GENETIC COMPETENCE IN STREPTOCCUS MUTANS BIOFILMS

变异链球菌生物膜的遗传能力

• 批准号: 6379936
• 负责人: Dennis G. Cvitkovitch
• 金额: $ 11.59万
• 依托单位: UNIVERSITY OF TORONTO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6379936
• 关键词: Streptococcus mutans; bacterial genetics; cell adhesion; confocal scanning microscopy; dental plaque; growth media; human tissue; messenger RNA; microorganism culture; molecular film; mutant; polymerase chain reaction

This proposal adresses mechanisms of genetic transformation of Streptococcus mutans, a principal etiologic agent of dental caries, in biofilms. To understand gene transfer in biofilms, it is necessary to determine the conditions which favor a physiologic state ( competence ) that allows bacteria to incorporate foreign DNA, permitting horizontal gene transfer among species. Though competence is significant in the emergence of antibiotic resistant pathogens, it has never been studied in biofilms. S. mutans will be grown in a continuous culture biofilm fermentor that controls growth rate, pH and growth-limiting nutrient, to find conditions for its optimum transformation as determined by conferment of antibiotic resistance via transformation with plasmids. To define the mechanisms of competence, Tn917-lac transposon mutagenesis will be used to generate mutants defective in competence; affected genes will be analyzed. A search for genes expressed under conditions of biofilm growth will also be executed using two novel approaches. One involves the comparison of mRNA extracted from biofilm and planktonic cells. Genes expressed during biofilm growth are identified by differential display PCR and cloned. The other uses a modification of the in vivo expression technology (IVET) system that treats the biofilm state as an in vivo state. It relies on construction of a heterodiploid mutant library of S. mutans with a chloramphenicol resistance-amylase gene fusion (cat-amy) under control of randomly inserted S. mutans promoters. Clones surviving in a biofilm with chloramphenicol but sensitive to it on agar plates will allow the cloning of genes that turn on preferentially during biofilm growth. Genes exhibiting sequence homology to known competence or transoport genes will be chosen for further study; they will be inactivated in the parent strain and their phenotypic properties assessed. The chosen genes will also be used to construct lacZ fusion reporter strains and tested for expression during adhesion and biofilm accumulation, detected by fluorecence imaging. The program should elucidate the environmental conditions, physiology, and genetics fostering gene transfer among S. mutans cells growing in biofilms.

该提案解决了生物膜中变形链球菌（龋齿的主要病原体）的遗传转化机制。 为了了解生物膜中的基因转移，有必要确定有利于细菌掺入外源DNA的生理状态（能力）的条件，从而允许物种之间的水平基因转移。 尽管能力对于抗生素抗性病原体的出现很重要，但从未在生物膜中进行过研究。 变形链球菌将在连续培养生物膜发酵罐中生长，该发酵罐控制生长速率、pH 和生长限制营养物质，以通过质粒转化赋予抗生素抗性来确定其最佳转化条件。 为了定义能力机制，Tn917-lac 转座子诱变将用于产生能力缺陷的突变体；将分析受影响的基因。 还将使用两种新方法来寻找在生物膜生长条件下表达的基因。 其中之一涉及从生物膜和浮游细胞中提取的 mRNA 的比较。 通过差异显示 PCR 鉴定并克隆生物膜生长期间表达的基因。 另一种使用体内表达技术（IVET）系统的修改，将生物膜状态视为体内状态。 它依赖于在随机插入的变形链球菌启动子的控制下构建具有氯霉素抗性-淀粉酶基因融合体(cat-amy)的变形链球菌异二倍体突变体文库。 在含有氯霉素的生物膜中存活但在琼脂平板上对其敏感的克隆将允许克隆在生物膜生长期间优先开启的基因。 将选择与已知能力或转运基因表现出序列同源性的基因进行进一步研究；它们将在亲本菌株中失活并评估其表型特性。 所选基因还将用于构建 lacZ 融合报告菌株，并通过荧光成像检测粘附和生物膜积累过程中的表达情况。 该计划应阐明促进生物膜中生长的变形链球菌细胞之间基因转移的环境条件、生理学和遗传学。