Study On Generation Of LuxS Reporter Construct And Its Expression Property In Streptococcus Mutans

Plaque biofilm, a well-organized microbial community located on tooth surface, has been recognized as a major factor in the pathogenesis of dental caries. Compared with their planktonic counterparts, the bacteria harbored in biofilms exhibit the different gene expression and phenotype, as well as an increased resistance to antimicrobials, presumably due to the regulation effect exerted by the signal transduction systems.With various techniques of molecular biology, a preliminary understanding on the role of signal transduction systems in biofilm formation has been achieved.The quorum sensing system of S.mutans has been proven to play a crucial role in the formation of biofilm and maintenance of steady-state. Especially, AI-2/LuxS may be involved in the regulation of S.mutans biofilm formation, glucose metabolism, antiacid capacity, and the production of virulence factors,thus, contributing significantly to the survival of S.mutans in the complex environment. Moreover, AI-2/LuxS mediated quorum sensing can be utilized or interrupted by the interaction of S.mutans and other oral bacteria, directly influencing the balance of oral bacteria flora. However, little has been known about the synthesis and secretion of AI-2, as well as its adjusting means and regulation mechanism on the activity of bacteria adjacent within biofilm. Therefore further investigation on the mechanism of LuxS-mediated induction in S.mutans is of great importance for reducing the cariogenesis of oral biofilm and providing possible solution for the resistance of bacteria to antimicrobials.This study successfully constructed S.mutans LuxS gene green fluorescent protein (gfp) fusion report plasmid through polymerase chain reaction (PCR) technique, and transferred this plasmid into S.mutans to verify it's expression. 1. Construction of a vector expressing LuxS gene promoter and gfp LuxS promoter and gfp region was amplified by PCR from S.mutans DNA and pEGFP-N1 vector, respectively, then cloned into the pUC19 vector following routine procedures by using molecular cloning technique.Identification by enzyme digestion, PCR and sequencing, confirmed the validity of the recombinant vector pUC19-pLuxS-gfp. The successful construction of the recombinant plasmid pUC19-pLuxS-gfp will be further studied in this field.2. The phenotype detection of the fusion reporter strainTransfer the prokaryotic expression recombinant plasmid pUC19-pLuxS-gfp into S.mutansUA159, using selection with 30μg ampicillin ml-1and isolating the transformants.Fluorescence microscope observationCultures of wild-type S. mutans strains and the reporter strain were grown in the brain heart infusion(BHI) which contain 3 % Horse serum and supplemented with 0.5% sucrose.The cell cultures were analyzed directly by using phase-contrast fluorescent microscopy. Fluorescence was readily detected in the plasmid-containing strain but not in the wild-type.CLSM of GFP-expressing S. mutans cells and biofilm morphology The collection teeth were cut into thin slice with the slow cut saw, and then placed in 96-well plates after disinfection, The 96-well plates contained the brain heart infusion(BHI) which supplemented 3 % Horse serum 0.5 % sucrose.After 24h incubation the reporter strain cells formed several large amorphous microcolonies and many small microcolonies were interspersed across the biofilm, The micrographs of single sections indicated an increase of fluorescence in the biofilm as a function of sucrose. These results demonstrated that: the recombinant establisthed can expressing in S.mutans, laying a foundation for further study.