YERSINIA-PESTIS LCRV FORMS A STABLE COMPLEX WITH LCRG AND MAY HAVE A SECRETION-RELATED REGULATORY ROLE IN THE LOW-CA2+ RESPONSE

Yersinia pestis contains a virulence plasmid, pCD1, that encodes many virulence-associated traits, such as the Yops (Yersinia outer proteins ) and the bifunctional LcrV, which has both regulatory and antihost fu nctions, In addition to LcrV and the Yops, pCD1 encodes a type III sec retion system that is responsible for Yop and LcrV secretion, The Yop- LcrV secretion mechanism is believed to regulate transcription of lcrV and yop operons indirectly by controlling the intracellular concentra tion of a secreted repressor, The activity of the secretion mechanism and consequently the expression of LcrV and Yops are negatively regula ted in response to environmental conditions such as Ca2+ concentration by LcrE and, additionally, by LcrG, both of which have been proposed to block the secretion mechanism, This block is removed by the absence of Ca2+ or by contact with eukaryotic cells, and some Yops are then t ranslocated into the cells, Regulation of LcrV and Yop expression also is positively affected by LcrV. Previously, LcrG was shown to be secr eted from bacterial cells when the growth medium lacks added Ca2+, alt hough most of the LcrG remains cell associated, In the present study, we showed that the cell-associated LcrG is cytoplasmically localized. We demonstrated that LcrG interacts with LcrV to form a heterodimeric complex by using chemical cross-linking and copurification of LcrG and LcrV. Additionally, we found that small amounts of LcrV and YopE can be detected in periplasmic fractions isolated by cold osmotic shock an d spheroplast formation, indicating that their secretion pathway is ac cessible to the periplasm or to these procedures for obtaining peripla smic fractions, We propose that the cytoplasmically localized LcrG blo cks the Yop secretion apparatus from the cytoplasmic side and that Lcr V is required to remove the LcrG secretion block to yield full inducti on of Yop and LcrV secretion and expression.