Identification And Mutation Of Two-component Systems And Functional Analysis Of KdpD-KdpE Of Lonsdalea Quercina Subsp.populi

The bark canker disease caused by Lonsdalea quercina subsp.populi has erupted in large areas in Henan Province and Shandong Province,where were mainly planted with poplar trees.The disease has also emerged in Tianjin Province.It is a new type of poplar bacterial disease found in recent years in China and the area of disease has been expanding year by year.However,the molecular mechanism of the pathogenic bacteria remains uncharacterized.The two-component signal transduction systems(TCSTS)are known as the "intelligence" of bacteria,and they are the one of important molecular mechanism of bacterial specific regulation of pathogenic gene expression and related cell behavior.In order to research the related molecular mechanisms of the TCSTS of L.quercina subsp.populi N-5-1 and understand the relationship between the pathogen and host,we would analyze TCSTS in its genome by bioinformatics.Secondly,by constructing insertionally inactivated mutants systematically and screening for pathogenicity-related phenotypes,genes with significant effects on the growth rate,motility and toxicity of pathogenic bacteria would be selected to construct defective mutants and complementary strains.Then,the downstream genes regulated by the target RR would be searched to analyze the molecular mechanism of the pathogenicity.The results of the study are as follows:(1)According to the genome sequence of L.quercina subsp.populi N-5-1,bioinformatics analysis and identifying of conservative domains associated with HK and RR phosphorylation,we annotated a total of 18 histidine kinases and 24 response regulators.(2)42 recombinant vectors were successfully constructed,and 32 insertional inactivation mutants were successfully obtained by the method of insertion inactivation.A large-scale mutational analysis revealed that 19 TCSTS genes regulated bacterial virulence against poplar trees.(3)KdpD-KdpE was selected for further study because the insertion and inactivation of kdpE severely affected the growth,motility and virulence of the pathogen.Additionally,the deletion of kdpE or overexpression of kdpD resulted in significant reduction of bacterial virulence.We observed that kdpE and kdpD formed a bicistronic operon.KdpD could physically bind to KdpE(Kd?5.73±0.64 ?M).It was inferred that KdpD-KdpE potentially forms a two-component signal transduction system.KdpD had autokinase activity as a histidine protein kinase,but its phosphotransferase activity on the downstream response regulator protein KdpE was not detected.(4)In order to reveal the regulatory mechanism of KdpE on downstream genes,a total of 44 regulated genes in 14 categories were obtained by using ChIP-seq,which are mainly involved in pathogenicity,fitness,material transport,cell structure,and material energy metabolism.EMSA confirmed that KdpE directly binds to the promoter regions of these 44 genes through an imperfect palindromic sequence.Three genes related to pathogenicity or adversity stress responses were selected.RT-PCR revealed that KdpE positively regulates the transcription levels of these three genes,suggesting that KdpE plays a role as a transcription factor to positively regulate the transcriptional levels of these genes.Overexpression of these three downstream genes restored the resistance of the kdpE deletion mutant to chloramphenicol stress,demonstrating the upstream and downstream signaling pathways in terms of biological function.The systematic analysis of the TCSTS is conducive to understanding the pathogenic molecular mechanism of L.quercina subsp.populi,which provides theoretical basis for the prevention and treatment of the disease.