Functional Analysis Of LysR-type Transcriptional Regulators In Ralstonia Solanacearum

The LysR-type transcriptional regulator(LTTR)represents an auto-induced regulator that is widely distributed in all kinds of microbes.Due to the particular structure domains,LTTR is conserved in bacteria,archaea and eukaryotic organisms.Previous works demonstrated that the LTTRs are involved in a large range of biological processes,including metabolism,quorum sensing,bacterial virulence,and cell motility.Ralstonia solanacearum is the causal agent of bacterial wilt disease,a severe threat to vegetables and economic crops.However,the function of LTTRs is known with few exceptions in R.solanacearum.In this thesis,79 lysR genes from R.solanacearum GMI1000 were mutated by using a site-directed mutagenesis strategy.A systematical phenotype analysis was employed to assess their relationship with bacterial virulence.Those included nutrient utilization,cell motility,biofilm and pathogenicity.Eventually,lysR37(RS16695)mutant was found to be enhanced in swimming motility and virulence.To confirm the phenotype,a deletion mutant of lysR37 was produced by using a suicide vector.Real-time PCR and promoter-GusA fusion experiments indicated that the transcriptional level of flagellum regulatory gene flhD was increased in lysR37 mutant.Yeast one-hybrid and EMSA proved that LysR37 protein binds directly to flhD promoter sequence.On the other hand,Real-time PCR experiment indicated that the transcriptional level of hrpG and hrpB was increased in lysR37 mutant.In conclusion,this thesis systemically mutated the lysR family genes in R.solanacearum.Through a set of phenotypic analysis,the LysR37 was found to be negatively controls the expression of flagellum regulatory gene flhD.The research on LTTRs provides an evidence to understand the infection mechanisms of R.solanacearum when interaction with host plants.