Screening And Identification Of Mycobacterium Tuberculosis Biofilm Formation Related Genes

Objective To screen and identify the related genes which may be associated withbiofilm formation of Mycobacterium tuberculosis (M. tuberculosis) based on the changes ofcolony phenotype and biofilm defects.Methods A random mutagenesis library was constructed by using the MycoMarT7transposon system. Mutants with colony morphological changes on solid agar plate or withdefects in pellicle biofilm formation in liquid culture medium were selected. In order todetermine the locus of mutation gene, the flanking fragments of gene were obtained using T-Acloning and resistance marker recovery method. Then, the function of mutated genes wasanalyzed and predicted by bioinformatics methods.Results39mutants with colony morphological changes or biofilm defects wereselected and the loci interrupted of16mutants were successfully identified. Compared withthe wild-type strain, the ability to form biofilms which can be formed biofilm in the20~25dof8strains has not occurred any change in the biofilm. The P7C8mutant was the biofilmcompletely deficient strain, there were still no biofilm formation phenotype which observedafter cultured7weeks. The remaining7mutant had the phenomenon of biofilm growth ormaturation delayed, one of the two mutant formed biofilms had taken up to30days,5strainsof about had began to be formed the thin film layer which can clearly be seen by the nakedeye in the surface of the gas-liquid after40days. Among the16genes,5genes wereassociated with the regulation of lipid metabolism,4were participated in cell wall and cellprocesses,2were related to intermediary metabolism and respiration, the product of1genemight be a regulatory protein,1was reported as virulence gene in previous studies,1was PE/ PPE family gene and the function of other genes were unknown. Among these genes,8genesmight be related to deficient in forming pellicle biofilm.Conclusion In this study, a Mycobacterium tuberculosis transposon insertion mutantlibrary of approximately10,000mutants was successfully constructed, pellicle biofilmformation defects were selected and biofilm formation related genes were identified. Toidentify genetic determinants of biofilm formation in Mycobacterium tuberculosis, weperformed a small-scale transposon screen using colony morphological changes and pelliclebiofilm formation assay in Mycobacterium tuberculosis strain H37Ra. This is a new methodfor isolating and identifying genes related to biofilm formation of Mycobacteriumtuberculosis. And new candidate genes obtained in this research will be the basis for thefurther study of biofilm formation regulatory mechanism and pathogenesis of Mycobacteriumtuberculosis. All results will be the basis of the further research of biofilm formationmechanism in Mycobacterium tuberculosis.