Development of mutant generation system of Streptococcus mutans, and analysis of the functional domain of glucosyltransferases using the mutant.

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The erythromycin resistant gene (erm) which works in Streptococcus mutans was cloned from Streptococcus-E.coli shuttle vector pVA838. pVA838 was digested with Hind III and Cla I, then inserted into a cloning vector pUC19.The DNA sequence of the insert was revealed that the essential part of the erm was app. 800 bp.Then PCR primers were designed to add restriction enzyme sites at the end of the essential gene.A smaller (830 bp) erythromycin resistant cassette was generated by PCR.The kanamycin resistant gene (aphA) was cloned from streptococcal transposon Tn 1545 by the similar procedure.DNA sequence of the 1.6 kb insert containing the aphA revealed that an open reading frame of the aphA was app. 1000 bp.From the sequence of the aphA gene, PCR primers were also synthesized.A smaller (1070 bp) kanamycin resistant cassette was produced by PCR.Furthermore, the tetracycline resistant gene was cloned from Tn 1545.On other hand, randomly mutagenesis was performed to investigate the localization mechanism of the cell-associated glucosyltransferase (CA-GTase).The chromosomal DNA gene banks from S.mutans strains MT8148 and GS-5 were generated with pVA891.The gene bank was randomly transformed into S.mutans.The erythromycin resistant transformants were screened from Mitis-Salivarius agar.Then expression of CA-GTase was examined by the Western blot analysis.Although over 1000 mutants were tested, the localization of the CA-GTase was not altered.