LuxS Gene Defect Affects Biological Characters And Biofilm Formation Of Streptococcus Mutans

Objective: This experiment use conventional biochemical identification,identifica--tion of Gram stain smear,determination of growth curve and other methods to describe biological characters between Streptococcus mutans standard strain and LuxS mutant strain. Furthermore, through stained with crystal violet dye,observe the architecture of Streptococcus mutans luxS mutant biofilms utilizing of an experimental model of luxS mutant biofilms. In this study,we observe the biofilms formed by S. mutans LuxS mutant strain and standard strain using scanning electron microscope. It can give a significant support to the further study of the signal system of mutants LuxS, whose adjustment mechanism can cause dental caries.Methods:Cultivate the standard strain and LuxS mutant strain respectively in TSA solid medium which contain erycin or not, in order to observe the growth state of two kinds of colonial and take a routine biochemistry test and also compare the growth characteristic of two kinds of strains by Gram staining. Let LuxS mutant strain and standard strain recover as usual and make them turbid liquid as reserved at the absorbance of 1.0 (A600=1.0) with an ultraviolet spectrophotometer. After 1000-fold dilution, the above bacterial suspension was inoculated into BHI liquid medium in constant temperature shaker, A timing measurement of the value of sample was observed to compare the differences in the growth curve. LuxS mutant and standard strain were grown in BHI medium in 96-well micro-titer plate. After 48h, to observe the adherent biofilms, which stained with crystal violet dye. Biofilms were grown on enamel slabs that were deposited in the wells of 6-well cell culture clusters and sorted by No.1-6 well,placing 4 pieces every well.And 1-ml portions of bacterial suspension were added respectively to the surfaces of enamel slabs in each well(in which No.1 and No.4 were standard strains, No.2,3,5,6 were LuxS mutant strains), after 2 min 5-ml portions of TPY liquid medium were added to each well(No.1-3 of which contained TPY medium supplemented with 2% glucose, No.4-6 contained TPY medium supplemented with 2% sucrose),and then separately added 500μl culture supernatant of standard strain to the two wells of No.3,6.A11 S.mutans strains were grown anaerobically at 37℃in order to form biofilms on the surfaces of enamel slabs, and then twenty-four-hour biofilms of the two strains were observed using scanning electron microscope in order to evaluate the overall comparison of the two strains biofilm formation.Results:In the TSA-Eymr solid medium contained erycin, the standard strain can't live and the growth state of LuxS mutant strain is generally normal.The colonial morphology of two kinds of strains don't have obviously difference and the thalline of standard strain present a long catenation and relational twisting form while mutants LuxS present a short catenation form under the microscope when they are cultivated in the TSA solid medium which don't contain erycin. From the growing curve,we found that there was no significant difference in growth mode between the two strains. But after entering the stable phase, there were some differences in degree of saturation, the standard strain got more cell growth. Both standard strain and mutant cells were able to form biofilms in BHI medium. However, there was a significant difference between them:LuxS mutant biofilms had a very rough appearance, rather than the relatively smooth, confluent layer seen in the standard strain. Both standard strain and mutant cells were able to form biofilms in BHI medium. However, there was a significant difference between them:LuxS mutant biofilms had a very rough appearance, rather than the relatively smooth, confluent layer seen in the standard strain. The LuxS mutant showed no significant qualitative difference in the biofilm phenotypes compared to the Streptococcus mutans standard strain in the medium supplemented with 2%glucose.In contrast,the LuxS mutant formed bilfilms that markedly differed from the standard strain when grown with 2% sucrose.The biofilms of standard strain appeared smooth and more evenly distributed,whereas the biofilms formed by the LuxS mutant were loose,hive-like,and had large gaps between the biofilm matrix,and the cell aggregates appeared muth larger. The mutant phenotypes were complemented partly by exposure to supernatants with no cells from standard strain cultures,and the capacity of mutant strain to form bilfilm was enhanced.Conclusions:Significant difference of growth characteristic is observed between standard strain and LuxS mutant strain through compare with tow kinds of strains, LuxS mutation can inhibit the growth of Streptococcus mutans. Both Streptococcus mutans standard strain and LuxS mutant strain could form the biofilms, but the architecture of biofilms formed by the LuxS mutant appeared to be altered; LuxS-Based quorum sensing could affect Streptococcus mutans biofilm formation.