MOLECULAR BIOLOGICAL CHARACTERIZATION OF ORAL BACTERIA

口腔细菌的分子生物学特征

• 批准号: 3896720
• 负责人: B M CHASSY
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3896720
• 关键词: Bacillus subtilis; Escherichia coli; Lactobacillaceae; Lactobacillus casei; Salmonella typhimurium; Staphylococcus aureus; Streptococcus lactis; bacterial DNA; bacterial genetics; bacterial proteins; beta galactosidase; electroporation; gene expression; gene therapy; genetic manipulation; genetic regulation; heterochromatin; host organism interaction; lac operon; molecular cloning; molecular pathology; mutant; nucleic acid sequence; oral bacteria; plasmids; site directed mutagenesis; transfection; transposon /insertion element; virulence

This project seeks to use genetic, biochemical and physiological approaches to investigate the pathogenicity of oral bacteria. The two specific areas of investigation are: 1) characterization of genes isolated from oral bacteria; and 2) development of systems for genetic exchange in oral bacteria. The amino acid residue that participates in transphosphorylation at the active center EnzymeII(lac) of Lactobacillus casei was determined. Replacement of each of the histidine and cysteine residues in EII with other amino acid by site-directed mutagenesis, followed by an analysis of their ability to phosphorylate lactose using an in vitro assay system, revealed that only cysteine-483 is required for enzymatic activity. Using the HOST-VECTOR system previously developed for the genetic manipulation of lactobacilli, the expression of a number of heterologous genes has been examined. Of particular interest, the genes encoding the EnzymeII(lac), p-beta-gal and Factor III(lac) of L. casei were transcriptionally fused to the strong constitutive beta- galactosidase promoted isolated from L. bulgaricus to create an operon- like structure which was inserted into the shuttle vector pBS19 forming pBS914. This construction was transformed into lactose negative L. casei strains; transformants were selected by their ability to grow on lactose. Efforts are now directed at construction of food grade vectors with lactobacilli used in food fermentations, or isolated from the gastrointestinal tract, for gene delivery and antigen presentation to humans.

该项目试图使用遗传，生化和生理学 研究口腔细菌致病性的方法。 两个 特定研究领域是：1）基因的表征 从口腔细菌中分离出来； 2）开发遗传系统 在口腔细菌中交换。 参与的氨基酸残基 乳酸杆菌的活性中心酶（LAC）的磷酸化 确定了壳。替换每个组氨酸和半胱氨酸 EII中与其他氨基酸中的残留物，通过位置定向诱变， 然后分析其使用乳糖磷酸化的能力 一个体外测定系统，发现只有半胱氨酸-483才需要 酶活性。 使用先前开发的宿主向量系统 对于乳杆菌的遗传操纵，数量的表达 已经检查了异源基因。 特别有趣的是 编码L。 酪蛋白在转录融合到强的本构β- 从保加利亚分离出的半乳糖苷酶创建了操纵子 - 就像插入穿梭矢量PBS19形成的结构一样 PBS914。 该结构转化为乳糖负L. casei 菌株；选择转化子是根据其增长的能力来选择的 乳糖。 现在努力进行食品级向量的建设 用用于食品发酵的乳酸杆菌或与 胃肠道，用于基因递送和抗原表现为 人类。