MEMBRANES OF THE DENTAL PATHOGEN STREPTOCOCCUS MUTANS

描述（由申请人提供）：在上一个资助周期中，在变形链球菌中发现了一个名为sat的5基因操纵子，用于分泌和耐酸性。该操纵子位于opuA β耐受性操纵子的下游，含有编码Ffh的ffh，Ffh是细菌信号识别颗粒（SRP）的关键组分。S.变异株FFH突变体表现出酸不耐受性，并且鉴定了酸诱导型启动子，其允许整个SAT基因座作为单个mRNA转录物协调表达。突变体显示H+/ATP酶水平显着降低，在隔离的膜导致的信念，酸耐受性反应涉及膜蛋白的共翻译易位的SRP。在本研究中，我们将通过北方印迹和真实的时间RT-PCR分析进一步研究sat基因座的调控及其与opuA的调控关系，并研究由y/xM直接上游offfh编码的YlxM作为推定的调控蛋白的作用。我们将确认在S.变异的，并确定其相似性SRPs描述从其他原核生物通过搜索已知的复合成分，Ffh和小细胞质（4.5S）RNA，沿着膜受体，FtsY，在复合物免疫沉淀从菌株NG 8细胞提取物。我们将采用ffh突变体MK 4和野生型菌株NG 8来鉴定需要SRP进行易位的膜蛋白。在pH 7或5的稳态（恒化器）条件下生长的细胞将用作细胞级分的来源。采用双向凝胶电泳、酵母双杂交、Ffh-GST亲和层析和亲和层析等方法。主要的生理参数，包括糖酵解和pH稳态，和遗传能力将在这个突变体已被证明有重大改变，这些特征进行了研究。同样在上一个资助周期中，在S.在大肠杆菌中不分泌。将评估CLP、dnaK和groE操纵子内编码的基因产物在细胞内周转和伴侣蛋白介导的P1表面表达中的作用，并评价稳定性和易位所需的P1结构域的分子内相互作用。通过对E. coliSec途径突变体，并利用NG 8基因文库，对S.将鉴定Sec组分的变异株同源物/类似物。免疫沉淀P1和相关的细胞蛋白与抗P1抗体将被用来确定分子伴侣或修饰因子参与出口的这一关键粘附素。本项目继续研究膜生物发生和一个主要的表面定位蛋白的转运。变异人

DESCRIPTION (provided by applicant): 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.