LuxS Gene Defect Affects Growth Characteristics,Acid Resistance And Protein Expression Of Streptococcus Mutans

Objective:This experiment use PCR and other methods to appraise Streptococcus mutants LuxS.Through colony culture and viewing under the microscope,it can compare the growth characteristics of the standard strain with which of mutants LuxS. To compare the acid resistance of the two kinds of strains,after pre-acidification, standard strain and mutants LuxS were cultivated in BHI liquid culture medium with a series of pH values.By using two dimensional electrophoretic techniques,it can compare the protein expression differences of two strains.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:Using definite PCR to amplify the genome DNA of mutants LuxS and standard strain.The PCR result can be evaluation by using electrophoresis.Cultivate the standard strain and mutants LuxS 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.Confect a series of BHI liquid culture medium with pH values ranging from 3.5 to 7.0 at the interval of 0.5 and cultivate standard strain and mutants LuxS in these culture medium respectively and then centrifuge.Dilute the sediments of germs with sterile physiological saline water,and determine the A value of absorbance at the point of 600nm in the ultraviolet spectrophotometer. Compare the growth situation of two kinds of strains.Cultivate the two kinds of strains in BHI liquid culture medium with pH value at 5.5,and then inoculate the pre-acidified germ liquid into BHI liquid culture medium with pH value at 3.0. Respectively dilute the sample before and after acidifying,and smear diluted germ liquid on BHI agar culture medium,and finally cultivate it and make a counting. Extrapolate the corresponding viscosity of germ liquid,and figure out the ratio of the viscosities of germ liquid before and after acidifying.For to study the expression difference,extract the total protein of the standard strain and mutants LuxS and find out the difference points by using two dimensional electrophoretic technique. Results:After amplifying,mutants LuxS doesn't have the amplification fragment of gene LuxS which standard strain has.While it has resistance gene fragment of erycin which standard strain doesn't have.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.In the TSA-Eym~r solid medium contained erycin,the standard strain can't live and the growth state of mutants LuxS is generally normal.When pH is 5.0,the growth of mutants LuxS is markedly restrained,while the standard strain can keep growing to some extent.When pH is 4.5,the growth of the standard strain is markedly restrained,and almost no overt growth can be observed on mutants LuxS. Pre-acidified in culture medium with pH value at 5.5,the resistance of the two kinds of strains against the destruction of lethal pH environment can be intensified. Compare the 2-DE atlas of two kinds of strains,there are 61 expression quantity changed protein points can be find in atlas of mutants LuxS,36 of which are step up and 25 are cut down.Conclusions:By using PCR amplification,mutants LuxS are identified.Significant difference of growth characteristic is observed between standard strain and mutants LuxS through compare with tow kinds of strains,it is sure that the construction of mutants LuxS is succeed and expression is lasting.The acid resistance of standard strain is stronger than which of mutants LuxS.The two kinds of strains both displayed the capability of acid tolerance responses,mutants LuxS is more sensitive to acid inactivation,but the capability of acid tolerance responses existed still.The obviously different of the protein point staining shade in 2-DE atlas suggest that there has a quantitative alleosis of total protein in mutants LuxS.