GENETIC COMPETENCE IN STREPTOCOCCUS MUTANS BIOFILMS

这一建议解决的遗传转化的变形链球菌，龋齿的主要病原体，在生物膜的机制。 为了了解生物膜中的基因转移，有必要确定有利于允许细菌掺入外源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。 尽管感受态在抗生素耐药病原体的出现中具有重要意义，但从未在生物膜中进行过研究。 S.将变形杆菌在控制生长速率、pH和生长限制性营养素的连续培养生物膜发酵罐中生长，以找到其最佳转化的条件，所述最佳转化通过经由质粒转化赋予抗生素抗性来确定。 为了定义感受态的机制，将使用Tn 917-lac转座子诱变来产生感受态缺陷的突变体;将分析受影响的基因。 还将使用两种新方法搜索在生物膜生长条件下表达的基因。 一个涉及从生物膜和增殖细胞中提取的mRNA的比较。 通过差异显示PCR鉴定生物膜生长期间表达的基因并克隆。 另一种是使用体内表达技术（IVET）系统的修改，将生物膜状态视为体内状态。 它依赖于异源二倍体突变体库的构建。在随机插入的S.变形杆菌启动子。 在氯霉素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打开的基因。 将选择与已知感受态或转座子基因具有序列同源性的基因进行进一步研究;将其在亲本菌株中灭活，并评估其表型特性。 所选择的基因也将用于构建lacZ融合报告菌株，并测试粘附和生物膜积累期间的表达，通过荧光成像检测。 该计划应阐明环境条件，生理学和遗传学促进基因转移之间的S。变形细胞在生物膜中生长。

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.