Virulent Yersinia species possess a common plasmid that encodes essential virulence determinants (Yops) which are regulated by the extracellular stimuli Ca2+ and temperature. The V antigen operon was recently shown to be involved in the Ca21_regulated negative pathway (A. Forsberg and H. Wolf-Watz, Mol. Microbiol. 2:121-133, 1988). We show here that the V antigen-containing operon of Yersinia pseudotuberculosis is a polycistronic operon having the gene order IcrGVH-yopBD. DNA sequencing analysis of lcrGVH revealed a high homology to the corresponding genes of Yersinia pestis. LcrG was conserved and LcrH showed only one amino acid difference, while LcrV showed only 96.6% identity. The amino acid substitutions of LcrV occurred in the central domain of the protein, while the two ends of the protein were conserved. Northern (RNA) blotting experiments showed that the operon is regulated at the transcriptional level by the extracellular stimuli temperature and calcium. One 4.6-kb transcriptional product of the operon was identified. This mRNA is rapidly processed at its 5' end, resulting in different mRNA species of variable stability. By genetic analysis, the kcrV and kcrH gene products were found to be regulatory proteins having important roles in the Ca2 -controlled regulation of Yop expression. The activity of LcrH is modulated by a gene product of the operon that inhibits the negative action of LcrH on yop transcription in the absence of Ca2+.

The virulence plasmids pYV019, pYV8081, and pIBl from Yersinia pestis, Yersinia enterocolitica, and Yersinia pseudotuberculosis, respectively, were characterized by restriction endonuclease analysis. The three plasmids exhibited a region of common DNA previously shown to encode determinants which confer Ca>2 dependence. The plasmids from Y. pestis and Y. pseudotuberculosis were similar throughout their genomes. In contrast, a region of the plasmid from Y. enterocolitica which contained an origin of replication differed from the other two plasmids as determined by DNA homology and replication properties. Plasmidassociated outer membrane proteins from all three species of Yersinia were characterized by polyacrylamide gel electrophoresis. There were no differences in the outer membrane protein profiles between plasmid-containing and homogenic strains lacking the plasmid after growth at 28°C. After growth at 37°C, both Y. enterocolitica and Y. pseudotuberculosis showed at least four major plasmid-associated outer membrane proteins. Y. pestis did not show any discernible changes after growth at 37°C. It was shown by using E. coli minicell analysis that the plasmid DNA from all three species of Yersinia contained the coding capacity for production of the novel outer membrane proteins.

Virulence plasmid-containing cells of Yersinia pseudotuberculosis had the ability to inhibit phagocytosis by mouse peritoneal macrophages cultured in vitro, but cells of its plasmid-cured derivative did not. Inhibition was most pronounced when the pathogen was incubated under Ca2-deficient conditions, which allowed a high level of expression of outer membrane proteins (Yops). The addition of 2.5 mM Ca2+ to the growth medium reduced the degree of inhibition by the pathogen, but it was still significantly higher than that of the plasmid-cured strain. An avirulent mutant strain, from which the entire yopH gene was deleted, was impaired in its phagocytosis inhibition ability. This mutant could be trans-complemented by the yopH+ gene back to the wild-type phenotype with respect to virulence, as well as the ability to inhibit phagocytosis, demonstrating that the ability to inhibit phagocytosis is an important virulence function. The mutant strain was still cytotoxic for HeLa cells, indicating that inhibition of phagocytosis can be genetically separated from the ability to cause a

A strain of Yersinia pseudotuberculosis which harbors a 63-kilobase plasmid was found to cause a lethal infection in Swiss albino mice. The rate of infection paralleled the ability of the pathogenic organism to attach to a monolayer of HeLa cells. One novel outer membrane protein (protein 1) with a molecular weight of 140,000 was found to be associated with the possession of the 63-kilobase plasmid not at 26 degrees C, and expression was moderately affected by the concentration of calcium in the growth medium. Moreover, it was found that synthesis of protein 1 associated outer membrane protein showing similar properties was also found to be expressed in plasmid-containing strains of Yersinia enterocolitica. The properties of protein 1 indicate that it could be identical to the previously described virulence W antigen.

The basic Yop2b protein, encoded by the virulence plasmid pIBI of Yersinia pseudotuberculosis, is produced under Ca2+-deficient conditions. A mutant deleted for the entire yopH gene, which encodes the Yop2b protein, was found to be avirulent. Virulence could be restored by trans-complementation. The DNA-sequence of yopH predicted a 50 737 D polypeptide lacking a typical signal peptide. Transcription of yopH is regulated by both temperature and Ca2+-concentration. Mutations within the region of the virulence plasmid known to be involved in regulating gene expression in response to Ca2+ abolished transcription of yopH. Other temperature-sensitive mutations in the Ca2+-regulatory locus showed a high level of transcription regardless of Ca2+-concentration. These responses were similar to those of the yopE gene. The promoter region of the yopE and yopH genes were compared and four conserved motifs identified.