Yersinia pestis, the plague agent, is a naturally nonureolytic microorganism, while all other Yersinia species display a potent urease activity. In this report we demonstrate that Y. pestis harbors a complete urease locus composed of three structural (ureABC) and four accessory (ureEFGD) genes. Absence of ureolytic activity is due to the presence of one additional G residue in a poly(G) stretch, which introduces a premature stop codon in ureD. The presence of the same additional G in eight other Y. pestis isolates indicates that this mutation is species specific. Spontaneous excision of the extra G occurs at a frequency of 10 ؊4 to 10 ؊5 and restores a ureolytic phenotype to Y. pestis. The virulence of two independent ureolytic clones of Y. pestis injected either intravenously, subcutaneously, or intragastrically did not differ from that of the parental strain in the mouse infection model. Coinfection experiments with an equal number of ureolytic and nonureolytic bacteria did not evidence any difference in the ability of the two variants to multiply in vivo and to cause a lethal infection. Altogether our results demonstrate that variation of one extra G residue in ureD determines the ureolytic activity of Y. pestis but does not affect its virulence for mice or its ability to multiply and disseminate.Yersinia pestis and Yersinia pseudotuberculosis are gram-negative bacteria pathogenic for animals and humans. The former is transmitted by fleas and is responsible for plague, a fatal systemic infectious disease, whereas the latter causes a selflimiting mesenteric lymphadenitis and is transmitted by the oral route (10). Although Y. pestis and Y. pseudotuberculosis have distinct cycles of transmission and induce infection with different clinical manifestations, they are genetically closely related, with a DNA relatedness superior to 90% as determined by DNA-DNA hybridization (4). Sequence comparisons of homologous genes from both microorganisms revealed nucleotide identities ranging from 97 to 99% (1,9,28,32,33,35,36,39,40), and recent results suggest that Y. pestis is a clone of Y. pseudotuberculosis that emerged less than 20,000 years ago (1). Strikingly, several genes present in both species are intact in Y. pseudotuberculosis but mutated in Y. pestis. These include the virulence-associated plasmid (pYV)-borne gene yadA and the chromosomal invasion genes inv and ail. Nonexpression of yadA is due to a frameshift mutation in its coding sequence (33), while inactivation of inv and ail results from the disruption of their open reading frames by insertion sequences IS1541 (35) and IS285 (S. D. Torosian and R. M. Zsigray, Abstr. 96th Gen. Meet. Am. Soc. Microbiol. 1996Microbiol. , abstr. B-213, 1996, respectively. Furthermore, several phenotypic properties such as motility at 28°C; synthesis of a complete LPS molecule; and ability to ferment rhamnose and melibiose, to synthesize some amino acids (methionine, phenylalanine, threonine-glycine, and isoleucine-valine), and to degrade urea are expressed in Y. pseudotubercul...

Yersinia pseudotuberculosis produces YPM (Y. pseudotuberculosis-derived mitogen), a superantigenic toxin that exacerbates the virulence of the bacterium in vivo. To date, three alleles of the superantigen gene (ypmA, ypmB, and ypmC) have been described. These genes are not found in all Y. pseudotuberculosis strains and have a low GC content, suggesting their location on mobile genetic elements. To elucidate this question, the genetic environment of the superantigen-encoding genes was characterized and 11 open reading frames (ORFs) were defined. Sequence analysis revealed that the ypm genes were not associated with plasmids, phages, transposons, or pathogenicity islands and that the superantigen genes were always located in the chromosome between ORF3 and ORF4. Nonsuperantigenic strains exhibited the same genetic organization of the locus but lacked the ypm gene between ORF3 and ORF4. A new insertion sequence, designated IS1398, which displays features of the Tn3 family, was characterized downstream of the ypmA and ypmC genes. A 13.3-kb region containing the ypm genes was not found in the genome of Y. pestis (CO92 and KIM 5 strains). We experimentally induced deletion of the ypm gene from a superantigen-expressing Y. pseudotuberculosis: using the association of aph(3)-IIIa and sacB genes, we demonstrated that when these reporter genes were present in the ypm locus, deletion of these genes was about 250 times more frequent than when they were located in another region of the Y. pseudotuberculosis chromosome. These results indicate that unlike other superantigenic toxin genes, the Yersinia ypm genes are not associated with mobile genetic elements but are inserted in an unstable locus of the genome.Yersinia pseudotuberculosis, a microorganism causing gastrointestinal diseases and immunopathological complications such as reactive arthritis and erythema nodosum (10,47,59,60), is at present the only gram-negative bacteria known to produce a superantigenic toxin. This molecule (designated YPM for Y. pseudotuberculosis-derived mitogen) is a 14.5-kDa protein able to induce proliferation of human T lymphocytes bearing V␤3, V␤9, V␤13.1, and V␤13.2 T-cell receptor variable regions (1, 61). In vivo, YPM induces lethal shock in mice (44) and exacerbates the virulence of Y. pseudotuberculosis in a murine experimental model of systemic infection (13). To date, three YPM variants (YPMa, YPMb, and YPMc) have been described. YPMa displays 83% identity with YPMb (11, 50) and differs from YPMc only by a single substitution at position 51 of the mature protein (12). A phylogenetic analysis based on the amino acid sequences of various bacterial superantigenic toxins indicated that the YPM variants belong to a new type of bacterial superantigen family (12, 46). The ypm genes encoding the YPM toxins have not been found in the genome of Yersinia pestis, a genetically related species (49). Yersinia enterocolitica, another pathogenic Yersinia species, has been described as a mitogen-producing microorganism (57, 58); however, the Tcell speci...

The genome of Yersinia pestis, the causative agent of plague, contains at least 30 copies of an element, designated IS1541, which is structurally related to IS200(85% identity). One such element is inserted within the chromosomalinv gene (M. Simonet, B. Riot, N. Fortineau, and P. Berche, Infect. Immun. 64:375–379, 1996). We characterized other IS1541 insertions by cloning 14 different Y. pestis 6/69M loci carrying a single copy of this insertion sequence (IS) into Escherichia coli and, for each element, sequencing 250 bp of both flanking regions. In no case was this IS element inserted into large open reading frames; however, in eight cases, it was detected downstream (17 to 139 bp) of genes thought to be transcribed monocistronically or which constituted the last gene of an operon, and in only one case was it detected upstream (37 bp) of the first gene of an operon. Sequence analysis revealed stem-loop structures (ΔG, <−10 kcal) resembling rho-independent transcription terminators in 8 of the 14 insertion sites. These motifs might constitute hot spots for insertion of this IS1541element within the Y. pestis genome.

We characterized Yersinia enterocolitica and Yersinia pseudotuberculosis insertion sequences related to insertion sequence 1541, recently identified in Yersinia pestis. For each of the two species, two insertion sequence copies were cloned and sequenced. Genetic elements from Y. pseudotuberculosis were almost identical to insertion sequence 1541, whereas these from Y. enterocolitica were less related. Phylogenetic analysis of the putative transposases encoded by insertion sequences from the three pathogenic members of the genus Yersinia showed that they clustered with those encoded by Escherichia coli and Salmonella enterica elements belonging to the insertion sequence 200/insertion sequence 605 group. Insertion sequences originating from Y. pestis and Y. pseudotuberculosis constitute a monophyletic lineage distinct from that of Y. enterocolitica. ß