Cloning Of S.mutans F-ATPase β Subunit Gene And Analysis Of F-ATPase Activity

Objective: Streptococcus mutans(S.mutans) is a component of the dental plaque biofilm and a major casual agent of dental caries. The F-ATPase functions to regulate the internal pH of oral streptococci, so the F-ATPase has important effects on the bacteria of dental plaque. In this study, the S.mutans F-ATPase β -subunit gene was cloned and analyzed, in order to investigate further the roles of F-ATPase β -subunit gene for S.mutans acid tolerance and caries generation by gene methods. Additionally, construction of the recombinant plasmid containing S.mutans F-ATPase β -subunit gene may assist in construction of homologues recombinant mutant by transformation of Streptococcus mutans. In a word, the study on enzyme including F-ATPase related caries is important to finding new treatment of dental caries.Methods: At first, S. mutans was grown in anaerobe condition, and S. mutans chromosomal DNA were extracted;a region at the 5' terminus of the S.mutans F-ATPase β subunit gene was amplified by PCR, the PCR product was inserted into vector pVA891,yielding recombinant plasmid;the plasmid was transformed into E.coli and the transformed E.coli was analyzed on F-ATPase activity;transformation ability was compared and analyzed between the S.mutans Ingbritt strain and the fluoride-resistance strain.Results: The DNA sequence of the recombinant plasmid was identified correct in whole by restriction endonuclease and DNA sequence techniques . The recombinant plasmid of S.mutans DNA was cloned in effect in E.coli, and theF-ATPase activity of transformed E.coli was determined different with the parent strain. In initial study, transformation in fluoride-resistance strain is significantly more frequent than Ingbritt strain.Conclusion: In this study, the recombinant plasmid was constructed and cloned efficiently in E.coli. hi addition, the F-ATPase activity of transformed E.coli containing F-ATPase 0 subunit gene was analyzed and demonstrated F-ATPase P subunit gene is important to the F-ATPase activity of transformed E.coli. So the important effect of S.mutans F-ATPase P subunit gene was confirmed further by gene methods. The ability to examine individual genes or their products remains technically more difficult than for other transformable systems and is still not possible for strains in each serotype of S. mutans. It is possible that recombinant plasmid may assist in construction of homologues recombinant mutant by transformation of Streptococcus mutans.