变异链球菌luxS基因缺陷突变株生物学性状的初步观察

Observation on biological characters of Streptococcus mutans luxS gene knockout mutant strain

目的为研究口腔主要致龋菌变异链球菌的种属间密度感应（quorum sensing）相关基因luxS对口腔生态的影响，构建此菌的luxS基因缺失突变株。方法采用同源重组的方法，利用红霉素耐药基因erm置换变异链球菌基因组中的luxS基因，并在含抗性标记的选择性培养基上筛选出阳性克隆。初步研究该基因的缺失突变对变异链球菌在生长与生物被膜成熟分化中的作用。结果经PCR鉴定，luxS基因的置换突变位置正确，并能在体外稳定传代，突变株与野生株相比在达静止期后总菌数量上有明显差别，但在生物被膜的形成与分化上并未发现显著差异。结论通过此研究建立了可稳定传代的变异链球菌luxS基因的缺失突变株，生长表型和生物被膜的形成与野生株无显著差异，为进一步研究此基因功能与调控机制提供了技术平台。

Objective To investigate the construction of Streptococcus mutans luxS gene knockout mutant which can act as the technical platform for following researches on luxS quorum sensing function in oral ecosystem. Methods Erythromycin resistance gene was inserted between two 1 kb fragments containing regions of DNA immediately upstream and downstream of the luxS translational start and stop codons. The resuiting construct was linearized and electro-transformed into Streptococcus mutans cells. After allelic exchange, the luxS gene knockout mutant strains were selected on 10μg/ml erythromycin plates, and compared the growth and binfilms formation of luxS knockout mutant with wild type strains. Results The luxS knockout mutant was confirmed by PCR, and it was also confirmed that this gene mutant could be stably passed through in vitro. The growth mode of luxS knockout mutant showed obvious differences against that of wild type at stationary phase, the knockout mutant gained more bacteria coils growth. Conclusion Strepto-coccus mutans luxS gene has been successfully disrupted with allelic exchange. This mutant strains showed higher growth ability which could be the consequence of quorum sensing mutant.