GENETIC COMPETENCE IN STREPTOCCUS MUTANS BIOFILMS

这项建议阐述了变形链球菌在生物膜中的遗传转化机制，变形链球菌是龋齿的主要病原菌。为了了解生物膜中的基因转移，有必要确定有利于细菌整合外来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。尽管能力在抗生素耐药病原体的出现中具有重要意义，但在生物膜中从未进行过研究。变形链球菌将在连续培养的生物膜发酵罐中生长，该生物膜发酵罐控制生长速度、pH和限制生长的营养物质，以寻找其最佳转化条件，该条件是通过用质粒转化产生抗生素耐药性来确定的。为了确定能力的机制，将使用Tn917-lac转座子突变来产生能力缺陷的突变；将分析受影响的基因。寻找在生物膜生长条件下表达的基因也将使用两种新的方法。其中一个涉及从生物膜和浮游细胞中提取的mRNA的比较。用差异显示聚合酶链式反应鉴定并克隆了生物膜生长过程中表达的基因。另一种是使用体内表达技术(IVET)系统的改进，该系统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(CAT-AMY)的变形链球菌异二倍体突变体库。在含有氯霉素的生物膜中存活，但在琼脂平板上对氯霉素敏感的克隆将允许克隆在生物膜生长过程中优先打开的基因。将选择与已知的能力或转位基因序列同源的基因进行进一步研究；它们将在亲本菌株中失活，并对其表型特性进行评估。选定的基因还将用于构建LacZ融合报告菌株，并通过荧光成像检测在黏附和生物膜积累过程中的表达。该计划应阐明在生物膜中生长的变形链球菌细胞之间的环境条件、生理和遗传学促进基因转移。

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.