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.

Construction of the markless clpP-deletion mutant of Streptococcus mutans / 中华微生物学和免疫学杂志

Objective To construct markless gene deletion mutant at the clpP loci on the chromosome of Streptococcus mutans(S.mutans).Methods ASp resistance gene was amplified by PCR,to construct the Sp resistance cassette where the Sp resistance gene was flanked with two loxP site.After the clpP gene was cloned into the pGEM-T-Easy TA cloning vector,it was digested and linked with the Sp resistance cassette,yielding homologous recombination vector pIB △ clpP-Sp.The vector was linearized and used for the transformation of S.mutans UA159,with transformants selected on TPY plates containing Sp.The selected strain was transformed with the thermosensitive plasmid pCrePA to excise the Sp resistance gene.The pCre-PA was then easily eliminated at nonpermissive temperature,resulting in a markless mutant strain carrying a deletion at the clpP loci,which was verified by PCR and DNA sequencing.Results The result of the PCR analysis and DNA sequencing indicated that a part of the clpP gene was deleted.There was a loxP at this loci without the Sp resistance gene.Conclusion The markless clpP-deletion mutant of S.mutans was constructed successfully,which laid a foundation for further study of its biological function and its influence on the cariogenicity of S.mutans.

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.

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

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.

The construction of phIFN-?-2B shuttle plasmid and its expression in BCG / 中华泌尿外科杂志

Objective To construct a new secreting recombinant hIFN-?-2B-BCG to provide a new tool for the treatment of bladder tumor. Methods BCG was genetically engineered to secrete recombinant human interferon-alpha 2B by transforming of shuttle plasmid phIFN-?-2B. Expression of hIFN-? was readily detectable by ELISA. Results The phIFN-?-2B was transformed in BCG correctly,and the value of hIFN-?-2B in supernatant of recombinant BCG culture was calculated to approximately 997.2 pg/ml. Conclusions This study demonstrates that the recombinant phIFN-?-2B can be expressed in BCG secretively. As the construction of the plasmid and its transformation and expression in BCG were accomplished successfully, a foundation of reformed BCG and new vaccine will be established.