Characterization of the fliA flgM flhF genes in Xanthomonas campestris pv. campestris

[[abstract]]Xanthomonas campestris pv. campestris (XCC) is a Gram negative, rod-shaped bacterium. It is the causative agent of black rot disease in cruciferous plants. The disease results in heavy loss in agriculture worldwide. Symptoms include marginal leaf chlorosis and darkening of vascular tissue, accompanied by extensive and necrosis. Full leaf yellowing, wilting, and necrosis occur as the disease advances. XCC has a single unipolar flagellum. From the completed genome sequence of XCC, more than 40 genes are predicted to be involved in the flagellar biogenesis. Research results in our laboratory showed that FleQ and RpoN2 positively regulate and FleN negatively regulates the XCC flagellar biosynthesis by up- or downregulating the promoter activity of seven flagellar operons, fliQ, flhF, flgG, fliL, fliC, flgB and fliE, respectively. The purpose of we were to elucidate the role of fliA, encoding a putative σ28 factor, flgM, encoding a putative anti-σ28 factor and flhF which was reported to be a positive regulator of flagellar biogenesis in Pseudomonas. Mutants of fliA, flgM or flhF were constructed by insertional mutagenesis and the phenotypes of the mutant were analyzed. The results showed that 1) fliA, flgM and flhF mutants have a decreased motility, 2) deficiency in motility of flgM mutant can be restored by a plasmid-born flgM, 3) as shown by transmission electron microscopy (TEM) fliA mutants have no flagellum; flgM mutants have a polar but shorter flagellum and flhF mutants have one to two lateral flagella, 4) mutations in any of these genes affect the growth rate in a poor medium, 5) mutations in fliA, flgM or flhF also decrease the pathogenicity. 6) all the three mutants are still sensitive to the infection of bacteriophage φLf and φL7, and 7) none of the mutation changed activity of extracellular enzymes. The results of transcriptional fusion assays demonstrated that 1) FliA positively regulates the expression of fliC and negatively regulated the expression of the fliQ, flhF and flgG operons, 2) FlgM positively regulates the expression of flgB, fliQ, flhF and fliL operons and negatively regulate the expression of the fliC and flgG operons, and 3) FlhF positively regulates the expression of fliE and pilA1 and negatively regulates the expression of flgB, fliQ and fliL. The results of Western blot demonstrated that 1) RpoN2 positively regulated the expression of FliA, and 2) expression of fliC is positively regulated by FliA and RpoN2, but is negatively regulated by FlgM