GENETIC COMPETENCE IN STREPTOCOCCUS MUTANS BIOFILMS

这一建议解决了变异链球菌的遗传转化机制，龋齿的主要病原，在生物膜。为了理解生物膜中的基因转移，有必要确定有利于细菌吸收外源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。虽然能力在抗生素耐药病原体的出现中很重要，但从未在生物膜中进行过研究。变形链球菌将在连续培养的生物膜发酵罐中生长，该发酵罐控制生长速率、pH值和生长限制性营养物质，通过与质粒转化来确定其抗生素抗性，从而找到最佳转化条件。为了明确能力的机制，将使用Tn917-lac转座子诱变产生能力缺陷的突变体；受影响的基因将被分析。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来执行。其中一个涉及从生物膜和浮游细胞中提取的mRNA的比较。利用差异显示PCR技术对生物膜生长过程中表达的基因进行鉴定和克隆。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文库。克隆体在含有氯霉素的生物膜中存活，但在琼脂板上对氯霉素敏感，这将允许克隆在生物膜生长过程中优先开启的基因。选择与已知能力或转运基因序列同源的基因进行进一步研究；它们将在亲本株中灭活，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，并通过荧光成像检测在粘附和生物膜积累过程中的表达。该计划应阐明环境条件，生理和遗传学促进变异链球菌细胞之间的基因转移生长在生物膜。

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.