title: Mutations in yscC, yscD, and yscG prevent high-level expression and secretion of V antigen and Yops in Yersinia pestis.
abstract: The Yersinia pestis low-Ca2+ response stimulon is responsible for the temperature- and Ca(2+)-regulated expression and secretion of plasmid pCD1-encoded antihost proteins (V antigen and Yops). We have previously shown that lcrD and yscR encode proteins that are essential for high-level expression and secretion of V antigen and Yops at 37 degrees C in the absence of Ca2+. In this study, we constructed and characterized mutants with in-frame deletions in yscC, yscD, and yscG of the ysc operon that contains yscA through yscM. All three mutants lost the Ca2+ requirement for growth at 37 degrees c, expressed only basal levels of V antigen and YopM in the presence or absence of Ca2+, and failed to secrete these proteins to the culture supernatant. Overproduction of YopM in these mutants failed to restore YopM export, showing that the mutations had a direct effect on secretion. The protein products of yscC, yscD, and yscG were identified and localized by immunoblot analysis. YscC was localized to the outer membrane of Y. pestis, while YscD was found in the inner membrane. YscG was distributed equally between the soluble and total membrane fractions. Double mutants were characterized to assess where YscC and YscD act in low-Ca2+ response (LCR) regulation. lcrH::cat-yscC and lcrH::cat-yscD double mutants were constitutively induced for expression of V antigen and YopM; however, these proteins were not exported. This finding showed that the ysc mutations did not directly decrease induction of LCR stimulon genes. In contrast, lcrE-yscC, lcrG-yscC, lcrE-yscD, and lcrG-yscD double mutants as well as an lcrE-lcrD double mutant expressed only basal levels of V antigen and YopM and also failed to secrete these proteins to the culture supernatant. These results indicated that a functional LCR secretion system was necessary for high-level expression of LCR stimulon proteins in the lcrE and lcrG mutants but not in an lcrH::cat mutant. Possible models of regulation which incorporate these results are discussed.
authors.affiliation: Department of Microbiology and Immunology, Chandler Medical Center, University of Kentucky, Lexington 40536, USA.
authors.forename: G V
authors.initials: GV
authors.lastname: Plano
authors.forename: S C
authors.initials: SC
authors.lastname: Straley
mesh_terms: D000942:Antigens, Bacterial / Q000096:biosynthesis*; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D001692:Biological Transport / Q000235:genetics; D002118:Calcium / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics*; D006358:Hot Temperature; D008565:Membrane Proteins / Q000235:genetics*; D026901:Membrane Transport Proteins; D008954:Models, Biological; D008958:Models, Molecular; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010641:Phenotype; D017354:Point Mutation; D052899:Pore Forming Cytotoxic Proteins; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D015010:Yersinia pestis / Q000235:genetics* / Q000254:growth & development / Q000276:immunology
chemical_list: D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; C088009:LcrG protein, Yersinia; C043908:LcrV protein, Yersinia; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D018832:Molecular Chaperones; D052899:Pore Forming Cytotoxic Proteins; C094424:YscC protein, Yersinia pestis; C094426:YscG protein, Yersinia; C060772:yopM protein, Yersinia; D002118:Calcium