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 Mr 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 theori 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.

A 4332 nt MluI-PstI DNA fragment, earlier termed parVP, carries the incompatibility and partition determinants of the virulence plasmid, pSLT, of Salmonella typhimurium. This fragment was sequenced and the genes and regions responsible for incompatibility and partition phenotypes were identified by reference to previously published work. Incompatibility with pSLT, mediated by the 4332 nt fragment, requires both a homolog of the parS region of the Escherichia coli plasmids P1/P7 and a new incompatibility determinant, termed incR. The two genes identified in the sequence were also close homologs of the parA and parB loci of P1/P7. Unlike the situation in P1/P7, however, the parA and parB genes appear to be independently transcribed. Effective partition of pJRD158-based plasmids carrying fragments of the sequenced DNA required ParA and tire incR region; ParB and parS were not necessary. It appears that pSLT may possess two related partitioning mechanisms, an analog of the recognized ParA/ParB/parS system and another system which requires ParA and incR.

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.

Outer membranes were isolated, by sodium lauryl sulphate extraction, from the American type strain, five Australian, and four English isolates of Campylobacter hyointestinalis. On SDS-PAGE examination, the protein profiles of seven strains (including the type strain) were similar, and were dominated by two major proteins of 47 and 50 kDa. Three other isolates had unique major protein profiles. The largest of these proteins was heat-modifiable in these isolates, and in the type strain. The flagellin of three isolates screened was of similar Mr to that of Campylobacter jejuni/Campylobacter coli. The lipopolysaccharides of C. hyointestinalis isolates were heterogeneous in structure; 5/10 isolates synthesised material of Mr value greater than that of the low MrC. jejuni/C. coli lipopolysaccharide. By gel excision and re-electrophoresis, it was shown that the higher Mr materials of one isolate were not artifactual aggregates of lower Mr species.

EX645 is a derivative of Salmonella typhi Ty21a which carries a plasmid specifying production of Vibrio cholerae O antigen. When cultured with exogenous galactose to overcome the galE defect of the vector, EX645 also synthesizes S. typhi O antigen, and this can result in the masking of the shorter V. cholerae O antigen on the bacterial surface. To determine whether the potential for such masking at least partly underlies the inconsistency of anti-V. cholerae responses elicited by EX645, a derivative of this strain has been isolated, characterized, and tested for immunogenicity in human volunteers. EX880 has an rfb defect which prevents synthesis of S. typhi O antigen, and consequently V. cholerae O antigen is still detectable on the surface of the clone following growth in the presence of galactose. Compared with EX645, EX880 more consistently elicited significant rises in serum bactericidal antibody levels, although individual responses within a cohort still varied widely.

The pmi gene, encoding phosphomannose isomerase, of Salmonella typhimurium, was cloned in Escherichia coli K-12, and the protein product visualised in minicells. The cloned gene was sequenced; there was 77.4% nucleotide homology between the cloned pmi gene and the analogous manA gene of E. coli K-12, and 86.2% amino acid sequence homology between their presumptive gene products.

Insertion mutations were constructed in cloned pmi and rfc genes of Salmonella typhimurium, and these mutations were recombined (singly) into the chromosome of mouse-virulent S. typhimurium C5, displacing the wild-type alleles. Phage sensitivity profiles, lipopolysaccharide analysis, and DNA blotting all confirmed that the replacement events had occurred. The mutations were complemented by plasmid-borne wild-type alleles, as judged by the restoration of wild-type phage plaquing profiles and lipopolysaccharide production (both mutants) and the restoration of pmi-encoded enzyme production (pmi mutant). The virulence, persistence, and immunizing capacities of the mutants fed to mice were compared with those of the wild-type strain and complemented mutants. Both mutants were much reduced in virulence, with the rfc mutant being avirulent even at 10(9) bacteria per mouse. This mutant was also avirulent at up to 10(6) bacteria per mouse when administered intraperitoneally. Both the rfc and pmi mutant strains persisted in the Peyer's patches of the gut after feeding and were capable of colonizing the deeper tissues of the mice from such initial infective foci. Both mutant strains were effective as live oral vaccines (10(7) bacteria or more) against oral S. typhimurium challenge (10(4) 50% lethal doses; 6 x 10(8) bacteria) in mice.

The rfc gene of Salmonella typhimurium was located in a 1.75-kb HindIII fragment and restored wild-type lipopolysaccharide synthesis ability to both an older rfc point mutant and new rfc::IS10 mutants. DNA sequencing of the HindIII fragment revealed an open reading frame which could encode a protein of 407 amino acids with an M(r) of 47,472 and also revealed potential translation signals. Modulator codons accounted for 12.5% of the total codon content, providing a possible explanation for the nondetectability of the protein in subcellular systems. Secondary structure analysis suggested the presence of transmembrane beta-sheet structures, implying a possible role for the protein in translocation of hydrophilic O-antigen-containing materials. Salmonella strains of groups A, B, and D1 contained rfc-homologous DNA, but strains of groups C1, C2, C3, D2, and E2 did not.

There continues to be considerable interest in the development of a safe, effective, live, oral vaccine to combat typhoid fever of humans. Such a vaccine may be a derivative of the causative agent of the disease, Salmonella typhi. The prototype of such a vaccine, Ty21a, is not ideal, but no replacement for Ty21a is yet obvious. The construction and trial of bivalent vaccines, in which an attenuated Salmonella strain expresses determinants from another pathogen, awaits the development of a suitably attenuated derivative. In parallel with vaccine development programmes, a variety of techniques have been designed to effect stable association between Salmonella carrier and introduced cloned DNA.

The incompatibility functions (inc) of the virulence plasmid of Salmonella typhimurium LT2 were initially located in a 4.3 kb region near the repA locus of the plasmid. Expression of inc required the presence, in cis or in trans, of two distinct DNA regions of the fragment. These regions, maximally 0.3 kb and 0.6 kb in size, were separated in the fragment by c. 3.0 kb. This intervening DNA encoded two proteins, of Mr values 37 kDa and 40 kDa. The promoter for the 37 kDa protein lay in or near one of the inc regions (incL). No function was assigned to this protein, however, it may be the product of a rep gene. The 40 kDa protein may have a partition (par) function, and may bind to a centromeric site in or near the other inc region (incR). An inc+ par− derivative of the original plasmid clone was used in a simple method, not involving the use of curing agents/mutagens, to eliminate virulence plasmid DNA from Salmonella typhimurium, Salmonella dublin, Salmonella enteritidis, and Salmonella cholerae-suis. The par function served to stabilise pJRD158B-based plasmid >106-fold in Escherichia coli and the virulence plasmid-cured Salmonella strains.

We have developed a system whereby heterologous DNA encoding an antigen from an enteropathogen may be recombined into the chromosome of an attenuated Salmonella carrier strain. The system involves two steps; (i) integration of a hisOG deletion mutation into the chromosome; (ii) replacement of the hisOG deletion by the complete hisOG region and the segment of heterologous DNA which encodes the antigen of interest. Recombinants may be selected (his+). The system was used to integrate the genes encoding K88 fimbriae from enterotoxigenic Escherichia coli into the chromosome of a galE mutant of Salmonella typhimurium (LT2H1). Recombinants were detected at a frequency of between 1.0 × 10−3 and 1.5 × 10−3. A variety of tests confirmed that the K88 genes were integrated into the chromosome of LT2H1 and were expressed. The stability of the recombinant was tested both in vivo and in vitro. When administered orally to mice, the recombinant elicited a serum antibody response to K88, and retained the Salmonella vaccine potential of the vector strain.

We have recently described the construction of a galE derivative of Salmonella typhi Ty2 (Ty2H1) which had a 0.4-kilobase deletion in the galE gene and was sensitive to galactose-induced lysis when cultured with greater than or equal to 0.06 mM galactose (D. M. Hone, R. Morona, S. Attridge, and J. Hackett, J. Infect. Dis. 156:167-174, 1987). We now report the selection of a rifampin-resistant, via derivative of Ty2H1, EX462. Compared with the Ty2 parent strain, EX462 was serum sensitive and highly attenuated in the mouse mucin virulence assay. When four human volunteers ingested 7 X 10(8) viable EX462, two became ill and developed a typhoidlike disease with fever and bacteremia. Blood isolates from these individuals were indistinguishable from the vaccine strain by a variety of criteria. We concluded that, even in a via background, the galE mutation was not attenuating for S. typhi in humans.

Some strains of Salmonella, when fed to mice, establish a nonlethal, limited infection in the Peyer's patches of the small intestine. When such mice are later challenged orally with a normally lethal dose of virulent Salmonella typhimurium, a protective effect of the prior vaccination is seen. In an effort to characterize the determinant(s) of the avirulent strains responsible for this protective effect, we examined the cell envelope protein profiles of five such protective strains and of eight strains of Salmonella that were both nonpersistent and nonprotective when fed to mice. The protective strains produced high levels of flagellin. We made otherwise isogenic fla+ and fla- derivatives of two such strains and showed that although the fla- derivatives colonized mice as well as did the fla+ strains, the fla- derivatives given orally showed a much reduced ability to protect mice from S. typhimurium challenge. Both fla+ and fla- strains induced cellular immunity in vaccinated mice.

A live oral vaccine has been developed against scouring induced in piglets by enterotoxigenic Escherichia coli (ETEC). An attenuated strain of Salmonella typhimurium, G30, has been used as a vector for plasmids encoding the production of the fimbrial colonization factors of porcine ETEC. Initial studies with clones expressing K88 or K99 fimbriae have shown them to be well tolerated when administered orally in very high doses. The clones elicited serum, colostral and milk antibodies to the fimbrial antigens, and a challenge trial indicated that such responses were sufficient to ensure the passive transfer of protective immunity to suckling piglets. The possible advantages of this approach are discussed.

The prophylactic significance of the nonlipopolysaccharide (non-LPS) antigens of Vibrio cholerae was investigated further with use of the infant mouse cholera model. Of 16 strains examined to date, 12-including eight recent field isolates of both biotypes and both common serotypes-express common non-LPS protective antigens. The exceptional strains are four old isolates of El Tor biotype, the outer membrane proteins of which are not atypical when analyzed by immunoblotting. The protective activities of antibodies to the shared non-LPS components correlated with their capacities to inhibit the in vitro attachment of Vibrio organisms to isolated murine enterocytes.

We describe the molecular cloning of the gal operon of Salmonella typhimurium LT2 and the localization of the gal promoter and the genes galE, galT, and galK. The order of the genes and the direction of transcription was the same as in Escherichia coli K12. A restriction enzyme map of the operon was obtained, and the approximate termini of the gal genes were located by using transposon insertion mutagenesis and minicell analysis. We constructed a plasmid that contained a defined 0.4-kilobase deletion in the galE but still expressed galT and galK activities from the gal promoter. This galE deletion was recombined into the chromosomal gal operons of S. typhimurium and Salmonella typhi Ty2. The resulting strains were vigorous, nonreverting galE mutants that were sensitive to galactose-induced lysis at 0.2 mM galactose. The S. typhimurium galE derivatives were avirulent and protective in mice.

A cosmid bank of the DNA (including cryptic plasmid DNA) of a virulent strain of Salmonella typhimurium was prepared in Escherichia coli K12, and clones that contained cryptic plasmid DNA were detected by probing. Two such clones expressed a new outer membrane protein of 11 kilodaltons (kDa) and were serum resistant (E. coli K12 is serum sensitive). The gene encoding the 11-kDa protein was subcloned in a 2.1-kilobase fragment and shown to mediate serum resistance in both E. coli K12 and a cryptic plasmid-free (serum-sensitive) strain of S. typhimurium. The cryptic plasmid-free S. typhimurium strain did not express normal lipopolysaccharide, but introduction of the 11-kDa protein gene into the strain rendered the strain serum resistant without restoration of normal lipopolysaccharide synthesis. The 11-kDa protein gene was not sufficient to restore either macrophage resistance or virulence to a cryptic plasmid-free strain of S. typhimurium.

We cured a strain of Salmonella typhimurium of its cryptic plasmid and confirmed that orally administered cured strains lost virulence for mice. Loss of the cryptic plasmid rendered the S. typhimurium strain sensitive to the bactericidal action of normal human serum. However, loss of the plasmid did not change the ability of the strain to associate with HeLa cells in tissue culture. Furthermore, when administered orally to mice, both the plasmid-containing and plasmid-free strains invaded the Peyer's patches of the small intestine to the same extent, and both were capable of inducing resistance to oral challenge with virulent S. typhimurium. When injected intraperitoneally, the cured strain was eliminated rapidly, whereas the parental strain persisted. We also showed that the cured strain did not contain a plasmid copy in the chromosome. We propose that although the plasmid-cured strain of S. typhimurium is able to colonize Peyer's patches, it cannot survive when administered intraperitoneally because it is susceptible to elimination by macrophages.

We present the nucleotide sequence of the tolC gene of Escherichia coli K12, and the amino acid sequence of the TolC protein (an outer membrane protein) as deduced from it. The mature TolC protein comprises 467 amino acid residues, and, as previously reported (1), a signal sequence of 22 amino acid residues is attached to the N-terminus. The C-terminus of the gene is followed by a stem-loop structure (8 base pair stem, 4 base loop) which may be a rho-independent termination signal. The codon usage of the gene is nonrandom; the major isoaccepting species of tRNA are preferentially utilised, or, among synonomous codons recognized by the same tRNA, those codons are used which can interact better with the anticodon (2,3). In contrast to the codon usage for other outer membrane proteins of E. coli (4) the rare arginine codons AGA and AGG are used once and twice respectively.

We examined the biosynthesis of the TolC protein of Escherichia coli K12 in a pulse-chase experiment, followed by immunoprecipitation with anti-TolC antibody and SDS-PAGE of the immunoprecipitate. This showed that TolC protein was originally synthesised in a precursor form (Mr 54 500) which could be chased into the mature form (Mr 52 000). DNA sequencing of a portion of the cloned tolC gene showed that the N-terminus of the mature rotein was preceded by a typical signal sequence of 22 residues (Mr 2542). The initiator Met was preceded by a Shine-Dalgarno sequence, with the correct spacing.