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 low-calcium response (lcr) is strongly conserved among the pathogenic Yersinia species and is observed when the pathogen is grown at 37 degrees C in Ca(2+)-depleted medium. This response is characterized by a general metabolic downshift and by a specific induction of virulence-plasmid-encoded yop genes. Regulation of yop expression is exerted at transcriptional level by a temperature-regulated activator and by Ca(2+)-regulated negative elements. The yopN gene was shown to encode a protein (formerly also designated Yop4b) which is surface-located when Yersinia is grown at 37 degrees C. yopN was found to be part of an operon that is induced during the low-calcium response. Insertional inactivation of the yopN gene resulted in derepressed transcription of yop genes. A hybrid plasmid containing the yopN gene under the control of the tac promoter fully restored the wild-type phenotype of the yopN mutant. Thus the surface-located YopN somehow senses the calcium concentration and transmits a signal to shut off yop transcription when the calcium concentration is high.

Transmission by flea bite is a relatively recent adaptation that distinguishes Yersinia pestis, the plague bacillus, from closely related enteric bacteria. Here, a plasmid-encoded phospholipase D (PLD), previously characterized as Yersinia murine toxin (Ymt), was shown to be required for survival of Y. pestis in the midgut of its principal vector, the rat flea Xenopsylla cheopis. Intracellular PLD activity appeared to protect Y. pestis from a cytotoxic digestion product of blood plasma in the flea gut. By enabling colonization of the flea midgut, acquisition of this PLD may have precipitated the transition of Y. pestis to obligate arthropod-borne transmission.