Construction and identification of a LuxS-deleted mutant strain of S.mutans / 中华微生物学和免疫学杂志

ABSTRACT

Objective To knock out the entire LuxS gene of Streptococcus mutans UA159 strain via homologous recombination and construct a LuxS-deleted mutant strain of S.mutans.Methods The erythromycin resistance gene(Eymr)was inserted between the two DNA fragments located in the upper and downstream of LuxS gene that had been amplified by PCR.Then the two DNA fragments along with the inserted Eymr were engineered into pUCl9 plasmid to construct the recombination plasmid pUCluxKO.Electrotransformation of S. mutans cells with pUCluxKO-mutant resulted in the isolation of erythromycin resistant S.mutans,transformants,which was then subjected to polymerase chain reaction,Vibrio harveyi BBl70 luminescence bioassay and sequencing analysis.Results Restriction endonuclease analysis showed that pUCluxKOmutant vector had been successfully recombined.The deletion of LuxS of S. mutans mutants was confirmed bv PCR with primers specific for the genes of LuxS and the erythromycin resistance.S.mutans mutant could not induce bioluminescence.indicating the mutant had been successfully recombined.The constructed Chinese S.mutans showed good stability after 20 generations of cultivation.Conclusion The S.mutans gene allelic exchange plasmid is constructed correctively and a LuxS-negative mutant of S.mutans has been constructed.which can be helpful for further study of the role of LuxS in the pathogenesis of S.mutans.