Effect of luxS mutation on the oxidative stress of Streptococcus mutans / 中华微生物学和免疫学杂志

Objective To investigate the effect of luxS inactivation on the oxidative stress of Streptococcus mutans and perform preliminary analysis of potential mechanism.Methods Strains were grown to mid-logarithmic phase and divided into three groups,one was used as control and inoculated into normal TPY medium,and the other two groups were experimental groups,and there were separately inoculated into TPY containing 58.8 mmol/L hydrogen peroxideor TPY containing 58.8 mmol/L hydrogen peroxide and 0.1 mmol/L 2,2'-dipyridyl.The survival rate of strain was calculated at 0.5,1,and 2 h.All the data were statistically analyzed.Results Compared with the control group,the survival rate of luxS mutation was always higher than standard strain at all pre-determined time inexperimental groups (P＜0.05),and compared with experimental group without iron chelator,the survival rate of strains was not raised with the added of iron chelator (P＞0.05).Conclusion luxS gene is involved in oxidative stress tolerance of Streptococcus mutans,and the oxidative stress tolerance is not achieved by avoiding the toxic effects of the Fenton reaction

Construction of LuxS gene deletion of Streptococcus mutans and evaluation of the acid tolerance of the mutant / 中华微生物学和免疫学杂志

Objective To construct Streptococcus mutans UA159 mutants with deletion of LuxS gene related to quorum-sensing pathway and evaluate the aciduricity of the mutants. Methods Using S. mutans UA159 as materials, the PCR fragments of the upstream and downstream regions of LuxS and erythromycin resistance(Eymr) gene of PJT10 were cloned into plasmid PUC19. The resulting constructs were integrated into the chromosome of S. mutans. LuxS gene deletion mutant was then constructed in S. mutans by means of allelic exchange and selected for resistance to erythromycin. The aciduric ability of the mutant under different pH was measured and S. mutans UA159 was used as control. Results The LuxS-deleted status of S. mutans mutants were confirmed by various PCR and DNA sequencing. The results showed that Eymr gene take the place of LuxS gene, while the mutant can not induce bioluminescenece. The LuxS mutant strain displayed a decreased growth ability with the decreasing pH values compared to those of the wild-type strain UA159. Conclusion A LuxS-negative mutants of S. mutans is constructed. The LuxS quorum sensing system is involved in the regulation of aciduricity of S. mutans UA159.

Effects of LuxS gene deletion on the acid resistance ability of Streptococcus mutans / 实用口腔医学杂志

Objective: To study the difference between the acid resistance of Streptococcus mulans Ingbritt C international standard strain and the acid resistance of LuxS mutant strain. Methods: Solutions of Streptococcus mulans standard strain and LuxS mutant strain with same density were prepared and cultured at pH 3. 5 to 7. 0 BH1 liquid for same period. Terminal growth situation was compared. After being acidized in pH 5.5 BHI liquid, the two strains were cultured at pH 3.0 BHI liquid. The acid tolerance responses of the two strains were compared. Results; (DAt pH 6.0 to 7. 0, the difference of growth between Streptococcus mulans standard strain and LuxS mutant strain was not significant at the same pH value, and the differences of bacterial growth situation under three different pH values were not significant. (1)At pH 4.5 to 5.5, the difference of growth between the two strains was significant. (2)At pH 3.0,the survival rate of LuxS mutant strain(0.006 5% )was significantly lower than the standard strain (0.078% ). (3)At pH 5.5, the survival rate of LuxS mutant strain(0.747% ) was lower than the standard strain(8.65% )by about 10 times after the pre-acidification. Conclusion; (4)At sub-lethal pH value, there is significant difference of aciduricity between Streptococcus mu-tans standard strain and LuxS mutant strain. The acid resistance of standard strain is stronger than that of LuxS mutant strain. The two strains both display the capability of acid tolerance responses. LuxS mutant strain is more sensitive to acid inactivation, but the capability of acid tolerance responses still exists.

Observation on biological characters of Streptococcus mutans luxS gene knockout mutant strain / 中华微生物学和免疫学杂志

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 resuhing 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 biofilms formation of luxS knockout mutant with wild type strains.Results The luxSknockout 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 difierences against that of wild type at stationary phase,the knockout mutant gained more bacteria cells growth.Conclusion Streptococcus mutans luxS gene has been successfully disrupted with allelic exchange.This mutant strains showed higher growth abilitv which could be the consequence of quorum sensing mutant.

Construction of a recombinant plasmid for S.mutants luxS gene knock-out / 实用口腔医学杂志

Objective:To construct a recombination plasmid containing a kanamycin resistance gene,the upstream and downstream fragment of luxS of Streptococcus mutans so that luxS can be knock out by transforming the plasmid into S.mutans later.Methods:Kanamycin resistance gene,the upstream and downstream of luxS were cloned respectively by using plasmid pEGFP-N1 and DNA of Streptococcus mutans as template.Then the genes were ligated into Multiple Cloning Site(MCS) of vector pMD19-T in certain order and transformed into E.coli Competent Cells.Finally transformants were selected for resistance to kanamycin and ampicillin.Results:Kanamycin resistance gene and the upstream and downstream of luxS were successfully ligated into accurate enzyme digestion site of vector pMD19-T,and restriction digests analysis and sequencing result was correct.Conclusion:LuxS gene knock-out of Streptococcus Mutans recombinant plasmid is constructed and built a base of constructing Streptococcus Mutans luxS mutans in the future.