The causative agent of plague, Yersinia pestis, contains a 75-kb plasmid, pCD1, which carries a virulencerelated stimulon called the low-Ca2+ response stimulon (LCRS). LCRS operons are regulated by the environmental signals of temperature and Ca2+. This study characterized a portion of the lkrB region of pCD1, known to contain at least one gene necessary for the regulation of LCRS operons by Ca2+. Ca2" or nucleotides (11,35,78). These genes, which belong to the LCR stimulon (LCRS), encode secreted factors such as V antigen (12, 79) and a set of about 10 virulence proteins called Yops (9,58,76,77,79). Proteins regulating the expression and secretion of V antigen and Yops are encoded within a ca. 25-kb region of the LCR plasmid termed the Ca2' dependence region (9,13,24,57,78,83).One manifestation of the LCR is the requirement for millimolar calcium or 10 to 20 mM nucleotides, such as ATP, for growth above 34°C (28,84,85). Although growth occurs independently of Ca2+ or ATP at 26°C, a shift in incubation temperature to 37°C in the absence of Ca2+ or ATP results in a cessation of growth within a few generations (28,84). This phenomenon is termed growth restriction. Paradoxically, restriction is concurrent with maximal induction of LCRS operons and secretion of LCRS-encoded proteins.V antigen is a soluble protein encoded by the kcrV gene of the lcrGVH-yopBD operon (5,52,59). Although a specific antihost function has not been assigned to V antigen, it is a protective antigen and is believed to be required for full virulence (10,36,73 (65, 66), and sequestration of thrombin (38, 64). V antigen and Yops lack cleavable N-terminal signal sequences and are secreted by an LCRS-encoded mechanism (45). N-terminal sequences have been implicated in directing transport from the cell (43).Previously, two loci within the Ca2" dependence region have been shown to affect both LCRS operon transcription and secretion. LcrD of the lcrDR operon is necessary for maximal induction of LCRS operons and secretion of LCRS-encoded virulence proteins (24,54,55). LcrD has homology to several proteins associated with flagellar biogenesis, invasion of epithelial cells, and plant pathogenesis in taxonomically divergent bacteria (54,78). The LcrD homolog is thought to be a component of a novel secretion system involved in each of these diverse processes.The 13-cistronysc locus is also necessary for the secretion of the V antigen and Yops (27,44). Polar insertion mutants of this region are defective in LCR-specific secretion and show altered regulation of LCRS operon transcription (24,27,44). However, the dominant effects on regulation are the opposite between Y pestis and the enteropathogenic yersiniae, with constitutive downregulation being seen in the former and constitutive induction being seen in the latter (24,27,44). This could arise from different extents of polarity in the respective ysc loci, and sorting out these differences will require analysis of nonpolar mutants in the various ysc genes.Early studies involving the mutagenesis of the Y...