Salmonella enterica serovar Typhi and some strains (Vi+) of serovar Dublin use type IVB pili to facilitate bacterial self-association, but only when the PilV proteins (potential minor pilus proteins) are not synthesized. Pilus-mediated self-association may be important in the pathogenesis of enteric fever. We have shown previously that the extent of DNA supercoiling controls the rate of Rci-catalyzed inversion of a DNA fragment which includes the C-terminal portions of the PilV proteins. This inversion therefore controls PilV synthesis as a high inversion rate prohibits transcription of pilV-encoding DNA. Here, we describe the manner in which PilV protein expression inhibits bacterial self-association and present data which suggest that incorporation of one or a few PilV protein molecules into a growing pilus, comprised of PilS subunits, causes the pilus to detach at the bacterial membrane. The bacteria are then unable to self-associate. We suggest that this phenomenon may be relevant to the pathogenesis of typhoid fever.

Salmonella enterica serovar Typhi uses type IVB pili to facilitate eukaryotic cell invasion. Here, we compare environmental and genetic controls on pil operon transcription with those regulating viaB genes required for Vi antigen expression. Transcription of pil occurs only in the late logarithmic and stationary phases of bacterial growth while viaB expression occurs in the logarithmic growth phase. Expression of both viaB and pil was, however, optimal at 100mM NaCl, and mutations in envZ/ompR, rcsB/rcsC, (but not rcsA), tviA, ihfB or fis affected transcription of both viaB and pil DNA. As both Vi antigen and Type IVB pili facilitate serovar Typhi invasion of human monocytes, an overlap of production controls is logical. It appears that Vi antigen synthesis precedes pilus production.

Some strains of Salmonella enterica serovar Dublin are Vi antigen-positive. S. enterica serovar Typhi uses Type IVB pili, encoded adjacent to the viaB locus required for Vi antigen synthesis, to facilitate both eukaryotic cell attachment and bacterial self-association under conditions that favour DNA supercoiling. These pilus-mediated events may be important in typhoid fever pathogenesis. A survey of 17 isolates of S. enterica serovar Dublin showed that all strains which carried the viaB region also carried a serovar Typhi-like Type IVB pil operon, and all serovar Dublin Vi antigen-negative isolates lacked the pil operon. The pil operon was completely sequenced from one of the Vi+ serovar Dublin strains, and was almost identical (4 nt changes; 3 aa changes, in over 10 kb) to that of serovar Typhi. A pilS mutant of one serovar Dublin strain was constructed, and shown to invade cultured human intestinal INT407 cells to an extent only 20% that of the wild-type parent. Purified prePilS protein inhibited INT407 cell entry by serovar Dublin. The wild-type serovar Dublin strain, but not the pilS mutant, self-associated. The data suggest that the serovar Dublin Type IVB pil operon may increase the human-invasiveness of serovar Dublin, compared to pil-free strains.

Salmonella typhimurium G30 was used as a vector to express the ETEC (enterotoxigenic Escherichia coli) fimbrial antigens K88 and K99. Two plasmids encoding K88 or K99 production and having a non-antibiotic selection marker (thyA+) were constructed. These were introduced into a thyA G30 derivative to give the vaccine strains EX841 and EX603, which were shown to express surface K88 or K99, respectively. When administered orally to adult pigs, a dose of 1011 vaccine organisms elicited significant serum antibody responses to the respective fimbrial antigens. Two such immunizations with EX841 generated serum antibody levels comparable to those obtained with intramuscular injection of killed organisms. Attenuated salmonellae can thus be used to deliver ETEC fimbrial antigens to the porcine intestinal immune system.

In the past year, the importance of secretory IgA has been emphasized as fundamental to protection against oral Salmonella infection. In several human trials, aro mutants of Salmonella typhi were highly immunogenic, but still retained the capacity to proceed beyond the gut wall after ingestion. Epitopes of Shiga toxin and influenza hemagglutinin have been expressed in Salmonella surface proteins in work aimed at the construction of hybrid vaccines. Eukaryotic cell involvement in the process of Salmonella attachment/invasion appears to be triggered by host cell phospholipase activation. Our understanding of the number and functions of Salmonella genes involved in the attachment/invasion process has increased considerably--different gene sets are required for invasion of different cell types.

Mobilization of a potential Campylobacter shuttle vector from Escherichia coli K-12 into C. hyointestinalis generated a small plasmid harboring a hybrid oriT nick region. This is presumed to have resulted from the cleavage and religation of the oriT of RP4 and the putative nick region of the shuttle vector. These data imply that two distinct DNA cleavage reactions occur at the oriT site of RP4 during conjugal transfer.

We have previously described the cosmid cloning of the genes determining the biosynthesis of the Inaba and Ogawa O-antigens of the lipopolysaccharides of Vibrio cholerae 01 (Manning et al., 1986). By Southern hybridization analysis of chromosomal and cosmid DNA, and heteroduplex analysis between the clones we have been able to precisely define the region of contiguous chromosomal DNA in the vicinity of the O-antigen-encoding region. These data and comparison of end points of clones and of deletion derivatives demonstrate that at least 16kb of a 19-kb SstI fragment is required to encode O-antigen biosynthesis. Expression of O-antigen is independent of the orientation of this SstI fragment with respect to cloning vectors suggesting that its regulatory region has been cloned intact. No detectable differences were observed in the restriction patterns of the Inaba and Ogawa coding regions implying that only minor changes are involved when serotype conversion (Inaba to Ogawa or vice versa) occurs. Bhaskaran [Ind. J. Med. Res. 47 (1959) 253–260] originally defined this region associated with O-antigen biosynthesis oag; however, to be consistent with other organisms [Hitchcock et al., J. Bacteriol. 166 (1986) 699–705], it is suggested this be changed to rfb.

A cosmid bank of the DNA of Salmonella typhimurium LT2 was prepared in Escherichia coli K12, and probed with radiolabelled cryptic plasmid from the same Salmonella strain. 2 of the 7 clones thus detected expressed an obvious new outer membrane protein of 23 kDa. The gene encoding the polypeptide was subcloned, in a 6.6-kb ClaI fragment, from one of the cosmids into pBR322. The clone was mapped with restriction enzymes. The gene location and the direction of transcription were defined with the aid of transposon Tn1725 insertions. The gene product was detected in minicells. The protein was partially peptidoglycan-associated in E. coli.

Using antisera prepared against live Vibrio cholerae we have selected several recombinant DNA clones, plasmids pPM440, pPM450 and pPM460, encoding the gene for a 22-kDal V. cholerae peptidoglycan-associated-outer-membrane protein. This is a minor protein in V. cholerae but is expressed in large amounts when the cloned gene is present in Escherichia coli K-12, where it is exposed on the cell surface as judged by ELISA. We have localized the gene within the cloned DNA by transposon mutagenesis and deletion analysis followed by analysis of whole cells and minicells to identify the plasmid-encoded proteins. The DNA region encoding the protein seems to be conserved between El Tor and Classical strains as judged by Southern DNA hybridization.

A group of ompA mutants of Escherichia coli K12 are described which were sensitive to bacteriophage K3 in a background wild-type for lipopolysaccharide (LPS). With mutant LPS in vivo (lacking some core sugar residues), however, the ompA mutations gave resistance to K3. Outer membrane levels of OmpA protein were normal or near-normal when the mutations resided in either wild-type or mutant LPS backgrounds. Strains in which the mutations occurred in a wild-type LPS background adsorbed K3 phage at the same initial rate and to the same extent as a wild-type strain, but the efficiency of plaquing of the adsorbed K3 was reduced to 25–50% of wild-type levels. Under conditions where a wild-type strain irreversibly adsorbed over 90% of available phage K3 within 3 min, double mutants (ompA mutant, LPS mutant) left 90% of the phage viable after 1h. The 10% of inactivated phage did not form plaques.

The regulatory genes phoB and phoR of the Escherichia coli K-12 pho regulon have been cloned using two types of selection. The genes were localized on one of the hybrid plasmids, pJP50, by analysis of mutant plasmids generated by Tn5 insertions. Restriction mapping of the other plasmid, pPR20, indicated that the gene order in this region of the chromosome is phoB phoR tsx. Analysis of plasmid-coded proteins in a minicell system showed that: (1) the products of phoB and phoR are proteins with apparent molecular weights of 30,000 and 47,000, respectively; and (2) the synthesis of the phoB gene product is derepressed by phoR mutations. The implications of this observation for the model of pho regulation are discussed.