Construction Of LuxS Gene Deficient Streptococcus Mutans And Detection Of AI-2 Quorum-sensing Pathway

Objective: We used molecular biological techniques to detect the possible AI-2quorum-sensing pathway in streptococcus mutans and construct the luxS gene allelicexchange plasmid for constructing the luxS gene knockout mutant, then provide thebasis for further study of the cariogenic virulence regulation mechanism by AI-2quorum sensing pathway of streptococcus mutans.Method: 1.Extract the DNA of streptococcus mutans and Erythromycinresistance plasmid PJT10 and design the primers using the Premier5.0 primer designsoftware. The upstream and downstream flank DNA fragments of S.mutans luxSgene and the E.coli Erythromycin gene were enriched with "nest PCR" methods.2.These fragments were ligated into PUC19 vector with double endonucleasereaction sequentially via T4 ligation enzyme. The homologous recombinationplasmid is constructed according to the "upstream sequence of luxs～Eym-resistance gene～downstream sequence of luxs" gene sequence.3.Electrotransformation of S.mutans with pUCluxKO-mutant resulted in isolation ofErythromycin resistant S.mutans transformants, which was identified by PCR andV.harveyi luminescence bioassay. 4.Streptococcus mutans and V.harveyi BB170were cultured and cell-free conditioned medium of S.mutans were prepared, LBmedium served as a negative control, V.harveyi BB170 was employed as thereporyer strain to detect AI-2 pathway in Streptococcus mutans and Compared theluminescence ability at different phases of growth.Results: 1. The upstream and downstream flank DNA fragments of S.mutansluxS gene and the E.coli Erythromycin gene were enriched with "nest PCR"methods. The PCR products showes a single Bright band analyzing throughSDS-PAGE, non-specific amplification and no tail, molecular weight is the same asexpected. 2. The construction of the Erythromycin resistance plasmid, pUCluxKO,with expected open frame was confirmed through restriction enzymemapping analysis and DNA sequencing. The E.coli containingErythromycin-resistant strains could grow well in LB medium with Erythromycinresistance and the Erythromycin resistance gene could be expressed in vitro. 3.TheLuxS-deleted status of S.mutans was confirmed by PCR with primers specific forthe genes of LuxS and Erythromycin resistance. After 20 generations of culture, theconstructed S.mutans mutants were confirmed stable. 4. Streptococcus mutansUA159 supernatant of the culture medium could induce V.harveyi BB170 reporterstrain bioluminescence activity, indicating that the S.mutans strains have the LuxSgene responsible for synthesizing the AI-2, which suggested the presence of AI-2quorum-sensing pathway in S.mutans. But the S.mutans mutant can not inducebioluminescence, indiating the mutant had been successfully recombined.Conclusions: This research detect the AI-2 in culture supematants of S.mutansby the means of V.harveyi luminescence bioassay, indicating that the S.mutansstrains have the LuxS gene which is thought to be responsible for synthesizing theAI-2. The nest polymerase chain reaction(PCR) is more veracity than direct PCR foramplifying the special DNA fragments, avoiding non special amplification derivedfrom the interference of the restriction enzyme sites spatial structure and improvingthe efficiency and accuracy of the PCR. The S.mutans gene allelic exchange plasmidis constructed correctively in this research and the Erythromycin resistance genecould be expressed in vitro. A LuxS-negative mutants of S.mutans is constructed,which can help to further study the role of LuxS in the pathogenesis of S.mutans.