Abstract:
There is provided a method for modulation of polysaccharide adhesin synthesis involving products of the ycdSRQP gene operon in bacteria, depicted in SEQ. ID. NO. 1 and 2. Also provided is the use of an inhibitor of a product of the ycdSRQP operon in improving the response of a mammalian patient suffering from a bacterial infection.

Description:
CROSS-REFERENCE TO RELATED APPLICATION  
       [0001]    This application claims priority from U.S. provisional application No. 60/414,352, filed Sep. 30, 2002, which is pending. 
     
    
     
       FIELD OF THE INVENTION  
         [0002]    The invention relates to methods for polysaccharide adhesin modulation and particularly adhesin synthesis relating to biofilm formation.  
         BACKGROUND OF THE INVENTION  
         [0003]    Microorganisms commonly attach to living and nonliving surfaces, including those of indwelling medical devices, and form biofilms made up of extracellular polymers. In this state, microorganisms are highly resistant to antimicrobial treatment and are tenaciously bound to the surface. Biofilms represent a distinct physiological state, designed to provide a protected environment for survival under hostile conditions. Many chronic infections that are difficult or impossible to eliminate with conventional antibiotic therapies are known to involve biofilms. A partial list of the infections that involve biofilms includes: otitis media, prostatitis, vascular endocarditis, cystic fibrosis pneumonia, meliodosis, necrotizing faciitis, osteomyelitis, peridontitis, biliary tract infection, struvite kidney stone and host of nosocomial infections.  
           [0004]    Biofilm formation is a two-step process that requires the adhesion of bacteria to a substrate surface followed by cell-to-cell adhesion, forming the multiple layers of the biofilm. Bacterial or microorganism adherence is thought to be the first crucial step in the pathogenesis and biofilm formation. A number of factors influence an organism&#39;s ability to adhere to a surface. The early stages of adherence are influenced by non-specific forces such as surface charge, polarity and hydrophobic interactions. Later stages of adherence are thought to involve more specific interactions between adhesins and receptors. Studies on the adherence of bacteria to a biotic or abiotic surface are focused in part on the role of the extracellular polysaccharide or glyocalyx, also known as slime. Currently, extracellular polysaccharide is thought to play a role in the later stages of adherence and persistence of infections. It may serve as an ion-exchange resin to optimize a local nutritional environment, prevent penetration of antibiotics into the macrocolony, and protect bacteria from host defense mechanisms. Extracellular polysaccharide appears in the later stages of attachment and is not present during the initial phase of adherence. However, study of exopolysaccharide has lended little to prevention of initial adherence by the bacteria.  
           [0005]    Several studies have examined biofilm components and/or genetic factors in biofilm formation.  
           [0006]    Potential adhesins in bacteria such as  Staphylococcus epidermidis  have been identified, including the polysaccharide adhesin (PS/A). PS/A contains a complex mixture of monosaccharides and purified PS/A blocks adherence of PS/A producing strains of  S. epidermidis . It appears that PS/A and SAA (slime associated antigen) are distinct. It has been hypothesized that each functions in different stages of the adherence process with one or more of these adhesins responsible for initial attraction while others are needed for aggregation to form the macrocolonies.  
           [0007]    The polysaccharide intercellular adhesin (PIA) is composed of linear β-1,6-linked glucosaminylglycans in  Staphylococcus epidermidis  and  Staphylococcus aureus . Mack, D., et al., J. Bacteriol., 178: 175-183 (1996); Crampton, S. E., et al., Infect. Immun., 67: 5427-5433 (1999).  
           [0008]    Polymeric β-1,6-N-acetylglucosamine has only been reported in Staphylococci. No such polymer is believed to have been previously reported in any gram-negative species.  
           [0009]    Genetic factors in biofilm formation have been considered for Staphylococci (Gerke,  J. Biol. Chem.,  273: 18586 (1998)) and  Yersinia pestis  (Hare,  J. Bacteriol.,  181:4896 (1999)).  
           [0010]    Studies by others have failed to provide substantive evidence of unique metabolic requirements for biofilm formation.  
           [0011]    Other microbial adhesins have been reported. Such adhesins include: polysaccharide antigen from  Pseudomonas aeruginosa  slime (U.S. Pat. No. 4,285,936; U.S. Pat. No. 4,528,458);  Escherichia coli  fimbrial protein adhesins (Orskov, I., et al., Infect. Immun., 47: 191-200, 1985; Chanter, H., J. Gen. Microbiol. 125: 225-243 (1983) and Moch, T., et al., Proc, Natl, Acad, Sci., 84: 3462-3466 (1987)); lectin-like glycoprotein adhesin ( Bacteroides fragilis  group); a 70 kDa adhesin (Rogemond, V., et al., Infect. Immun., 53: 99-102 (1986)); and, uroepithelial cell adhesin protein of 17.5 kDa ( Proteus mirabilis ) (Wray, S. K., et al., Infect. Immun., 54: 43-49 (1986)).  
           [0012]    Crude extracellular products from the slime of homologous strains of  Staphylococcus epidermidis  inhibit the adherence of homologous bacterial cells to polymeric materials used as catheters and prostheses. Materials derived from the surface of such cells have been used as vaccines to produce antibodies directed against homologous bacteria. For example, Frank (French Patent Application 85-07315, Nov. 21, 1986); Pier, (U.S. Pat. No. 5,055,455 Oct. 8, 1991; U.S. Pat. No. 4,443,549; U.S. Pat. No. 4,652,498); and McKenny (Canadian Pat. No. CA2,333,931, Jan. 12, 2001).  
           [0013]    The complete genome of  E. coli  K12 was reported by Blattner (Science 277: 1453 (1997). However, this report failed to suggest any function for the region encoding the ycdSRQP operon. Information is also provided in Hare, J. M. and McDonough, K. A., J. Bacteriol. 181: 4896-4904 (1999).  
           [0014]    Thus, it is an object of the invention to provide an improved method for polysaccharide adhesin modulation.  
         SUMMARY OF THE INVENTION  
         [0015]    An embodiment of the invention provides, inter alia, the ycdSRQP operon, products thereof and methods and uses therefore. This operon was identified by independent insertions in ycdS (SEQ ID NO: 1), ycdR (SEQ ID NO: 2) and ycdQ (SEQ ID NO: 3), which severely decreased biofilm formation in  E. coli  wild type strain MG1655.  
           [0016]    YcdQ of  E. coli  appears to be associated with the inner membrane and contains 5 putative membrane-spanning domains. YcdR appears to have a function as a polysaccharide deacetylase. YcdR is also believed to be involved in the transport of PIA. YcdR is believed to be a lipoprotein in its active form. YcdS of  E. coli  is a putative outer membrane protein believed to be involved in the extracellular localization/transport of the PIA polymer and/or as a docking protein to assist in the formation of an intercellular bridge between cells.  
           [0017]    An embodiment of the invention provides ycdS, ycdR and ycdQ polynucleotides and polypeptides and uses and methods relating thereto.  
           [0018]    While the invention is not limited to any particular mechanism of action, it appears that the genes of this operon are involved in the production and biological function of a linear β-1,6-N-acetylglucosamine polymer that functions as an adhesin in biofilm formation. Biofilm formation is believed to depend on the production of a polysaccharide intercellular adhesin (PIA). The PIA represents and mediates the intercellular adherence of bacteria to each other and accumulation of a multilayered biofilm.  
                             TABLE 1                           Metabolic Conversion of Glycogen to PIA in  E. coil                  Steps   Gene products                       1. Glycogen → Glucose-1-Phosphate   GlgP, GlgX           2. Glucose-1-Phosphate → Glucose-6-Phosphate   Pgm           3. Glucose-6-Phosphate → Fructose-6-Phosphate   Pgi           4. Fructose-6-Phosphate → GlcN-6-P   GlmS           5. GlcN-6-P → GlcN-1-P   GlmM           6. GlcN-1-P → GlcNAc-1-P   GlmU           7. GlcNAc-1-P → UDP-GlcNAc   GlmU           8. UDP-GlcNAc →β-1,6-GlcNAc (n + 1)   YcdQ                      
 
           [0019]    Table 1. Pathway for converting glycogen into PIA in  E. coli . GlgX is the glycogen debranching enzyme, which hydrolyzes the 1,6-linkages of glycogen, and thereby enhances the conversion of glycogen to glucose-1-phosphate by glycogen phosphorylase (GlgP). GlmU is required to both the aceylation of GlcN-1-P and the UDP-GlcNAc pyrophosphorylase reaction.  
           [0020]    In an embodiment of the invention there are provided products of the ycdSRQP operon.  
           [0021]    In an embodiment of the invention there is provided a method of identifying inhibitors of products of the ycdSRQP operon.  
           [0022]    In an embodiment of the invention there is provided a method of decreasing biofilm formation by biofilm-forming bacteria by decreasing expression of one or more products of the ycdSRQP operon.  
           [0023]    In an embodiment of the invention there is provided the use of a product of the ycdSRQP operon to modulate polysaccharide adhesin synthesis.  
           [0024]    In an embodiment of the invention there is provided the use of a product of the ycdSRQP operon to modulate biofilm formation.  
           [0025]    In an embodiment of the invention there is provided use of a product of the ycdSRQP operon in improving the response of a mammalian patient suffering from a bacterial infection by biofilm forming bacteria.  
           [0026]    In an embodiment of the invention there is provided a method of inhibiting polysaccharide deacetylation by reducing YcdR activity.  
           [0027]    In an embodiment of the invention there is provided a method of inhibiting adhesin transport by reducing YcdR activity.  
           [0028]    In an embodiment of the invention there is provided a method of reducing extracellular adhesin binding in  E. coli  by reducing YcdS activity.  
           [0029]    In an embodiment of the invention there is provided a method of improving the response of a mammalian patient suffering from a bacterial infection to antibiotics for treatment of said bacterial infection comprising reducing biofilm formation by infecting the bacteria.  
           [0030]    In an embodiment of the invention there is provided a method of facilitating the reduction of bacterial load in a mammalian patient suffering from bacterial infection, comprising inhibiting the activity of a product of the ycd operon in at least some of the infecting bacteria.  
           [0031]    In an embodiment of the invention there is provided a method of decreasing cell to cell biofilm links by reducing YcdS activity.  
           [0032]    In an embodiment of the invention there is provided a method of reducing adhesin synthesis in  E. coli  by reducing YcdQ activity.  
           [0033]    In an embodiment of the invention there is provided a method of reducing 13-1,6-N-acetylglucosamine (13-1,6Glc NAc) polymer synthesis by reducing YcdQ activity.  
           [0034]    In an embodiment of the invention there is provided a method of reducing glycosyltransferase activity in  E. coli  by reducing YcdQ activity.  
           [0035]    In an embodiment of the invention there are provided antibodies to  E. coli  β-1,6Glc NAc.  
           [0036]    In an embodiment of the invention there is provided a use and method of using antibodies to  E. coli  β-1,6Glc NAc in an assay to identify biofilm production and an assay to identify biofilm reduction.  
           [0037]    In an embodiment of the invention there is provided a method of reducing biofilm formation by reducing the activity of YcdQ in a plurality of bacterial cells.  
           [0038]    In an embodiment of the invention there is provided a method of reducing biofilm formation by reducing the activity of YcdS in a plurality of bacterial cells.  
           [0039]    In an embodiment of the invention there is provided a method of reducing biofilm formation by reducing the activity of YcdR in a plurality of bacterial cells.  
           [0040]    In an embodiment of the invention there is provided a method of reducing biofilm formation by reducing the activity of YcdP in a plurality of bacterial cells.  
           [0041]    There are provided products of the ycdSRQP operon and uses and methods for using these products in the production of antibodies to the products of these genes. These antibodies may be useful diagnostically in identifying aberrations in proteins encoded by this operon and therapeutically to reduce cell-cell interactions mediated by these products of the ycdSRQP operon, and particularly YcdS. Additionally, these gene products may be used in screening tests for inhibitors of these products.  
           [0042]    There is provided a method of identifying inhibitors of products of ycdSRQP operon comprising selecting a gene product of interest, assaying the activity of that gene product under control conditions, adding a potential inhibitor of the gene product, assaying the activity of the gene product in the presence of the potential inhibitor, and ascertaining whether the presence of the potential inhibitor resulted in an inhibition of the function of that gene product.  
           [0043]    There is provided a use and a method of decreasing biofilm formation. This may be accomplished by a variety of means, including using antisense RNA sequences to decrease expression of the products of the genes of ycdSRQP operon.  
           [0044]    There is provided a use and a method of using antisense sequences to genes, or portions thereof, of the ycdSRQP operon to reduce the rate of conversion of UDP-GlcNAc to β-1,6GlcNAa polymeric units in an  E. coli  containing environment. This may be accomplished by reducing the expression or activity of one or more genes of the ycd operon involved in biofilm formation. For example, antisense sequences complementary to mRNA encoding YcdS or YcdQ may be employed to reduce translation of the corresponding protein, and thus the activity of that protein.  
           [0045]    Antisense sequences may be administered exogenously in bacterial culture, by administration to a patient suffering from  E. coli  infection, or by gene therapy to introduce genetic material encoding the antisense sequence directly into  E. coli , and/or into the patient in a form which it can be excreted from the cell, and taken up by the invading  E. coli.    
           [0046]    In some instances, the bacteria is at least one of  E. coli  or Staphylococcus.  
           [0047]    In some instances, the  E. coli  is  E. coli  K12.  
           [0048]    In some instances, the  E. coli  is any member of the  E. coli  species.  
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0049]    [0049]FIG. 1 is a graph showing plasmid clones (pUCPGA372) stimulate biofilm formation in a variety of  E. coli  strains. Bar graph A shows the effects in MG1655 for various isogenic strains represented by bars 1 to 7. Bar graph B shows the effects of ycd genes in TRMG1655 (csrA::kanrR) for various strains represented by bars 1 to 7.  
         [0050]    [0050]FIG. 2 is a graph showing the fractionation of polysaccharide adhesion by gel filtration FPLC, cell extract from strain TRMG1655 cpsE ycdQ containing pUCPG372 (graph A) or pUC19 (graph B). 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
     EXAMPLE 1  
     Molecular Cloning of ycd Operon  
       [0051]    Plasmid clones (pUCPGA372) of this operon complement ycdQ and ycdS mutations and stimulate biofilm formation in a variety of  E. coli  strains. FIG. 1 shows the effect of ycd genes on biofilm formation. Bar graph A shows the effects in MG1655. Isogenic strains represented by bars 1 to 7 are MG1655. ycdQ mutant, ycdS mutant, ycdQ mutants containing pUC19 or pUCPGA372 (cloned ycdSRQP) and ycdS mutant containing pUC19 or pUCPGA372, respectively. Bar graph B shows the effects of ycd genes in TRMG1655 (csrA::kanrR). Strain identities for bar 1 to 7 are TRMG1655, ycdQ mutant, ycdS mutant, ycdQ mutants containing pUC19 or pUCPGA372, and ycdS mutant containing pUC19 or pUCPGA372, respectively.  
         [0052]    A purification protocol was designed, which yielded a highly enriched polymeric GlcN fraction from a strain containing the ycdSRQP plasmid clone. FIG. 2 shows the fractionation of polysaccharide adhesion by gel filtration FPLC. Cell extract from strain TRMG1655 cpsE ycdQ containing pUCPG372 (graph A) or pUC19 (graph B) was fractionated using a Sephacryl S-200 (16/60) column. Fractions of 2 ml were collected and analyzed for total carbohydrate (triangle) and, after hydrolysis, for glucosamine (square). The straight line on each of graph A and B indicates the void volume of the column and was determined using 2-MDa blue dextran.  
         [0053]    The polysaccharide was used for routing polyclonal antibody production and for affinity-column purification of the antibodies.  
         [0054]    The antisera are used to develop a simple quantitative assay for the polymer, including ELISA. There is a correlation between ycd gene expression, β-1,6GlcNAc synthesis, and biofilm formation in  E. coli.    
         [0055]    Mutations in Cloned ycd Operon Carried by pUCPGA372.  
         [0056]    The ycd genes were cloned and were found to differ from the sequence reported by Blattner as follows.  
         [0057]    In the ycdR gene, nucleotide 723 was changed from A to G, and the codon was changed from GTT (Leu) to GCT (Ser). Two other mutations in ycdS gene, in which nucleotide 582 and 389 were changed from T to C, and the codons were changed from TAA (Asn) to TAG (Asp), and AAC (Gln) to AGC (Arg) respectively.  
         [0058]    With reference to SEQ ID NO: 6, the numbering for the full DNA sequence of ycdS starts at the A of the ATG initiation codon. Individual mutations are numbered from the start codons of each gene. In SEQ ID NO: 6, underlining indicates codons affected by point mutations and the insertion sites for the various transposon mutants are shown by downward facing arrows.  
       EXAMPLE 2A  
     Involvement of yCdSRQP Operon in the Biosynthesis of Unbranched β-1,6-GlcNAc (Polysaccharid Intercellular Adhesin)  
       [0059]    The ycdSRQP operon, which encodes proteins needed for the production and function of a biofilm polysaccharide adhesin, was cloned and sequenced, and mutants were prepared.  
         [0060]    Methods:  
         [0061]    Plasmid Construction. The ycd operon was amplified by polymerase chain reaction from chromosomal DNA of MG1655 using the oligonucleotide primers TACAGTTMGTGTGTTATCGGTGCAGAGCC (SEQ ID NO: 4) and CTCMCGCCTGGCTGATTAAACCMCTATTC (SEQ ID NO: 5). The PCR product, a 6.9 kb fragment, was purified by QIAquick Gel Extraction Kit (QIAGEN) and cloned into vector pCR-XL-TOPO (Invitrogen) using D H5α as the host for transformation. Approximately 120 clones were screened for increased biofilm production. One clone pCRPGA37, increasing biofilm ˜6-fold when expressed in DH5α was subsequently treated with HindIII and Xbal, and the insert DNA was subcloned into pUCl9 to yield plasmid pUCPGA372. PCRPGA37 was sequenced.  
         [0062]    Transposon Mutagenesis. Transposon mutants were generated by infecting TRMG1655ΔfimB-HΔmotB with λNK1324 at a multiplicity of infection of 0.2, essentially as described in Romeo et al.,  J. Bacteriol.  175: 4744 (1993) and Kleckner,  Meth. Enzymol.  204:139 (1991). The insertion mutants were selected on Kornberg agar containing 30 ug/ml chloramphenicol. Chloramphenicol-resistant colonies were picked and grown at 26° C. in 96-well, polystyrene microtiter plate containing CFA with 30 ug/ul chloramphenicol. After 24 hr, the cells were subculture into corresponding wells in 96-well microtiter plates containing CFA with 30 ug/ul chloramphenicol and incubated at 26° C. for 24 hr. Turbidity in the wells was determined to avoid isolation of mutants with growth defects, and biofilm by the mutants was measured. Mutants with altered ability to form biofilms were saved. These candidate mutants were streaked to isolate single colonies on Kornberg agar and retested for their ability to form biofilm. Candidate insertion mutations were transferred by P1vir transduction into the original parent strain or related strains and retested for the biofilm development. Stock cultures were saved at −80° C.  
         [0063]    Purification of the Polysaccharide Adhesin.  E. coli  strains containing pUCPGA372 or pUCl9 were grown for 24 hours at 37° C. with shaking at 250 rpm in CFA medium containing 100 μg/ml ampicillin. Bacterial cells were harvested and resuspended in 50 mM Tris.HCl (pH 8.0). Cell extracts were prepared by lysozyme-EDTA treatment in the presence of DNase, RNase and α-amylase (Sigma) and were phenol extracted (Wolf-Watz, H., J. Bacteriol., 115: 1191-1197, 1973; Westphal, O. and Jann, K., J., Methods Carbohyd. Chem., 1964). The aqueous phase was extracted with chloroform, concentrated in an Amicon cell with a YM10 membrane and fractionated by FPLC on Sephacryl 5-200. The column was equilibrated with 0.1 M PBS (pH 7.4) and eluted with the same buffer. The GlcNAc-containing polysaccharide was detected by the MBTH assay following hydrolysis for 2 hours at 110° C. in 0.5M HCl (Smith, R. L. and Gilkerson, E., Anal. Biochem., 98: 478-480, 1979). Total carbohydrate was measured by phenol-sulfuric acid assay (Dubois, M., et al., Anal. Chem. 28: 350-356, 1959).  
         [0064]    Quantitative Biofilm Assay. Bacterial overnight cultures were inoculated 1:100 dilution into 96-well microtiter plate containing 200 □l/well fresh medium plus appropriate antibiotics. The plates were incubated at 26° C. for 24 hours. Biofilm was measured by discarding the medium, rinsing the wells with water (three times), and staining bound cells with crystal violet (BBL). The dye was solubilized with 33% acetic acid, and absorbance at 630 nm was determined using a microtiter plate reader. Background staining was corrected. All comparative analyses were conducted by incubating strains within the same microtiter plate to minimize variability. Each experiment was performed at least in triplicate.  
       EXAMPLE 2B  
     Precursor-Product Relationship of Glycogen to PIA by  13 C NMR  
       [0065]    Direct evidence for the precursor-product relationship of glycogen to PIA is established using  13 C glucose pulse labelling at the transition to a stationary phase. During this time, replication and growth decline, while glycogen synthesis remains active. Thus,  13 C incorporation into glycogen is efficient. NMR spectra of growing cultures are monitored in real time for glycogen and PIA. The availability of a strain disrupted in YcdQ is a powerful asset for these studies, and allows the precursor-product relationship to be firmly established. YcdQ blocks PIA synthesis, but not glycogen synthesis. Glucose differentially labeled in carbons 1, 2 or 6 is used to follow the conversion to glycogen and PIA. The commercial availability of these substrates allows monitoring of bacterial metabolism.  
       EXAMPLE 3  
     YcdQ for the Cell-Free Synthesis of Poly B-1,6-GlcNAc (PIA)  
       [0066]    To assess the potential role of ycdQ and the other ycd genes in synthesis of β-1,6-GlcNAc, membranes are prepared from wild type and nonpolar mutants, incubated with UDP-N-acetyl-D-[U-14C] glucosamine. The resulting oligosaccharides are separated by thin-layer chromatography and detected by autoradiography (Gerke, C et al, J. Biol. Chem. 273: 18586-18593, 1998). YcdQ is a N-acetylglucosamine transferase which adds N-acetylglucosamine to the growing polymer. Thus, YcdQ is very important for cell-free synthesis of PIA, although other ycd genes can affect the reaction rate and/or extent of the polymerization reaction.  
       EXAMPLE 4  
     The Roles of ycd Genes in PIA Transport and PIA-Dependent Adhesion  
       [0067]    There is a mechanism by which PIA traverses the outer membrane of  E. coli . In some instances, YcdS is involved in PIA export. To show this, PIA is synthesized in isolated membranes from an ycdS nonpolar mutant. This PIA is detectable in cell lysates, but is not found on the cell surface using antibody binding to whole cells. YcdS is involved in the formation of cell to cell biofilm links. In some instances YcdS also plays a role as an anchor protein that helps to attach PIA to the cell surface. In such instances, significant amounts of PIA are observed in extracellular fractions, but little cell bound materials is present.  
         [0068]    YcdR plays a role in polysaccharide deacetylation. This is evaluated by NMR studies. The role of YcdR in transit is proven by immunolocation studies.  
         [0069]    YcdQ is involved in adhesin synthesis. This is shown by the reduction of biofilm formation following disruption of the ycdQ gene.  
         [0070]    Thus, the invention provides, in one embodiment, a mutation of the ycdR gene, sufficient to alter YCdR activity: The mutation is a non-conservative mutation, disrupting expression of the normal gene product. In some instances the mutation changes the encoded amino acid from an aliphatic amino acid to a hydrophilic amino acid. In some instances the mutation enables the encoded amino acid to engage in hydrogen bonding, which the wild type encoded amino acid was unable to engage in. In some instances the mutation is a frame shift mutation resulting in a loss of the downstream encoded gene product. In some instances the mutation introduces a stop codon into the gene prior to the normal stop position, resulting in a truncated gene product.  
         [0071]    In an embodiment of the invention there are provided non conservative mutants, of the ycdS gene.  
         [0072]    In some instances, the mutation in ycdS gene is a non-conservative mutation resulting in coding for an uncharged amino acid (at physiological pH) where a charged amino acid appears in the wild type. In some instances, the mutation results in the replacement of a negatively charged amino acid with an uncharged amino acid (at physiological pH). In some instances, the mutation results in the replacement of an amino acid generally uninvolved in hydrogen bonding, with one capable of forming a hydrogen bond at physiological pH. In some instances the mutation is a frame shift mutation resulting in a loss of the downstream encoded gene product. In some instances the mutation introduces a stop codon into the gene prior to the normal stop position, resulting in a truncated gene product.  
         [0073]    In some instances, the mutation in the ycdS gene results in the replacement of an uncharged amino acid (at physiological pH) with a charged amino acid. In some instances, this mutation results in the replacement of an uncharged amino acid with a positively charged (at physiological pH) amino acid. In some instances, the mutation results in the replacement of an amino acid having a side chain capable of acting as a hydrogen bond acceptor with an amino acid incapable of acting as a hydrogen bond acceptor (at physiological pH).  
         [0074]    Mutation of the YcdP gene substantially prevents biofilm formation. Thus, YcdP is needed for biofilm formation.  
       EXAMPLE 5  
     Inhibition of Biofilm Formation Through Interference with the Activity of Proteins Encoded by the ycd Operon  
       [0075]    YcdQ is involved in the polymerization of UDP-N-acetylglucosamine to form β-1,6-N-acetylglucosamine polymer known as PIA (polysaccharide intercellular adhesin) from UDP-N-acetylglucosamine, which is required for biofilm formation.  
                         
 
         [0076]    Crude Enzyme Preparation:  
         [0077]    Crude membrane-bound N-acetylglucosaminyltransferase is prepared from overproducing strain of  E. coli  according to the method, described by Gerke, et al. (J. Biol. Chem., 273: 18586-18593, 1998). The overnight culture of  E. coli  is harvested by centrifugation, and the cell pellets, are resuspended in buffer A (50 mM Tris HCL pH 7.5, 10 mM MgCl 2  and 4 mM dithiothreitol; 2 μl/mg of cell wet weight). Grinding in a mortar disrupts DNase 1 (20 μg/ml) is added before breaking the cells. Unbroken cells are sedimented (2000×g, 10 min and the supernatant is saved. The procedure is repeated one to three times and all the supernatants are pooled. Membranes are sedimented from the crude extract by ultracentrifugation (200,000×g, 20 min) and resuspended in buffer A at a protein concentration of 5 mg/ml (5-fold concentration of the membrane proteins over the crude extract). For further purification, the crude membranes are extracted with 2% (w/v) Triton X-100 (in buffer A) for 2 h with gentle shaking, sedimented again, washed once with buffer A, and resuspended in the same volume of buffer A as the crude membranes. Protein concentration is determined by the method Bradford (Anal. Biochem., 72: 248-254, 1976).  
         [0078]    Enzyme Assay:  
         [0079]    In vitro reactions to analyze N-acetylglucosaminyltransferase activity are performed by incubating crude extracts with 0.4 mM UDP N-acetylglucosamine. In vitro synthesis of peptidoglycan is repressed by adding 50 μg/ml D-cycloserine (Lugtenberg, et al., J. Bacteriol., 109: 326-335,1972). For radiolabeling, 10 μM UDP-N-acetyl-D-(U- 14 C) glucosamine is added. Analytical mixture is carried out in a total volume of 50 μl. Reaction mixture is incubated for 12 h at 20° C. The reaction is stopped by the addition of 200 μl of water and boiling for 3 min. After centrifugation, the supernatant is loaded on a Sephadex A-25 anion-exchange column (gel volume, 300-500 μl) equilibrated with water. The column is washed with 2 ml of water. The unbound fraction (flowthrough and wash) is lyophilized. Radioactive products purified by Sephadex A-25 are subjected to gel filtration on a Bio-Gel P-2 column (90×1.5 cm) equilibrated with 0.1 M pyridine acetate (pH 6) at a flow rate of 0.3 ml/min. Fractions of 2 ml are collected and radioactivity is measured by liquid scintillation counting (Geremia, et al., Proc. Natl. Acad. Sci., USA., 91: 2669-2673,1994).  
         [0080]    Identification and Selection of Enzyme Inhibitors  
         [0081]    For all ycd proteins of interest, combinatorial libraries are screened to identify inhibitors. In addition, known inhibitors of key enzymes are tested using appropriate concentrations as reported in the literature. These inhibitors include natural or synthetic compounds and some analogues. These compounds are obtained from routine suppliers of reagent grade chemicals. The compounds showing maximum inhibition will be selected for determining their antibiofilm activity. Alternatively or additionally, libraries of compounds are tested for antibiofilm activity. Antibiofilm activity can include inhibiting YcdQ activity acid inhibiting biofilm formation by an  E. coli  culture.  
         [0082]    Known deacetylase inhibitors and variants of such inhibitors are used to study their inhibitory effects on YcdR.  
         [0083]    Short oligosaccharides of beta-1,6-GlcAc and synthetic/semisynthetic compounds capable of binding YcdS under physiological conditions are used to study their inhibitory effects on YcdS.  
         [0084]    Known glycosyltransferase inhibitors, such as tunicamycin, bacitracin, isofagomine and azafagomine are used to study their inhibitory effects on N-acetylglucosaminyltransferase (YcdQ). In addition, variants of such inhibitors are examined. (For example, having acyl substitutions of a different size or having one or more altered or additional side groups.) N-acetylglucosaminyltransferase in a crude extract is incubated with different concentrations of inhibitors in the presence of 0.4 mM UDP-N-acetylglucosamine. In vitro synthesis of peptidoglycan is repressed by adding 50 μg/ml D-cycloserine (Lugtenberg, et al, J. Bacteriol., 109: 326-335, 1972). For radiolabeling, 10 μM UDP-N-acetyl-D-(U- 14 C) glucosamine will be added. The reaction is carried out in a total volume of 50 μl. The reaction mixture is incubated for 12 h at 20° C. The reaction is stopped by the addition of 200 μl of water and boiling for 3 min. After centrifugation, the supernatant is loaded on a Sephadex A-25 anion-exchange column (gel volume, 300-500 μl) equilibrated with water. The column is washed with 2 ml of water. The unbound fraction (flowthrough and wash) is lyophilized. Radioactive products purified by Sephadex A-25 a subjected to gel filtration on a Bio-Gel P-2 column (90×1.5 cm) equilibrated with 0.1 M pyridine acetate (pH 6) at a flow rate of 0.3 ml/min. Fractions of 2 ml are collected and radioactivity is measured by liquid scintillation counting (Geremia, et al., Proc. Natl. Acad. Sd., USA., 91: 2669-2673,1994).  
         [0085]    Determining the Antibiofilm Activity of Selected Enzyme Inhibitors  
         [0086]    The antibiofilm activity of selected enzyme inhibitors is evaluated using a microtiter plate format biofilm assay as described below.  E. coli  are used for biofilm inhibition assay. (The biofilm assay can be automated using robotics, if desired.) Further, the compounds showing significant antibiofilm activity are tested for their ability to block biofilm formation on commonly used medical devices.  
         [0087]    Biofilm Assay:  
         [0088]    Cultures of  E. coli  for biofilm assay are grown in Luria-Bertani (LB) at 37° C. Biofilm assays are carried out in colony-forming antigen (CFA) medium. Overnight cultures are inoculated 1:100 into fresh medium. In the microtiter plate assay, inoculated cultures are grown in a 96-well polystyrene microtiter plate for 24 h at 26° C. Growth of planktonic cells are determined by absorbance at 600 nm or total protein assay using a ELISA plate reader. Biofilm is measured by discarding the medium, rinsing the wells with water (three times), and staining bound cells with crystal violet (BBL). The dye is solubilized with 33% acetic acid, and absorbance at 630 nm is determined using a microtiter plate reader. For each experiment, background staining is corrected by subtracting the crystal violet bound to uninoculated controls. All comparative analyses are conducted by incubating 25 strains within the same microtiter plate to minimize the variability.  
         [0089]    Biofilm Inhibition Studies:  
         [0090]    At least two compounds from each enzyme inhibition study are selected for evaluation of their antibiofilm activity. The biofilm inhibition assay is performed for each compound. In the microtiter plate assay, inoculated cultures are grown in a 96-well polystyrene plate in the presence and absence (control) of selected enzyme inhibitors at different concentrations at 26° C. The plates are incubated for 24 h at 37° C. Biofilm is measured by discarding the medium, rinsing the wells with water (three times), and staining bound cells with crystal violet. The dye is solubilized with 33% acetic acid, and absorbance at 630 nm is corrected by subtracting the crystal violet bound to uninoculated controls. Each assay is performed 3-5 times. The concentrations of each enzyme inhibitor used for the assay is plotted against 0 D obtained for biofilm growth in order to indicate the percentage of inhibition in comparison with the control.  
         [0091]    The compounds that inhibit biofilm formation on a microtiter plate are tested for their inhibitory effects on biofilm formation of  E. coli  in medical devices like urinary catheters.  
         [0092]    The above methods are also applied, with suitable modifications employed in identifying, inhibitors of other products of the ycd operon, including YcdR and YcdS.  
         [0093]    YcdR  
         [0094]    In one approach, YcdR activity is determined by assaying the production of acetate from polysaccharide by HPLC. In one approach, radiolabeled PIA and its precursors are provided and the release of radiolabeled acetate is measured. Such release is proportional to YcdR activity.  
       EXAMPLE 6  
     Alternative Approach to Inhibitor Selection and/or Design  
       [0095]    Method A:  
         [0096]    (i) The proteins encoded by the genes of the ycd operon are purified by routine means, and their crystal structure is determined.  
         [0097]    (ii) The structure of the region surrounding the amino acids in the YcdR which binds the polysaccharide is examined to identify the characteristics of molecules likely to interact specifically with that region.  
         [0098]    (iii) Compounds having the general characteristics identified are screened for an ability to bind to the identified region in YcdR when immobilized in solution at physiological pH, tonicity and temperature.  
         [0099]    (iv) Compounds showing an ability to bind to YcdR are identified. These compounds are, individually, added to  E. coli  cultures, and their effect on biofilm formation is determined.  
         [0100]    Compounds capable of reducing biofilm formation in  E. coli  cultures are inhibitors of the YcdR protein.  
         [0101]    Method B:  
         [0102]    Steps (i) and (ii) of Method B are omitted.  
         [0103]    (i) YcR is immobilized.  
         [0104]    (ii) Large libraries of compounds are screened for an ability to bind to YcdR when immobilized.  
         [0105]    (iii) Binding compounds are examined with respect to their ability to decrease biofilm formation in  E. coli  culture.  
         [0106]    Either one of Method A or B is applied with suitable modification to identify inhibitors of YcdQ and YcdS. Modification will involve immobilizing the gene product of interest and, for Method A, step (ii), examining the structure of the region surrounding the amino acid by the codon containing a nucleotide mutation of which reduces biofilm formation an  E. coli  containing environment.  
         [0107]    In some instances, inhibitors of products of the ycd operon may be encapsulated or otherwise treated to facilitate entry into  E. coli  cells, for example by liposome encapsulation including specific factors encouraging uptake by  E. coli  cells.  
                                 TABLE 2                       Polynucleotide and Polypeptide Sequences of ycdS,           ycdR and ycdQ (Sequences from  Escherichia coil ).       (Note: Sequence numbering differs. Examples       and discussions refer to numbering of SEQ ID NO: 6.)                                SEQ ID NO: 1               1 (ycdS)         ATG TATTCAAGTAGCAGAAAAAGGTGCCCGAAAACCAAATGGGCTTTGAAACTTCTTACT   300       M  Y  S  S  S  R  K  R  C  P  K  T  K  W  A  L  K  L  L  T                 GCCGCATTTTTAGCAGCGAGTCCCGCGGCGAAGAGTGCTGTTAATAACGCCTATGATGCA   360       A  A  F  L  A  A  S  P  A  A  K  S  A  V  N  N  A  Y  D  A                 TTGATTATTGAAGCTCGCAAGGGTAATACTCAGCCAGCTTTGTCATGGTTTGCACTAAAA   420       L  I  I  E  A  R  K  G  N  T  Q  P  A  L  S  W  F  A  L  K                 TCAGCACTCAGCAATAACCAAATTGCTGACTGGTTACAGATTGCCTTATGGGCCGGGCAA   480       S  A  L  S  N  N  Q  I  A  D  W  L  Q  I  A  L  W  A  G  Q                 GATAAACAGGTTATTACCGTTTACAACCGCTACCGTCATCAGCAATTACCAGCGCGTGGT   540       D  K  Q  V  I  T  V  Y  N  R  Y  R  H  Q  Q  L  P  A  R  G                 TATGCAGCTGTCGCCGTCGCTTATCGTAACCTGCAACAATGGCAAAACTCGCTTACACTG   600       Y  A  A  V  A  V  A  Y  R  N  L  Q  Q  W  Q  N  S  L  T  L                 TGGCAAAAGGCGCTCTCTCTGGAGCCGCAAAATAAGGATTATCAACGGGGACAAATTTTA   660       W  Q  K  A  L  S  L  E  P  Q  N  K  D  Y  Q  R  G  Q  I  L                 ACCCTGGCAGATGCTGGTCACTATGATACTGCGCTGGTTAAACTTAAGCAGCTTAACTCT   720       T  L  A  D  A  G  H  Y  D  T  A  L  V  K  L  K  Q  L  N  S                 GGAGCACCGGACAAAGCCAATTTACTCGCAGAAGCCTATATCTATAAACTGGCGGGGCGT   780       G  A  P  D  K  A  N  L  L  A  E  A  Y  I  Y  K  L  A  G  R                 CATCAGGATGAATTACGGGCGATGACAGAGTCATTACCTGAAAATGCATCTACGCAACAA   840       H  Q  D  E  L  R  A  M  T  E  S  L  P  E  N  A  S  T  Q  Q                 TATCCCACAGAATACGTGCAGGCATTACGTAATAATCAACTTGCTGCCGCGATTGACGAT   900       Y  P  T  E 75   Y  V  Q  A  L  R  N  N  Q  L  A  A  A  I  D  D                 GCCAATTTAAC GC CAGATATTCGCGCTGATATTCATGCCGAACTGGTCAGACTGTCGTTT   960       A  N  L  T  P  D  I  R  A  D  I  H  A  E  L  V  R  L  S  F                 ATGCCTACGCGCAGTGAAAGTGAACGTTATGCCATTGCCGATCGCGCCCTCGCCCAATAC   1020       M  P  T  R  S  E  S  E  R  Y  A  I  A  D  R  A  L  A  Q  Y                 GCTGCATTAGAAATTCTGTGGCACGATAACCCAGACCGCACTGCCCAGTACCAGCGTATT   1080       A  A  L  E  I  L  W  H  D  N  P  D  R  T  A  Q  Y  Q  R  I                 CAGGTTGATCATCTTGGCGCGTTATTAACTCGCGATCGTTATAAAGACGTTATTTCTCAC   1140       Q  V  D  H  L  G  A  L  L  T  R  D  R  Y  K  D  V  I  S  H                TATCAGCGATTAAAAAAGACGGGGCAAATTATTCCGCCCTGGGGGCAATATTGGGTTGCA   1200       Y  Q  R  L  K  K  T  G  Q  I  I  P  P  W  G  Q  Y  W  V  A                 TCGGCTTATCTCAAAGATCATCAGCCGAAAAAAGCACAGTCAATAATGACCGAGCTCTTT   1260       S  A  Y  L  K  D  H  Q  P  K  K  A  Q  S  I  M  T  E  L  F                 TATCACAAGGAGACCATTGCCCCGGATTTATCCGATGAAGAACTTGCGGATCTCTTTTAC   1320       Y  H  K  E  T  I  A  P  D  L  S  D  E  E  L  A  D  L  F  Y                 AGCCACCTGGAGAGTGAAAATTATCCGGGCGCGCTAACTGTCACCCAACATACCATTAAT   1380       S  H  L  E  S  E  N  Y  P  G  A  L  T  V  T  Q  H  T  I  N                 ACTTCGCCGCCTTTCCTTCGGTTAATGGGCACGCCTACGAGCATCCCGAATGATACCTGG   1440       T  S  P  P  F  L  R  L  M  G  T  P  T  S  I  P  N  D  T  W                 TTACAGGGGCATTCGTTTCTCTCAACCGTAGCAAAATATAGTAATGATCTTCCTCAGGCT   1500       L  Q  G  H  S  F  L  S  T  V  A  K  Y  S  N  D  L  P  Q  A                 GAAATGACAGCCAGAGAGCTTGCTTATAACGCACCAGGAAATCAGGGACTGCGCATTGAT   1560       E  M  T  A  R  E  L  A  Y  N  A  P  G  N  Q  G  L  R  I  D                 TACGCGAGTGTGTTACAAGCCCGCGGTTGGCCTCGTGCAGCAGAAAATGAATTAAAAAAA   1620       Y  A  S  V  L  Q  A  R  G  W  P  R  A  A  E  N  E  L  K  K                 GCAGAAGTGATCGAGCCACGTAATATTAATCTGGAGGTTGAACAAGCCTGGACAGCATTA   1680       A  E  V  I  E  P  R  N  I  N  L  E  V  E  Q  A  W  T  A  L                 ACGTTACAAGAATGGCAGCAGGCAGCTGTCTTAACGCACGATGTTGTCGAACGTGAACCG   1740       T  L  Q  E  W  Q  Q  A  A  V  L  T  H  D  V  V  E  R  E  P                 CAAGATCCCGGCGTTGTACGATTAAAACGTGCGGTTGATGTACATAATCTTGCAGAGCTT   1800       Q  D  P  G  V  V  R  L  K  R  A  V  D  V  H  N  L  A  E  L                 CGTATCGCTGGCTCAACAGGAATTGATGCCGAAGGCCCGGATAGTGGTAAACATGATGTC   1860       R  I  A  G  S  T  G  I  D  A  E  G  P  D  S  G  K  H  D  V                 GACTTAACCACCATCGTTTATTCACCACCGCTGAAGGATAACTGGCGCGGTTTTGCTGGA   1920       D  L  T  T  I  V  Y  S  P  P  L  K  D  N  W  R  G  F  A  G                 TTCGGTTATGCCGATGGACAATTTAGCGAAGGAAAAGGGATTGTTCGCGACTGGCTTGCG   1980       F  G  Y  A  D  G  Q  F  S  E  G  K  G  I  V  R  D  W  L  A                 GGTGTTGAGTGGCGGTCACGTAATATCTGGCTCGAGGCAGAGTACGCTGAACGCGTTTTC   2040       G  V  E  W  R  S  R  N  I  W  L  E  A  E  Y  A  E  R  V  F                 AATCATGAGCATAAACCCGGCGCGCGCCTGTCTGGCTGGTATGATTTTAATGATAACTGG   2100       N  H  E  H  K  P  G  A  R  L  S  G  W  Y  D  F  N  D  N  W                 CGTATTGGTTCGCAACTGGAACGCCTCTCTCACCGCGTTCCATTACGGGCAATGAAAAAT   2160       R  I  G  S  Q  L  E  R  L  S  H  R  V  P  L  R  A  M  K  N                 GGTGTTACAGGCAACAGTGCTCAGGCTTATGTTCGCTGGTATCAAAATGAGCGGCGTAAG   2220       G  V  T  G  N  S  A  Q  A  Y  V  R  W  Y  Q  N  E  R  R  K                 TACGGTGTCTCCTGGGCTTTCACTGATTTTTCCGACAGTAACCAGCGTCATGAAGTCTCA   2280       Y  G  V  S  W  A  F  T  D  F  S  D  S  N  Q  R  H  E  V  S                 CTTGAGGGTCAGGAACGCATCTGGTCTTCACCATATTTGATTGTCGATTTCCTACCCAGT   2340       L  E  G  Q  E  R  I  W  S  S  P  Y  L  I  V  D  F  L  P  S                 CTGTATTACGAACAAAATACAGAACACGATACCCCATACTACAACCCTATAAAAACGTTC   2400       L  Y  Y  E  Q  N  T  E  H  D  T  P  Y  Y  N  P  I  K  T  F                 GATATTGTTCCGGCATTTGAGGCAAGCCATTTGTTATGGCGAAGCTATGAAAATAGCTGG   2460       D  I  V  P  A  F  E  A  S  H  L  L  W  R  S  Y  E  N  S  W                 GAGCAAATATTCAGCGCAGGTGTTGGTGCCTCCTGGCAAAAACATTATGGCACGGATGTC   2520       E  Q  I  F  S  A  G  V  G  A  S  W  Q  K  H  Y  G  T  D  V                 GTCACCCAACTCGGCTACGGGCAACGCATTAGTTGGAATGACGTGATTGATGCTGGCGCA   2580       V  T  Q  L  G  Y  G  Q  R  I  S  W  N  D  V  I  D  A  G  A                 ACGCTACGCTGGGAAAAACGACCTTATGACGGTGACAGAGAACACAACTTATACGTTGAA   2640       T  L  R  W  E  K  R  P  Y  D  G  D  R  E  H  N  L  Y  V  E                 TTCGATATGACATTCAGATTT TAA GGAT AAATATGTTACG TAATGGAAATAAATATCTCC   2700       F  D  M  T  F  R  F  *               SEQ ID NO: 2               ***         1(YCDR)       TTAAGGATAAATATGTTACGTAATGGAAATAAATATCTCCTGATGCTGGTGAGTATAATT   60         M  L  R  N  G  N  K  Y  L  L  ML  V  S  I  I                             ATGCTCACCGCGTGCATTAGCCAGTCAAGAACATCATTTATACCGCCACAGGATCGCGAA   120       M  L  T  A  C  I  S  Q  S  R  T  S  F  I  P  P  Q  D  R  E                 TCTTTACTCGCCGAGCAACCGTGGCCGCATAATGGTTTTGTAGCGATTTCATGGCATAAC   180       S  L  L  A  E  Q  P  W  P  H  N  G  F  V  A  I  S  W  H  N                 GTTGAAGACGAAGCTGCCGACCAGCGTTTTATGTCAGTGCGGACATCAGCACTGCGTGAA   240       V  E  D  E  A  A  D  Q  R  F  M  S  V  R  T  S  A  L  R  E                 CAATTTGCCTGGCTGCGCGAGAACGGTTATCAACCGGTCAGTATTGCTCAAATTCGTGAA   300       Q  F  A  W  L  R  E  N  G  Y  Q  P  V  S  I  A  Q  I  R  E                 GCACATCGAGGAGGAAAACCGCTACCGGAAAAAGCTGTAGTGCTGACTTTTGATGACGGC   360       A  H  R  G  G  K  P  L  P  E  K  A  V  V  L  T  F  D  D  G                 TACCAGAGTTTTTATACCCGCGTCTTCCCAATTCTTCAGGCCTTCCAGTGGCCTGCTGTA   420       Y  Q  S  F  Y  T  R  V  F  P  I  L  Q  A  F  Q  W  P  A  V                 TGGGCCCCCGTCGGCAGTTGGGTCGATACGCCAGCGGATAAACAAGTAAAATTTGGCGAT   480       W  A  P  V  G  S  W  V  D  T  P  A  D  K  Q  V  K  F  G  D                 GAGTTGGTCGATCGAGAATATTTTGCCACGTGGCAACAAGTGCGAGAAGTTGCGCGTTCC   540       E  L  V  D  R  E  Y  F  A  T  W  Q  Q  V  R  E  V  A  R  S                 CGGCTCGTTGAGCTCGCTTCTCATACATGGAATTCTCACTACGGTATTCAGGCTAATGCC   600       R  L  V  E  L  A  S  H  T  W  N  S  H  Y  G  I  Q  A  N  A                 ACCGGCAGCTTATTGCCTGTATATGTAAATCGTGCATATTTTACTGACCACGCACGGTAT   660       T  G  S  L  L  P  V  Y  V  N  R  A  Y  F  T  D  H  A  R  Y                 GAAACCGCAGCAGAATACCGGGAAAGAATTCGTCTGGATGCTGTAAAAATGACGGAATAC   720       E  T  A  A  E  Y  R  E  R  I  R  L  D  A  V  K  M  T  E  Y                 CTGCGTACAAAGGTTGAGGTAAATCCACACGTTTTTGTTTGGCCTTATGGCGAAGCGAAT   780       L  R  T  K  V  E  V  N  P  H  V  F  V  W  P  Y  G  E  A  N                 GGCATAGCGATAGAGGAATTAAAAAAACTCGGTTATGACATGTTCTTCACCCTTGAATCA   840       G  I  A  I  E  E  L  K  K  L  G  Y  D  M  F  F  T  L  E  S                 GGTTTGGCAAATGCGTCGCAATTGGATTCCATTCCGCGGGTATTAATCGCCAATAATCCC   900       G  L  A  N  A  S  Q  L  D  S  I  P  R  V  L  I  A  N  N  P                 TCATTAAAAGAGTTTGCCCAGCAAATTATTACCGTACAGGAAAAATCACCACAACGGATA   960       S  L  K  E  F  A  Q  Q  I  I  T  V  Q  E  K  S  P  Q  R  I                 ATGCATATCGATCTTGATTACGTTTATGACGAAAACCTCCAGCAAATGGATCGCAATATT   1020       M  H  I  D  L  D  Y  V  Y  D  E  N  L  Q  Q  M  D  R  N  I                 GATGTGCTAATTCAGCGGGTGAAAGATATGCAAATATCAACCGTGTATTTGCAGGCATTT   1080       D  V  L  I  Q  R  V  K  D  M  Q  I  S  T  V  Y  L  Q  A  F                 GCTGATCCCGATGGTGATGGGCTGGTCAAAGAGGTCTGGTTTCCAAATCGTTTGCTACCA   1140       A  D  P  D  G  D  G  L  V  K  E  V  W  F  P  N  R  L  L  P                 ATGAAAGCAGATATTTTTAGTCGGGTTGCCTGGCAATTACGTACCCGCTCAGGTGTAAAC   1200       M  K  A  D  I  F  S  R  V  A  W  Q  L  R  T  R  S  G  V  N                 ATCTATGCGTGGATGCCGGTATTAAGCTGGGATTTAGATCCCACATTAACGCGAGTAAAA   1260       I  Y  A  W  M  P  V  L  S  W  D  L  D  P  T  L  T  R  V  K         TACTTACCAACAGGGGAGAAAAAAGCACAAATTCATCCTGAACAATATCACCGTCTCTCT   1320       Y  L  P  T  G  E  K  K  A  Q  I  H  P  E  Q  Y  H  R  L  S                 CCTTTCGATGACAGAGTCAGAGCACAAGTTGGCATGTTATATGAAGATCTTGCCGGACAT   1380       P  F  D  D  R  V  R  A  Q  V  G  M  L  Y  E  D  L  A  G  H                 GCTGCTTTTGATGGCATATTGTTCCACGATGATGCTTTGCTTTCAGATTATGAAGATGCC   1440       A  A  F  D  G  I  L  F  H  D  D  A  L  L  S  D  Y  E  D  A                 AGTGCACCGGCTATCACGGCTTATCAGCAAGCAGGCTTTAGCGGGAGTCTGAGCGAAATT   1500       S  A  P  A  I  T  A  Y  Q  Q  A  G  F  S  G  S  L  S  E  I                 CGACAAAACCCGGAGCAATTTAAACAGTGGGCCCGCTTTAAAAGTCGTGCGTTAACTGAC   1560       R  Q  N  P  E  Q  F  K  Q  W  A  R  F  K  S  R  A  L  T  D                 TTCACTTTAGAACTTAGTGCGCGCGTAAAAGCCATTCGCGGTCCACATATTAAAACTGCA   1620       F  T  L  E  L  S  A  R  V  K  A  I  R  G  P  H  I  K  T  A                 CGAAATATTTTTGCACTTCCGGTAATACAACCTGAAAGTGAAGCCTGGTTTGCACAGAAT   1680       R  N  I  F  A  L  P  V  I  Q  P  E  S  E  A  W  F  A  Q  N                 TATGCTGATTTCCTAAAAAGCTATGACTGGACCGCTATTATGGCTATGCCTTATCTGGAA   1740       Y  A  D  F  L  K  S  Y  D  W  T  A  I  M  A  M  P  Y  L  E                 GGTGTCGCAGAAAAATCGGCTGACCAATGGTTAATACAATTGACCAATCAAATTAAAAAC   1800       G  V  A  E  K  S  A  D  Q  W  L  I  Q  L  T  N  Q  I  K  N                 ATCCCTCAGGCTAAAGACAAATCTATTTTAGAATTACAGGCACAAAACTGGCAGAAAAAT   1860       I  P  Q  A  K  D  K  S  I  L  E  L  Q  A  Q  N  W  Q  K  N                 GGTCAGCATCAGGCTATTTCTTCGCAACAACTCGCTCACTGGATGAGCCTATTACAACTG   1920       G  Q  H  Q  A  I  S  S  Q  Q  L  A  H  W  M  S  L  L  Q  L                 AATGGAGTGAAAAACTATGGTTATTATCCCGACAATTTTCTGCATAACCAACCTGAAATA   1980       N  G  V  K  N  Y  G  Y  Y  P  D  N  F  L  H  N  Q  P  E  I                 GACCTTATTCGTCCTGAGTTTTCAACAGCCTGGTATCCGAAAAATGATTAA   2031       D  L  I  R  P  E  F  S  T  A  W  Y  P  K  N  D  ***       (YCDR STOP CODON)       (YCDQ START CODON)               SEQ ID NO: 3                1 (ycdQ) *       AA   AAT   GATT AA TCGCATCGTATCGTTTTTTATATTATGTCTGGTGTTATGCATACCCCTA   240        M  I  N  R  I  V  S  F  F  I  L  C  L  V  L  C  I  P  L                   TGCGTAGCGTACTTTCACTCTGGTGAACTGATGATGAGGTTCGTTTTCTTCTGGCCGTTT   300       C  V  A  Y  F  H  S  G  E  L  M  M  R  F  V  F  F  W  P  F                 TTTATGTCCATTATGTGGATTGTTGGCGGCGTCTATTTCTGGGTCTATCGTGAACGCCAC   360       F  M  S  I  M  W  I  V  G  G  V  Y  F  W  V  Y  R  E  R  H                 TGGCCGTGGGGAGAAAACGCACCAGCTCCCCAGTTGAAAGATAATCCGTCTATCTCCATT   420       W  P  W  G  E  N  A  P  A  P  Q  L  K  D  N  P  S  I  S  I                 ATCATTCCCTGTTTTAATGAGGAGAAAAACGTTGAGGAAACCATACACGCCGCTTTAGCA   480       I  I  P  C  F  N  E  E  K  N  V  E  E  T  I  H  A  A  L  A                 CAGCGTTATGAGAACATTGAAGTTATTGCCGTAAATGACGGTTCAACAGATAAAACCCGT 540       Q  R  Y  E  N  I  E  V  I  A  V  N  D  G  S  T  D  K  T  R                 GCCATCCTGGATCGCATGGCTGCACAAATTCCCCATTTGCGGGTCATTCATCTGGCGCAA   600       A  I  L  D  R  M  A  A  Q  I  P  H  L  R  V  I  H  L  A  Q                 AACCAGGGGAAAGCCATTGCGCTTAAAACCGGAGCTGCCGCGGCGAAAAGTGAATATCTG   660       N  Q  G  K  A  I  A  L  K  T  G  A  A  A  A  K  S  E  Y  L                 GTGTGCATTGATGGCGATGCGTTATTAGACCGCGATGCGGCGGCATATATTGTGGAACCG   720       V  C  I  D  G  D  A  L  L  D  R  D  A  A  A  Y  I  V  E  P                 ATGTTGTACAACCCGCGTGTGGGTGCCGTAACCGGTAATCCTCGTATTCGAACACGTTCT   780       M  L  Y  N  P  R  V  G  A  V  T  G  N  P  R  I  R  T  R  S                 ACCCTGGTGGGTAAAATTCAGGTTGGCGAGTATTCCTCAATTATTGGTTTGATCAAGCGA   840       T  L  V  G  K  I  Q  V  G  E  Y  S  S  I  I  G  L  I  K  R                 ACCCAGCGTATCTATGGAAACGTATTTACCGTTTCCGGTGTTATTGCCGCATTTCGTCGC   900       T  Q  R  I  Y  G  N  V  F  T  V  S  G  V  I  A  A  F  R  R                 AGCGCCCTGGCAGAAGTGGGTTACTGGAGTGACGATATGATCACCGAAGATATTGATATT   960       S  A  L  A  E  V  G  Y  W  S  D  D  M  I  T  E  D  I  D  I                 AGCTGGAAGCTGCAGTTGAATCAGTGGACGATTTTTTACGAGCCACGGGCACTGTGCTGG   1020       S  W  K  L  Q  L  N  Q  W  T  I  F  Y  E ↓  P  R  A  L  C  W                 ATATTAATGCCTGAAACGTTAAAAGGGCTGTGGAAACAGCGC CT GCGCTGGGCTCAGGGC   1080       I  L  M  P  E  T  L  K  G  L  W  K  Q  R  L  R  W  A  Q  G                 GGTGCAGAAGTATTCCTCAAAAATATGACAAGGTTGTGGCGCAAAGAAAACTTTCGAATG   1140       G  A  E  V  F  L  K  N  M  T  R  L  W  R  K  E  N  F  R  M                 TGGCCGCTGTTTTTTGAATACTGCCTGACGACAATATGGGCCTTCACCTGCCTGGTCGGT   1200       W  P  L  F  F  E  Y  C  L  T  T  I  W  A  F  T  C  L  V  G                 TTCATTATTTACGCAGTCCAACTTGCCGGTGTACCGTTAAATATTGAATTGACACATATC   1260       F  I  I  Y  A  V  Q  L  A  G  V  P  L  N  I  E  L  T  H  I                 GCTGCGACACATACTGCCGGAATATTATTGTGTACGTTATGTTTACTGCAATTTATTGTC   1320       A  A  T  H  T  A  G  I  L  L  C  T  L  C  L  L  Q  F  I  V                 AGCCTGATGATCGAGAATCGCTATGAGCATAATCTGACTTCATCGCTTTTCTGGATTATT   1380       S  L  M  I  E  N  R  Y  E  H  N  L  T  S  S  L  F  W  I  I                 TGGTTCCCGGTTATTTTCTGGATGCTGAGCCTGGCAACGACATTGGTATCATTTACACGA   1440       W  F  P  V  I  F  W  M  L  S  L  A  T  T  L  V  S  F  T  R                 GTCATGTTGATGCCTAAAAAGCAACGCGCCCGTTGGGTAAGTCCCGATCGCGGGATTCTG   1500       V  M  L  M  P  K  K  Q  R  A  R  W  V  S  P  D  R  G  I  L                 AGAGGT TAA T ATG AACAATTTAATTATTACGACCCGACAATCACCAGTACGTTTACTGGT   1560       R  G  *   M  N  N   L(ycdp)               SEQ ID NO: 6               ycdS(+1)         ATG TATTCAAGTAGCAGAAAAAGGTGCCCGAAAACCAAATGGGCTTTGAAACTTCTTACT               GCCGCATTTTTAGCAGCGAGTCCCGCGGCGAAGAGTGCTGTTAATAACGCCTATGATGCA               TTGATTATTGAAGCTCGCAAGGGTAATACTCAGCCAGCTTTGTCATGGTTTGCACTAAAA               TCAGCACTCAGCAATAACCAAATTGCTGACTGGTTACAGATTGCCTTATGGGCCGGGCAA               GATAAACAGGTTATTACCGTTTACAACCGCTACCGTCATCAGCAATTACCAGCGCGTGGT   300               TATGCAGCTGTCGCCGTCGCTTATCGTAACCTGCAACAATGGCAAAACTCGCTTACACTG                  389                                                     TGGCAAAAGGCGCTCTCTCTGGA GCC G C         AAA TAAGGATTATCAACGGGGACAAATTTTA               ACCCTGGCAGATGCTGGTCACTATGATACTGCGCTGGTTAAACTTAAGCAGCTTAACTCT               GGAGCACCGGACAAAGCCAATTTACTCGCAGAAGCCTATATCTATAAACTGGCGGGGCGT                      583                                                 CATCAGGATGAATTACGGGCGATGACAGAGTCATTA CCT GAA z,801 AT GCATCTACGCAACAA   600               TATCCCACAGAATACGTGCAGGCATTACGTAATAATCAACTTGCTGCCGCGATTGACGAT                ↓                                                         GCCAATTTAAC GC CAGATATTCGCGCTGATATTCATGCCGAACTGGTCAGACTGTCGTTT               ATGCCTACGCGCAGTGAAAGTGAACGTTATGCCATTGCCGATCGCGCCCTCGCCCAATAC               GCTGCATTAGAAATTCTGTGGCACGATAACCCAGACCGCACTGCCCAGTACCAGCGTATT               CAGGTTGATCATCTTGGCGCGTTATTAACTCGCGATCGTTATAAAGACGTTATTTCTCAC   900               TATCAGCGATTAAAAAAGACGGGGCAAATTATTCCGCCCTGGGGGCAATATTGGGTTGCA               TCGGCTTATCTCAAAGATCATCAGCCGAAAAAAGCACAGTCAATAATGACCGAGCTCTTT               TATCACAAGGAGACCATTGCCCCGGATTTTATCCGATGAAGAACTTGCGGATCTCTTTTAC               AGCCACCTGGAGAGTGAAAATTATCCGGGCGCGCTAACTGTCACCCAACATACCATTAAT               ACTTCGCCGCCTTTCCTTCGGTTAATGGGCACGCCTACGAGCATCCCGAATGATACCTGG   1200               TTACAGGGGCATTCGTTTCTCTCAACCGTAGCAAAATATAGTAATGATCTTCCTCAGGCT               GAAATGACAGCCAGAGAGCTTGCTTATAACGCACCAGGAAATCAGGGACTGCGCATTGAT               TACGCGAGTGTGTTACAAGCCCGCGGTTGGCCTCGTGCAGCAGAAAATGAATTAAAAAAA               GCAGAAGTGATCGAGCCACGTAATATTAATCTGGAGGTTGAACAAGCCTGGACAGCATTA               ACGTTACAAGAATGGCAGCAGGCAGCTGTCTTAACGCACGATGTTGTCGAACGTGAACCG   1500               CAAGATCCCGGCGTTGTACGATTAAAACGTGCGGTTGATGTACATAATCTTGCAGAGCTT               CGTATCGCTGGCTCAACAGGAATTGATGCCGAAGGCCCGGATAGTGGTAAACATGATGTC               GACTTAACCACCATCGTTTATTCACCACCGCTGAAGGATAACTGGCGCGGTTTTGCTGGA               TTCGGTTATGCCGATGGACAATTTAGCGAAGGAAAAGGGATTGTTCGCGACTGGCTTGCG               GGTGTTGAGTGGCGGTCACGTAATATCTGGCTCGAGGCAGAGTACGCTGAACGCGTTTTC   1800               AATCATGAGCATAAACCCGGCGCGCGCCTGTCTGGCTGGTATGATTTTAATGATAACTGG               CGTATTGGTTCGCAACTGGAACGCCTCTCTCACCGCGTTCCATTACGGGCAATGAAAAAT               GGTGTTACAGGCAACAGTGCTCAGGCTTATGTTCGCTGGTATCAAAATGAGCGGCGTAAG               TACGGTGTCTCCTGGGCTTTCACTGATTTTTCCGACAGTAACCAGCGTCATGAAGTCTCA               CTTGAGGGTCAGGAACGCATCTGGTCTTCACCATATTTGATTGTCGATTTCCTACCCAGT   2100               CTGTATTACGAACAAAATACAGAACACGATACCCCATACTACAACCCTATAAAAACGTTC               GATATTGTTCCGGCATTTGAGGCAAGCCATTTGTTATGGCGAAGCTATGAAAATAGCTGG               GAGCAAATATTCAGCGCAGGTGTTGGTGCCTCCTGGCAAAAACATTATGGCACGGATGTC               GTCACCCAACTCGGCTACGGGCAACGCATTAGTTGGAATGACGTGATTGATGCTGGCGCA               ACGCTACGCTGGGAAAAACGACCTTATGACGGTGACAGAGAACACAACTTATACGTTGAA   2400                  ycdR(+1)                                             TTCGATATGACATTCAGATTT TAA GGATAAAT ATG TTACGTAATGGAAATAAATATCTCC               TGATGCTGGTGAGTATAATTATGCTCACCGCGTGCATTAGCCAGTCAAGAACATCATTTA               TACCGCCACAGGATCGCGAATCTTTACTCGCCGAGCAACCGTGGCCGCATAATGGTTTTG               TAGCGATTTCATGGCATAACGTTGAAGACGAAGCTGCCGACCAGCGTTTTATGTCAGTGC               GGACATCAGCACTGCGTGAACAATTTGCCTGGCTGCGCGAGAACGGTTATCAACCGGTCA   2700               GTATTGCTCAAATTCGTGAAGCACATCGAGGAGGAAAACCGCTACCGGAAAAAGCTGTAG               TGCTGACTTTTGATGACGGCTACCAGAGTTTTTATACCCGCGTCTTCCCAATTCTTCAGG               CCTTCCAGTGGCCTGCTGTATGGGCCCCCGTCGGCAGTTGGGTCGATACGCCAGCGGATA               AACAAGTAAAATTTGGCGATGAGTTGGTCGATCGAGAATATTTTGCCACGTGGCAACAAG        ↓                                                                 T GC GAGAAGTTGCGCGTTCCCGGCTCGTTGAGCTCGCTTCTCATACATGGAATTCTCACT   3000               ACGGTATTCAGGCTAATGCCACCGGCAGCTTATTGCCTGTATATGTAAATCGTGCATATT               TTACTGACCACGCACGGTATGAAACCGCAGCAGAATACCGGGAAAGAATTCGTCTGGATG                   723                                                    CTGTAAAAATGACGGAATACCTGCGTAC AAAG G T         G AGGTAAATCCACACGTTTTTGTTT               GGCCTTATGGCGAAGCGAATGGCATAGCGATAGAGGAATTAAAAAAACTCGGTTATGACA               TGTTCTTCACCCTTGAATCAGGTTTGGCAAATGCGTCGCAATTGGATTCCATTCCGCGGG   3300               TATTAATCGCCAATAATCCCTCATTAAAAGAGTTTGCCCAGCAAATTATTACCGTACAGG               AAAAATCACCACAACGGATAATGCATATCGATCTTGATTACGTTTATGACGAAAACCTCC               AGCAAATGGATCGCAATATTGATGTGCTAATTCAGCGGGTGAAAGATATGCAAATATCAA               CCGTGTATTTGCAGGCATTTGCTGATCCCGATGGTGATGGGCTGGTCAAAGAGGTCTGGT               TTCCAAATCGTTTGCTACCAATGAAAGCAGATATTTTTAGTCGGGTTGCCTGGCAATTAC   3600               GTACCCGCTCAGGTGTAAACATCTATGCGTGGATGCCGGTATTAAGCTGGGATTTAGATC               CCACATTAACGCGAGTAAAATACTTACCAACAGGGGAGAAAAAAGCACAAATTCATCCTG               AACAATATCACCGTCTCTCTCCTTTCGATGACAGAGTCAGAGCACAAGTTGGCATGTTAT               ATGAAGATCTTGCCGGACATGCTGCTTTTGATGGCATATTGTTCCACGATGATGCTTTGC               TTTCAGATTATGAAGATGCCAGTGCACCGGCTATCACGGCTTATCAGCAAGCAGGCTTTA   3900               GCGGGAGTCTGAGCGAAATTCGACAAAACCCGGAGCAATTTAAACAGTGGGCCCGCTTTA               AAAGTCGTGCGTTAACTGACTTCACTTTAGAACTTAGTGCGCGCGTAAAAGCCATTCGCG               GTCCACATATTAAAACTGCACGAAATATTTTTGCACTTCCGGTAATACAACCTGAAAGTG               AAGCCTGGTTTGCACAGAATTATGCTGATTTCCTAAAAAGCTATGACTGGACCGCTATTA               TGGCTATGCCTTATCTGGAAGGTGTCGCAGAAAAATCGGCTGACCAATGGTTAATACAAT   4200               TGACCAATCAAATTAAAAACATCCCTCAGGCTAAAGACAAATCTATTTTAGAATTACAGG               CACAAAACTGGCAGAAAAATGGTCAGCATCAGGCTATTTCTTCGCAACAACTCGCTCACT               GGATGAGCCTATTACAACTGAATGGAGTGAAAAACTATGGTTATTATCCCGACAATTTTC               TGCATAACCAACCTGAAATAGACCTTATTCGTCCTGAGTTTTCAACAGCCTGGTATCCGA        ycdQ(+1)                                                       AAA ATG AT TAA TCGCATCGTATCGTTTTTTATATTATGTCTGGTGTTATGCATACCCCTA   4500               TGCGTAGCGTACTTTCACTCTGGTGAACTGATGATGAGGTTCGTTTTCTTCTGGCCGTTT               TTTATGTCCATTATGTGGATTGTTGGCGGCGTCTATTTCTGGGTCTATCGTGAACGCCAC               TGGCCGTGGGGAGAAAACGCACCAGCTCCCCAGTTGAAAGATAATCCGTCTATCTCCATT               ATCATTCCCTGTTTTAATGAGGAGAAAAACGTTGAGGAAACCATACACGCCGCTTTAGCA               CAGCGTTATGAGAACATTGAAGTTATTGCCGTAAATGACGGTTCAACAGATAAAACCCGT   4800               GCCATCCTGGATCGCATGGCTGCACAAATTCCCCATTTGCGGGTCATTCATCTGGCGCAA               AACCAGGGGAAAGCCATTGCGCTTAAAACCGGAGCTGCCGCGGCGAAAAGTGAATATCTG               GTGTGCATTGATGGCGATGCGTTATTAGACCGCGATGCGGCGGCATATATTGTGGAACCG               ATGTTGTACAACCCGCGTGTGGGTGCCGTAACCGGTAATCCTCGTATTCGAACACGTTCT               ACCCTGGTGGGTAAAATTCAGGTTGGCGAGTATTCCTCAATTATTGGTTTGATCAAGCGA   5100               ACCCAGCGTATCTATGGAAACGTATTTACCGTTTCCGGTGTTATTGCCGCATTTCGTCGC               AGCGCCCTGGCAGAAGTGGGTTACTGGAGTGACGATATGATCACCGAAGATATTGATATT               AGCTGGAAGCTGCAGTTGAATCAGTGGACGATTTTTTACGAGCCACGGGCACTGTGCTGG                                           ↓                              ATATTAATGCCTGAAACGTTAAAAGGGCTGTGGAAACAGCGC CT GCGCTGGGCTCAGGGC               GGTGCAGAAGTATTCCTCAAAAATATGACAAGGTTGTGGCGCAAAGAAAACTTTCGAATG   5400               TGGCCGCTGTTTTTTGAATACTGCCTGACGACAATATGGGCCTTCACCTGCCTGGTCGGT               TTCATTATTTACGCAGTCCAACTTGCCGGTGTACCGTTAAATATTGAATTGACACATATC               GCTGCGACACATACTGCCGGAATATTATTGTGTACGTTATGTTTACTGCAATTTATTGTC               AGCCTGATGATCGAGAATCGCTATGAGCATAATCTGACTTCATCGCTTTTCTGGATTATT               TGGTTCCCGGTTATTTTCTGGATGCTGAGCCTGGCAACGACATTGGTATCATTTACACGA   5700               GTCATGTTGATGCCTAAAAAGCAACGCGCCCGTTGGGTAAGTCCCGATCGCGGGATTCTG           ycdP(+1)                                                    AGAGGT TAA T ATG AACAATTTAATTATTACGACCCGACAATCACCAGTACGTTTACTGGT               TGATTATGTTGCCACAACCATCTTGTGGACATTATTTGCGTTGTTCATATTCTTATTCGC                                      ↓                                   CATGGATCTGCTGACGGGTTATTACTGGCAAAGCG AG GCCAGAAGCCGACTTCAGTTCTA               TTTTTTGCTGGCAGTGGCGAATGCCGTCGTGTTAATTGTCTGGGCGCTGTACAATAAGCT   6000               GCGTTTTCAAAAACAGCAGCATCATGCAGCCTACCAATATACGCCGCAAGAATATGCAGA               GAGCTTAGCAATACCTGATGAGCTCTATCAGCAACTACAAAAAAGCCACAGGATGAGCGT               ACACTTCACCAGCCAGGGGCAAATAAAAATGGTTGTTTCAGAAAAAGCGCTAGTCCGGGC               A TAA ACACCCAAAACAAAGCCCGGTTCGCCCGGGCTCTGCACCGATAACACACTTAACTG               TAGGCATGCAGCGTACGTTGGCAAAGTGCCGAACGTACGCAGTCCTCTTTACCGAACCGG   6300               ACGATCCCAACCATTTCATCTTCTTCGAAACGTTCCAGCGCGTCACTTAATCCGGAGCAC               ACGCCGCGAGGCAAATCGCATTGCGTGATATCACCGTTGACGATAACCGTCACGTTCTCC               CCGAGGCGGGTTAAAAACATTTTCATTTGCGCGGCAGTCACATTCTGCGCCTCGTCAAGA               ATGACGACTGCATTTTCAAAGGTACGTCCACGCATATAGGCGAACGGCGCAATTTCCACC               TTCCCTATTTCCGGTCGCAGGCAGTACTGCATAAAGGAAGCCCCTAAGCGCCGGACCAGC   6600               ACGTCGTAGACCGGGCGAAAATAGGGAGCAAACTTTTCTGCGATATCTCCAGGTAAGAAG               CCAAGATCTTCATCGGCTTGCAGAACTGGACGGGTGACGATAATCCTGTCGACATCCTTA               TGTATCAGGGCCTCTGCCGCTTTTGCTGCGCTGATCCAGGTTTTTCCGCACCCGGCTTCG               CCCGTGGCGAATATCAGCTGCTTACTCTCAATAGCCTTCAGATAGTGCAATTGCGCTTCA               TTTCGCGCGAGGATGGGCGAAGTATCGCGACTGTCGCGGGCCATACCAATGGCTTCTACG   6900               CCGCCCATCTGCACAAGCGAGGTGACCGATTCTTCTTCACGCTGCTTATGGCTGCGCGAA               TCCCGTCTCAGCACACGTTTTGCCTCGCGACGAGCTTTGATCACTGCTTTTTGTCTTCCC               ATGGAGAGCACCTTGAGTTGTTTGTATTCATCACACGCGCCGTTGGCAGCGCGATTATGC               GCACGAACATCAGAGGGTTGGCTTCCTTGTAAGCCATAGTTTGCTTTTGGATAAAATGCC               GAAAAACGGCTACGCGCACCGTTTACGGCGTCGGTAACACATGAAAAGAAAGGATGAGGT   7200               TGAAAATGCAAAGTGACGAGATGACTACCGGAGGAGAAAACTCCGCGAGTGGTGGCGCGT               TGATTATCTAAAACATGTCCAGTACAGGACGTTACCATCCGCGATCTCCATAGTGACTGA               CTATCACTGCCGGGAACTTCCGCTGCTACTTAATAAGTACAACAGATCTCGCATTTATTG               CAACAATATATTTACTTATATTTAACT ATAA AACACCATTTCAGTGACATTAGTTTCTAC               TGGAAAGATGACAGAGTGATGACAGTGATGAAAAAAGCTGTGTGCTTTCAGCAGGATTTG   7500                                          
 
         [0108]    Mutations in cloned ycd operon carried by pUCPGA372. One mutation is in the ycdR gene, in which nucleotide 723 was changed from T to C, and the codon was changed from TTG (Leu) to TCG (Ser). The other two mutations are in ycdS gene, in which nucleotide 583 and 389 were changed from A to G, and the codons were changed from AAT (Asn) to GAT (Asp), and CAA (Gin) to CGA (Arg) respectively.  
     
       
       
         1 
         
           
             9  
           
           
             1  
             2460  
             DNA  
             Escherichia coli  
           
            1 

atgtattcaa gtagcagaaa aaggtgcccg aaaaccaaat gggctttgaa acttcttact     60 

gccgcatttt tagcagcgag tcccgcggcg aagagtgctg ttaataacgc ctatgatgca    120 

ttgattattg aagctcgcaa gggtaatact cagccagctt tgtcatggtt tgcactaaaa    180 

tcagcactca gcaataacca aattgctgac tggttacaga ttgccttatg ggccgggcaa    240 

gataaacagg ttattaccgt ttacaaccgc taccgtcatc agcaattacc agcgcgtggt    300 

tatgcagctg tcgccgtcgc ttatcgtaac ctgcaacaat ggcaaaactc gcttacactg    360 

tggcaaaagg cgctctctct ggagccgcaa aataaggatt atcaacgggg acaaatttta    420 

accctggcag atgctggtca ctatgatact gcgctggtta aacttaagca gcttaactct    480 

ggagcaccgg acaaagccaa tttactcgca gaagcctata tctataaact ggcggggcgt    540 

catcaggatg aattacgggc gatgacagag tcattacctg aaaatgcatc tacgcaacaa    600 

tatcccacag aatacgtgca ggcattacgt aataatcaac ttgctgccgc gattgacgat    660 

gccaatttaa cgccagatat tcgcgctgat attcatgccg aactggtcag actgtcgttt    720 

atgcctacgc gcagtgaaag tgaacgttat gccattgccg atcgcgccct cgcccaatac    780 

gctgcattag aaattctgtg gcacgataac ccagaccgca ctgcccagta ccagcgtatt    840 

caggttgatc atcttggcgc gttattaact cgcgatcgtt ataaagacgt tatttctcac    900 

tatcagcgat taaaaaagac ggggcaaatt attccgccct gggggcaata ttgggttgca    960 

tcggcttatc tcaaagatca tcagccgaaa aaagcacagt caataatgac cgagctcttt   1020 

tatcacaagg agaccattgc cccggattta tccgatgaag aacttgcgga tctcttttac   1080 

agccacctgg agagtgaaaa ttatccgggc gcgctaactg tcacccaaca taccattaat   1140 

acttcgccgc ctttccttcg gttaatgggc acgcctacga gcatcccgaa tgatacctgg   1200 

ttacaggggc attcgtttct ctcaaccgta gcaaaatata gtaatgatct tcctcaggct   1260 

gaaatgacag ccagagagct tgcttataac gcaccaggaa atcagggact gcgcattgat   1320 

tacgcgagtg tgttacaagc ccgcggttgg cctcgtgcag cagaaaatga attaaaaaaa   1380 

gcagaagtga tcgagccacg taatattaat ctggaggttg aacaagcctg gacagcatta   1440 

acgttacaag aatggcagca ggcagctgtc ttaacgcacg atgttgtcga acgtgaaccg   1500 

caagatcccg gcgttgtacg attaaaacgt gcggttgatg tacataatct tgcagagctt   1560 

cgtatcgctg gctcaacagg aattgatgcc gaaggcccgg atagtggtaa acatgatgtc   1620 

gacttaacca ccatcgttta ttcaccaccg ctgaaggata actggcgcgg ttttgctgga   1680 

ttcggttatg ccgatggaca atttagcgaa ggaaaaggga ttgttcgcga ctggcttgcg   1740 

ggtgttgagt ggcggtcacg taatatctgg ctcgaggcag agtacgctga acgcgttttc   1800 

aatcatgagc ataaacccgg cgcgcgcctg tctggctggt atgattttaa tgataactgg   1860 

cgtattggtt cgcaactgga acgcctctct caccgcgttc cattacgggc aatgaaaaat   1920 

ggtgttacag gcaacagtgc tcaggcttat gttcgctggt atcaaaatga gcggcgtaag   1980 

tacggtgtct cctgggcttt cactgatttt tccgacagta accagcgtca tgaagtctca   2040 

cttgagggtc aggaacgcat ctggtcttca ccatatttga ttgtcgattt cctacccagt   2100 

ctgtattacg aacaaaatac agaacacgat accccatact acaaccctat aaaaacgttc   2160 

gatattgttc cggcatttga ggcaagccat ttgttatggc gaagctatga aaatagctgg   2220 

gagcaaatat tcagcgcagg tgttggtgcc tcctggcaaa aacattatgg cacggatgtc   2280 

gtcacccaac tcggctacgg gcaacgcatt agttggaatg acgtgattga tgctggcgca   2340 

acgctacgct gggaaaaacg accttatgac ggtgacagag aacacaactt atacgttgaa   2400 

ttcgatatga cattcagatt ttaaggataa atatgttacg taatggaaat aaatatctcc   2460 

 
           
             2  
             807  
             PRT  
             Escherichia coli  
           
            2 

Met Tyr Ser Ser Ser Arg Lys Arg Cys Pro Lys Thr Lys Trp Ala Leu 
1               5                   10                  15 

Lys Leu Leu Thr Ala Ala Phe Leu Ala Ala Ser Pro Ala Ala Lys Ser 
            20                  25                  30 

Ala Val Asn Asn Ala Tyr Asp Ala Leu Ile Ile Glu Ala Arg Lys Gly 
        35                  40                  45 

Asn Thr Gln Pro Ala Leu Ser Trp Phe Ala Leu Lys Ser Ala Leu Ser 
    50                  55                  60 

Asn Asn Gln Ile Ala Asp Trp Leu Gln Ile Ala Leu Trp Ala Gly Gln 
65                  70                  75                  80 

Asp Lys Gln Val Ile Thr Val Tyr Asn Arg Tyr Arg His Gln Gln Leu 
                85                  90                  95 

Pro Ala Arg Gly Tyr Ala Ala Val Ala Val Ala Tyr Arg Asn Leu Gln 
            100                 105                 110 

Gln Trp Gln Asn Ser Leu Thr Leu Trp Gln Lys Ala Leu Ser Leu Glu 
        115                 120                 125 

Pro Gln Asn Lys Asp Tyr Gln Arg Gly Gln Ile Leu Thr Leu Ala Asp 
    130                 135                 140 

Ala Gly His Tyr Asp Thr Ala Leu Val Lys Leu Lys Gln Leu Asn Ser 
145                 150                 155                 160 

Gly Ala Pro Asp Lys Ala Asn Leu Leu Ala Glu Ala Tyr Ile Tyr Lys 
                165                 170                 175 

Leu Ala Gly Arg His Gln Asp Glu Leu Arg Ala Met Thr Glu Ser Leu 
            180                 185                 190 

Pro Glu Asn Ala Ser Thr Gln Gln Tyr Pro Thr Glu Tyr Val Gln Ala 
        195                 200                 205 

Leu Arg Asn Asn Gln Leu Ala Ala Ala Ile Asp Asp Ala Asn Leu Thr 
    210                 215                 220 

Pro Asp Ile Arg Ala Asp Ile His Ala Glu Leu Val Arg Leu Ser Phe 
225                 230                 235                 240 

Met Pro Thr Arg Ser Glu Ser Glu Arg Tyr Ala Ile Ala Asp Arg Ala 
                245                 250                 255 

Leu Ala Gln Tyr Ala Ala Leu Glu Ile Leu Trp His Asp Asn Pro Asp 
            260                 265                 270 

Arg Thr Ala Gln Tyr Gln Arg Ile Gln Val Asp His Leu Gly Ala Leu 
        275                 280                 285 

Leu Thr Arg Asp Arg Tyr Lys Asp Val Ile Ser His Tyr Gln Arg Leu 
    290                 295                 300 

Lys Lys Thr Gly Gln Ile Ile Pro Pro Trp Gly Gln Tyr Trp Val Ala 
305                 310                 315                 320 

Ser Ala Tyr Leu Lys Asp His Gln Pro Lys Lys Ala Gln Ser Ile Met 
                325                 330                 335 

Thr Glu Leu Phe Tyr His Lys Glu Thr Ile Ala Pro Asp Leu Ser Asp 
            340                 345                 350 

Glu Glu Leu Ala Asp Leu Phe Tyr Ser His Leu Glu Ser Glu Asn Tyr 
        355                 360                 365 

Pro Gly Ala Leu Thr Val Thr Gln His Thr Ile Asn Thr Ser Pro Pro 
    370                 375                 380 

Phe Leu Arg Leu Met Gly Thr Pro Thr Ser Ile Pro Asn Asp Thr Trp 
385                 390                 395                 400 

Leu Gln Gly His Ser Phe Leu Ser Thr Val Ala Lys Tyr Ser Asn Asp 
                405                 410                 415 

Leu Pro Gln Ala Glu Met Thr Ala Arg Glu Leu Ala Tyr Asn Ala Pro 
            420                 425                 430 

Gly Asn Gln Gly Leu Arg Ile Asp Tyr Ala Ser Val Leu Gln Ala Arg 
        435                 440                 445 

Gly Trp Pro Arg Ala Ala Glu Asn Glu Leu Lys Lys Ala Glu Val Ile 
    450                 455                 460 

Glu Pro Arg Asn Ile Asn Leu Glu Val Glu Gln Ala Trp Thr Ala Leu 
465                 470                 475                 480 

Thr Leu Gln Glu Trp Gln Gln Ala Ala Val Leu Thr His Asp Val Val 
                485                 490                 495 

Glu Arg Glu Pro Gln Asp Pro Gly Val Val Arg Leu Lys Arg Ala Val 
            500                 505                 510 

Asp Val His Asn Leu Ala Glu Leu Arg Ile Ala Gly Ser Thr Gly Ile 
        515                 520                 525 

Asp Ala Glu Gly Pro Asp Ser Gly Lys His Asp Val Asp Leu Thr Thr 
    530                 535                 540 

Ile Val Tyr Ser Pro Pro Leu Lys Asp Asn Trp Arg Gly Phe Ala Gly 
545                 550                 555                 560 

Phe Gly Tyr Ala Asp Gly Gln Phe Ser Glu Gly Lys Gly Ile Val Arg 
                565                 570                 575 

Asp Trp Leu Ala Gly Val Glu Trp Arg Ser Arg Asn Ile Trp Leu Glu 
            580                 585                 590 

Ala Glu Tyr Ala Glu Arg Val Phe Asn His Glu His Lys Pro Gly Ala 
        595                 600                 605 

Arg Leu Ser Gly Trp Tyr Asp Phe Asn Asp Asn Trp Arg Ile Gly Ser 
    610                 615                 620 

Gln Leu Glu Arg Leu Ser His Arg Val Pro Leu Arg Ala Met Lys Asn 
625                 630                 635                 640 

Gly Val Thr Gly Asn Ser Ala Gln Ala Tyr Val Arg Trp Tyr Gln Asn 
                645                 650                 655 

Glu Arg Arg Lys Tyr Gly Val Ser Trp Ala Phe Thr Asp Phe Ser Asp 
            660                 665                 670 

Ser Asn Gln Arg His Glu Val Ser Leu Glu Gly Gln Glu Arg Ile Trp 
        675                 680                 685 

Ser Ser Pro Tyr Leu Ile Val Asp Phe Leu Pro Ser Leu Tyr Tyr Glu 
    690                 695                 700 

Gln Asn Thr Glu His Asp Thr Pro Tyr Tyr Asn Pro Ile Lys Thr Phe 
705                 710                 715                 720 

Asp Ile Val Pro Ala Phe Glu Ala Ser His Leu Leu Trp Arg Ser Tyr 
                725                 730                 735 

Glu Asn Ser Trp Glu Gln Ile Phe Ser Ala Gly Val Gly Ala Ser Trp 
            740                 745                 750 

Gln Lys His Tyr Gly Thr Asp Val Val Thr Gln Leu Gly Tyr Gly Gln 
        755                 760                 765 

Arg Ile Ser Trp Asn Asp Val Ile Asp Ala Gly Ala Thr Leu Arg Trp 
    770                 775                 780 

Glu Lys Arg Pro Tyr Asp Gly Asp Arg Glu His Asn Leu Tyr Val Glu 
785                 790                 795                 800 

Phe Asp Met Thr Phe Arg Phe 
                805 

 
           
             3  
             2031  
             DNA  
             Escherichia coli  
           
            3 

ttaaggataa atatgttacg taatggaaat aaatatctcc tgatgctggt gagtataatt     60 

atgctcaccg cgtgcattag ccagtcaaga acatcattta taccgccaca ggatcgcgaa    120 

tctttactcg ccgagcaacc gtggccgcat aatggttttg tagcgatttc atggcataac    180 

gttgaagacg aagctgccga ccagcgtttt atgtcagtgc ggacatcagc actgcgtgaa    240 

caatttgcct ggctgcgcga gaacggttat caaccggtca gtattgctca aattcgtgaa    300 

gcacatcgag gaggaaaacc gctaccggaa aaagctgtag tgctgacttt tgatgacggc    360 

taccagagtt tttatacccg cgtcttccca attcttcagg ccttccagtg gcctgctgta    420 

tgggcccccg tcggcagttg ggtcgatacg ccagcggata aacaagtaaa atttggcgat    480 

gagttggtcg atcgagaata ttttgccacg tggcaacaag tgcgagaagt tgcgcgttcc    540 

cggctcgttg agctcgcttc tcatacatgg aattctcact acggtattca ggctaatgcc    600 

accggcagct tattgcctgt atatgtaaat cgtgcatatt ttactgacca cgcacggtat    660 

gaaaccgcag cagaataccg ggaaagaatt cgtctggatg ctgtaaaaat gacggaatac    720 

ctgcgtacaa aggttgaggt aaatccacac gtttttgttt ggccttatgg cgaagcgaat    780 

ggcatagcga tagaggaatt aaaaaaactc ggttatgaca tgttcttcac ccttgaatca    840 

ggtttggcaa atgcgtcgca attggattcc attccgcggg tattaatcgc caataatccc    900 

tcattaaaag agtttgccca gcaaattatt accgtacagg aaaaatcacc acaacggata    960 

atgcatatcg atcttgatta cgtttatgac gaaaacctcc agcaaatgga tcgcaatatt   1020 

gatgtgctaa ttcagcgggt gaaagatatg caaatatcaa ccgtgtattt gcaggcattt   1080 

gctgatcccg atggtgatgg gctggtcaaa gaggtctggt ttccaaatcg tttgctacca   1140 

atgaaagcag atatttttag tcgggttgcc tggcaattac gtacccgctc aggtgtaaac   1200 

atctatgcgt ggatgccggt attaagctgg gatttagatc ccacattaac gcgagtaaaa   1260 

tacttaccaa caggggagaa aaaagcacaa attcatcctg aacaatatca ccgtctctct   1320 

cctttcgatg acagagtcag agcacaagtt ggcatgttat atgaagatct tgccggacat   1380 

gctgcttttg atggcatatt gttccacgat gatgctttgc tttcagatta tgaagatgcc   1440 

agtgcaccgg ctatcacggc ttatcagcaa gcaggcttta gcgggagtct gagcgaaatt   1500 

cgacaaaacc cggagcaatt taaacagtgg gcccgcttta aaagtcgtgc gttaactgac   1560 

ttcactttag aacttagtgc gcgcgtaaaa gccattcgcg gtccacatat taaaactgca   1620 

cgaaatattt ttgcacttcc ggtaatacaa cctgaaagtg aagcctggtt tgcacagaat   1680 

tatgctgatt tcctaaaaag ctatgactgg accgctatta tggctatgcc ttatctggaa   1740 

ggtgtcgcag aaaaatcggc tgaccaatgg ttaatacaat tgaccaatca aattaaaaac   1800 

atccctcagg ctaaagacaa atctatttta gaattacagg cacaaaactg gcagaaaaat   1860 

ggtcagcatc aggctatttc ttcgcaacaa ctcgctcact ggatgagcct attacaactg   1920 

aatggagtga aaaactatgg ttattatccc gacaattttc tgcataacca acctgaaata   1980 

gaccttattc gtcctgagtt ttcaacagcc tggtatccga aaaatgatta a            2031 

 
           
             4  
             672  
             PRT  
             Escherichia coli  
           
            4 

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

Met Leu Thr Ala Cys Ile Ser Gln Ser Arg Thr Ser Phe Ile Pro Pro 
            20                  25                  30 

Gln Asp Arg Glu Ser Leu Leu Ala Glu Gln Pro Trp Pro His Asn Gly 
        35                  40                  45 

Phe Val Ala Ile Ser Trp His Asn Val Glu Asp Glu Ala Ala Asp Gln 
    50                  55                  60 

Arg Phe Met Ser Val Arg Thr Ser Ala Leu Arg Glu Gln Phe Ala Trp 
65                  70                  75                  80 

Leu Arg Glu Asn Gly Tyr Gln Pro Val Ser Ile Ala Gln Ile Arg Glu 
                85                  90                  95 

Ala His Arg Gly Gly Lys Pro Leu Pro Glu Lys Ala Val Val Leu Thr 
            100                 105                 110 

Phe Asp Asp Gly Tyr Gln Ser Phe Tyr Thr Arg Val Phe Pro Ile Leu 
        115                 120                 125 

Gln Ala Phe Gln Trp Pro Ala Val Trp Ala Pro Val Gly Ser Trp Val 
    130                 135                 140 

Asp Thr Pro Ala Asp Lys Gln Val Lys Phe Gly Asp Glu Leu Val Asp 
145                 150                 155                 160 

Arg Glu Tyr Phe Ala Thr Trp Gln Gln Val Arg Glu Val Ala Arg Ser 
                165                 170                 175 

Arg Leu Val Glu Leu Ala Ser His Thr Trp Asn Ser His Tyr Gly Ile 
            180                 185                 190 

Gln Ala Asn Ala Thr Gly Ser Leu Leu Pro Val Tyr Val Asn Arg Ala 
        195                 200                 205 

Tyr Phe Thr Asp His Ala Arg Tyr Glu Thr Ala Ala Glu Tyr Arg Glu 
    210                 215                 220 

Arg Ile Arg Leu Asp Ala Val Lys Met Thr Glu Tyr Leu Arg Thr Lys 
225                 230                 235                 240 

Val Glu Val Asn Pro His Val Phe Val Trp Pro Tyr Gly Glu Ala Asn 
                245                 250                 255 

Gly Ile Ala Ile Glu Glu Leu Lys Lys Leu Gly Tyr Asp Met Phe Phe 
            260                 265                 270 

Thr Leu Glu Ser Gly Leu Ala Asn Ala Ser Gln Leu Asp Ser Ile Pro 
        275                 280                 285 

Arg Val Leu Ile Ala Asn Asn Pro Ser Leu Lys Glu Phe Ala Gln Gln 
    290                 295                 300 

Ile Ile Thr Val Gln Glu Lys Ser Pro Gln Arg Ile Met His Ile Asp 
305                 310                 315                 320 

Leu Asp Tyr Val Tyr Asp Glu Asn Leu Gln Gln Met Asp Arg Asn Ile 
                325                 330                 335 

Asp Val Leu Ile Gln Arg Val Lys Asp Met Gln Ile Ser Thr Val Tyr 
            340                 345                 350 

Leu Gln Ala Phe Ala Asp Pro Asp Gly Asp Gly Leu Val Lys Glu Val 
        355                 360                 365 

Trp Phe Pro Asn Arg Leu Leu Pro Met Lys Ala Asp Ile Phe Ser Arg 
    370                 375                 380 

Val Ala Trp Gln Leu Arg Thr Arg Ser Gly Val Asn Ile Tyr Ala Trp 
385                 390                 395                 400 

Met Pro Val Leu Ser Trp Asp Leu Asp Pro Thr Leu Thr Arg Val Lys 
                405                 410                 415 

Tyr Leu Pro Thr Gly Glu Lys Lys Ala Gln Ile His Pro Glu Gln Tyr 
            420                 425                 430 

His Arg Leu Ser Pro Phe Asp Asp Arg Val Arg Ala Gln Val Gly Met 
        435                 440                 445 

Leu Tyr Glu Asp Leu Ala Gly His Ala Ala Phe Asp Gly Ile Leu Phe 
    450                 455                 460 

His Asp Asp Ala Leu Leu Ser Asp Tyr Glu Asp Ala Ser Ala Pro Ala 
465                 470                 475                 480 

Ile Thr Ala Tyr Gln Gln Ala Gly Phe Ser Gly Ser Leu Ser Glu Ile 
                485                 490                 495 

Arg Gln Asn Pro Glu Gln Phe Lys Gln Trp Ala Arg Phe Lys Ser Arg 
            500                 505                 510 

Ala Leu Thr Asp Phe Thr Leu Glu Leu Ser Ala Arg Val Lys Ala Ile 
        515                 520                 525 

Arg Gly Pro His Ile Lys Thr Ala Arg Asn Ile Phe Ala Leu Pro Val 
    530                 535                 540 

Ile Gln Pro Glu Ser Glu Ala Trp Phe Ala Gln Asn Tyr Ala Asp Phe 
545                 550                 555                 560 

Leu Lys Ser Tyr Asp Trp Thr Ala Ile Met Ala Met Pro Tyr Leu Glu 
                565                 570                 575 

Gly Val Ala Glu Lys Ser Ala Asp Gln Trp Leu Ile Gln Leu Thr Asn 
            580                 585                 590 

Gln Ile Lys Asn Ile Pro Gln Ala Lys Asp Lys Ser Ile Leu Glu Leu 
        595                 600                 605 

Gln Ala Gln Asn Trp Gln Lys Asn Gly Gln His Gln Ala Ile Ser Ser 
    610                 615                 620 

Gln Gln Leu Ala His Trp Met Ser Leu Leu Gln Leu Asn Gly Val Lys 
625                 630                 635                 640 

Asn Tyr Gly Tyr Tyr Pro Asp Asn Phe Leu His Asn Gln Pro Glu Ile 
                645                 650                 655 

Asp Leu Ile Arg Pro Glu Phe Ser Thr Ala Trp Tyr Pro Lys Asn Asp 
            660                 665                 670 

 
           
             5  
             1380  
             DNA  
             Escherichia coli  
           
            5 

aaaatgatta atcgcatcgt atcgtttttt atattatgtc tggtgttatg cataccccta     60 

tgcgtagcgt actttcactc tggtgaactg atgatgaggt tcgttttctt ctggccgttt    120 

tttatgtcca ttatgtggat tgttggcggc gtctatttct gggtctatcg tgaacgccac    180 

tggccgtggg gagaaaacgc accagctccc cagttgaaag ataatccgtc tatctccatt    240 

atcattccct gttttaatga ggagaaaaac gttgaggaaa ccatacacgc cgctttagca    300 

cagcgttatg agaacattga agttattgcc gtaaatgacg gttcaacaga taaaacccgt    360 

gccatcctgg atcgcatggc tgcacaaatt ccccatttgc gggtcattca tctggcgcaa    420 

aaccagggga aagccattgc gcttaaaacc ggagctgccg cggcgaaaag tgaatatctg    480 

gtgtgcattg atggcgatgc gttattagac cgcgatgcgg cggcatatat tgtggaaccg    540 

atgttgtaca acccgcgtgt gggtgccgta accggtaatc ctcgtattcg aacacgttct    600 

accctggtgg gtaaaattca ggttggcgag tattcctcaa ttattggttt gatcaagcga    660 

acccagcgta tctatggaaa cgtatttacc gtttccggtg ttattgccgc atttcgtcgc    720 

agcgccctgg cagaagtggg ttactggagt gacgatatga tcaccgaaga tattgatatt    780 

agctggaagc tgcagttgaa tcagtggacg attttttacg agccacgggc actgtgctgg    840 

atattaatgc ctgaaacgtt aaaagggctg tggaaacagc gcctgcgctg ggctcagggc    900 

ggtgcagaag tattcctcaa aaatatgaca aggttgtggc gcaaagaaaa ctttcgaatg    960 

tggccgctgt tttttgaata ctgcctgacg acaatatggg ccttcacctg cctggtcggt   1020 

ttcattattt acgcagtcca acttgccggt gtaccgttaa atattgaatt gacacatatc   1080 

gctgcgacac atactgccgg aatattattg tgtacgttat gtttactgca atttattgtc   1140 

agcctgatga tcgagaatcg ctatgagcat aatctgactt catcgctttt ctggattatt   1200 

tggttcccgg ttattttctg gatgctgagc ctggcaacga cattggtatc atttacacga   1260 

gtcatgttga tgcctaaaaa gcaacgcgcc cgttgggtaa gtcccgatcg cgggattctg   1320 

agaggttaat atgaacaatt taattattac gacccgacaa tcaccagtac gtttactggt   1380 

 
           
             6  
             445  
             PRT  
             Escherichia coli  
           
            6 

Met Ile Asn Arg Ile Val Ser Phe Phe Ile Leu Cys Leu Val Leu Cys 
1               5                   10                  15 

Ile Pro Leu Cys Val Ala Tyr Phe His Ser Gly Glu Leu Met Met Arg 
            20                  25                  30 

Phe Val Phe Phe Trp Pro Phe Phe Met Ser Ile Met Trp Ile Val Gly 
        35                  40                  45 

Gly Val Tyr Phe Trp Val Tyr Arg Glu Arg His Trp Pro Trp Gly Glu 
    50                  55                  60 

Asn Ala Pro Ala Pro Gln Leu Lys Asp Asn Pro Ser Ile Ser Ile Ile 
65                  70                  75                  80 

Ile Pro Cys Phe Asn Glu Glu Lys Asn Val Glu Glu Thr Ile His Ala 
                85                  90                  95 

Ala Leu Ala Gln Arg Tyr Glu Asn Ile Glu Val Ile Ala Val Asn Asp 
            100                 105                 110 

Gly Ser Thr Asp Lys Thr Arg Ala Ile Leu Asp Arg Met Ala Ala Gln 
        115                 120                 125 

Ile Pro His Leu Arg Val Ile His Leu Ala Gln Asn Gln Gly Lys Ala 
    130                 135                 140 

Ile Ala Leu Lys Thr Gly Ala Ala Ala Ala Lys Ser Glu Tyr Leu Val 
145                 150                 155                 160 

Cys Ile Asp Gly Asp Ala Leu Leu Asp Arg Asp Ala Ala Ala Tyr Ile 
                165                 170                 175 

Val Glu Pro Met Leu Tyr Asn Pro Arg Val Gly Ala Val Thr Gly Asn 
            180                 185                 190 

Pro Arg Ile Arg Thr Arg Ser Thr Leu Val Gly Lys Ile Gln Val Gly 
        195                 200                 205 

Glu Tyr Ser Ser Ile Ile Gly Leu Ile Lys Arg Thr Gln Arg Ile Tyr 
    210                 215                 220 

Gly Asn Val Phe Thr Val Ser Gly Val Ile Ala Ala Phe Arg Arg Ser 
225                 230                 235                 240 

Ala Leu Ala Glu Val Gly Tyr Trp Ser Asp Asp Met Ile Thr Glu Asp 
                245                 250                 255 

Ile Asp Ile Ser Trp Lys Leu Gln Leu Asn Gln Trp Thr Ile Phe Tyr 
            260                 265                 270 

Glu Pro Arg Ala Leu Cys Trp Ile Leu Met Pro Glu Thr Leu Lys Gly 
        275                 280                 285 

Leu Trp Lys Gln Arg Leu Arg Trp Ala Gln Gly Gly Ala Glu Val Phe 
    290                 295                 300 

Leu Lys Asn Met Thr Arg Leu Trp Arg Lys Glu Asn Phe Arg Met Trp 
305                 310                 315                 320 

Pro Leu Phe Phe Glu Tyr Cys Leu Thr Thr Ile Trp Ala Phe Thr Cys 
                325                 330                 335 

Leu Val Gly Phe Ile Ile Tyr Ala Val Gln Leu Ala Gly Val Pro Leu 
            340                 345                 350 

Asn Ile Glu Leu Thr His Ile Ala Ala Thr His Thr Ala Gly Ile Leu 
        355                 360                 365 

Leu Cys Thr Leu Cys Leu Leu Gln Phe Ile Val Ser Leu Met Ile Glu 
    370                 375                 380 

Asn Arg Tyr Glu His Asn Leu Thr Ser Ser Leu Phe Trp Ile Ile Trp 
385                 390                 395                 400 

Phe Pro Val Ile Phe Trp Met Leu Ser Leu Ala Thr Thr Leu Val Ser 
                405                 410                 415 

Phe Thr Arg Val Met Leu Met Pro Lys Lys Gln Arg Ala Arg Trp Val 
            420                 425                 430 

Ser Pro Asp Arg Gly Ile Leu Arg Gly Met Asn Asn Leu 
        435                 440                 445 

 
           
             7  
             30  
             DNA  
             Escherichia coli  
           
            7 

tacagttaag tgtgttatcg gtgcagagcc                                      30 

 
           
             8  
             31  
             DNA  
             Escherichia coli  
           
            8 

ctcaacgcct ggctgattaa accaactatt c                                    31 

 
           
             9  
             7500  
             DNA  
             Escherichia coli  
           
            9 

atgtattcaa gtagcagaaa aaggtgcccg aaaaccaaat gggctttgaa acttcttact     60 

gccgcatttt tagcagcgag tcccgcggcg aagagtgctg ttaataacgc ctatgatgca    120 

ttgattattg aagctcgcaa gggtaatact cagccagctt tgtcatggtt tgcactaaaa    180 

tcagcactca gcaataacca aattgctgac tggttacaga ttgccttatg ggccgggcaa    240 

gataaacagg ttattaccgt ttacaaccgc taccgtcatc agcaattacc agcgcgtggt    300 

tatgcagctg tcgccgtcgc ttatcgtaac ctgcaacaat ggcaaaactc gcttacactg    360 

tggcaaaagg cgctctctct ggagccgcaa aataaggatt atcaacgggg acaaatttta    420 

accctggcag atgctggtca ctatgatact gcgctggtta aacttaagca gcttaactct    480 

ggagcaccgg acaaagccaa tttactcgca gaagcctata tctataaact ggcggggcgt    540 

catcaggatg aattacgggc gatgacagag tcattacctg aaaatgcatc tacgcaacaa    600 

tatcccacag aatacgtgca ggcattacgt aataatcaac ttgctgccgc gattgacgat    660 

gccaatttaa cgccagatat tcgcgctgat attcatgccg aactggtcag actgtcgttt    720 

atgcctacgc gcagtgaaag tgaacgttat gccattgccg atcgcgccct cgcccaatac    780 

gctgcattag aaattctgtg gcacgataac ccagaccgca ctgcccagta ccagcgtatt    840 

caggttgatc atcttggcgc gttattaact cgcgatcgtt ataaagacgt tatttctcac    900 

tatcagcgat taaaaaagac ggggcaaatt attccgccct gggggcaata ttgggttgca    960 

tcggcttatc tcaaagatca tcagccgaaa aaagcacagt caataatgac cgagctcttt   1020 

tatcacaagg agaccattgc cccggattta tccgatgaag aacttgcgga tctcttttac   1080 

agccacctgg agagtgaaaa ttatccgggc gcgctaactg tcacccaaca taccattaat   1140 

acttcgccgc ctttccttcg gttaatgggc acgcctacga gcatcccgaa tgatacctgg   1200 

ttacaggggc attcgtttct ctcaaccgta gcaaaatata gtaatgatct tcctcaggct   1260 

gaaatgacag ccagagagct tgcttataac gcaccaggaa atcagggact gcgcattgat   1320 

tacgcgagtg tgttacaagc ccgcggttgg cctcgtgcag cagaaaatga attaaaaaaa   1380 

gcagaagtga tcgagccacg taatattaat ctggaggttg aacaagcctg gacagcatta   1440 

acgttacaag aatggcagca ggcagctgtc ttaacgcacg atgttgtcga acgtgaaccg   1500 

caagatcccg gcgttgtacg attaaaacgt gcggttgatg tacataatct tgcagagctt   1560 

cgtatcgctg gctcaacagg aattgatgcc gaaggcccgg atagtggtaa acatgatgtc   1620 

gacttaacca ccatcgttta ttcaccaccg ctgaaggata actggcgcgg ttttgctgga   1680 

ttcggttatg ccgatggaca atttagcgaa ggaaaaggga ttgttcgcga ctggcttgcg   1740 

ggtgttgagt ggcggtcacg taatatctgg ctcgaggcag agtacgctga acgcgttttc   1800 

aatcatgagc ataaacccgg cgcgcgcctg tctggctggt atgattttaa tgataactgg   1860 

cgtattggtt cgcaactgga acgcctctct caccgcgttc cattacgggc aatgaaaaat   1920 

ggtgttacag gcaacagtgc tcaggcttat gttcgctggt atcaaaatga gcggcgtaag   1980 

tacggtgtct cctgggcttt cactgatttt tccgacagta accagcgtca tgaagtctca   2040 

cttgagggtc aggaacgcat ctggtcttca ccatatttga ttgtcgattt cctacccagt   2100 

ctgtattacg aacaaaatac agaacacgat accccatact acaaccctat aaaaacgttc   2160 

gatattgttc cggcatttga ggcaagccat ttgttatggc gaagctatga aaatagctgg   2220 

gagcaaatat tcagcgcagg tgttggtgcc tcctggcaaa aacattatgg cacggatgtc   2280 

gtcacccaac tcggctacgg gcaacgcatt agttggaatg acgtgattga tgctggcgca   2340 

acgctacgct gggaaaaacg accttatgac ggtgacagag aacacaactt atacgttgaa   2400 

ttcgatatga cattcagatt ttaaggataa atatgttacg taatggaaat aaatatctcc   2460 

tgatgctggt gagtataatt atgctcaccg cgtgcattag ccagtcaaga acatcattta   2520 

taccgccaca ggatcgcgaa tctttactcg ccgagcaacc gtggccgcat aatggttttg   2580 

tagcgatttc atggcataac gttgaagacg aagctgccga ccagcgtttt atgtcagtgc   2640 

ggacatcagc actgcgtgaa caatttgcct ggctgcgcga gaacggttat caaccggtca   2700 

gtattgctca aattcgtgaa gcacatcgag gaggaaaacc gctaccggaa aaagctgtag   2760 

tgctgacttt tgatgacggc taccagagtt tttatacccg cgtcttccca attcttcagg   2820 

ccttccagtg gcctgctgta tgggcccccg tcggcagttg ggtcgatacg ccagcggata   2880 

aacaagtaaa atttggcgat gagttggtcg atcgagaata ttttgccacg tggcaacaag   2940 

tgcgagaagt tgcgcgttcc cggctcgttg agctcgcttc tcatacatgg aattctcact   3000 

acggtattca ggctaatgcc accggcagct tattgcctgt atatgtaaat cgtgcatatt   3060 

ttactgacca cgcacggtat gaaaccgcag cagaataccg ggaaagaatt cgtctggatg   3120 

ctgtaaaaat gacggaatac ctgcgtacaa aggttgaggt aaatccacac gtttttgttt   3180 

ggccttatgg cgaagcgaat ggcatagcga tagaggaatt aaaaaaactc ggttatgaca   3240 

tgttcttcac ccttgaatca ggtttggcaa atgcgtcgca attggattcc attccgcggg   3300 

tattaatcgc caataatccc tcattaaaag agtttgccca gcaaattatt accgtacagg   3360 

aaaaatcacc acaacggata atgcatatcg atcttgatta cgtttatgac gaaaacctcc   3420 

agcaaatgga tcgcaatatt gatgtgctaa ttcagcgggt gaaagatatg caaatatcaa   3480 

ccgtgtattt gcaggcattt gctgatcccg atggtgatgg gctggtcaaa gaggtctggt   3540 

ttccaaatcg tttgctacca atgaaagcag atatttttag tcgggttgcc tggcaattac   3600 

gtacccgctc aggtgtaaac atctatgcgt ggatgccggt attaagctgg gatttagatc   3660 

ccacattaac gcgagtaaaa tacttaccaa caggggagaa aaaagcacaa attcatcctg   3720 

aacaatatca ccgtctctct cctttcgatg acagagtcag agcacaagtt ggcatgttat   3780 

atgaagatct tgccggacat gctgcttttg atggcatatt gttccacgat gatgctttgc   3840 

tttcagatta tgaagatgcc agtgcaccgg ctatcacggc ttatcagcaa gcaggcttta   3900 

gcgggagtct gagcgaaatt cgacaaaacc cggagcaatt taaacagtgg gcccgcttta   3960 

aaagtcgtgc gttaactgac ttcactttag aacttagtgc gcgcgtaaaa gccattcgcg   4020 

gtccacatat taaaactgca cgaaatattt ttgcacttcc ggtaatacaa cctgaaagtg   4080 

aagcctggtt tgcacagaat tatgctgatt tcctaaaaag ctatgactgg accgctatta   4140 

tggctatgcc ttatctggaa ggtgtcgcag aaaaatcggc tgaccaatgg ttaatacaat   4200 

tgaccaatca aattaaaaac atccctcagg ctaaagacaa atctatttta gaattacagg   4260 

cacaaaactg gcagaaaaat ggtcagcatc aggctatttc ttcgcaacaa ctcgctcact   4320 

ggatgagcct attacaactg aatggagtga aaaactatgg ttattatccc gacaattttc   4380 

tgcataacca acctgaaata gaccttattc gtcctgagtt ttcaacagcc tggtatccga   4440 

aaaatgatta atcgcatcgt atcgtttttt atattatgtc tggtgttatg cataccccta   4500 

tgcgtagcgt actttcactc tggtgaactg atgatgaggt tcgttttctt ctggccgttt   4560 

tttatgtcca ttatgtggat tgttggcggc gtctatttct gggtctatcg tgaacgccac   4620 

tggccgtggg gagaaaacgc accagctccc cagttgaaag ataatccgtc tatctccatt   4680 

atcattccct gttttaatga ggagaaaaac gttgaggaaa ccatacacgc cgctttagca   4740 

cagcgttatg agaacattga agttattgcc gtaaatgacg gttcaacaga taaaacccgt   4800 

gccatcctgg atcgcatggc tgcacaaatt ccccatttgc gggtcattca tctggcgcaa   4860 

aaccagggga aagccattgc gcttaaaacc ggagctgccg cggcgaaaag tgaatatctg   4920 

gtgtgcattg atggcgatgc gttattagac cgcgatgcgg cggcatatat tgtggaaccg   4980 

atgttgtaca acccgcgtgt gggtgccgta accggtaatc ctcgtattcg aacacgttct   5040 

accctggtgg gtaaaattca ggttggcgag tattcctcaa ttattggttt gatcaagcga   5100 

acccagcgta tctatggaaa cgtatttacc gtttccggtg ttattgccgc atttcgtcgc   5160 

agcgccctgg cagaagtggg ttactggagt gacgatatga tcaccgaaga tattgatatt   5220 

agctggaagc tgcagttgaa tcagtggacg attttttacg agccacgggc actgtgctgg   5280 

atattaatgc ctgaaacgtt aaaagggctg tggaaacagc gcctgcgctg ggctcagggc   5340 

ggtgcagaag tattcctcaa aaatatgaca aggttgtggc gcaaagaaaa ctttcgaatg   5400 

tggccgctgt tttttgaata ctgcctgacg acaatatggg ccttcacctg cctggtcggt   5460 

ttcattattt acgcagtcca acttgccggt gtaccgttaa atattgaatt gacacatatc   5520 

gctgcgacac atactgccgg aatattattg tgtacgttat gtttactgca atttattgtc   5580 

agcctgatga tcgagaatcg ctatgagcat aatctgactt catcgctttt ctggattatt   5640 

tggttcccgg ttattttctg gatgctgagc ctggcaacga cattggtatc atttacacga   5700 

gtcatgttga tgcctaaaaa gcaacgcgcc cgttgggtaa gtcccgatcg cgggattctg   5760 

agaggttaat atgaacaatt taattattac gacccgacaa tcaccagtac gtttactggt   5820 

tgattatgtt gccacaacca tcttgtggac attatttgcg ttgttcatat tcttattcgc   5880 

catggatctg ctgacgggtt attactggca aagcgaggcc agaagccgac ttcagttcta   5940 

ttttttgctg gcagtggcga atgccgtcgt gttaattgtc tgggcgctgt acaataagct   6000 

gcgttttcaa aaacagcagc atcatgcagc ctaccaatat acgccgcaag aatatgcaga   6060 

gagcttagca atacctgatg agctctatca gcaactacaa aaaagccaca ggatgagcgt   6120 

acacttcacc agccaggggc aaataaaaat ggttgtttca gaaaaagcgc tagtccgggc   6180 

ataaacaccc aaaacaaagc ccggttcgcc cgggctctgc accgataaca cacttaactg   6240 

taggcatgca gcgtacgttg gcaaagtgcc gaacgtacgc agtcctcttt accgaaccgg   6300 

acgatcccaa ccatttcatc ttcttcgaaa cgttccagcg cgtcacttaa tccggagcac   6360 

acgccgcgag gcaaatcgca ttgcgtgata tcaccgttga cgataaccgt cacgttctcc   6420 

ccgaggcggg ttaaaaacat tttcatttgc gcggcagtca cattctgcgc ctcgtcaaga   6480 

atgacgactg cattttcaaa ggtacgtcca cgcatatagg cgaacggcgc aatttccacc   6540 

ttccctattt ccggtcgcag gcagtactgc ataaaggaag cccctaagcg ccggaccagc   6600 

acgtcgtaga ccgggcgaaa atagggagca aacttttctg cgatatctcc aggtaagaag   6660 

ccaagatctt catcggcttg cagaactgga cgggtgacga taatcctgtc gacatcctta   6720 

tgtatcaggg cctctgccgc ttttgctgcg ctgatccagg tttttccgca cccggcttcg   6780 

cccgtggcga atatcagctg cttactctca atagccttca gatagtgcaa ttgcgcttca   6840 

tttcgcgcga ggatgggcga agtatcgcga ctgtcgcggg ccataccaat ggcttctacg   6900 

ccgcccatct gcacaagcga ggtgaccgat tcttcttcac gctgcttatg gctgcgcgaa   6960 

tcccgtctca gcacacgttt tgcctcgcga cgagctttga tcactgcttt ttgtcttccc   7020 

atggagagca ccttgagttg tttgtattca tcacacgcgc cgttggcagc gcgattatgc   7080 

gcacgaacat cagagggttg gcttccttgt aagccatagt ttgcttttgg ataaaatgcc   7140 

gaaaaacggc tacgcgcacc gtttacggcg tcggtaacac atgaaaagaa aggatgaggt   7200 

tgaaaatgca aagtgacgag atgactaccg gaggagaaaa ctccgcgagt ggtggcgcgt   7260 

tgattatcta aaacatgtcc agtacaggac gttaccatcc gcgatctcca tagtgactga   7320 

ctatcactgc cgggaacttc cgctgctact taataagtac aacagatctc gcatttattg   7380 

caacaatata tttacttata tttaactata aaacaccatt tcagtgacat tagtttctac   7440 

tggaaagatg acagagtgat gacagtgatg aaaaaagctg tgtgctttca gcaggatttg   7500