The genome of the typhoid fever bacterium, Salmonella typhi, contains at least 3 large insertions ("pathogenicity islands") relative to the chromosome of Salmonella typhimurium (which is normally non-invasive for humans)[Liu and Sanderson, Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 1018-1022]. DNA encoding a site-specific recombinase (the rci gene) and an adjacent putative pilus-tip adhesin protein (the pilV gene) was located (near min 94) in the major pathogenicity island of the S. typhi chromosome, cloned, and sequenced. It was shown that the Rci protein inverted a DNA segment of 490 bp, between two 19-bp inverted repeat elements, to place either of 2 possible C-termini on a constant N-terminal region of the PilV protein. Both possible PilV proteins were seen when the alternative pilV ge...[ Read more ]

The genome of the typhoid fever bacterium, Salmonella typhi, contains at least 3 large insertions ("pathogenicity islands") relative to the chromosome of Salmonella typhimurium (which is normally non-invasive for humans)[Liu and Sanderson, Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 1018-1022]. DNA encoding a site-specific recombinase (the rci gene) and an adjacent putative pilus-tip adhesin protein (the pilV gene) was located (near min 94) in the major pathogenicity island of the S. typhi chromosome, cloned, and sequenced. It was shown that the Rci protein inverted a DNA segment of 490 bp, between two 19-bp inverted repeat elements, to place either of 2 possible C-termini on a constant N-terminal region of the PilV protein. Both possible PilV proteins were seen when the alternative pilV genes were transcribed from the T7 promoter and translated in vivo. Both the rci and the N-terminal region of the pilVgene show high homology to genes encoded by the IncI2 plasmid R721 and the IncIl plasmid R64. One of the possible pilV C-termini (in the pilV1 gene) is highly homologous to shufflon C (one of the possible PilV C-termini) of R64; the other possible pilV C-terminus (in the pilV2 gene) shows no homology to any published shufflon. In the R64 plasmid, the PilV proteins are pilus-tip adhesins; different PilV proteins recognize different potential recipient bacterial strains as a prelude to mating in liquid culture [Komano et al.,J.Mol.Biol.243 (1994) 6-9]. It seemed likely that S. typhi encodes pili bearing 2 alternative PilV proteins as tip adhesins, one of which recognizes, specifically, a membrane component of Escherichia coli K-12, while the specificity of the other PilV protein is not known.

Next, DNA sequencing upstream of the pilV and rci genes described above identified intact pilR, pilS, pilT, and pilU genes. Partial DNA sequencing also located pilO, pilP, and pilQ genes upstream of the pilR-pilV cluster. Probing of DNA regions upstream of the pilO-pilV region showed that cosmid pUST90 contained DNA homologous to the plasmid R64 truB, traC, pilK, pilL, pilM and pilN genes. It thus seemed that S. typhi might synthesize Type IV pili, with PilS protein as the major structural component, and that the pilin operon was similar to that in the R64 plasmid of E. coli in gene organization, nucleotide sequence, protein sequences, and hydrophilicity profiles of the proteins examined. PilS protein was purified, by thrombin cleavage of a fusion protein in which PilS was attached to GST, and an anti-PilS antiserum raised in mice. Also, a deletion mutant of S. typhi, lacking the pil operon, was constructed. When the pil⁺ and pil^- strains were grown in shaking liquid culture, the pil⁺ strain expressed PilS protein externally, as shown by Western blotting of material pelletted by ultrahigh-speed centrifugation of the culture supematant. The pil^- strain did not express PiIS. PCR was used to show that only S. typhi, of a range of Salmonella serovars tested, contained a pilS gene. By deletion mutagenesis of rci, S. typhi strains expressing either pilV1 or pilV2, but not both, were constructed, as was a strain lacking both rci and pilV. These three strains, marked with a Km-resistance cassette in rci DNA, were tested for an ability to transfer Km-resistance to a putative E. coli recipient, or to S. typhi. No transfer of Km-resistance was found. It appears that DNA including the rci gene may not be self-transmissible from S. typhi. The functions of the S. typhi Type IV pili remain to be ascertained.