Investigation Of The Mechanism By High Hfq Regulats Cell Motility In Xanthomonas Campestris Pathovar Campestris

As a global post transcriptional regulator,Hfq(Host factor for Qβ)plays an important role in bacterial cell motility,chemotaxis,pathogenicity and quorum sensing.Previously,our lab found that deletion of hfq,a gene encoding Hfq,leads to almost loss of swarming motility of Xanthomonas campestris pv.campestris(Xcc),but the mechanism still unknown.Transcriptome analysis showed that the m RNA level of four putative motility-related genes(fim T,pilV,pilY and pilE1)were significantly increased in the hfq deletion mutant.This study speculates that Hfq may control the swarming motility of Xcc by regulating the expression of the four genes.To verify this hypothesis,in this study,firstly,the transcriptome data was confirmed by RT-PCR.The results showed that the m RNA level of these four motility-related genes fim T,pilV,pilY and pilE1 were significantly increased in the hfq deletion mutant,compared to that in the wild type strain,confirming the correctness of the transcriptome data and indicating that fim T,pilV,pilY and pilE1 are negatively regulated by Hfq.Then,the protein expression of fim T,pilV,pilY and pilE1 was detected by western blotting.The results further support this conclusion.To clarify whether fim T,pilV,pilY and pilE1 are really related to the motility of Xcc,the "deletion mutant construction → phenotype analysis→ functional complementation" strategy was used to identify the biological functions of these genes.The results showed that the swarming motility of fim T,pilV,pilY and pilE1 deletion mutants was significantly enhanced than that of the wild type strain,while the swarming motility of the complementary strains were similar to that of the wild type strain,demonstrating that fim T,pilV,pilY and pilE1 can repress the swarming motility of Xcc.These results explain the decreased swarming motility phenotype of the hfq deletion mutant: hfq deletion leads to increased expression of fim T,pilV,pilY and pilE1,four genes have swarming motility inhibiting function,thus resulting in a decreased swarming motility of Xcc.In addition,this study found that the pathogenicity of pilV deletion mutant was significantly lower than that of the wild type strain,and the complementary strain was similar to that of wild type strain.The results showed that the motility-related gene pilV plays a positive role in the virulence of Xcc.The swimming motility of the motility-related gene pilE1 deletion mutant was significantly enhanced than that of wild type strain,and the swimming motility of the complementary strain was similar to that of wild type strain,which indicating that motility-related gene pilE1 has the function of inhibiting Xcc swimming motility.In summary,this study obtained the following innovative results:(1)uncover of a mechanism by which Hfq regulates Xcc motility;(2)identify four new motility-related genes in Xcc;(3)demonstration of pilV is involved in Xcc virulence.All together,these results increased the understanding of the mechanism of Hfq action,the cell motility regulation and the virulence of Xcc.