZ expression. When transcription of mucA and mucB on pMJStac84AB was further induced by IPTG, algD::lacZ expression was completely abrogated (Fig. 7) , bringing the levels of jS-galactosidase activity significantly below the values observed in the control strains (e.g. VD1870 harboring pUC12, Fig. 6). These experiments suggest that: (i) mucA and mucB play a direct negative regulatory role in algD transcription; and (ii) they exert this effect via algU, since loss of algU was epistatic to the inactivation of mucA.

Effects of AlgR on algD Transcription in E. coli Many different regulatory elements have been proposed to cooperate in the process of algD activation. One of the better studied factors is the response regulator AlgR, which has been shown to bind to three sites within the algD promoter (Fig. 5B) defined by the recognition sequence ACCGTTGTC. In order to examine whether algR could affect the function of the reconstituted system of algD transcription in E. coli, the algR gene was subcloned on a vector compatible with pUC12. This construct, pMJStac84R, was introduced into the algD::lacZ lysogen VD1870 and the resulting strain secondarily transformed with pDM76. The presence of algR additionally stimulated algD transcription over the

levels obtained with pDM76 alone (Fig. 8) . However, the algR-dependent transcription of algD was significantly lower vis-a-vis algU-dependent stimulation of algD expression (Fig. 8) . Moreover, when the medium was supplemented with 1 mM IPTG to increase algR expression, this resulted in a decreased algD transcription. The reasons for this are currently not known, but the status of AlgR-phosphorylation and a number of other phenomena could account for this observation.

EXAMPLE 7 instability of mucoidy in P. aeruginosa and second site suppressor mutations in algU

The role of mutations at the algU loci were also conducted. Further support for the mechanism of reversion to nonmucoidy in P. aeruginosa, a phenomenon frequently referred to as the instability of mucoid phenotype, was found. Spontaneous nonmucoid derivatives of the mucoid strain PA0578 carrying the mucA22 mutation were examined for the presence of alterations within the algU mucA mucB locus. Point mutations which inactivated algU were detected in some, but not all, nonmucoid revertants. No reversion of the original mucA22 mutation (a deletion of one C) was observed in any of the investigated strains. This suggests that the process of conversion to nonmucoidy can be explained, at least partially, by second site suppressor mutations, and that a fraction of such mutations occurs in algU.

The mucoid phenotype of P. aeruginosa is frequently unstable and nonmucoid variants can be observed upon repeated passages on standard laboratory media (Govan, (1988), and Ohman, et al . , (1990). The fact that conversion to mucoid phenotype in some strains is a result of mucA inactivation (Martin, et al . (1993))

suggests that in such mutants the presence of uncontrolled AlgU could interfere with the normal physiology of the cell. It is thus not surprising that in the absence of selective pressure to maintain mucoid phenotype, e.g. once the strains are extracted form the CF lung, nonmucoid variants emerge. Based on the studies with the epistatic nature of algU relative to mucA mutations presented here, and the fact that algU mutants are viable in the laboratory, it was hypothesized that conversion to nonmucoid phenotype could be due to mutations in algU.

In order to demonstrate this possibility, the strain PA0578 was chosen for further study. PA0578 has the following relevant characteristics: (i) It consistently displays a relatively high level instability of the mucoid phenotype and nonmucoid variants can be readily obtained; and (ii) it carries the fully characterized mucA22 mutation (a deletion of a single nucleotide within a string of 5 Cs in the coding region of mucA) , which has been shown in the previous Examples, by gene replacements to cause conversion to mucoidy. In the process of isolating spontaneous nonmucoid variants, it was observed that PA0578 gave rise to at least 3 morphologically distinct types differing from the mucoid appearance of the parent PA0578. The different morphotypes of PA0578 and its derivatives were classified based on their phenotype on two media, PIA and LB: Type I was mucoid on PIA and LB [the original PA0578]; type II was mucoid on PIA, but lost its mucoid appearance on LB; type III was nonmucoid on both PIA and LB; and type IV displayed a very low level but detectable mucoidy on both media after prolonged incubation. This suggested that there were several ways the system could adjust to the presence of a mucA mutation. Type III was further examined to investigate mechanisms contributing to

complete conversion to nonmucoidy, and to test whether the original mutation in mucA had been repaired in such strains.

Three randomly chosen, independently obtained type III isolates were subjected to further analysis. First, the levels of alginate production and algD transcription were determined, in order to rule out mutations that did not affect algD expression (e.g., altered export of the exopolysaccharide, etc.). The strains, PA057874,

PAO578160, displayed dramatic reduction in detectable uronic acids, and algD transcription relative to PA0578 (Table 9) .

TABLE 9. Alginate production and algD transcription in PA0578 and its spontaneous nonmucoid derivatives PA057874 and PAO578160

algU mucA Alginate production CDO ^c

Strain ³ Phenotype* ³ alleles (μg/mg of wet cell weight) (U/mg)

PA0578 M algU+ 100.33±19.2 60.51+21.1 mucA22

I

PA057874 NM algU74 0.57±0.065 0.014±0.001 -j -o mucA22 I

PAO578160 NM algU160 0.60±0.23 0.017±0.003 mucA22

PAO578160 and PA057874 are spontaneous, type III (see text) nonmucoid derivatives of

PA0578. b Phenotype was scored as mucoid (M) or nonmucoid (NM) on PIA. c For transcriptional fusion measurements, all strains harbored pPAOM3 carrying an algD::xylE transcriptional fusion. Activity was expressed as units of catechol 2, 3 deoxygenase (CDO; the xylE gene product) per mg of crude protein extracts ±SE.

This was also the case with the third isolate studied, PAO57800. Next, the chromosomal regions encoding algU and mucA were amplified by PCR from these strains, and subjected to direct nucleotide sequencing of the PCR products. When the sequences of mucA were examined, all strains displayed the presence of the original mucA22 mutation. In contrast, when algU was sequenced, the results of these analyses showed that the strains PA057874 and PAO578160 had acquired new mutations in algU that were not present in the parental mucoid strain PA0578 (Fig. 9) . The lesion in PA057874 was a nonsense mutation, which resulted in conversion of the codon specifying Trp74 into a stop codon . The strain PA0578160 had a frameshift mutation (deletion of a T residue within the codon specifying Aspl60) which brought in frame a premature stop codon 10 nucleotides downstream of the deletion. However, the third strain tested, PAO57800 , did not have any alterations within the algU coding region. This strain still carried the original mucA22 mutation, thus ruling out the possibility that its change into the nonmucoid phenotype was due to the repair of the lesion in mucA.

To ascertain whether the detected algU lesions were responsible for conversion to nonmucoidy in PA057874 and PAO578160, gene replacements of the respective algU alleles with the wild type algU gene from PAOl were carried out. The plasmid pDMUM13 carrying the wild type algU mucA genes were introduced into PA057874 and PAO578160 by triparental filter matings, and Tcr exconjugants selected. The resulting strains were nonmucoid, but gave rise to rare mucoid colonies detectable on a lawn of nonmucoid cells harboring the plasmid. Such mucoid derivatives were candidates for gene replacements of the mutant chromosomal algU gene (alleles algU74 and algU176) with the wild type copy of algU

(algU+) . This was verified by plasmid curing, purification of mucoid derivatives which were TcS and thus had lost the plasmid, followed by amplification of the algU sequences and their direct sequencing. The results of these experiments confirmed that the chromosomal alleles algU74 and algU160 had been replaced by the functional wild type copies of algU in all mucoid colonies that were tested. This served as a demonstration that the algU mutations in PA057874 and PAO578160 were responsible for conversion to nonmucoidy. These findings suggest, that although multiple mechanisms exists, one pathway of reverting to nonmucoid phenotype in P. aeruginosa is through inactivation of algU.

The present inventors have also achieved the algU-dependent algD transcription in a heterologous host. This work provides further characterization of the gene cluster controlling conversion to mucoidy in P. aeruginosa and provides the means for identifying compounds that may inhibit alginate production. The genetic reconstitution experiments, presented herewith, have been carried out in a heterologous host which does not produce alginate, and therefore is expected to lack the necessary elements for expression of alg genes. This strongly suggests that AlgU acts directly on algD.

Efforts to overproduce and purify AlgU have indicated that expression clones of the algU gene are highly unstable. The reasons for this are currently not known, but one possible explanation is that the gene product is toxic to bacterial cells. The present inventoras have noticed that E. coli strains transformed with high copy number plasmids carrying algU can form colonies on selection media but are difficult or impossible to propagate. It is worth mentioning that the initial analysis of the algU mucA mucB locus via deletion

subcloniϊig approach has resulted in a series of deletions from both the 5' and 3' ends of the algU mucA mucB cluster. The deletion clones generated from the 5' end represent an unbiased series of overlapping clones with more or less evenly distributed deletion end points.

Interestingly, in contrast to this the 3' end deletions never extended into the mucA gene while leaving algU intact on the same fragment. Thus, the only recovered deletion clones from the 3' end of the cluster either contained the complete algU and mucA genes, or had deletion end points encroaching on the coding region of algU. This further supports the notion that the algU gene product may be toxic to the cells, and also suggests that this putative toxicity is counteracted by the presence of mucA (and possibly mucB) .

One explanation for this effect of mucA is that it suppresses the activity of AlgU. Such negative regulation of algU by mucA has been addressed and established in the studies presented here. For example, insertional inactivation of mucA derepresses algU-dependent algD transcription in the genetically reconstructed system in E. coli. This is in keeping with the observations that mucoid strains of P. aeruginosa can emerge both in vitro (Fyfe, et al . (1980) and in vivo (Martin, et al . (1993A)) via spontaneous frameshift or nonsense mutations in mucA on the chromosome. One could then expect that mutations which inactivate mucA could generate strong selective pressure to contain or suppress the unattended and presumably toxic AlgU. Thus, it is not surprising that many mucoid CF isolates, and laboratory strains which have been converted to mucoidy via genetic manipulations of mucA or mucB (Martin, et al . (1993B) , in absence of selective pressures to maintain mucoid phenotype, show tendency to convert into nonmucoid or attenuated alginate producing forms. One mechanism underlying these processes

has been presented herewith, and is based on ocurrence of second site suppressor mutations in algU which can override the effects of mucA mutations. However, this is not the only mechanism of conversion to nonmucoid phenotype. The analysis of PA0578 variants displaying various medium-dependent patterns and attenuated levels of alginate production, and the absence of algU mutation in the nonmucoid derivative type III PAO57800, strongly suggest that pseudoreversion to a nonmucoid state can occur via several alternative pathways. These mechanisms of attenuating constitutive alginate production are currently investigated since they may help uncover additional factors that interact with AlgU.

PROPHETIC EXAMPLE 8 Screening for Candidate Mucoid Inhibitors

The present invention also provides candidate substance screening methods which are based upon whole cell assays and which, preferably, employ the LacZ reporter gene that confers on its recombinant hosts a readily detectable phenotype that emerges only under conditions where the protein products of algU, mucA and mucB, control transcription of the algD gene. Generally, reporter genes encode a polypeptide not otherwise produced by the host cell which is detectable by analysis of the cell culture, e.g., by fluorometric, radioisotopic or spectrophotometric analysis of the bacterial culture. Exemplary enzymes include esterases, phosphatases, proteases (tissue plasminogen activator or urokinase) and other enzymes capable of being detected by their activity, as will be known to those skilled in the art. A preferred example is E . coli beta-galactosidase, which produces a color change upon cleavage of an indigogenic substrate.

The polypeptide products of the reporter gene are secreted, intracellular or, as noted above, membrane bound polypeptides. If the polypeptide is not ordinarily secreted it is fused to a heterologous signal sequence for processing and secretion. In other circumstance the signal is modified in order to remove sequences that interdict secretion. Preferably, however, the products of the reporter gene are lodged in the intracellular or membrane compartments, and may be released as described in Example 7 through sonication. Then they can be fixed to the culture container, e.g., microtiter wells, in which they are grown, followed by addition of a detectable signal generating substance such as a chromogenic substrate for reporter enzymes.

In general, the proteins of the present invention are employed to control transcription and expression of the reporter gene. The process which in its entirety leads to enhanced transcriptional promotion is termed "activation." The mechanism by which a successful candidate substance acts is not material since the objective is to promote the expression of wild-type algU, mucA and mucB in order to inhibit algD expression or function, and particularly the activity of the algD gene product in alginate sysnthesis, by whatever means will function to do so. The use of the entire gene loci is preferred as it will most closely model the therapeutic target, and given the surprising difficulties in expressing algU alone.

The host cells used in the screening assay herein generally are E. coli cells, and are preferably cell lines which may be used in connection with Example 6. These bacteria should be relatively easy to grow in large scale culture, and may be tested in microtiter plates.

Also, as provided for in Example 8, the native background

can be easily accounted for, and may serve to examine both agonists and antagonists of algD gene expression.

The screening assay typically is conducted by growing the transformants to confluency in microtiter wells, adding serial molar proportions of candidate substances to a series of wells, and the signal level determined after an incubation period that is sufficient to demonstrate /3-galactosidase activity in controls incubated alone. The throughput of samples can be greatly enhanced through the use of automated ELISA readers, scanning at an optical density of 410nm. The wells containing varying proportions of candidate are then evaluated for signal activation. Candidates that demonstrate dose related enhancement or repression of reporter gene transcription or expression are then selected for further evaluation as clinical therapeutic agents. The stimulation of transcription may be observed in the absence of added candidate, in which case the candidate compound might be a positive stimulator of algD gene transcription. Alternatively, the candidate compound might only give a stimulation in the presence of algU, mucA and mucB, which would indicate that it functions to oppose the algU-mediated suppression of the algD promoter. Candidate compounds of either class might be useful therapeutic agents that would stimulate production of algU and thereby preclude P. aeruginoεa mucoidy in CF patients.

It should be understood that the screening method herein is useful notwithstanding that effective candidates may not be found, since it would be a practical utility to know that algU activators do not exist. The invention consists of providing a method for screening for such candidates, not in finding them.

The term "capable of activating algU-mucA-mucB mediated inhibition of algD" means that upon contacting the bacteria, with the algU-mucA-mucB repressed algD reporter system, for example, under appropriate conditions of ionic strength, temperature, pH and the like, specific binding will occur, and thereby repression of algD promoter driven transcription. The interaction may occur due to specific electrostatic, hydrophobic, entropic or other interaction of certain amino acid residues of the one protein, with specific amino acid residues of the second protein, particularly algU binding to mucA and mubB, to form a stable complex under the conditions effective to promote the interaction. The interaction may alter the three dimensional conformation of either or both proteins or polypeptides involved in the interaction and it may also alter the function or activity of either or both proteins or polypeptides involved in the interaction. For example, the interaction of algU with other protein may alter the ability of the RNA polymerase complex to bind other proteins, such as sigma factors, and to bind or recognize specific regions of DNA, to act as a substrate for a phosphorylation or dephosphorylation or other enzymatic reaction, or to catalyze or effect any enzymatic reaction involving other substrates and reactants.

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will

be apparent to those of skill in the art that variations may be applied to the composition, methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Terry, J. M. , Pina, S. E. , and Mattingly, S. J. (1991) Environmental conditions which influence mucoid conversion in Pseudomonas aeruginosa PAOl. Infect. Immun. 59:471-477.

Tosi, M. F., Zakem, H. , and Berger, M. (1990) Neutrophil elastase cleaves C3bi on opsonized Pseudomonas as well as CRl on neutrophils to create a functionally important opsonin receptor mismatch. J. Clin. Invest. 86:300-308.

Totten, P. A., Lara, J. C, and Lory, S. (1990) The rpoN gene product of Pseudomonas aeruginosa is required for expression of diverse genes, including the flagellin gene. J. Bacteriol. 172:389-396.

Trempy, J. E. , Morrison-Plummer, J., and Haldenwang, W. G. (1985) Synthesis of _ ²⁹ , and RNA polymerase specificity determinant, is a developmentally regulated event in Bacillus subtilis. J. Bacteriol, 161:340-346.

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SEOUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

NAME: BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM STREET: 201 West 7th Street

CITY: Austin

STATE: Texas

COUNTRY: United States of America

POSTAL CODE: 78701 TELEPHONE NO: (512)499-4462

TELEFAX: (512)499-4523

(ii) INVENTORS

DERETIC, Vojo MARTIN, Daniel W.

(iii) TITLE OF INVENTION: DETECTION OF CONVERSION TO

MUCOIDY IN PSEUDOMONAS AERUGINOSA INFECTING CYSTIC FIBROSIS PATIENTS

(iv) NUMBER OF SEQUENCES: 19

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Arnold, White & Durkee

(B) STREET: P. O. Box 4433

(C) CITY: Houston

(D) STATE: Texas (E) COUNTRY: USA

(F) ZIP: 77210

(V) COMPUTER READABLE FORM: (A) MEDIUM TYPE: FLOPPY DISK

(B) COMPUTER: IBM PC COMPATIBLE

(C) OPERATING SYSTEM: PC-DOS/MS-DOS/ASCII

(Vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: UNKNOWN

(B) FILING DATE: UNKNOWN

(C) CLASSIFICATION: UNKNOWN (vϋ) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 08/017,114

(B) FILING DATE: 12 February 1993 (12.02.93)

(C) CLASSIFICATION: UNKNOWN

(vii) ATTORNEY/AGENT INFORMATION:

(A) NAME: HODGINS, DANIEL S.

(B) REGISTRATION NUMBER: 31,026 (C) REFERENCE/DOCKET NUMBER: UTFK231P-

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 512/320-7200 (B) TELEFAX: 713/789-2679

(C) TELEX: 79-0924

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 595 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

AGGTATCGCT ATGAGTCGTG AAGCCCTGCA GGAAACTCTG TCCGCTGTGA TGGATAACGA 60

AGCGGATGAA CTCGAGTTGC GGCGGGTGCT CGCAGCTTGC GGCGAGGATG CCGAGCTGCG 120

TTCCACCTGG TCGCGTTACC AGTTGGCGCG GTCCGTCATG CACCGCGAGC CTACCCTGCC 180

GAAGCTGGAT ATCGCTGCGG CGGTCTCTGC TGCCCTGGCC GACGAGGCCG CTCCGCCGAA 240

10

AGCGGAGAAG GGACCGTGGC GGATGGTCGG TCGCCTGGCG GTCGCTGCCT CGGTGACCCT 300

GGCGGTGCTG GCCGGCGTGC GTCTGTACAA CCAGAACGAC GCCCTGCCGC AAATGGCGCA 360

15 ACAGGGGACC ACCCCGCAGA TCGCCCTGCC TCAGGTGAAA GGCCCGGCCG TGCTGGCCGG 420 -Λ o

CTACAGCGAA GAGCAGGGGG CGCCGCAGGT GATCACCAAC TCCTCGTCCA GCGATACCCG 480 V

CTGGCATGAG CAGCGTCTGC CGATCTACCT GCGTCAGCAC GTGCAACAAT CCGCCGTCAG 540

20 TGGTACAGAG AGCGCGCTGC CCTACGCTCG GGCAGCCAGC CTGGAAAACC GCTGA 595

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GGGACCCCCC GCA 13

(2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

GAGCAGGGGC GCC 13

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CAGGGGGCCA GGGGGC 16

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

-109-

(A) LENGTH: 16 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

GCCGCACGTC ACGAGC 16

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: GAGTCCATCC GCTTCG 16

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CTGTCCGCTG TGATGG 16

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: CGCCCCTGCT CCTCGA 16

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 647 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

GTCTATCTTG GCAAGACGAT TCGCTGGGAC GCTCGAAGCT CCTCCAGGTT CGAAGAGGAG 60

CTTTCATGCT AACCCAGGAA CAGGATCAGC AACTGGTTGA ACGGGTACAG CGCGGAGACA 120

AGCGGGCTTT CGATCTGCTG GTACTGAAAT ACCAGCACAA GATACTGGGA TTGATCGTGC 180

GGTTCGTGCA CGACGCCCAG GAAGCCCAGG ACGTAGCGCA GGAAGCCTTC ATCAAGGCAT 240

10

ACCGTGCGCT CGGCAATTTC CGCGGCGATA GTGCTTTTTA TACCTGGCTG TATCGGATCG 300

CCATCAACAC CGCGAAGAAC CACCTGGTCG CTCGCGGGCG TCGGCCACCG GACAGCGATG 360

15 TGACCGCAGA GGATGCGGAG TTCTTCGAGG GCGACCACGC CCTGAAGGAC ATCGAGTCGC 420

CGGAACGGGC GATGTTGCGG GATGAGATCG AGGCCACCGT GCACCAGACC ATCCAGCAGT 480

TGCCCGAGGA TTTGCGCACG GCCCTGACCC TGCGCGAGTT CGAAGGTTTG AGTTACGAAG 540

20

ATATCGCCAC CGTGATGCAG TGTCCGGTGG GGACGGTACG GTCGCGGATC TTCCGCGCTC 600

GTGAAGCAAT CGACAAAGCT CTGCAGCCTT TGTTGCGAGA AGCCTGA 647

25

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1800 base pairs 30 (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

TTTGTTGCGA GAAGCCTGAC ACAGCGGCAA ATGCCAAGAG AGGTATCGCT ATGAGTCGTG 60

AAGCCCTGCA GGAAACTCTG TCCGCTGTGA TGGATAACGA AGCGGATGAA CTCGAGTTGC 120

GGCGGGTGCT CGCAGCTTGC GGCGAGGATG CCGAGCTGCG TTCCACCTGG TCGCGTTACC 180

10

AGTTGGCGCG GTCCGTCATG CACCGCGAGC CTACCCTGCC GAAGCTGGAT ATCGCTGCGG 240

CGGTCTCTGC TGCCCTGGCC GACGAGGCCG CTCCGCCGAA AGCGGAGAAG GGACCGTGGG 300

15 GGATGGTCGG TCGCCTGGCG GTCGCTGCTC GGTGACCCTG GCGGTGCTGG CCGGCGTGCG 360 -

P"

TCTGTACAAC CAGAACGACG CCCTGCCGCA AATGGCGCAA CAGGGGACCA CCCCGCAGAT 420 *\

CGCCCTGCCT CAGGTGAAAG GCCCGGCCGT GCTGGCCGGC TACAGCGAAG AGCAGGGGGC 480

20

GCCGCAGGTG ATCACCAACT CCTCGTCCAG CGATACCCGC TGGCATGAGC AGCGTCTGCC 540

CGATCTACCT GCGTCAGCAC GTGCAACAAT CCGCCGTCAG TGGTACAGAG AGCGCGCTGC 600

25 CCTACGCTCG GGCAGCCAGC CTGGAAAACC GCTGAGGAGA GACATGCGCA CCACCTCCCT 660

GTTGCTTTTG CTTGGCAGCC TGATGGCGGT TCCCGCCACT CAGGCTGCCG ACGCTTCCGA 720

CTGGCTGAAT CGTCTCGCCG AGGCCGATCG CCAGAACAGT TTCCAAGGCA CCTTCGTCTA 780

30

CGAGCGCAAT GGCAGCTTCT CCACCCATGA GATCTGGCAT CGCGTGGAGA GCGATGGTGC 840

GGTTCGCGAG CGCCTGCTCC AGCTCGACGG CGCGCGCCAG GAAGTGGTCC GGGTCGACGG 900

GCGCACCCAG TGCATCAGCG GCGGCCTTGC CGACCAACTG GCCGATGCCC AGCTGTGGCC 960

GGTGCGCAAG TTCGATCCCT CCCAGCTGGC TTCCTGGTAC GACCTGCGCC TGGTCGGGGA 1020

ATCCCGTGTC GCCGGCCGCC CGGCAGTGGT CCTTGCGGTG ACTCCGCGCG ACCAGCATCG 1080

CTACGGCTTC GAGCTGCACC TGGACCGCGA CACCGGCCTG CCGTTGAAGT CGCTGCTGCT 1140

GAACGAGAAG GGGCAGTTGC TCGAGCGCTT CCAGTTCACC CAGTTGAATA CCGGCGCGGC 1200

10

ACCTGCCGAA GACCAGTTGC AGGCGGGCGC CGAATGCCAG GTCGTCGGCC CGGCCAAGGC 1260

CGACGGGGAG AAGACCGTGG CCTGGCGCTC GGAATGGCTG CCGCCAGGTT TCACCCTGAC 1320

15 CCGCAGTTTC ATGCGTCGCA GTCCGGTCAC CCCCGATCCG GTCGCCTGCC TGACCTATGG 1380 -

CGATGGCCTG GCACGATTCT CGGTGTTCAT CGAGCCGCTG CACGGTGCCA TGGTTGGCGA 1440 V

CGCGCGCAGC CAGCTCGGCC CGACCGTGGT GGTTTCCAAG CGCCTGCAGA CCGATGACGG 1500

20

CGGCCAGATG GTGACCGTCG TCGGCGAAGT GCCGCTGGGC ACCGCCGAGC GGGTGGCGCT 1560

GTCCATCCGG CCCGAGGCCG CCGCCCAGAA ATGATCGAGG AGCAGGGGCG AGTGGTGGCG 1620

25 ACCGAGCCGG GAGCGGTATG GGTCGAGACC GTGCGCCGAG TACCTGCTCG TCCTGCTCGG 1680

CCAATGCCGG TTGCGGCCAG GGGCTGATGC AGCGCCTGGG CGTCGGCGCG GGGCGTGCCC 1740

GGGTGCGCGC GTTGAGCGAC CTGAGCCTGC GGGTCGGCGA TGCGGTCGTC CTAGGAATTC 1800

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: AGCGAAGAGC AGGGGGCGCC GCAGGTGATC A 31

(2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

GAGCAGGGGG CGCCG 15

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: AACAGGGGAC CACCCCGCAG ATCGCC 26

(2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

GGGACCACCC CGC 13

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 194 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

Met Ser Arg Glu Ala Leu Gin Glu Thr Leu Ser Ala Val Met Asp Asn 1 5 10 15

5

Glu Ala Asp Glu Leu Glu Leu Arg Arg Val Leu Ala Ala Cys Gly Glu 20 25 30

Asp Ala Glu Leu Arg Ser Thr Trp Ser Arg Tyr Gin Leu Ala Arg Ser 10 35 40 45

Val Met His Arg Glu Pro Thr Leu Pro Lys Leu Asp lie Ala Ala Ala 50 55 60

15 Val Ser Ala Ala Leu Ala Asp Glu Ala Ala Pro Pro Lys Ala Glu Lys →

Gly Pro Trp Arg Met Val Gly Arg Leu Ala Val Ala Ala Ser Val Thr

85 90 95

20

Leu Ala Val Leu Ala Gly Val Arg Leu Tyr Asn Gin Asn Asp Ala Leu 100 105 110

Pro Gin Met Ala Gin Gin Gly Thr Thr Pro Gin lie Ala Leu Pro Gin 25 115 120 125

Val Lys Gly Pro Ala Val Leu Ala Gly Tyr Ser Glu Glu Gin Gly Ala

130 135 140

30 Pro Gin Val lie Thr Asn Ser Ser Ser Ser Asp Thr Arg Trp His Glu

145 150 155 160

Gin Arg Leu Pro lie Tyr Leu Arg Gin His Val Gin Gin Ser Ala Val

165 170 175

Ser Gly Thr Glu Ser Ala Leu Pro Tyr Ala Arg Ala Ala Ser Leu Glu

180 185 190

Asn Arg 5

(2) INFORMATION FOR SEQ ID NO: 16:

10 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

15 I p»

(ii) MOLECULE TYPE: peptide P>

*v]

20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

Leu Leu Arg Glu Ala 1 5

25 (2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 194 amino acids

(B) TYPE: amino acid

30 (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

Met Ser Arg Glu Ala Leu Gin Glu Thr Leu Ser Ala Val Met Asp Asn 5 1 5 10 15

Glu Ala Asp Glu Leu Glu Leu Arg Arg Val Leu Ala Ala Cys Gly Glu 20 25 30

10 Asp Ala Glu Leu Arg Ser Thr Trp Ser Arg Tyr Gin Leu Ala Arg Ser

35 40 45

Val Met His Arg Glu Pro Thr Leu Pro Lys Leu Asp lie Ala Ala Ala 50 55 60

15

Val Ser Ala Ala Leu Ala Asp Glu Ala Ala Pro Pro Lys Ala Glu Lys 00

65 70 75 80 ϊ

Gly Pro Trp Arg Met Val Gly Arg Leu Ala Val Ala Ala Ser Val Thr 20 85 90 95

Leu Ala Val Leu Ala Gly Val Arg Leu Tyr Asn Gin Asn Asp Ala Leu 100 105 110

25 Pro Gin Met Ala Gin Gin Gly Thr Thr Pro Gin lie Ala Leu Pro Gin

115 120 125

Val Lys Gly Pro Ala Val Leu Ala Gly Tyr Ser Glu Glu Gin Gly Ala 130 135 140

30

Pro Gin Val lie Thr Asn Ser Ser Ser Ser Asp Thr Arg Trp His Glu

Gin Arg Leu Pro lie Tyr Leu Arg Gin His Val Gin Gin Ser Ala Val

165 170 175

Ser Gly Thr Glu Ser Ala Leu Pro Tyr Ala Arg Ala Ala Ser Leu Glu 180 185 190

Asn Arg

(2) INFORMATION FOR SEQ ID NO:18

10

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 316 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single 15 (D) TOPOLOGY: linear I

H H

(ii) MOLECULE TYPE: peptide f

20

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

Met Arg Thr Thr Ser Leu Leu Leu Leu Leu Gly Ser Leu Met Ala Val 1 5 10 15

25

Pro Ala Thr Gin Ala Ala Asp Ala Ser Asp Trp Leu Asn Arg Leu Ala 20 25 30

Glu Ala Asp Arg Gin Asn Ser Phe Gin Gly Thr Phe Val Tyr Glu Arg 30 35 40 45

Asn Gly Ser Phe Ser Thr His Glu lie Trp His Arg Val Glu Ser Asp

50 55 60

Gly Ala Val Arg Glu Arg Leu Leu Gin Leu Asp Gly Ala Arg Gin Glu 65 70 75 80

Val Val Arg Val Asp Gly Arg Thr Gin Cys He Ser Gly Gly Leu Ala 85 90 95

Asp Gin Leu Ala Asp Ala Gin Leu Trp Pro Val Arg Lys Phe Asp Pro 100 105 110

10 Ser Gin Leu Ala Ser Trp Tyr Asp Leu Arg Leu Val Gly Glu Ser Arg 115 120 125

Val Ala Gly Arg Pro Ala Val Val Leu Ala Val Thr Pro Arg Asp Gin 130 135 140

15 I

H

His Arg Tyr Gly Phe Glu Leu His Leu Asp Arg Asp Thr Gly Leu Pro O 145 150 155 160 I

Leu Lys Ser Leu Leu Leu Asn Glu Lys Gly Gin Leu Leu Glu Arg Phe

20 165 170 175

Gin Phe Thr Gin Leu Asn Thr Gly Ala Ala Pro Ala Glu Asp Gin Leu 180 185 190

25 Gin Ala Gly Ala Glu Cys Gin Val Val Gly Pro Ala Lys Ala Asp Gly 195 200 205

Glu Lys Thr Val Ala Trp Arg Ser Glu Trp Leu Pro Pro Gly Phe Thr 210 215 220

30

Leu Thr Arg Ser Phe Met Arg Arg Ser Pro Val Thr Pro Asp Pro Val 225 230 235 240

Ala Cys Leu Thr Tyr Gly Asp Gly Leu Ala Arg Phe Ser Val Phe He

245 250 255

Glu Pro Leu His Gly Ala Met Val Gly Asp Ala Arg Ser Gin Leu Gly 260 265 270

5

Pro Thr Val Val Val Ser Lys Arg Leu Gin Thr Asp Asp Gly Gly Gin 275 280 285

Met Val Thr Val Val Gly Glu Val Pro Leu Gly Thr Ala Glu Arg Val 10 290 295 300

Ala Leu Ser He Arg Pro Glu Ala Ala Ala Gin Lys 305 310 315

15

(2) INFORMATION FOR SEQ ID NO:19: ^M

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 193 amino acids 20 (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

25

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

30 Met Leu Thr Gin Glu Gin Asp Gin Gin Leu Val Glu Arg Val Gin Arg

1 5 10 15

Gly Asp Lys Arg Ala Phe Asp Leu Leu Val Leu Lys Tyr Gin His Lys

20 25 30

He Leu Gly Leu He Val Arg Phe Val His Asp Ala Gin Glu Ala Gin 35 40 45

Asp Val Ala Gin Glu Ala Phe He Lys Ala Tyr Arg Ala Leu Gly Asn 50 55 60

Phe Arg Gly Asp Ser Ala Phe Tyr Thr Trp Leu Tyr Arg He Ala He 65 70 75 80

10 Asn Thr Ala Lys Asn His Leu Val Ala Arg Gly Arg Arg Pro Pro Asp 85 90 95

Ser Asp Val Thr Ala Glu Asp Ala Glu Phe Phe Glu Gly Asp His Ala 100 105 110

15 I p»

Leu Lys Asp He Glu Ser Pro Glu Arg Ala Met Leu Arg Asp Glu He t

CO 115 120 125 I

Glu Ala Thr Val His Gin Thr He Gin Gin Leu Pro Glu Asp Leu Arg

20 130 135 140

Thr Ala Leu Thr Leu Arg Glu Phe Glu Gly Leu Ser Tyr Glu Asp He 145 150 155 160

25 Ala Thr Val Met Gin Cys Pro Val Gly Thr Val Arg Ser Arg He Phe 165 170 175

Arg Ala Arg Glu Ala He Asp Lys Ala Leu Gin Pro Leu Leu Arg Glu 180 185 190

30

Ala

(2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

10

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 15 GCCGCACGTC ACGAGC 16 t ω I

(2) INFORMATION FOR SEQ ID NO:21:

20 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

25

(ii) MOLECULE TYPE: DNA (genomic)

30 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

CGCGAACCGC ACCATCGCTC 20 ( 2 ) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

10

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: ATCGCTGTCC GGTCGGC 17

15 I

H

(2) INFORMATION FOR SEQ ID NO:23: to I

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 base pairs

20 (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

25

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

30 GAGTTCATCC GCTTCG 16