Construction of a mutant strain of Streptococcus mutans with clpC-deletion to study the role of clpC ;gene in genetic competence / 中华微生物学和免疫学杂志

ABSTRACT

Objective To construct a mutant strain of Streptococcus mutans ( S.mutans ) with clpC-deletion and to investigate the role of clpC gene in genetic competence.Methods The fragment of clpC gene and the kanamycin resistant cassette flanked by two loxP sites were amplified by PCR.The purified fragment of clpC gene was cloned into pMD-19T simple vector to construct pCKX1.The pCKX1 vector was digested with ClaⅠ/EcoRⅠ, then blunted and introduced into lox71-KMR-lox66 to obtain pCKX2 vector via homologous recombination.The pCKX2 vector was linearized with SalⅠ and transformed into S.mutans UA159 strain.The positive strains constructed via homologous recombination were screened with kanamycin and transformed with the thermosensitive plasmid pCrePA.The KMR cassette was excised after incubating at 30℃ for 48 hours.Then the pCrePA plasmid was removed after overnight incubating at 37℃for the prepara-tion of clpC-deletion mutant.Total RNA were extracted from the S.mutans UA159 strain and the clpC-dele-tion mutant strain respectively, and then reverse transcribed into first strand cDNA.The target gene frag-ments were amplified by RT-PCR and analyzed by the agarose gel electrophoresis and sequencing.After be-ing verified by PCR and sequencing, the S.mutans UA159 strain and the clpC-deletion mutant strain were re-spectively transformed with E.coli-S.mutans shuttle vector pDL276 to observe the competence development induced by the competence-stimulating peptide (CSP).Results The PCR and sequencing results showed that the pCKX2 vector and the mutant strain with clpC-deletion were constructed successfully via homologous recombination.No clpC gene was detected in the clpC-deletion mutant as indicated by RT-PCR analysis.The formation of competent clpC-deletion mutant was delayed and the competence state was prolonged as com-pared with its parent strains.Conclusion The clpC gene negatively regulated the formation of competent S.mutans.