Construction of a mutant strain of Streptococcus mutans with clpC-deletion to study the role of clpC ;gene in genetic competence

• DOI: 10.3760/cma.j.issn.0254-5101.2015.04.002
• Publication Date: 2015-01-01
• Authors: Qiaoli Xu; Huihua Rao; Xiaobo Ma; Chaoyang Huang; Gangsen Zheng; Jiaqin Zhang; Xiuyu Song

Objective To construct a mutant strain of Streptococcus mutans (S.mutans) with clpC-deletion and to investigate the role clpC gene in genetic competence.

Methods The fragment kanamycin resistant cassette flanked by two loxP sites were amplified PCR. The purified was cloned into pMD-19T simple vector pCKX1. pCKX1 digested ClaⅠ/EcoRⅠ, then blunted introduced lox71-KMR-lox66 obtain pCKX2 via homologous recombination. linearized SalⅠ transformed S. UA159 strain. positive strains constructed recombination screened thermosensitive plasmid pCrePA. KMR excised after incubating at 30℃ for 48 hours. Then pCrePA removed overnight 37℃ preparation mutant. Total RNA extracted from respectively, reverse transcribed first strand cDNA. target fragments RT-PCR analyzed agarose gel electrophoresis sequencing. After being verified PCR sequencing, respectively E. coli-S.mutans shuttle pDL276 observe competence development induced competence-stimulating peptide (CSP).

Results The sequencing results showed that successfully No detected as indicated analysis. formation competent delayed state prolonged compared its parent strains.

Conclusion The negatively regulated mutans.

Key words: Streptococcus mutans; Competence; clpC; Cre/loxP