【摘要】 目的:通过同源重组法构建变异链球菌LuxS基因缺陷突变菌株,比较其与变异链球菌UA159标准株在生物膜形成上的差异。方法:运用基因同源重组方法将氯霉素抗性基因(Cmr)与LuxS基因上下游区域的2个基因片段按一定顺序重组到PUC载体的多克隆位点中,构建出带氯霉素抗性标志的重组质粒,将载体质粒转化到含完整LuxS基因的变异链球菌UA159中,利用氯霉素抗性筛选出LuxS基因缺陷的突变株,并利用聚合酶链式反应(PCR)和哈氏弧菌发光实验进行检测。以釉质磨片为载体,在扫描电镜下观察上述两菌株含有20 g/L葡萄糖、20 g/L蔗糖的乳酪消化胨酵母(TPY)液体培养基中形成24 h的生物膜。结果:PCR基因扩增结果显示:突变株LuxS基因已被Cmr基因完全替换,成功的构建了变异链球菌LuxS基因突变株,并且突变株不能诱导哈氏弧菌BB170的生物发光。当用葡萄糖作为补充糖源时,变异链球菌UA159标准株和LuxS基因突变株所形成的生物膜表现型未见明显差异;而用蔗糖作为补充糖源时,两菌株所形成的生物膜有明显不同,UA159生物膜相对平滑均质,而LuxS基因突变株生物膜呈松散的蜂房状,基质间有较大的间隙,形成较大的团簇状菌落。结论:成功构建出LuxS基因缺陷的变异链球菌突变株,与变异链球菌UA159标准株其生物膜结构发生改变,提示LuxS会影响变异链球菌生物膜的形成。更多还原

【Abstract】 AIM: To knock out the entire Luxs gene of Streptococcus mutans UA159 strain via homologous recombination,and to construct a Luxs-deleted mutant strain of Streptococcus mutans and study the difference of biofilm formation between the wild type and the muted strains.METHODS: Two DNA fragments located in the upper and downstream of LuxS gene were amplified and a chloromycetin resistance gene of PJT10 between them was engineered into PUC19 plasmid for constructing the recombinant plasmid pUCluxKO.Electrotransformation of S.mutans cells with pUCluxKO-mutant resulted in isolation of chloromycetin resistant Streptococcus mutans transformants,which was identified by polymerase chain reaction,V.harveyi BB170 luminescence bioassay.The two strains were cultured on enamel slabs in TPY liquid medium containing 20 g/L glucose or sucrose for 24 h.Biofilm formation was observed by scanning electron microscope.RESULTS: The LuxS-deleted status of S.mutants was confirmed by PCR.Cmr gene completely took the place of LuxS gene,and the mutant strain could not induce bioluminescence.The LuxS mutant showed no significant qualitative difference in the biofilm phenotypes compared to the Streptococcus mutans standard strain in the medium supplemented with 20 g/L glucose.In contrast,the LuxS mutant formed biofilm that markedly different from the standard strain when grown with 20 g/L sucrose.The biofilm of standard strain appeared smooth and more evenly distributed,whereas the biofilm formed by the LuxS mutant was loose,hive-like,and had large gaps between the biofilm matrix,and the cell aggregates appeared much larger.CONCLUSION: The S.mutans gene allelic exchange plasmid was constructed correctly and a LuxS-negative mutants of S.mutans was constructed.Both Streptococcus mutans standard strain and LuxS mutant strain could form biofilm,but the architecture of the biofilm formed by LuxS mutant appeared to be altered;LuxS-Based quorum sensing could affect Streptococcus mutans biofilm formation.更多还原