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。该操纵子位于opuA渗透耐受操纵子的下游，含有编码细菌信号识别粒子（SRP）关键成分ffh的ffh。S. mutans ffh突变体表现出酸不耐受，并且鉴定出一个酸诱导启动子，允许整个sat位点作为单个mRNA转录物进行坐标表达。突变体在分离膜中表现出明显降低的H+/ atp酶水平，导致人们认为酸耐受性反应涉及SRP对膜蛋白的共翻译易位。在本研究中，我们将通过Northern blot和实时RT-PCR分析进一步研究sat位点的调控及其与opuA的调控关系，并研究由y/xM直接上游编码的YlxM作为一种推定的调控蛋白的作用。我们将通过从菌株NG8细胞提取物中免疫沉淀的复合物中寻找已知的络合成分Ffh和小细胞质（4.5S） RNA以及膜受体FtsY，来确认变形链球菌中SRP的存在，并确定其与其他原核生物中描述的SRP的相似性。我们将使用ffh突变株MK4和野生型菌株NG8来鉴定需要SRP进行易位的膜蛋白。在pH值为7或5的稳态（趋化）条件下生长的细胞将作为细胞组分的来源。将采用二维凝胶电泳、酵母双杂交、Ffh-GST亲和层析和亲和层析。主要的生理参数，包括糖酵解和pH稳态，以及遗传能力将在这个突变体中进行研究，该突变体已被证明在这些特征上有重大改变。在之前的资助周期中，也描述了在S. mutans中粘附素P1 （I/II）表面表达而不是在大肠杆菌中分泌的中心富含脯氨酸重复结构域的必要性。将评估clp、dnaK和groE操纵子编码的基因产物在细胞内转换和伴侣介导的P1表面表达中的作用，并评估P1结构域稳定和易位所需的分子内相互作用。通过将大肠杆菌Sec途径突变体与NG8基因文库进行互补，可以鉴定出Sec成分的突变s同源物/类似物。P1和相关细胞蛋白与抗P1抗体的免疫沉淀将用于鉴定参与这一关键黏附素输出的伴侣或修饰因子。在这个提议中描述的项目继续研究变形链球菌的膜生物发生和主要表面定位蛋白的易位。

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.