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.
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