During the previous funding cycle a 5-gene operon named sat, for secretion and acid tolerance, was identified in Streptococcus mutans. This operon, located immediately downstream from the opuA osmotolerance operon, contains ffh that encodes Ffh, a key component of the bacterial signal recognition particle (SRP). S. mutans ffh mutants displayed acid intolerance and an acid inducible promoter was identified that allowed coordinate expression of the entire sat locus as a single mRNA transcript. Mutants displayed markedly reduced levels of H+/ATPase in isolated membranes leading to the belief that an acid tolerance response involves cotranslational translocation of membrane proteins by the SRP. In the present proposal, we will study further regulation of the sat locus, and its regulatory relationship to opuA, by means of Northern blot and real time RT-PCR analyses and investigate the role of YlxM, encoded by y/xM immediately upstream offfh, as a putative regulatory protein. We will confirm the presence of the SRP in S. mutans and determine its similarity to SRPs described from other prokaryotes by searching for the known complexed components, Ffh and the small cytoplasmic (4.5S) RNA, along with the membrane receptor, FtsY, in complexes immunoprecipitated from strain NG8 cellular extracts. We will employ the ffh mutant, MK4, and the wild-type strain NG8 to identify membrane proteins that require the SRP for translocation. Cells grown under steady-state (chemostat) conditions at pH 7 or 5 will serve as the source of cell fractions. Two-D gel electrophoresis, yeast two-hybrid, Ffh-GST affinity chromatography and affinity chromatography will be employed. Major physiologic parameters, including glycolysis and pH homeostasis, and genetic competence will be studied in this mutant which has been shown to have major alterations in these characteristics. Also during the previous funding cycle, the necessity of the central proline-rich repeat domain for surface expression of adhesin P1 (I/II) in S. mutans, but not secretion in E coli, was described. The role of gene products encoded within the clp, dnaK and groE operons in the intra-cellular turnover and chaperone-mediated surface expression of P1 will be assessed and intra-molecular interactions of P1 domains necessary for stability and translocation will be evaluated. By complementation of E. coli Sec-pathway mutants with an NG8 gene library, S. mutans homologues/analogues of Sec components will be identified. Immunoprecipitation of P1 and associated cellular proteins with anti-P1 antibodies will be used to identify chaperones or modifying factors involved in the export of this key adhesin. The project described in this proposal continues studies of membrane biogenesis and translocation of a major surface-localized protein of S. mutans.
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