Characterization of six flagellin genes in the H3, H53 and H54 standard strains of Escherichia coli

Six flagellin genes in three H standard Escherichia coli strains for H3, H53 and H54 were characterized. Each strain has two flagellin genes, one of which is expressed as its standard H antigen. A pair of flagellin genes flkA3 (encoding for H3 antigen) and fliC16 (H16) was cloned from Bi7327-41, flkA53 (H53) and fliC-53 from E480-68, and flmA54 (H54) and fliC-54 from E223-69. Two fliC genes, fliC-53 and fliC-54, are nonfunctional owing to the insertions of IS1 and IS1222, respectively. The flkA and flmA regions are located in the 3' end of the rnpB gene and near the nlpA gene, respectively. Each of them is followed by a gene homologous to fljA, which is known to repress the expression of fliC(i) in Salmonella enterica serovar Typhimurium. These results suggest that they are derived from the same origin of the fljBA operon. However, these regions contain neither the hin gene nor the invertible H segment. The four flagellin genes, fliC16, flkA3, flkA53 and flmA54, share high homology in nucleotide and amino-acid sequences with one another and with the S. enterica serovar Typhimurium flagellin genes. The promoter sequence of fliC16 is homologous to that of fliC(i), whereas the promoter sequences of flkA and flmA are homologous to that of fljB. The terminator sequences of the fliC16, fliC-53 and fliC-54 genes are conserved among themselves and identical with that of the E. coli fliC48 gene. Three FljA repressors, FljA3, FljA53 and FljA54, are homologous highly with one another and moderately with FljA of Salmonella. These results indicate that six flagellin genes analyzed are markedly similar to the Salmonella flagellin genes, suggesting their lateral transfer from Salmonella.     

Identification of H-specific determinants in flagellin of four Escherichia coli strains

The H serogroup of Escherichia coli is determined by the flagellar antigen, flagellin. Sequence analysis of the flagellin gene, fliC, reveals a central variable region and the highly conserved N- and C-termini. This variable region has been shown to encode both H-specific and cross-reactive epitopes. Using polyclonal antibodies, we mapped the linear H-specific determinants in flagellin from four E. coli serotypes O157:H10, 0138:H14, O157:H42 and O157:H43. The specificity of all potential fragments was verified with 52 ECRC (Escherichia coli Reference Center) H-specific antisera. Our results indicated that: (a) a specific determinant of H10 flagellin (1263 bp long) maps to the region covering amino acid residues 305-331; (b) a specific determinant of H14 flagellin (1653 bp long) maps to the region covering amino acid residues 430-461; (c) a specific determinant of H42 flagellin (1281 bp long) maps to a region covering amino acid residues 171-201; and (d) a specific determinant of H43 flagellin (1506 bp long) maps to a region covering amino acid residues 200-260.     

Maintenance of the cellobiose utilization genes of Escherichia coli in a cryptic state

The genes for cellobiose utilization are normally cryptic in Escherichia coli. The cellobiose system was used as a model to understand the process by which silent genes are maintained in microbial populations. Previously reported was (1) the isolation of a mutant strain that expresses the cellobiose-utilization (Cel) genes and (2) that expression of those genes allows utilization of three beta- glucoside sugars: cellobiose, arbutin, and salicin. The Cel gene cluster has now been cloned from that mutant strain. In the course of locating the Cel genes within the cloned DNA segment, it was discovered that inactivation of the Cel-encoded hydrolase rendered the host strain sensitive to all three beta-glucosides as potent inhibitors. This sensitivity arises from the accumulation of the phosphorylated beta- glucosides. Because even the fully active genes conferred some degree of beta-glucoside sensitivity, the effects of cellobiose on a series of five Cel+ mutants of independent origin were investigated. Although each of those strains utilizes cellobiose as a sole carbon and energy source, cellobiose also acts as a potent inhibitor that reduces the growth rate on glycerol 2.5-16.5-fold. On the other hand, wild-type strains that cannot utilize cellobiose are not inhibited. The observation that the same compound can serve either as a nutrient or as an inhibitor suggests that, under most conditions in which cellobiose will be present together with other resources, there is a strong selective advantage to having the cryptic (Cel0) allele. In those environments in which cellobiose is the sole, or the best, resource, mutants that express the genes (Cel+) will have a strong selective advantage. It is suggested that temporal alternation between these two conditions is a major factor in the maintenance of these genes in E. coli populations. This alternation of environments and fitnesses was predicted by the model for cryptic-gene maintenance that was previously published.      

Characterization of cryptic flagellin genes in Shigella boydii and Shigella dysenteriae.

Flagellin (fliC) genes of 12 Shigella boydii and five Shigella dysenteriae strains were characterized. Though these strains are nonmotile, the cryptic fliCSB gene, cloned from S. boydii strain C3, is functional for expression of flagellin. It consists of 1,704 bp, and encodes 568 amino acid residues (57,918 Da). The fliCSD gene from S. dysenteriae strain 16 consists of 1,650 bp encoding 549 amino acid residues (57,591 Da) and contains an IS1 element inserted in its 3' end. The two genes are composed of the 5'-constant, central variable and 3'-constant sequences, like other known fliC genes. The two genes share high homology in nucleotide and amino acid sequences with each other and also with the Escherichia coli fliCE gene, indicating that both genes are closely related to the fliCE gene. Comparison of the central variable sequences of six different fliC genes showed that the fliCSB and fliCSD genes share low homology in amino acid sequence with the other fliC genes, suggesting that they encode antigenic determinants intrinsic to respective subgroups. However, Southern blotting using as probes the central variable sequences of several fliC genes showed that four of 12 S. boydii strains have a fliC gene similar to that of Shigella flexneri, and that among five fliC genes from S. dysenteriae strains, one is similar to that of S. flexneri, two are similar to that of S. boydii, and only one is unique to S. dysenteriae. Some of these variant alleles were verified by immunoblotting with flagellins produced from cloned fliC genes. The presence of variant fliC alleles in S. boydii and S. dysenteriae indicates that subdivision into subgroups does not reflect the ancestral flagella H antigenic relationships. These data will be useful in considering the evolutionary divergence of the Shigella spp..     

Molecular characterization of intact, but cryptic, flagellin genes in the genus Shigella

Flagellin genes (fliC) were detected in two species of the genus Shigella. The fliC_SF gene cloned from Shigella flexneri produced normal-type flagella in an Escherichia coli δ fliC strain while the fliC_SS genes from two Shigella sonnei strains produced curly-type flagella and their expression is repressible by Salmonella FljA repressor. The fliC_SF gene (1650bp) shared high similarity with the E. coli fliC_E gene not only in the 5′ and 3′ constant sequences but also in the upstream and downstream sequences. The fliC_S genes (1572 bp) shared high similarity with the Salmonella typhimurium fliCs gene in the operator and 3’constant sequences and also shared high similarity with the fliC_E gene in the downstream sequence, suggesting that the fliC gene has undergone horizontal transfer and recombination. Differences In nucleotide sequences of the central variable regions among the four fliC genes, including fliC_E and fliC_S, suggest that they started differentiation in each lineage approximately 80 million years ago. Loss of motility in Shigella seems to be evolutionarily a recent event.     

Fragmentation of Escherichia coli type 1 fimbriae exposes cryptic D-mannose-binding sites.

Cells of the gram-negative bacterium Escherichia coli are able to attach to various host cells by means of a mannose-specific adhesin associated with type 1 fimbriae. Here we show that fragmentation of type 1 fimbriae by freezing and thawing results in increased mannose-binding activity as demonstrated by increased hemagglutination, increased stimulation of human lymphocyte proliferation, and increased binding of the mannose-containing enzyme horseradish peroxidase. Increased activity in all three assays was mannose sensitive and was not exhibited by FimH- mutant type 1 fimbriae lacking the adhesin. Scatchard analysis of the data from peroxidase binding assays showed that unfrozen and frozen fimbriae contain binding sites displaying two classes of affinity. Frozen and thawed fimbriae expressed an increase in the number of high-affinity binding sites. These results show that fragmentation of the fimbrial structure exposes cryptic mannose-binding activity associated with type 1 fimbriae, presumably that of internally located adhesin molecules. Our data support earlier observations that adhesin moieties of type 1 fimbriae are located both at the tips and at intervals along the length of the fimbriae. In addition, our data suggest that only the adhesin moieties that are located at the fimbrial tips are functional in binding mannose. Adhesins located along the length of the fimbriae have their mannose-binding activity buried within the fimbrial structure and hence are not functional. We propose an updated model for the structure of type 1 fimbriae that is in agreement with the above observations.     

The beta-glucoside genes of Klebsiella aerogenes: conservation and divergence in relation to the cryptic bgl genes of Escherichia coli

The ability to metabolize aromatic beta-glucosides such as salicin and arbutin varies among members of the Enterobacteriaceae. The ability of Escherichia coli to degrade salicin and arbutin appears to be cryptic, subject to activation of the bgl genes, whereas many members of the Klebsiella genus can metabolize these sugars. We have examined the genetic basis for beta-glucoside utilization in Klebsiella aerogenes. The Klebsiella equivalents of bglG, bglB and bglR have been cloned using the genome sequence database of Klebsiella pneumoniae. Nucleotide sequencing shows that the K. aerogenes bgl genes show substantial similarities to the E. coli counterparts. The K. aerogenes bgl genes in multiple copies can also complement E. coli mutants deficient in bglG encoding the antiterminator and bglB encoding the phospho-beta-glucosidase, suggesting that they are functional homologues. The regulatory region bglR of K. aerogenes shows a high degree of similarity of the sequences involved in BglG-mediated regulation. Interestingly, the regions corresponding to the negative elements present in the E. coli regulatory region show substantial divergence in K. aerogenes. The possible evolutionary implications of the results are discussed.     

Electrophoretic mobilities of Escherichia coli O157:H7 and wild-type Escherichia coli strains.

The electrophoretic mobilities (EPMs) of a number of Escherichia coli O157:H7 and wild-type E. coli strains were measured. The effects of pH and ionic strength on the EPMs were investigated. The EPMs of E. coli O157:H7 strains differed from those of wild-type strains. As the suspension pH decreased, the EPMs of both types of strains increased.     

Identification by the phage-display technique of peptides that bind to H7 flagellin of Escherichia coli

The four peptides interacting with H7 flagellin of Escherichia coli were selected from a phage display library. The library was selected four times, and the interacting phage peptides were competitively eluted with H7 flagellin. An enzyme-linked immunosorbent assay (ELISA) showed that these peptides were reactive with the H7 flagellin in a dose-dependent manner. Among them, a D1 phage clone showed the highest binding affinity to the H7 flagellin. We synthesized the D1 peptide (LHIHRPTLSIQG) corresponding to the peptide-encoding region of the D1 phage clone. The synthetic peptide showed micro-molar affinity (EC(50) value=1.9 microM) for the H7 flagellin. Furthermore, this D1 peptide interacted more specifically with the H7 flagellin than with the other flagellins (H1, H5, H12, or H23) of E. coli. In situ hybridization clearly showed that the peptide only detected those cells harboring the H7 flagellin gene (fliC). The peptide may specifically bind to the H7 flagellin on the cell surface. These results suggest that the phage-display technique could be used as a tool for identifying peptides as an alternative to using a ligand as a diagnostic reagent in food products or in clinical testing.     

Cloning and expression of Pseudomonas aeruginosa flagellin in Escherichia coli.

The flagellin gene was isolated from a Pseudomonas aeruginosa PAO1 genomic bank by conjugation into a PA103 Fla- strain. Flagellin DNA was transferred from motile recipient PA103 Fla+ cells by transformation into Escherichia coli. We show that transformed E. coli expresses flagellin protein. Export of flagellin to the E. coli cell surface was suggested by positive colony blots of unlysed cells and by isolation of flagellin protein from E. coli supernatants.     

Characterization of Escherichia coli strains with gapA and gapB genes deleted.

We obtained a series of Escherichia coli strains in which gapA, gapB, or both had been deleted. Delta gapA strains do not revert on glucose, while delta gapB strains grow on glycerol or glucose. We showed that gapB-encoded protein is expressed but at a very low level. Together, these results confirm the essential role for gapA in glycolysis and show that gapB is dispensable for both glycolysis and the pyridoxal biosynthesis pathway.     

Species-wide variation in the Escherichia coli flagellin (H-antigen) gene

Escherichia coli is a clonal species. The best-understood components of its clonal variation are the flagellar (H) and polysaccharide (O) antigens, both well documented since the mid-1930s because of their use in serotyping. Flagellin is the protein subunit of the flagellum that carries H-antigen specificity. We show that 43 of the 54 H-antigen specificities of E. coli map to the flagellin gene at fliC and sequenced all 43 forms and confirmed specificity of each by cloning and expression. This is, to our knowledge, the first time that all known forms of such a highly polymorphic gene have been fully sequenced and characterized for any species. The established distinction between a highly variable central region and more conserved flanking regions is upheld. The sequences fall into two groups, one of which may be derived from the fliC gene of the E. coli/Salmonella enterica common ancestor, the other perhaps obtained by lateral transfer since species divergence. Comparison of sequences revealed that both horizontal DNA transfer and fixation of mutations under diversifying selection pressure contributed to polymorphism in this locus.     

Production of a monoclonal antibody specific to Escherichia coli H33 flagellin by using predetermined polypeptides

The Escherichia coli H serogroup is determined by flagellin, which has both H-type-specific and cross-reactive epitopes. The cross-reactive epitopes are responsible for the cross-reaction found in agglutination. To identify the specific epitope in H33 flagellin, the H33 flagellin gene was sequenced and the encoded central variable region (CVR) was determined. Four overlapping fragments of the CVR were prepared and their specificity was verified using different H-type antisera. Short fragments carrying potential H-type-specific determinants were selected, and monoclonal antibodies (MAbs) against these fragments were prepared. A murine MAb of subtype IgG1 showing specificity to H33 flagellin was produced. The epitope of the MAb was mapped to amino acid residues 250-260.     

Construction of a minimum-size functional flagellin of Escherichia coli.

Various deletions were introduced into the central region of Escherichia coli flagellin (497 residues) without destroying its ability to form flagellar filaments. The smallest flagellin retained only the N-terminal 193 residues and the C-terminal 117 residues, which are suggested to be the domains essential for filament formation.     

Isolation and characterization of mutant strains of Escherichia coli altered in H2 metabolism

A positive selection procedure is described for the isolation of hydrogenase-defective mutant strains of Escherichia coli. Mutant strains isolated by this procedure can be divided into two major classes. Class I mutants produced hydrogenase activity (determined by using a tritium-exchange assay) and formate hydrogenlyase activity but lacked the ability to reduce benzyl viologen or fumarate with H2 as the electron donor. Class II mutants failed to produce active hydrogenase and hydrogenase-dependent activities. All the mutant strains produced detectable levels of formate dehydrogenase-1 and -2 and fumarate reductase. The mutation in class I mutants mapped near 65 min of the E. coli chromosome, whereas the mutation in class II mutants mapped between srl and cys operons (58 and 59 min, respectively) in the genome. The class II Hyd mutants can be further subdivided into two groups (hydA and hydB) based on the cotransduction characteristics with cys and srl. These results indicate that there are two hyd operons and one hup operon in the E. coli chromosome. The two hyd operons are needed for the production of active hydrogenase, and all three are essential for hydrogen-dependent growth of the cell.