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Objective: To investigate the predominant contribution of methyl-metabolism pathway to the regulation of LuxS of Strecptococcus mutans., Methods: The differences in biofilm formation and aciduricity of Strecptococcus mutans among the methyl-metabolism-complementation strain (KO-S), the parental wide-type strain (WT) and the luxS null strain (KO) were observed by real-time PCR for monitoring the transcriptional level of genes related to biofilm formation (smu.238, gtfD) and aciduricity (smu.44, smu.46) of the studied strains, methyl thiazolyl tetrazolium (MTT) for quantifying the biofilm of the exhibited strains and confocal laser scanning microscopy for estimating the structure of the biofilm., Results: The transcriptional level of smu.44, smu.46, smu.238, gtfD in WT were 1.289 ± 0.051, 1.694 ± 0.140, 1.565 ± 0.107, 1.667 ± 0.196 respectively; in KO were 1.001 ± 0.045, 1.007 ± 0.151, 1.000 ± 0.021, 1.012 ± 0.196 respectively, downregulated compared with WT (P < 0.05); in KO-S were 4.662 ± 0.091, 5.019 ± 0.258, 3.462±0.029, 3.071 ± 0.136 respectively, upregulated compared both with KO and with WT (P < 0.05). The quantity of biofilms formed by the studied strains were WT (1.592 ± 0.213), KO (0.939 ± 0.029), KO- S (2.177 ± 0.226), KO- P (1.020 ± 0.093), respectively, representing a less quantity by KO and KO-P than WT (P < 0.05) and a more quantity by KO-S than other three stains (P < 0.05). According to the observation of biofilms texture by confocal laser scanning microscopy, the WT biofilm was condensed and even. In contrast, fissures and gaps were found scattered in biofilms of KO, KO-P while lessened in that of KO-S, in which high-density bacterial aggregates were observed. The acid assay indicated a smaller biofilm decrease by WT and KO-S than that by KO and KO- P(P < 0.05)., Conclusions: The methyl- metabolism pathway contributes to LuxS regulation on biofilm formation and auiduricity of Strecptococcus mutans.