Study On The Gene Related To The Acid Tolerance Of Streptococcus Mutans

Streptococcus mutans (S. mutans) plays a critical role in the onset and progression of dental caries. Its main virulent factors include adhesion to the tooth surface, acidogenicity and acid tolerance. The mechanisms about both the adherence and the acidogenicity are clear now while the molecular mechanism of acid tolerance is still under investigation. More and more researches are focused on the genes that may be related to the acid tolerance of this opportunistic oral pathogen.Studies have shown that the acid tolerance of S. mutans includes two parts, the constitutive acid tolerance and the acid tolerance response, both of which are related to the activity and optimum pH values of the proton translocating membrane ATPase (H+-ATPase). The more the acitivity and the lower the pH optima value of H+-ATPase is, the more acid tolerant the bacteria will be. With the development of modern molecular biology, more and more genes that may be responsible for the acid tolerance of 5. mutans are being discovered. Among them the most useful tool is transposon mutagenesis, that is, taking advantage of the mobile characteristic of transposon to induce its random insertion into the chromosome DNA of bacteria, select mutants with mutant phenotype followed by the recover of the mutant gene. If the phenotypic change is the result of single transposon insertion, then the interrupted gene should be related to this phenotype.Until now, S. mutans UA159 is the only strain of mutans Strptococci whose complete genome sequence has been reported. The successful sequencing of this strain provides a convenient pathway for the genetic study of 5. mutans. The advantage of using UA159 as an experimental strain for the genetic study of S. mutans with unknown genetic basis under specific phenotype is that once one obtained a short fragment of the gene flanking the insertion loci, the wholegene and its flanking sequences can be easily recovered and analyzed through Genbank. In this study, for the first time, we used transposon Tn917 to randomly mutagenize 5. mutans UA159 and to select an acid-sensitive mutant. Southern analysis and genetic back-cross experiment were performed to determine the association of Tn917 insertion to the mutant phenotype (acid-sensitivity). Comparison of the abilities to grow at low pH, the glycolytic pH drop, the activity of ATPase and the killing pH were performed between the acid-sensitive mutant and the parent strain. Asymmetric PCR were used to obtain the gene fragment flanking the Tn917 insertion site and blasts were performed to find the loci of the insertion site in the chromosome of UA159 followed by the analysis of the gene both upstream and downstream. We try to find a new gene that may be responsible for the acid tolerance of S. mutans.Part One Construction of mutant S.mutans defective in acid toleranceFirst, the temperature sensitive plasmid pTVl-OK harboring Tn917 was prepared by the alkaline lysis method and purified by polyethylene glycol precipitation. pTVl-OK was transformed into S. mutans UA159. Since S. mutans UA159 is Gram positive bacteria and is poorly transformable strain, the competence stimulating peptide (CSP) was used to induce the bacteria to permit the uptake and incorporation of foreign DNA more easily, and the transformation rate was 20 times improved. The second step is to induce the transposition of Tn917 into the genome of S. mutans UA159. Transformant harboring the pTVl-OK was incubated at 28 C for 48 hours. Saturated culture were subcultured at different dilutions into fresh THYE broth(containing 0.04 u g/ml erythromcin or not) followed by a shift to nonpermissive temperature(37-42C) and incubated overnight. The optimum condition was 1:100 or 1:200 dilution and incubation at 41-42C. The 3rd step is the selection of the mutant defective in acid tolerance by screening the mutant pools on pH 5.0 solid plates. From 2316 transporsants, one mutant defective in acid tolerance was successfully isolated and was named as b23. 4th, Souhtern blot analysis was performed with EcoR I (no cut...