The toxin-co-regulated pilus of Vibrio cholerae O1: a model for type 4 pilus biogenesis?

The toxin-co-regulated pilus (TCP), an important colonization factor of Vibrio cholerae, is similar to the type 4 pilus produced by a variety of pathogenic Gram-negative bacteria. The putative translocation and assembly machinery of TCP has broad similarities with known pilin and nonpilin export mechanisms.

Surface exclusion genes traS and traT of the F sex factor of Escherichia coli K-12. Determination of the nucleotide sequence and promoter and terminator activities.

The DNA encoding the surface exclusion genes traS and traT of the F sex factor of Escherichia coli K-12 has been sequenced and the biological activity of the various terminators and promoters determined. The data show that traS encodes a 16,861 Mr protein with no apparent signal sequence, as expected for its cytoplasmic membrane location. The protein is extremely hydrophobic. traS has its own promoter and a weak terminator region follows the gene. After the traS termination loop there is a small intergenic region before the traT promoter. The traT gene encodes a 25,932 Mr precursor for the 23,709 Mr mature protein. The amino-terminal signal peptide is 21 amino acid residues, consistent with it being an outer membrane lipoprotein. A very strong termination loop follows the gene and adjacent to this a further loop can be predicted from the sequence. These secondary structures would be expected to enhance the stability of the mRNA in the presence of 3' specific ribonucleases accounting for the apparent long half-life of the messenger. The amino acid sequence of the mature product of traT of F differs from that of R100 by only a single amino acid substitution (Gly for Ala at position 119), whereas that of pED208 (Folac) differs at 40 positions. traT lies in a region of heteroduplex homology between F and R100, and the nucleotide sequence confirms this and demonstrates that this homology breaks down immediately preceding and following the coding region. Sequence analysis shows that this is also so for pED208. Thus the entire traS of F, R100 and pED208 are very different at the DNA level. An open reading frame, preceded by a typical promoter sequence and a weak and poorly located Shine-Dalgarno sequence, follows traT and corresponds to the start of traD. Alone, this promoter appears to be inactive.

The tcp gene cluster of Vibrio cholerae.

The toxin co-regulated pilus (TCP) has been identified as a critical colonization factor in both animal models and humans for Vibrio cholerae O1. The major pilin subunit, TcpA (and also TcpB), is similar to type-4 pilins but TCP probably more appropriately belongs to a sub-class which includes the bundle-forming pilus of enteropathogenic Escherichia coli. The genes for TCP biosynthesis and assembly are clustered with the exception of housekeeping functions such as TcpG (=DsbA, a periplasmic disulfide bond epimerase). The nt sequences from El Tor and classical strains show only minor differences corresponding to the major regulatory regions and in TcpA itself. These differences are thought to account for the alternate conditions required for expression of TCP by the two biotypes and the antigenic variation and lack of cross-protection. Aside from the TcpA only a few of the proteins have had their roles in TCP biogenesis defined. Regulation of TCP is controlled by the ToxR regulon via ToxT with a possible involvement of TcpP and the cAMP-CRP system. Experiments using the infant mouse cholera model have now shown that TCP is a colonization factor and protective antigen for both classical and El Tor O1 strains and in the O139 Bengal serotype and that the mannose-sensitive haemagglutinin pilus does not appear to play a comparable role.

Extracellular proteins of Vibrio cholerae: molecular cloning, nucleotide sequence and characterization of the deoxyribonuclease (DNase) together with its periplasmic localization in Escherichia coli K-12.

The gene encoding the extracellular DNase of Vibrio cholerae was cloned into Escherichia coli K-12. A maximal coding region of 1.2 kb and a minimal region of 0.6 kb were determined by transposon mutagenesis and deletion analysis. The nucleotide sequence of this region contained a single open reading frame of 690 bp corresponding to a protein of Mr 26,389 with a typical N-terminal signal sequence of 18 aa which, when removed, would give a mature protein of Mr 24,163. This is in good agreement with the size of 24 kDa, calculated directly by Coomassie blue staining following sodium dodecyl sulphate-polyacrylamide gel electrophoresis and indirectly via a DNA-hydrolysis assay. The protein is located in the periplasmic space of E. coli K-12 unlike in V. cholerae where it is excreted into the extracellular medium. The introduction of the DNase gene into a periplasmic (tolA) leaky mutant of E. coli K-12 facilitates the release of the protein, further confirming the periplasmic location.

Genetic analysis of the export of an extracellular DNase of Vibrio cholerae using DNase-beta-lactamase fusions.

A series of C-terminal deletions of the dns-encoded extracellular deoxyribonuclease (DNS) of Vibrio cholerae, fused to the mature form TEM beta-lactamase (Bla) has been used to analyse the export of the DNase in both V. cholerae and Escherichia coli. All hybrid proteins were localized to the periplasmic space in E. coli and V. cholerae, with specific cleavage of the DNS-Bla fusion occurring in V. cholerae. Periplasmic accumulation of wt DNS was also seen in V. cholerae when present on a multicopy plasmid. DNS fusions retaining all six Cys residues of DNS displayed both DNase and Bla enzymatic activity. While hybrid proteins were unable to be secreted across the outer membrane in V. cholerae, the cleaved (active) DNS portion of these proteins was exported. Taken together, these data suggest that the periplasmic form seen in E. coli is a normal intermediate also seen in V. cholerae, and that the lack of secretion machinery in E. coli prevents further export across the outer membrane. Although the DNS portion of the protein fusions must be able to interact with secretion genes, the whole fusion proteins are not exported.

Translocation failure in a type-4 pilin operon: rfb and tcpT mutants in Vibrio cholerae.

Defined chromosomal mutations that lead to assembly failure of the toxin coregulated pilus (TCP) of Vibrio cholerae provide useful insights into the biogenesis of a type-4 pilus. Mutants in rfb affecting LPS O-antigen biosynthesis, and strains depleted of the cytoplasmic membrane-associated ATP-binding protein TcpT, provide contrasting TCP export-defective phenotypes acting at different locations. Mutants in the perosamine biosynthesis pathway of V. cholerae 569B result in an rfb phenotype with an LPS consisting only of core oligosaccharide and lipid A. Such strains are unable to assemble TCP, and TcpA subunits are found in the periplasm and membrane fractions. In both rfb and tcpT mutants, the export defect is specific and complete. TcpT is a member of a large family of cytoplasmic membrane-associated ATP-binding proteins which are essential in type-4 pilin systems and in many non-pilin outer membrane transporters in Gram-negative bacteria. The behaviour of translocation-arrested TcpA in rfb and tcpT mutants is indistinguishable from that within assembled pilus under a range of conditions including flotation in density gradients, chemical cross-linking, and detergent extraction experiments. From the data presently available, it would appear that TcpA requires TcpT-mediated translocation from the cytoplasmic membrane and that TcpT stabilizes the subunit at or immediately beyond this stage, before crossing the outer membrane.

Nucleotide sequence of the traD region in the Escherichia coli F sex factor.

The complete nucleotide sequence has been determined of a 3635-bp region, extending from the HpaI site in traT, at F coordinate 90.3 kb, to beyond the end of traD, of the F sex factor plasmid of Escherichia coli K-12. This region contains the C-terminal coding part of traT and the entire traD gene. An open reading frame (ORF) of 2148 bp within the sequence confirms that traD encodes an 81.4-kDa cytoplasmic membrane protein. The TraD protein has several regions with an unusually high pI (greater than 10), suggesting that they may correspond to the DNA-binding domains. Several other ORFs were detected within the region including the gene (ORF1) for a 26.3-kDa protein and ORF2, probably corresponding to traI, which continues to the end of the sequence. An ORF for an 8.5-kDa protein preceded by an excellent promoter and ribosome-binding site is present in the region following traD but on the opposite strand. This promoter is thought to correspond to the major RNA polymerase binding site in this region, implying that traI does not have its own promoter. The lack of a typical terminator following traD and ORF1 and the translational coupling provided by overlapping stop and start codons is consistent with this conclusion.

Inactivation of the Escherichia coli B41 (O101:K99/F41) rfb gene encoding an 80-kDa polypeptide results in the synthesis of an antigenically altered lipopolysaccharide in E. coli K-12.

The genetic organisation of the rfb region from Escherichia coli B41 (O101:K99/F41) which determines the biosynthesis of the O101 O-antigen of the lipopolysaccharide (LPS) has been examined in E. coli K-12. Maxicell analysis of the plasmid-encoded proteins facilitated the construction of a physical map of the rfb region, consisting of six proteins, designated A (87 kDa), B (80 kDa), C (49 kDa), D (38 kDa), E (36.5 kDa) and F (27 kDa). Proteins E and F are not required for O-antigen biosynthesis. The introduction of frameshift mutations within the region encoding protein B resulted in the synthesis of an antigenically altered LPS which is shorter than the wild-type LPS, as assessed by reaction to antisera in colony and Western immunoblots, and by silver staining of LPS separated on sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. The results demonstrate that protein B has a novel role in O-antigen biosynthesis associated with both the control of LPS chain length and antigenic structure. The nucleotide sequence of the rfb gene encoding protein B has been determined, confirming it to be a 697-amino acid protein of 78.9 kDa predicted to be located in the cytoplasmic membrane.

Homology of TcpN, a putative regulatory protein of Vibrio cholerae, to the AraC family of transcriptional activators.

The nucleotide sequence has been determined for the gene designated tcpN, encoding a putative regulatory protein within the tcp gene cluster associated with the biosynthesis and assembly of the toxin-coregulated pilus of Vibrio cholerae. It is preceded by a powerful transcriptional terminator which presumably delimits the major tcp operon, but at its 3' end is translationally coupled to the gene, tcpJ, encoding the TCP pilin signal peptidase. The tcpN gene encodes a putative 276-residue protein of 31,890 Da. This TcpN shows a high degree of homology to the transcriptional activators, Rns, associated with pilus biosynthesis in enterotoxigenic Escherichia coli, and to VirF, which controls the Yersinia virulence regulon. This homology also extends to the C termini of other members of the AraC family of transcriptional regulators, including RhaS, RhaR and CelD.

RNA-polymerase binding sites within the tra region of the F factor of Escherichia coli K-12.

Chimeric plasmids containing the tra operon of the Escherichia coli K-12 F factor were used to map by electron microscopy the RNA polymerase binding sites within the contiguous F EcoRI restriction fragments f6, f16, f1, f17, f19 and f2. [These fragments have been previously cloned in the EcoRI site of pSC101 to give the chimeric plasmids pRS27 (f6, f15), pRS29 (f15, f1) and pRS31 (f17, f19 and f2)]. The results may reflect the presence of a number of previously unrecognized promoters within the traY----Z operon.

Genetic analysis of the rfbX gene of Shigella flexneri.

Analysis of the nucleotide sequence of the rfbX gene of Shigella flexneri revealed that it contained a high proportion of rare codons, as previously observed in the analysis of the O-antigen polymerase-encoding gene rfc [Morona et al., J. Bacteriol. 176 (1994) 733-747]. The rfbX gene encodes a hydrophobic, 46-kDa protein, with 12 potential transmembrane-spanning domains, that shows structural homology with gene products encoded in many rfb regions, and with Orf0416 of the rff region of Escherichia coli K-12 which has also been identified as a member of this class of proteins. Attempts to clone rfbX independent of other rfb genes, and to identify the protein product of rfbX have proven unsuccessful. Analysis of plasmids containing various deletions within the rfb region suggest that the 5' end of rfbX plays an indirect regulatory role in expression of the dTDP-rhamnose biosynthetic enzymes, encoded by rfbBCAD. We speculate that RfbX is a cytoplasmic membrane protein which functions in the transport of the O-antigen repeat unit.

The oac gene encoding a lipopolysaccharide O-antigen acetylase maps adjacent to the integrase-encoding gene on the genome of Shigella flexneri bacteriophage Sf6.

Lysogens of Shigella flexneri harbouring the temperate bacteriophage, Sf6, have been previously shown to undergo a serotype conversion due to O-acetylation of the O-antigen of the lipopolysaccharide. A partial physical map of the phage genome has been constructed. Analysis of the phage DNA suggests that the phage packages by a headful mechanism and that the mature DNA molecules are terminally redundant. Cloning of the PstI fragments of Sf6 enabled the region encoding the serotype conversion to be localized, showing that this was clearly phage-encoded. The gene was further localized by mutagenesis with Tn5 and the nucleotide sequence of the entire 2693-bp PstI fragment was determined. Two major open reading frames (ORFs) were found capable of encoding proteins of 44.1 and 37.2 kDa. The latter corresponds to the O-antigen acetylase and its gene has been designated oac. The oac gene is capable of converting Sh. flexneri serotypes X, Y, 1a and 4a to 3a, 3b, 1b and 4b, respectively. The Oac protein bears a high degree of homology to the NodX protein of Rhizobium leguminosarum suggesting that it, too, may be a sugar acetylase. The second ORF immediately upstream from oac corresponds to the bacteriophage Sf6 integrase responsible for chromosomal integration and is highly homologous to the integrases of Escherichia coli bacteriophages P4 and phi 80, but less closely related to those of P1, P2, P22, 186 and lambda.

Isolation of enterohemolysin (Ehly2)-associated sequences encoded on temperate phages of Escherichia coli.

We have cloned and sequenced the enterohemolysin (ehl)-associated region from a temperate bacteriophage isolated from an Escherichia coli O26:H11 strain. Phage phi C3208 was isolated together with other temperate bacteriophages which transduce the enterohemolytic phenotype to non-hemolytic E. coli O26 strains. The nucleotide sequence of the 1245-bp phi C3208 DNA insert in plasmid pEO39, which mediates Ehly2 production in E. coli K-12, was determined and was found to be partially homologous to DNA of bacteriophage lambda but is completely unrelated to DNA sequences encoding the synthesis of Ehly1 [Stroeher et al. Gene 132 (1993), 89-94]. It was shown that part of this region can be used as an Hly2-associated specific DNA probe.

Characterization of the replication region of the small cryptic plasmid of Campylobacter hyointestinalis.

The complete nucleotide sequence of a 2.5-kb cryptic plasmid from Campylobacter hyointestinalis was determined. Only one open reading frame (ORF), encoding a polypeptide of M(r) 39,667, designated RepA, could be identified within the sequence. This was confirmed by minicell analysis. Analysis of the region upstream from the ORF showed an A+T-rich region followed by four 19-bp direct repeats. Together, these features are characteristic of other replication origins (ori(s)). The promoter sequence of the repA gene was identified by primer extension analysis and both the putative -10 and -35 regions were found to lie within two potential hairpin-loop structures. RepA showed marked amino acid sequence homology to a replication-initiation protein from the Neisseria gonorrhoeae plasmid, pFA3, and with other replication-initiation proteins over two conserved motifs. A putative partitioning (par) locus was identified upstream from the ori and consisted of a perfect 9-bp inverted repeat and six putative DNA gyrase-binding sites. A putative mobilization origin (oriT) region was identified. This featured a 19-bp imperfect inverted repeat adjacent to a sequence of 12 bp which showed strong homology to the consensus sequence of the 'nick regions' in a variety of oriTs of other plasmids.

Isolation of a restriction-less mutant and development of a shuttle vector for the genetic analysis of Campylobacter hyointestinalis.

A cosmid shuttle cloning vector, pCHI15, was constructed which could be mobilized from Escherichia coli K-12 to a putative restriction-less mutant of Campylobacter hyointestinalis, C. fetus subsp. fetus, and C. fetus subsp. venerealis at a frequency of 10(-4) transconjugants per donor. A previously described C. coli shuttle vector, pILL550, could not be mobilized into the C. hyointestinalis restriction-less mutant, implying that the C. coli replicon was not functional in a C. hyointestinalis host. The type strains of C. jejuni, C. coli, C. fetus subsp. fetus, and C. hyointestinalis were analysed for their ability to be transformed by plasmid DNA which had been modified by other Campylobacter species. Each Campylobacter species was found to be most efficiently transformed by plasmid DNA that had been previously passaged in the same species. pCHI15 could be mobilized from C. coli into C. fetus subsp. fetus and the putative restriction-less mutant of C. hyointestinalis at a frequency of 3.0 x 10(-4) and 2.5 x 10(-3) transconjugants per donor, respectively.

Regulation of tcp genes in classical and El Tor strains of Vibrio cholerae O1.

Expression of genes encoding the toxin-co-regulated pilus (TCP) varies between the two biotypes of Vibrio cholerae O1. Sequence analysis of the tcp locus from the classical and El Tor strains has revealed differences in the intergenic regions between tcpI and tcpP, and tcpH and tcpA, which may be involved in regulation. To investigate this possibility, transcription of tcpA, and the predicted upstream promoters for tcpI and tcpP, has been analysed in the classical and El Tor strains using promoter-cat (chloramphenicol acetyltransferase) fusions. Together with primer extension analyses, these studies indicate that the tcpA and tcpP promoters are toxR-dependent and suggest that TcpP may be involved in activation of both the tcpI and tcpP promoters. We conclude that differences in the level of tcpA expression in a classical and an El Tor strain are likely to be due to the effect of sequence variation on the ability of control factors to act on these regulatory regions.

Nucleotide sequence of the structural gene, tcpA, for a major pilin subunit of Vibrio cholerae.

The toxin co-regulated pilus (Tcp) of Vibrio cholerae appears to be a major protective antigen. By cosmid cloning we have isolated a number of clones capable of converting Tcp- El Tor strains of V. cholerae to Tcp+. A synthetic oligodeoxyribonucleotide probe based upon the N-terminal amino acid sequence of TcpA, has been used to localize the structural gene within the cosmid clones. Using suitable subclones, the nucleotide sequence of the tcpA gene has been determined. The gene encodes a 23.3-kDa pre-protein which in its mature form has a size of 20.3 kDa. The N-terminal leader peptide or signal sequence is atypical and does not conform with the usual rules of such sequences. The TcpA protein shows some similarities to the major pilins of the methylated phenylalanine type or type-4 pili from other bacteria; however, it is sufficiently different that it may represent a new class.

In Vibrio cholerae serogroup O1, rfaD is closely linked to the rfb operon.

The rfaD gene of Escherichia coli encodes ADP-L-glycero-D-mannoheptose-6- epimerase, an enzyme required for the biosynthesis of the lipopolysaccharide (LPS) precursor ADP-L-glycero-D- mannoheptose, associated with production of the core oligosaccharide. We have identified an rfaD homologue in Vibrio cholerae O1. This gene maps adjacent to the rfb region encoding O-antigen biosynthesis, but is transcribed divergently. The complete nucleotide sequence of rfaD and the flanking DNA has been determined, and rfaD would appear to be the only gene homologous to known LPS core biosynthesis genes in this region. Comparison with the E. coli rfaD shows many similar structural features such as the ADP-binding beta alpha beta fold at the N terminus, as well as a high degree of homology of both the nucleotide and amino-acid sequences. Based on homology, rfaD of V. cholerae may be transcribed using both sigma 70- and sigma 54-dependent promoters.

Genetic organization of the regions associated with surface polysaccharide synthesis in Vibrio cholerae O1, O139 and Vibrio anguillarum O1 and O2: a review.

Vibrio cholerae and V. anguillarum are recognized as aquatic-borne human and fish pathogens, respectively. Based upon analyses of several genes and the presence of novel genetic elements it seems that these two species are very closely related. Studies in this laboratory have identified an association of IS1358 with rfb and capsule loci in these two species. The most recent findings suggest that IS1358 is associated with the rfb region in V. cholerae O1 and O139 and in V. anguillarum O1 and O2. In addition, the rfb region in both V. cholerae serogroups and in V. anguillarum O1 is limited at one end by gmhD. These features make it feasible to envisage a mechanism by which the evolution of new rfb genes is taking place involving IS1358 and the region around gmhD. Furthermore, it is possible to envisage that there is or has been an exchange of genetic material between these species leading to new rfb/capsule regions. This review examines the genetics and biosynthesis of the O-antigen and capsule of V. cholerae O1 and O139, as well as the V. anguillarum serogroup O1 and the role of IS1358. Throughout this review we have used the new nomenclature for rfb genes proposed by.

Cloning of the structural gene (hly) for the haemolysin of Vibrio cholerae El Tor strain 017.

We have cloned the DNA encoding the haemolysin of Vibrio cholerae El Tor strain 017 into the plasmid vector pBR322. The resultant plasmid, pPM431, has a 6.2-kb PstI DNA insert which leads to the production of the haemolysin in Escherichia coli K-12. Deletion analysis and transposon mutagenesis have allowed us to localize several regions affecting haemolysin production. A number of these mutants have been analysed in E. coli K-12 minicells. Three proteins have been identified: A, 80 kDal; B, 71 kDal; and C, 22 kDal. A is the haemolysin which appears to be cell-associated in E. coli K-12, and B and C are required for its efficient production. We suggest that the genes for proteins A, B and C be designated hlyA, hlyB and hlyC, respectively.