Yersinia pestis, the causative agent of plague, contains a virulence plasmid pCD that encodes an adaptive mechanism called the low-Ca2+ response. This mechanism confers upon the bacteria the ability to use the environmental cues of temperature, Ca2+ and nucleotides to regulate growth and the expression of two proteins known as the plague virulence antigens V and M. The low-Ca2+ response is thought to ensure that virulence functions such as B and M are expressed in the proper environment within the mammalian host. Genetic studies have shown that a large region on pCD contains genes necessary for expression of the low-Ca2+ response, and 5 low-Ca2+ response (lcr) genes have been identified. Five other pCD genes are strongly regulated in their expression by Ca2+, nucleotides, and temperature. These are candidates for virulence functions regulated in the low-Ca2+ response. The specific aims of this project are to determine the identities, genetic arrangement, and functional relation of lcr genes to each other and to the pCD genes that are regulated by Ca2+ and nucleotides, using molecular genetic manipulations and mutagenesis techniques. The products of these genes will be identified with the aid of protein fusions to beta-galactosidase, specific antibody, and two-dimensional electrophoresis. The molecular basis of regulation of gene expression by Ca2+ and nucleotides will be probed by obtaining and characterizing mutant Y. pestis aberrant in these properties. The role of pCD gene products in pathogenesis will be studied by kinetically characterizing the fates of parent and mutant strains of Y. pestis in mouse peritoneal cavities and determining the classes of mammalian cells that are involved in the interactions. These studies have the long-range goal of understanding the molecular basis of the low-Ca2+ response and of the function of pCD genes in pathogenesis of a severe infectious disease. They also will enhance our understanding of regulatory mechanisms in enteric bacteria, since the regulation of bacterial growth by Ca2+ and nucleotides is novel.