Preliminary Study On Biological Character Alteration Of Streptococcus Mutans After The LuxS Was Knocked Out

【Objective】LuxS-based signaling is thought to represent an important means of intergeneric communication, especially in biofilms. To find the development of biofilms regulation and the changes in biological properties in this organism, the luxS mutant must be construction.【Methods】(1) Long flanking homology polymerase chain reaction(LFH-PCR) was introduced to generate a gene disruption construct consisting of Emr cassette with long flanking homology regions to the target gene. The electroporation competence of streptococcus mutans was then transformed with this PCR product. Then positive transformants were counted on selective agar which containing erythromycin and identified by PCR and sequencing. (2) The strains were grown in three different conditions(BHI,2% glucose-BHI,2% sucrose-BHI),and the growth curve was analyzed by 722-S visible range spectrophotometer. (3) The ability of S.mutans UA159 and luxS mutant biofilm formation was examined in early mature stage phases by scanning electron microscopy. (4) For biofilm formation, the strains in logarithmic growth phase were grown in BHI Supplement with 2%(W/V)sucrose. The cell soluble protein expression of Streptococcus mutans and its mutans were measured respectively by SDS-PAGE.【Results】(1) Identification by PCR and sequencing confirmed the validity of the LuxS deletion mutant of streptococcus mutans. (2) The growth of Streptococcus mutans was changed under three conditions. The growth of luxS-deficient Streptococcus Mutans is faster than Streptococcus mutans UA159. (3) Compared with S.mutans UA159, the LuxS mutant maintained in BHI medium containing 2% sucrose displayed an apparent defect in biofilm formation, while they showed no significant deviation in BHI medium containing 2% glucose. (4) There were no specific protein bands in cell soluble protein expression and no significant difference in quantity of protein expression among the different strains biofilm.【Conclusions】(1) The successful construction of the LuxS deletion mutant can be used in further functional genome research.(2) The LuxS quorum sensing was blocked,the growth of Streptococcus mutans will be change. While the environment is full with abundant nutrition, the quantity of bacterial was increase. (3) LuxS gene in S.mutans can play a role in dental plaque biofilm formation, and the LuxS gene is possible to regulate sucrose–dependent biofilm formation. (4)There were no significant difference in biofilm cell soluble protein expression between Streptococcus mutans and its mutans at early mature stage.