Abstract:
A method of treating or preventing Helicobacter infection in humans or animals comprising the step of administering a molecule, such as a molecule that can interact with UreI, capable of inhibiting the growth or survival of Helicobacter in vivo to a human or animal in need of such treatment.

Description:
[0001]    This invention relates to methods of screening molecules capable of inhibiting the survival of Helicobacter. particularly  Helicobacter pylori , in vivo by specifically inhibiting the activity of UreI, to the molecules identified by these methods, and to the use of these molecules to treat or prevent Helicobacter infection.  
         BACKGROUND OF INVENTION  
         [0002]    [0002] Helicobacter pylori  is a microaerophilic Gram-negative bacterium, which colonizes the gastric mucosa of humans (10).  H. pylori  is associated with gastritis and peptic ulcer disease and has been shown to increase the risk of gastric cancers. Urease is a major virulence factor of  H. pylori . It is involved in neutralizing the acidic microenvironment of the bacterium and also plays a role in  H. pylori  metabolism (11, 26).  
           [0003]    The urease region of the  H. pylori  genome is composed of two gene clusters common to all strains (9 and FIG. 1), one comprising the ureAB genes encoding the structural urease sub units and the other containing the ureEFGH genes encoding the accessory proteins required for nickel incorporation into the urease active site. The ureI gene lies immediately upstream from this latter gene cluster and is transcribed in the same direction (FIG. 1 ). The ureA, ureB, ureE, ureF, ureG, ureH, and ureI genes and gene products have been described and claimed in U.S. Pat. No. 5,695,931 and allowed patent application Ser. No. 08/472,285. both of which are specifically incorporated herein by reference.  
           [0004]    The distances separating ureI from ureE (one base pair, bp) and ureE from ureF (11 bp) suggest that ureI-ureE-ureF constitute an operon. Cotranscription of ureI and ureE has been demonstrated by northern blot analysis (1). An  H. pylori  N6 mutant with a ureI gene disrupted by a MiniTn3-Km transpose was previously described by Ferrero et al. (1994) (13). This strain (N6-ureI::TnKm-8) presented a urease negative phenotype, so it was concluded that ureI was an accessory gene required for full urease activity.  
           [0005]    The sequences of UreI from  H. pylori  and the AmiS proteins, encoded by the aliphatic amidase operons of  Pseudomonas aeruginosa  and Rhodococcus sp. R312, are similar (5, 27). Aliphatic amidases catalyze the intracellular hydrolysis of short-chain aliphatic amides to produce the corresponding organic acid and ammonia. It has been shown that  H. pylori  also has such an aliphatic amidase, which hydrolyzes acetamide and propionamide in vitro (23).  
           [0006]    In view of the sequence similarity between UreI and AmiS together with the very similar structures of the urease and amidase substrates (urea: NH 2 —CO—NH 2  and acetamide: CH 3 —CO—NH 2 ) and the production of ammonia by both enzymes, a better understanding of the function of the  H. pylori  UreI protein is required. This understanding will open new opportunities for the prevention and treatment of  H. pylori  infections.  
         SUMMARY OF THE INVENTION  
         [0007]    This invention provides methods for identifying molecules capable of inhibiting the growth and/or survival of Helicobacter species, particularly,  H. pylori , in vivo. In particular, the methods of this invention involve screening molecules that specifically inhibit UreI protein function.  
           [0008]    The invention encompasses the molecules identified by the methods of this invention and the use of the molecules by the methods of this invention to treat or prevent Helicobacter, and particularly  H. pylori , infection in humans and animals.  
           [0009]    Another aspect of this invention is a method of preventing or treating Helicobacter species infection by administration to a human or animal in need of such treatment a molecule capable of inhibiting the growth and/or survival of Helicobacter species in vivo. One such molecule according to the invention is characterized by a high affinity for UreI, which allows it (i) to be transported inside the Helicobacter cell, or (ii) to inhibit transport properties of UreI, or (iii ) to inhibit UreI function by inhibiting UreI interaction with urease or other Helicobacter proteins. By inhibiting UreI, such molecule renders the bacteria more sensitive to acidity.  
           [0010]    Yet another aspect of this invention is the production of immunogenic UreI antigens and their use as vaccines to prevent Helicobacter species infection and/or colonization of the stomach or the gut. Antibodies to these UreI antigens are also encompassed within the scope of this invention.  
           [0011]    This invention further relates to recombinant strains of  H. pylori  comprising a modified ureI gene, such that the products of the modified gene contribute to the attenuation of the bacteria&#39;s ability to survive in vivo and thus, its pathogenic effects.  
       
    
    
     BRIEF DESCRIPTION OF THE FIGURES  
       [0012]    [0012]FIG. 1 depicts the urease gene cluster of  H. pylori  parental strains N6 and SS1 and of the derived mutants deficient in UreI, strains N6-823, N6-834, and SS 1-834. The genes are indicated by boxes with an arrow showing the direction of their transcription. The distances between the ure genes are given in base pairs, bp. The site hybridizing to the primers used to confirm correct allelic exchange in strains N6-823, N6-834, and SS1-834 is shown. Blank boxes represent the cassettes containing the genes conferring resistance to Cm (cat) or to Km (aphA-3). The urease activity of these strains is given on the right-hand side of the figure. Urease activity was measured as the release of ammonia on crude extracts of bacteria grown 48 hours on blood agar plates as described previously (9). One unit corresponds to the amount of enzyme required to hydrolyze 1 μmol of urea min −1  mg −1  total protein. The data are means ± standard deviation calculated from 3 to 5 determinations.  
         [0013]    [0013]FIG. 2A depicts a restriction map of pILL823, pILL824, pILL833 and pILL834. Small boxes mark the vector of each plasmid, and large boxes correspond to genes. Ori indicates the position of the ColE1 origin of replication. Sp R  and Ap R  are the genes conferring resistance to spectinomycin and ampicillin, respectively. Cassettes inserted into ureI and conferring resistance to chloramphenicol (cat) or kanamycin (aphA-3) are also shown. The sequence of the DNA region comprising the ureI stop codon and the ureE start codon, including the BclI site where adaptor H19 was inserted, is given. Insertion of H19 into the BclI site of pILL824 produced pILL825, the resulting ureI-ureE intergenic region is also shown. The stop codon of ureI and the start codon of ureE are boxed and the ribosome binding site (RBS) is underlined. Brackets indicate the position of restriction sites removed by ligation.  
         [0014]    [0014]FIG. 2B depicts a restriction map of two  H. pylori/E. coli  shuttle plasmids: pILL845 and pILL850. Small boxes mark the vector of each plasmid, and large boxes correspond to genes. Ori indicates the position of the  E. coli  ColE 1 origin of replication and repA the gene coding for the RepA protein necessary for autonomous replication of the pHe12 in  H. pylori . Cm P.  marks the gene conferring resistance to chloramphenicol. The ureI promoter is represented by a “P” with an arrow indicating the direction of the transcription. The other symbols are as in FIG. 1.  
         [0015]    [0015]FIG. 3 shows the alignment of the amino acid sequence of UreI from  H. pylori  with those of similar proteins and prediction of the two-dimensional structure of members of the UreI/AmiS protein family. Residues identical at one position in, at least, four sequences are boxed. and dashes indicate gaps inserted to optimize alignment. The organisms from which the sequences originated and the degree of identity with the  H. pylori  UreI protein are: UreI-Hp,  Helicobacter pylori  (195 residues, accession No. M84338); UreI-Hf,  Helicobacter  felis (74% identity over 196 residues, accession No. A41012); UreI-Lacto,  Lactobacillus fermentum  (55% identity over the 46 residues-long partial sequence, accession No. D10605); UreI-Strepto.  Streptococcus salivarius  (54% identity over the 129 residues-long partial sequence, accession No. U35248); AmiS-Myco.  Mycobacterium smegmatis  (39% identity over 172 residues, accession No. X57175); AmiS-Rhod, Rhodococcus sp. R312 (37% identity over 172 residues accession No. Z46523) and AmiS-Pseudo,  Pseudomonas aeruginosa  (37% identity over 171 residues, accession No. X77161). Predicted transmembrane ∀-helices are shown as shaded boxes. The regions separating these boxes are hydrophilic loops labeled “IN” when predicted to be intracellular and “OUT” when predicted to be extracellular.  
         [0016]    [0016]FIG. 4 depicts the kinetics of ammonium release by the N6 parental strain (panel A) and the UreI-deficient strain N6-834 (panel B). Bacteria (2× 10   8 /ml) were harvested and washed (as described in Skouloubris et al. (30)) resuspended in 10 ml of phosphate saline buffer (PBS) at pH 7.5 or 2.2 in the presence of 10 mM urea. After 0, 3, 5 and 30 minutes, 0.5 ml were withdrawn and centrifuged to eliminate bacteria. The supernatant was kept on ice until ammonium concentration was measured using the assay commercialized by Sigma (kit reference #171). 
     
    
       [0017]    Table 2 shows the results obtained with the in vitro viability tests and the pH measurements.  
         [0018]    Table 3 gives the values of ammonium production by strain N6 and N6-834 presented on the graphs of FIG. 4.  
       DETAILED DESCRIPTION  
       [0019]    The urease cluster of Helicobacter species is unique among the many urease operons of Gram-negative bacteria that have been sequenced (20) in that it has an extra gene, ureI. The function of UreI has therefore been the subject of much speculation. It has mostly been attributed the function of an accessory protein required for nickel incorporation at the urease active site or a nickel transporter. A  H. pylori  strain carrying a deletion of ureI replaced by a non-polar cassette (Kanamycin resistance cassette) has been constructed and was named.N6-834 (30). The strain has been deposited at C.N.C.M. (Collection Nationale de Culture de Microorganismes, 25 rue du Docteur Roux, 75724 Paris Cédex 15, France) on Jun. 28, 1999. This is the first time that a non-polar cassette (19) has been shown to be functional in  H. pylori . These results provide a valuable tool for genetic analysis of complex  H. pylori  operons, such as Cag, a multigenic pathogenicity island.  
         [0020]    Studies with this strain demonstrated that UreI is not required for full activity of  H. pylori  urease as measured after in vitro growth at neutral pH. This result argues against UreI being involved in nickel transport since such a protein, NixA (3) already identified in  H. pylori , is necessary for full urease activity. Comparing ureases expressed from a UreI-deficient strain and the corresponding parental strain show that (i) they present the same activity optimum pH (pH 8); (ii) the urease structural sub units, UreA-B, are produced in equal amounts; and (iii) the urease cellular location is identical.  
         [0021]    It is demonstrated here that (i) UreI is essential for colonization of mice by  H. pylori ; (ii) UreI is important for survival of  H. pylori  at acidic pH; and (iii) UreI is necessary for urease “activation” at low pH.  
         [0022]    [0022] H. pylori  during the colonization process of the stomach has to deal with important pH variations and especially has to adapt rapidly to extremely acidic pH (as acidic as pH 1.4). We have shown that UreI is required for  H. pylori  adaptation to acidity, consistently with the absence of colonization of the mouse stomach. As an essential protein for the  H. pylori  resistance to acidity, UreI certainly plays a key role in the infection, establishment, and persistence of  H. pylori . UreI has a sequence similar to those of the AmiS proteins, proposed to be involved in the transport of short-chain amides (27), molecules structurally similar to urea. The UreI/AmiS proteins have the characteristics of integral membrane proteins, probably of the cytoplasmic membrane.  
         [0023]    Different roles for UreI can be proposed. For instance, UreI might be involved in transport (import or export) of urea or short chain amides specifically active at low pH. However, an essential role for UreI as an aside transporter is less likely because a SS1 mutant, deficient in aliphatic amidase, colonizes as efficiently as the parental strain in mouse colonization experiment. In addition, amidase activity is not significantly modified by the deletion of ureI in the N6-834 mutant strain (C.N.C.M. filed on Jun. 28,1999). Import or export of urea could be consistent with the existence of a urea cycle, which is one of the characteristics of  H. pylori  (28).  
         [0024]    Alternatively. UreI might be involved in an active ammonium export system. Finally, UreI might be involved in a mechanism of coupling urease activity to the periplasmic pH, allowing urease to become more active when extracellular pH is acidic.  
         [0025]    Our results are compatible with the first hypothesis of UreI being an urea transporter active at acidic pH values and the third hypothesis of UreI being a kind of sensor protein between the periplasmic pH and urease activity. We think that these two hypothesis are not exclusive. Whatever the role of UreI, as a membrane protein essential for the survival of  H. pylori  in vivo, it now provides a powerful target for a new eradication therapy and for vaccines against  H. pylori.    
         [0026]    Molecules capable of inhibiting the growth and/or survival of Helicobacter in vivo may be identified by contacting a parental Helicobacter strain with said molecule in a biological sample; testing and comparing, in the presence or absence of urea, the sensitivity to the extracellular pH of the parental strain to a strain deficient in UreI and to a UreI deficient strain complemented with ureI; selecting said molecules displaying a differential effect on the parental or complemented strain as compared to the UreI deficient strain; and collecting said active molecule.  
         [0027]    A molecule active specifically on UreI will be the one rendering  H. pylori  sensitive to acidic pH (pH 2.2) in the presence of urea without affecting the strain behavior at neutral pH. Sensitivity to acidity in the presence of urea can be tested on whole  H. pylori  cells following a protocol described in the examples and adapted from Clyne et al. (8). We are now trying to transpose this test in  E. coli  whole cells carrying the complete urease gene cluster on a plasmid (ureAB-ureIEFGH). Screening for a molecule rendering this recombinant  E. coli  more sensitive to acidity in the presence of urea will be performed as described for  H. pylori  in the examples. To distinguish between inhibitory molecules acting on UreI and those acting on urease, the medium pH after whole cell incubation at pH 7 in the presence of urea will be measured. Interesting molecules are those affecting response to acidity without inhibiting the alkalization of the medium observed after incubation at neutral pH.  
         [0028]    These methods may be used to identify molecules that inhibit any Helicobacter species carrying a UreI-homolog. This includes the gastric Helicobacter species:  Helicobacter pylori. Helicobacter felis, Helicobacter mustelae, Helicobacter muridaruni , and also  Helicobacter heiimannii, Helicobacter canis, Helicobacter bilis, Helicobacter heparicus , and  Helicobacter troguntum.    
         [0029]    The molecules identified by the methods of this invention will be capable of inhibiting UreI activity by (i) inhibiting transport of urea or short chain amides, (ii) inhibiting ammonium export, or (iii) inhibiting urease “activation” at low pH. The molecules according to point (i) and (ii) should be able to diffuse throughout the outer membrane and should be active even at low concentration. Suitable candidate molecules are structural analogs of urea or short chain amides, ammonium derivatives or urease inhibitors. For example, molecules derived from AHA (acetohydroxamic acid), hydroxyurea, hippuric acid, flurofamide, hydroxylamine, methylurea, thiourea (29), or methylammonium. The molecules according to point (iii) should inhibit the contact between UreI (probably inserted in the cytoplasmic membrane) and periplasmic, membrane, or cytoplasmic  H. pylori  proteins, which are necessary for urease “activation” at low pH. These proteins could be the structural sub units of urease itself, the accessory proteins, or other proteins. Molecules obtained according to this invention should not be urease competitive inhibitors, should not be toxic or mutagenic in vivo and could potentalize the action of antibiotics or bactericidal molecules. Validation of the action of such molecules could be performed in vivo in the mouse animal model with the pair of isolenic strains SS1 and SS1-834. as described in the examples.  
         [0030]    One example of a molecule according to this invention is a monoclonal or polyclonal antibody specific for UreI. Preferably, the antibody is capable of specifically inhibiting UreI activity.  
         [0031]    The molecules of this invention may be administered in combination with a pharmaceutically acceptable carrier to a patient suffering from a Helicobacter infection. Alternatively, immunogenic compositions comprising one or more molecules according to this invention may be administered in a vaccine composition to prevent infection by Helicobacter species.  
         [0032]    Immunogenic compositions according to this invention may also comprise all or part of the UreI protein. Preferably, the UreI fragments comprise at least 10 consecutive amino acids of the native UreI sequence and more preferably, the fragments comprise at least 18, 20, or 25 consecutive amino acids of the native UreI sequence. Other suitable UreI fragments may contain at least 40 or at least 100 consecutive amino acids of the native UreI sequence. Suitable fragments of  Helicobacter pylori  include, for example, fragments selected from the group consisting of amino acid residues 22 to 31, 49 to 74, 94 to 104, and 123 to 142 of  H. pylori  (GenBank accession No. M84338)  
         [0033]    Reference will now be made to the following Examples. The Examples are purely exemplary, of the invention and are not to be construed as limiting of the invention.  
       EXAMPLES  
       [0034]    Construction of Defined Mutations of the  H. Pylori  UreI Gene  
         [0035]    [0035] H. pylori  strains with defined mutations in ureI were generated by allelic exchange to determine whether the UreI protein was necessary for production of active urease. For this purpose, two plasmids (pILL823 and pILL834) with cassettes carrying antibiotic resistance genes inserted in ureI were constructed in  E. coli.    
         [0036]    In one plasmid. pILL823 (FIG. 2A), the ureI gene was inactivated by insertion of a promoterless cat gene. conferring resistance to chloramphenicol (Cm). A 780 bp blunt-ended BamHI restriction fragment containing the “cat cartridge” from pCM4 (Pharmacia, Sweden) was introduced into a unique HpaI site, between codons 21 and 22 of ureI, in pILL753 (9). In the resulting plasmid, pILL823 (FIG. 2A), cat is in the same orientation as ureI and is expressed under the control of the ureI promoter.  
         [0037]    The second plasmid, pILL834, carried a ureI gene in which all but the first 21 codons were deleted and replaced with a non-polar cassette composed of the aphA-3 kanamycin (Km) resistance gene (25), which has been deleted from its own promoter and terminator regions (19). In  Shigella flexneri  (19) and other organisms (such as  Yersinia enterocolitica , 2) this cassette has been shown not to affect the transcription of the genes downstream within an operon as long as these distal genes have intact translation signals. There is only one base pair separating ureI from ureE (FIG. 1) and ureE does not have an RBS (ribosome binding site) of its own, so the expression of ureI and ureE is transcriptionally and translationally coupled. Therefore, a ureI deletion was accompanied by the addition of an RBS immediately upstream from ureE. Three intermediates, pILL824, pILL825 and pILL833 (FIG. 2A), were constructed in order to produce the final plasmid, pILL834 (FIG. 2A). A 1.8 Kb HpaI-HindIII restriction fragment from pILL753 (9) was inserted between the EcoRV and HindIII sites of pBR322, to give pILL824. Insertion of the H19 adaptor (carrying an RBS and ATG in frame with ureE, Table 1) into a BclI site overlapping the two first codons of ureE in pILL824 produced pILL825 (FIG. 2A). The BamHI fragment of pILL825 was then replaced by a 1.3 Kb blunt-ended PvuIl-BamHI fragment from pILL753. This resulted in the reconstitution of a complete ureI gene, and this plasmid was called pILL833. Finally, pILL834 was obtained by replacement of the HpaI-BglII fragment of pILL833 (thereby deleting all but the first 21 codons of ureI) with an 850 bp blunt-ended EcoRI-BamHI fragment of pUC18K2 containing the non-polar Km cassette (19).  
                                 TABLE 1                           Name and nucleotide sequence of oligonucleotides            Primer       Oligodeoxynucleozide sequence                   (5′ to 3′)               H17   TTTGACTTACTGGGGATCAAGCCTG   (SEQ ID NO:1)           H19*   GATCATTTATTCCTCCAGATCTGGAGGAATAAAT   (SEQ ID NO:2)       H28   GAAGATCTCTAGGACTTGTATTGTTATAT   (SEQ ID NO:3)       H34   TATCAACGGTGGTATATCCAGTG   (SEQ ID NO:4)       H35   GCAGTTATTGGTGCCCTTAAACG   (SEQ ID NO:5)       H50   CCGGTGATATTCTCATTTTAGCC   (SEQ ID NO:6)       8A   GCGAGTATGTAGGTTCAGTA   (SEQ ID NO:7)       9B   GTGATACTTGAGCAATATCTTCAGC   (SEQ ID NO:8)       12B   CAAATCCACATAATCCACGCTGAAATC   (SEQ ID NO:9)                          
 
         [0038]    Introduction of UreI Mutations into  H. pylori    
         [0039]    [0039] H. pylori  ureI mutants were produced by allelic exchange following electroporation with a concentrated preparation of pILL823 and pILL834 as previously described by Skouloubris et al. (23) from  H. pylori  strain N6 (12) and from the mouse-adapted  H. pylori  strain, SS1 (Sydney Strain, 17). Bacteria with chromosomal allelic exchange with pILL823 were selected on Cm (4 μg/ml) and those with chromosomal allelic exchange with pILLS34 on Km (20 μg/ml). it was determined that the desired allelic exchange had taken place in strains N6-823, N6-834, and SS1-834 (FIG. 1) by performing PCR with the appropriate oligonucleotides (Table 1). The PCR products obtained with genomic DNA of these strains were as expected (i) for strain N6-823: 140 bp with primers H28-H34, 220 bp with H35-9B, and 1.2 Kb with H28-9B, and (ii) for strains N6-834 and SS1-834, 150 bp with primers H28-H50, 180 bp with H17-12B, and 1 Kb with H28-12B.  
         [0040]    The growth rate of strain N6-834 carrying a non-polar deletion of ureI was compared to that of the parental strain N6. No difference in the colony size was observed on blood agar medium plates. Identical doubling times and stationary phase OD were measured for both strains grown in BHI (Oxoid) liquid medium containing 0.2% ∃-cyclodextrin (Sigma). Thus, UreI is not essential for  H. pylori  growth in vitro.  
         [0041]    Urease Activity of  H. pylori  UreI Mutants  
         [0042]    The urease activity of strains N6-823, N6-834, and SS1-834 was measured in vitro as described previously by Cussac et al. (9) and compared to the activity of the parental strains, N6 and SS1 (FIG. 1). Urease activity was almost completely abolished in strain N6-823 (0.3±0.1 units). Strains N6-834 and SS1-834, with non-polar ureI mutations had wild-type levels of activity (N6-834 and SS1-834: 12±2 units; parental strains, N6: 10±1 and SS1: 12±0.4 units).  
         [0043]    The pH optimum of urease produced either from the N6 parental strain or from the UreI deficient strain N6-834 was measured and compared. For both strains, urease has a pH optimum of 8 which is consistent with the published data.  
         [0044]    These results strongly suggest that the urease-negative phenotype of the N6-ureI::TnKm-8 (13) and the very weak urease activity of N6-823 strains were due to a polar effect of the inserted cassettes on the expression of the downstream genes ureE and ureF (FIG. 1 ). This hypothesis was tested by measuring urease activity of strain N6-823 complemented in trans with an  E. coli/H. pylori  shuttle plasmid expressing the ureE-F genes. This plasmid, pILL845 (FIG. 2B), was obtained by insertion of a 2.8 Kb ClaI-BamHI fragment of pILLS834 (comprising the 3′-end of ureB, the non-polar deletion of ureI and intact ureE and ureF genes) into the corresponding sites of the shuttle vector pHe12 constructed by Heuermann and Haas (15). Strain N6-823 was electroporated with a DNA preparation of pILL845 as described by Skouloubris et al. (23), and transformants were selected on kanamycin (20 μg/ml) and chloramphenicol (4 μg/ml). In strain N6-823 harboring pILL845, wild type urease activity was recovered confirming that the very low urease activity of strain N6-823 was due to a polar effect on the expression of the accessory genes ureE-F. In  Klebsiella aerogenes , the absence of UreE has little effect on urease activity (4). In contrast. UreF, as part of the accessory protein complex (UreDFG), is absolutely required for the production of active urease (21). Thus, by analogy, it is likely that the phenotype of the  H. pylori  polar ureI mutants was due to the absence of ureF expression.  
         [0045]    The urease structural sub units, UreA and UreB, produced by strain N6 or strain N6-834 were compared with the Western blot technique using a mixture of antisera directed against each urease subunit. It was observed that the amount of each subunit produced by the two strains is identical. The possibility that urease cellular localization could be affected in the absence of UreI was examined after cellular fractionation (separating the soluble from the membrane associated proteins and from the supernatant) of strains N6 and N6-834. These experiments revealed no difference between the urease cellular localization in the wild type strain or in the UreI-deficient mutant. These results demonstrate that, at neutral pH, UreI is neither implicated in the stabilization of the urease structural sub units nor in a targeting process of urease to a specific cellular compartment.  
         [0046]    Colonization Test for the  H. pylori  SS1-834 Mutant in the Mouse Animal Model  
         [0047]    The mouse model for infection by the  H. pylori  SS1 strain (Sydney Strain, 17), validated by Chevalier et al. (7) and Ferrero et al. (14), was used to test the function of UreI in vivo. Mice were infected with the non-polar ureI mutant, SS1-834, and with the parental strain, SS1, (which had gone through an equivalent number of in vitro subcultures) as a positive control. This experiment was repeated three times and produced identical results (30). Two independently constructed SS1-834 mutants were used. The first mutant strain had gone through 30 in vitro subcultures, the second only 20. Under the same experimental conditions, strain SS1 can undergo more than 80 in vitro subcultures without losing its colonization capacity.  
         [0048]    In each experiment, aliquots (100 μl) containing 10 6    H. pylori  strain SS1 or SS1-834 bacteria prepared in peptone broth were administered orogastrically to 10 mice each (six to eight-weeks old Swiss specific-pathogen-free mice) as described by Ferrero et al. (14). Mice were killed four weeks after inoculation. The presence of  H. pylori  was tested with a direct urease test on biopsies performed on half the stomach (14). The remaining gastric tissues were used for quantitative culture of  H. pylori  as described by Ferrero et al. (14). In each experiment, the stomachs of the ten SS1-infected mice all tested positive for urease. The bacterial load was between 5×10 4  and 5×10 5  colony forming units (CFU) per g of stomach. None of the stomachs of the mice infected with strain SS1-834 tested positive for urease and no  H. pylori  cells were cultured from them. Thus, the UreI protein is essential for the  H. pylori  in vivo survival and/or colonization of the mouse stomach.  
         [0049]    UreI is Essential for  H. pylori  Resistance to Acidity  
         [0050]    Survival to acidic conditions in the presence or absence of 10 mM urea was tested with strains N6 and N6-834. The experimental procedures detailed in Skouloubris et al. (30) were based on those described in Clyne et al. (8). Exponentially grown bacteria were harvested, washed in PBS (phosphate buffer saline), and approximately 2×10 8  CFU/ml were resuspended in PBS of pH 2.2 or pH 7 in the presence or the absence of 10 mM urea and incubated at 37EC. After one hour incubation (i) quantitative cultures of the  H. pylori  strains were performed to evaluate bacterial survival, and (ii) the bacteria were centrifuged and the pH of the medium was measured. The results obtained are presented in Table 2. In the absence of urea, both strains N6 and N6-834 presented identical phenotype, i.e., they were killed at pH 2.2, and survived at pH 7 without modifying the final pH of the medium (Table 2). After incubation at pH 7 in the presence of urea, both strains were killed because the final pH rose to pH 9. At pH 2.2 in the presence of urea, the parental strain survived well since it was able to raise the pH to neutrality. Incontrast, a completely different phenotype was obtained with the UreI-deficient strain N6-834 which was unable to raise the pH and whose viability was seriously affected (Table 2).  
         [0051]    Complementation of the UreI-Deficient Strain N6-834 with Plasmid pILL850  
         [0052]    Direct implication of the UreI protein in the  H. pylori  capacity to resist to acidity has been confirmed by trans-complementation with plasmid pILL850 (FIG. 2B restriction map and details of construction). This plasmid [CNCM I-2245 filed on Jun. 28, 1999] is derived from the  H. pylori/E. coli  shuttle vector pHe12 (15). Plasmid pILL850 carries the ureI gene under the control of its own promoter and was constructed as follows: a 1.2 kb BclI restriction fragment of plasmid pILL753 (9) was introduced between the BamHI and BclI restriction sites of pHe12 (FIG. 2B). Strains N6 and N6-834 were transformed by this plasmid and the phenotype of the complemented strains in the acidity sensitivity test experiments described above was examined. As shown in Table 2, the phenotype of strain N6-834 complemented by pILL850 is identical to that of the parental strain N6. Interestingly, the urease activity of the complemented strains (measured on sonicated extracts as described in Skouloubris et al. (30)) has been found to be significantly higher as compared to that of the corresponding strains without pILL850. For the purpose of the deposit at the CNCM pILL850 is placed into an  E. coli  strain, MCl 061 (Wertman KF. et al, 1986, Gene 49: 253-262).  
         [0053]    Measurements of Ammonium Production  
         [0054]    The amount of ammonium produced in the extracellular medium of  H. pylori  whole cells was measured by an enzymatic assay commercialized by Sigma following the supplier&#39;s instructions. These experiments were performed after incubation of the cells in PBS at different pH values and after different incubation times. Such experiments gave an accurate, evaluation of ammonium production and excretion in different strains as well as a measure of the kinetics of this reaction. A control experiment showed that ammonium production was very low (10-20 μM) in the absence of urea.  
         [0055]    [0055]FIG. 4 depicts the kinetics (0, 3, 5, and 30 min. incubation time) of extracellular ammonium released by the N6 parental strain (panel A) and the UreI-deficient strain N6-834 (panel B) incubated in PBS at pH 2.2, pH 5, or pH 7 in the presence of 10 mM urea. The results obtained indicate that (i) ammonium is largely produced and rapidly released in the extracellular medium; and (ii) in the N6 wild type strain (FIG. 4, panel A and Table 3) ammonium production is significantly enhanced when the extracellular pH is acidic. This effect is already visible at pH 5 and is even stronger at pH 2.2. This last observation is consistent with the resuits of Scott et al. (31) who suggested urease activation at low pH. In our experiments, the rapidity of the response to acidity argues against urease activation depending on transcriptional regulation or on de novo protein synthesis.  
         [0056]    Ammonium production was then measured in the UreI-deficient strain N6-834 (FIG. 4, panel B and Table 3). At neutral pH, kinetics of ammonium production were similar to those of the wild type strain. In contrast, at pH 5 ammonium production was reduced and delayed as compared to the wild type strain. A dramatic effect of the absence of UreI was observed at pH 2.2, where the amount of ammonium was very low, which is consistent with our results showing that UreI is necessary for adaptation to acidity.  
         [0057]    Our results demonstrate that UreI is essential for the resistance of  H. pylori  to acidity. In the absence of UreI. urease, although present in huge amounts, is not able to protect the bacteria against the aggression of acidity. This is consistent with the essential role of UreI in vivo. During its passage in the acidic stomach lumen, the viability of the UreI-deficient strain is affected. As a consequence, the bacterial load becomes too low to permit colonization. The different roles proposed for UreI are presented in the “detailed description” section.  
         [0058]    Alignment of-the UreI and AmiS Protein Sequences and Two Dimensional Structure Prediction  
         [0059]    A systematic search for UreI homologs in the protein data banks was carried out. It was determined that  H. pylori  is not the only ureolytic bacterium with a ureI gene. Two phylogenetically related Gram-positive organisms,  Streptococcus salivarius , a dental plaque bacterium (6), and  Lactobacillus fermentum , a lactic acid bacterium (16), carry genes for UreI-homologs (FIG. 3) located immediately upstream from the urease structural genes. The ureI gene has been detected in various Helicobacter species; the  H. felis  ureI gene has been entirely sequenced (FIG. 3 and allowed U.S. patent application Ser. No. 08/467,822, the entire contents of which are incorporated herein by reference). PCR experiments have suggested that there is a ureI gene in  H. heilmannii  (24) and in  H. mustelae.    
         [0060]    Sequence similarities between the UreI protein of  H. pylori  and the AmiS proteins expressed by the aliphatic amidase operons from  P. aeruginosa  (27) and Rhodococcus sp. R312 (5) have been reported. In  Mycobacterium smegmatis , there is an additional AmiS-homolog encoded by a gene, ORF P3, located immediately upstream from an amidase gene (18).  
         [0061]    Alignment of these UreI/AmiS proteins [using the Clustal W(1. 60) program] defined strongly conserved stretches of amino acids (FIG. 3). All but one of these conserved blocks are in highly hydrophobic segments. These regions, each 17 to 22 residues long, are probably folded into transmembrane ∀-helices (FIG. 3). Six transmembrane regions were predicted for the protein; from  H. pylori, H. felis , and  P. aeruginosa  and seven for those from  Rhodococcus  sp. R312 and  M. smegmatis  (highly reliable predictions, performed with pHD, a profile fed neural network system as described by Rost et al. (22)). The orientation of the UreI/AmiS proteins in the membrane was deduced from the charges of the intercalated hydrophilic regions. which are short in these proteins (FIG. 3). The first five such regions are poorly conserved and of various length. The last interhelical segment common to these proteins is significantly more conserved than the others. This region predicted to be intracellular maybe be the active site of UreI or a site of multimerization or interaction with an intracellular partner. These results strongly suggest that the members of the UreI/AmiS family, found in both Gram-positive and -negative bacteria, are integral membrane proteins. These proteins have no signal sequence and should therefore be inserted into the cytoplasmic membrane in Gram-negative bacteria.  
         [0062]    Two peptides. selected from the UreI sequence, were synthesized and injected into two rabbits to obtain serum containing polyclonal antibodies directed against UreI. One peptide corresponds to the first predicted intracellular loop of UreI (from residue nB 15 to 31, see FIG. 3) and the second one to the second predicted extracellular loop of UreI (from residue nB 118 to 134 see FIG. 3. These sera are presently being tested and if proven to recognize the UreI protein will allow us to precisely define the localization of this protein and to verify the predicted UreI two-dimensional structure presented in FIG. 3.  
         [0063]    The references cited herein are specifically incorporated by reference in their entirety.  
                                                           TABLE 2                           Effect of the presence of urea at pH 7, 5 or 2.2 on       (i) the viability of different  H. pylori  strains and (ii) the       extracellular pH (indicated as final pH). The experimental       procedures are described in reference 30 and in the examples.       Strain N6 is the parental strain and strain N6-834 the       Urel-deficient mutant. Plasmid plLL850 is derived from a         E. coli / H. pylori  shuttle vector, it carries the       urel gene and complements the urel mutation of strain N6-834.                    final               strains   initial pH   pH   urea 10 mM     H. pylori  CFU/ml                    N6   2.2   2.26   −   0       N6   2.2   6.6   +     8 × 10 7         N6   7   6.98   −     2 × 10 8         N6   7   8.88   +   0       N6-834   2.2   2.2   −   0       N6-834   2.2   2.37   +     7 × 10 5         N6-834   7   7.1   −   3.5 × 10 7         N6-834   7   9.05   +   0       N6-834 + plLL850   2.2   2.3   −   0       N6-834 + plLL850   2.2   6.9   +   1.3 × 10 8         N6-834 + plLL850   7   7.1   −   1.7 × 10 8         N6-834 + plLL850   7   9   +   0                  
 
         [0064]    [0064]                                                         TABLE 3                                       medium       [NH4]           Strain   pH   minutes   mM                                        N6   7.0   0   3.5           N6   7.0   3   4.4           N6   7.0   5   3.1           N6   7.0   30   5.6           N6   5.0   0   12.8           N6   5.0   3   9.3           N6   5.0   5   11.8           N6   5.0   30   16.0           N6   2.2   0   6.7           N6   2.2   3   9.0           N6   2.2   5   11.0           N6   2.2   30   20.0           N6-834   7.0   0   2.7           N6-834   7.0   3   2.8           N6-834   7.0   5   3.8           N6-834   7.0   30   5.8           N6-834   5.0   0   1.4           N6-834   5.0   3   1.7           N6-834   5.0   5   2.9           N6-834   5.0   30   4.6           N6-834   2.2   0   0.9           N6-834   2.2   3   0.6           N6-834   2.2   5   0.7           N6-834   2.2   30   1.3                        
       REFERENCES  
       [0065]    1. Akada, J. K.. M. Shirai, H. Takeuchi, M. Tsuda, and T. Nakazawa. 1997. Transcriptional analvsis of urease structural gene and the ureI gene in  Helicobacter pylori . Gut. 41: A9.  
         [0066]    2. Allaoui, A., Schulte. R. and G. R. Cornelis. 1995. Mutational analysis of the  Yersinia euzerocolitica  virC operon: characterization of yscE, F, G, H, I, J, K required for Yop secretion and ysch encoding YopR. Mol. Microbiol. 18:343-355.  
         [0067]    3. Bauerfeind, P.. R. M. Garner, and H. L. T. Mobley. 1996. Allelic exchange mutagenesis of nixA in  Helicobacter pylori  results in reduced nickel transport and urease activity. Infect. Immun. 64:2877-2880.  
         [0068]    4. Brayman, T. G.. and R. T. Hausinger. 1996. Purification, characterization, and functional analysis of a truncated  Klebsiella aerogenes  UreE urease accessory protein lacking the Histidine-Rich carboxyl terminus. J. Bacteriol. 178:5410-5416.  
         [0069]    5. Chebrou, H.. F. Bigey, A. Arnaud, and P. Gaizy. 1996. Amide metabolism: a putative ABC transporter in Rhodococcus sp. R312. Gene. 182:215-218.  
         [0070]    6. Chen, Y.-Y. M.. K. A. Clancy, and R. A. Burne. 1996 . Streptococcus salivarius  urease: genetic and biochemical characterization and expression in a dental plaque Streptococcus. Infect. Immun. 64:585-592.  
         [0071]    7. Chevalier, C.. J.-M. Thiberge, R. L. Ferrero, and A. Labigne. 1999. Essential role of  Helicobacter pylori  g-Glutamyltranspeptidase (GGT) for the colonization of the gastric mucosa in mice. Mol. Microbiol. 31:1359-1372.  
         [0072]    8. Clyne, M., A. Labigne, and B. Drumm. 1995 . Helicobacter pylori  requires an acidic environment to survive in the presence of urea. Infect. Immun. 63:1669-1673.  
         [0073]    9. Cussac, V., R. L. Ferrero, and A. Labigne. 1992. Expression of  Helicobacter pylori  urease genes in  Escherichia coli  grown under nitrogen-limiting conditions. J. Bacteriol. 174:2466-2473.  
         [0074]    10. Dunn, B. E.. H. Cohen, and M. Blaser. 1997 . Helicobacter pylori . Clin. Microbiol. Rev. 10:720-741.  
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         [0076]    12. Ferrero, R. L.. V. Cussac, P. Courcoux, and A. Labigne. 1992. Construction of isogenic urease-negative mutants of  Helicobacter pylori  by allelic exchange. J. Bacteriol. 174:4212-4217.  
         [0077]    13. Ferrero, R. L.. V. Cussac, P. Courcoux, and A. Labigne. 1994. Construction of isogenic mutants of  Helicobacter pylori  deficient in urease activity. pp179-182. In Basic and Clinical Aspects of  H. pylori  infection. Springer-Verlag Berlin Heidelberg.  
         [0078]    14. Ferrero, R. L.. J.-M. Thiberge, M. Huerre, and A. Labigne. 1998. Immune responses of specific-pathogen-free mice to chronic  Helicobacter pylori  (strain SS 1) infection. Infect. Immun. 66:1349-1355.  
         [0079]    15. Heuermann, D.. and R. Haas. 1998. A stable shuttle vector system for efficient genetic complementation of  Helicobacter pylori  strains by complementation and conjugation. Mol. Gen. Genet. 257:519-528.  
         [0080]    16. Kakimoto, S.. Y. Sumino, K. Kawahara, E. Yamazaki, and I. Nakatsui. 1990. Purification and characterization of acid urease from  Lactobacillus fermentum . Appl. Microbiol. &amp; Biotechnol. 32:538-543.  
         [0081]    17. Lee, A., J. O&#39;Rourke, M. Corazon De Ungria, B. Robertson, G. Daskalopoulos, and M. F. Dixon. 1997. A standardized mouse model of  Helicobacter pylori  infection: introducing the Sydney Strain. Gastroenterology. 112:1386-1397.  
         [0082]    18. Mahenthiralingam, E., P. Draper, E. O. Davis, and M. J. Colston. 1993. Cloning and sequencing of the gene which encodes the highly inducible acetamidase of  Mycobacterium smegmatis . J. Gen. Microbiol. 139:575-583.  
         [0083]    19. Menard, R., P. J. Sansonetti, and C. Parsot. 1993. Nonpolar mutagenesis of the ipa genes defines IpaB, IpaC, and IpaD as effectors of  Shigella flexneri  entry into epithelial cells. J. Bacteriol. 175:5899-5906.  
         [0084]    20. Mobley, H. L. T.. M. D. Island, and R. P. Hausinger. 1995. Molecular biology of ureases. Microbiol. Rev. 59:451-480.  
         [0085]    21. Moncrief, M. B. C., and R. P. Hausinger. 1997. Characterization of UreG, identification of a UreD-UreF-UreG complex, and evidence suggesting that a nucleotide-binding site in UreG is required for in vivo metallocenter assembly of  Klebsiella aerogenes  urease. J. Bacteriol. 179:4081-4086.  
         [0086]    22. Rost. B., R. Casadio, P. Fariselli, and C. Sander. 1995. Prediction of helical transmembrane segments at 95% accuracy. Prot. Science. 4:521-533.  
         [0087]    23. Skouloubris, S.. A. Labigne, and H. De Reuse. 1997. Identification and characterization of an aliphatic amidase in  Helicobacter pylori . Mol. Microbiol. 25:989-998.  
         [0088]    24. Solnick, J. V.. J. O&#39;Rourke, A. Lee, and L. S. Tompkins. 1994. Molecular analysis of urease genes from a newly identified uncultured species of Helicobacter. Infect. Immun. 62:1631-1638.  
         [0089]    25. Trieu-Cuot, P.. G. Gerbaud, T. Lambert, and P. Counralin. 1985. In vivo transfer of genetic information between Gram-positive and Gram-negative bacteria. EMBO J. 4:3583-3587.  
         [0090]    26. Williams, C. L.. T. Preston, M. Hossack, C. Slater, and K. E. L. McColl. 1996 . Helicobacter pylori  utilizes urea for amino acid synthesis. FEMS Immunol. Med. Microbiol. 13:87-94.  
         [0091]    27. Wilson, S. A.. R. J. Williams, L. H. Pearl, and R. E. Drew. 1995. Identification of two new genes in the  Pseudomonas aeruginosa  amidase operon, encoding an ATPase (AmiB) and a putative integral membrane protein (AmiS). J. Biol. Chem. 270:18818-18824.  
         [0092]    28. Mendz, G. L. and S. L. Mazell. 1996. The Urea Cycle of  Helicobacter pylori  Microbiology 142:2959-2967.  
         [0093]    29. Nicholson, E. B.. E. A. Concaugh and H. L. T. Mobley. 1991 . Proteus mirabilis  urease: use of ureA-lacZ fusion demonstrates that induction is highly specific for urea. Infection and Immunity. 59(10):3360-3365.  
         [0094]    30. Skouloubris, S.. J.-M. Thiberge, A. Labigne and H. De Reuse (1998) The  Helicobacter pylori  UreI protein is not involved in urease activity but is essential for bacterial survival in vivo. Infect. Immun. 66: 451-74521.  
         [0095]    31. Scott, D. R., D. Weeks, C. Hong, S. Postius, K Melchers and G. Sachs (1998) The role of internal urease in acid resistance of  Helicobacter pylori . Gastroenterology. 114: 58-70.  
     
       
       
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             DNA  
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               Description of Artificial Sequence Primer  
             
           
            1 

tttgacttac tggggatcaa gcctg                                           25 

 
           
             2  
             34  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Adaptor 
      sequence  
             
           
            2 

gatcatttat tcctccagat ctggaggaat aaat                                 34 

 
           
             3  
             29  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Primer  
             
           
            3 

gaagatctct aggacttgta ttgttatat                                       29 

 
           
             4  
             23  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Primer  
             
           
            4 

tatcaacggt ggtatatcca gtg                                             23 

 
           
             5  
             23  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Primer  
             
           
            5 

gcagttattg gtgcccttaa acg                                             23 

 
           
             6  
             23  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Primer  
             
           
            6 

ccggtgatat tctcatttta gcc                                             23 

 
           
             7  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Primer  
             
           
            7 

gcgagtatgt aggttcagta                                                 20 

 
           
             8  
             25  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Primer  
             
           
            8 

gtgatacttg agcaatatct tcagc                                           25 

 
           
             9  
             27  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Primer  
             
           
            9 

caaatccaca taatccacgc tgaaatc                                         27 

 
           
             10  
             195  
             PRT  
             Helicobacter pylori  
           
            10 

Met Leu Gly Leu Val Leu Leu Tyr Val Gly Ile Val Leu Ile Ser Asn 
  1               5                  10                  15 

Gly Ile Cys Gly Leu Thr Lys Val Asp Pro Lys Ser Thr Ala Val Met 
             20                  25                  30 

Asn Phe Phe Val Gly Gly Leu Ser Ile Ile Cys Asn Val Val Val Ile 
         35                  40                  45 

Thr Tyr Ser Ala Leu Asn Pro Thr Ala Pro Val Glu Gly Ala Glu Asp 
     50                  55                  60 

Ile Ala Gln Val Ser His His Leu Thr Asn Phe Tyr Gly Pro Ala Thr 
 65                  70                  75                  80 

Gly Leu Leu Phe Gly Phe Thr Tyr Leu Tyr Ala Ala Ile Asn His Thr 
                 85                  90                  95 

Phe Gly Leu Asp Trp Arg Pro Tyr Ser Trp Tyr Ser Leu Phe Val Ala 
            100                 105                 110 

Ile Asn Thr Ile Pro Ala Ala Ile Leu Ser His Tyr Ser Asp Met Leu 
        115                 120                 125 

Asp Asp His Lys Val Leu Gly Ile Thr Glu Gly Asp Trp Trp Ala Ile 
    130                 135                 140 

Ile Trp Leu Ala Trp Gly Val Leu Trp Leu Thr Ala Phe Ile Glu Asn 
145                 150                 155                 160 

Ile Leu Lys Ile Pro Leu Gly Lys Phe Thr Pro Trp Leu Ala Ile Ile 
                165                 170                 175 

Glu Gly Ile Leu Thr Ala Trp Ile Pro Ala Trp Leu Leu Phe Ile Gln 
            180                 185                 190 

His Trp Val 
        195 

 
           
             11  
             196  
             PRT  
             Helicobacter felis  
           
            11 

Met Leu Gly Leu Val Leu Leu Tyr Val Ala Val Val Leu Ile Ser Asn 
  1               5                  10                  15 

Gly Val Ser Gly Leu Ala Asn Val Asp Ala Lys Ser Lys Ala Ile Met 
             20                  25                  30 

Asn Tyr Phe Val Gly Gly Asp Ser Pro Leu Cys Val Met Trp Ser Leu 
         35                  40                  45 

Ser Ser Tyr Ser Thr Phe His Pro Thr Pro Pro Ala Thr Gly Pro Glu 
     50                  55                  60 

Asp Val Ala Gln Val Ser Gln His Leu Ile Asn Phe Tyr Gly Pro Ala 
 65                  70                  75                  80 

Thr Gly Leu Leu Phe Gly Phe Thr Tyr Leu Tyr Ala Ala Ile Asn Asn 
                 85                  90                  95 

Thr Phe Asn Leu Asp Trp Lys Pro Tyr Gly Trp Tyr Cys Leu Phe Val 
            100                 105                 110 

Thr Ile Asn Thr Ile Pro Ala Ala Ile Leu Ser His Tyr Ser Asp Ala 
        115                 120                 125 

Leu Asp Asp His Arg Leu Leu Gly Ile Thr Glu Gly Asp Trp Trp Ala 
    130                 135                 140 

Phe Ile Trp Leu Ala Trp Gly Val Leu Trp Leu Thr Gly Trp Ile Glu 
145                 150                 155                 160 

Cys Ala Leu Gly Lys Ser Leu Gly Lys Phe Val Pro Trp Leu Ala Ile 
                165                 170                 175 

Val Glu Gly Val Ile Thr Ala Trp Ile Pro Ala Trp Leu Leu Phe Ile 
            180                 185                 190 

Gln His Trp Ser 
        195 

 
           
             12  
             46  
             PRT  
             Lactobacillus fermentum  
           
            12 

Ile Leu Trp Leu Thr Gly Phe Leu Thr Asn Asn Leu Lys Met Asn Leu 
  1               5                  10                  15 

Gly Lys Phe Pro Gly Tyr Leu Gly Ile Ile Glu Gly Ile Cys Thr Ala 
             20                  25                  30 

Trp Ile Pro Gly Phe Leu Met Leu Leu Asn Tyr Trp Pro Asn 
         35                  40                  45 

 
           
             13  
             129  
             PRT  
             Streptococcus salivarius  
           
            13 

Ile Leu Asn Ile Ile Val Ile Ala Tyr Gly Ala Cys Thr Gly Gln Gly 
  1               5                  10                  15 

Ala Glu Trp Phe Tyr Gly Ser Ala Thr Gly Leu Leu Phe Ala Phe Thr 
             20                  25                  30 

Tyr Leu Tyr Ser Ala Ile Asn Thr Ile Phe Asp Phe Asp Gln Arg Leu 
         35                  40                  45 

Tyr Gly Trp Phe Ser Leu Phe Val Ala Ile Asn Thr Leu Pro Ala Gly 
     50                  55                  60 

Ile Leu Cys Leu Thr Ser Gly Tyr Gly Gly Asn Ala Trp Tyr Gly Ile 
 65                  70                  75                  80 

Ile Trp Phe Leu Trp Gly Ile Leu Trp Leu Thr Ala Phe Ile Glu Ile 
                 85                  90                  95 

Asn Leu Lys Lys Asn Leu Gly Lys Phe Val Pro Tyr Leu Ala Ile Phe 
            100                 105                 110 

Glu Gly Ile Val Thr Ala Trp Ile Pro Gly Leu Leu Met Leu Trp Gly 
        115                 120                 125 

Lys 

 
           
             14  
             213  
             PRT  
             Mycobacterium smegmatis  
           
            14 

Met Gly Gly Val Gly Leu Phe Tyr Val Gly Ala Val Leu Ile Ile Asp 
  1               5                  10                  15 

Gly Leu Met Leu Leu Gly Arg Ile Ser Pro Arg Gly Ala Thr Pro Leu 
             20                  25                  30 

Asn Phe Phe Val Gly Gly Leu Gln Val Val Thr Pro Thr Val Leu Ile 
         35                  40                  45 

Leu Gln Ser Gly Gly Asp Ala Ala Val Ile Phe Ala Ala Ser Gly Leu 
     50                  55                  60 

Tyr Leu Phe Gly Phe Thr Tyr Leu Trp Val Ala Ile Asn Asn Val Thr 
 65                  70                  75                  80 

Asp Trp Asp Gly Glu Gly Leu Gly Trp Phe Ser Leu Phe Val Ala Ile 
                 85                  90                  95 

Ala Ala Leu Gly Tyr Ser Trp His Ala Phe Thr Ala Glu Ala Asp Pro 
            100                 105                 110 

Ala Phe Gly Val Ile Trp Leu Leu Trp Ala Val Leu Trp Phe Met Leu 
        115                 120                 125 

Phe Leu Leu Leu Gly Leu Gly His Asp Ala Leu Gly Pro Ala Val Gly 
    130                 135                 140 

Phe Val Ala Val Ala Glu Gly Val Ile Thr Ala Ala Val Pro Ala Phe 
145                 150                 155                 160 

Leu Ile Val Ser Gly Asn Trp Glu Thr Gly Pro Leu Pro Ala Ala Val 
                165                 170                 175 

Ile Ala Val Ile Gly Phe Ala Ala Val Val Leu Ala Tyr Pro Ile Gly 
            180                 185                 190 

Arg Arg Leu Ala Ala Pro Ser Val Thr Asn Pro Pro Pro Ala Ala Leu 
        195                 200                 205 

Ala Ala Thr Thr Arg 
    210 

 
           
             15  
             206  
             PRT  
             Rhodococcus sp.  
           
            15 

Met Gly Ser Val Gly Leu Leu Tyr Val Gly Ala Val Leu Phe Val Asn 
  1               5                  10                  15 

Gly Leu Met Leu Leu Gly Thr Val Pro Val Arg Ser Ala Ser Val Leu 
             20                  25                  30 

Asn Leu Phe Val Gly Ala Leu Gln Cys Val Val Pro Thr Val Met Leu 
         35                  40                  45 

Ile Gln Ala Gln Gly Asp Ser Ser Ala Val Leu Ala Ala Ser Gly Leu 
     50                  55                  60 

Tyr Leu Phe Gly Phe Thr Tyr Leu Tyr Val Gly Ile Ser Asn Leu Ala 
 65                  70                  75                  80 

Gly Phe Glu Pro Glu Gly Ile Gly Trp Phe Ser Leu Phe Val Ala Cys 
                 85                  90                  95 

Ala Ala Leu Val Tyr Ser Phe Leu Ser Phe Thr Val Ser Asn Asp Pro 
            100                 105                 110 

Val Phe Gly Val Ile Trp Leu Ala Trp Ala Ala Leu Trp Thr Leu Phe 
        115                 120                 125 

Phe Leu Val Leu Gly Leu Gly Arg Glu Asn Leu Ser Arg Phe Thr Gly 
    130                 135                 140 

Trp Ala Ala Ile Leu Leu Ser Gln Pro Thr Cys Thr Val Pro Ala Phe 
145                 150                 155                 160 

Leu Ile Leu Thr Gly Asn Phe His Thr Thr Pro Ala Val Ala Ala Gly 
                165                 170                 175 

Trp Ala Gly Ala Leu Leu Val Leu Leu Gly Leu Ala Lys Ile Leu Ala 
            180                 185                 190 

Ala Pro Lys Ala Ala Val Pro Gln Pro Arg Pro Val Phe Asn 
        195                 200                 205 

 
           
             16  
             171  
             PRT  
             Pseudomonas aeruginosa  
           
            16 

Met Leu Gly Leu Val Leu Leu Tyr Val Gly Ala Val Leu Phe Leu Asn 
  1               5                  10                  15 

Ala Val Trp Leu Leu Gly Lys Ile Ser Gly Arg Glu Val Ala Val Ile 
             20                  25                  30 

Asn Phe Leu Val Gly Val Leu Ser Ala Cys Val Ala Phe Tyr Leu Ile 
         35                  40                  45 

Phe Ser Ala Ala Ala Gly Gln Gly Ser Leu Lys Ala Gly Ala Leu Thr 
     50                  55                  60 

Leu Leu Phe Ala Phe Thr Tyr Leu Trp Val Ala Ala Asn Gln Phe Leu 
 65                  70                  75                  80 

Glu Val Asp Gly Lys Gly Leu Gly Trp Phe Cys Leu Phe Val Ser Leu 
                 85                  90                  95 

Thr Ala Cys Thr Val Ala Ile Glu Ser Phe Ala Gly Ala Ser Gly Pro 
            100                 105                 110 

Phe Gly Leu Trp Asn Ala Val Asn Trp Thr Val Trp Ala Leu Leu Trp 
        115                 120                 125 

Phe Cys Phe Phe Leu Leu Leu Gly Leu Ser Arg Gly Ile Gln Lys Pro 
    130                 135                 140 

Val Ala Tyr Leu Thr Leu Ala Ser Ala Ile Phe Thr Ala Trp Leu Pro 
145                 150                 155                 160 

Gly Leu Leu Leu Leu Gly Gln Val Leu Lys Ala 
                165                 170 

 
           
             17  
             19  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Primer  
             
           
            17 

tgggtgtgag atgatcata                                                  19 

 
           
             18  
             53  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Adaptor 
      sequence  
             
           
            18 

tgggtgtgag atgatcattt attcctccag atctggagga ataaatgatc ata            53