Functionl study of flhA and flhB genes in Xanthomonas campestris pv.campestris

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Title:	Functionl study of flhA and flhB genes in Xanthomonas campestris pv.campestris
Authors:	Yu-Wei leu
Contributors:	Department of Biotechnology
Keywords:	Yu-Wei leu;Rouh-Mei Hu;flhA and flhB
Date:	2008
Issue Date:	2009-11-06 06:11:12 (UTC+0)
Publisher:	Asia University
Abstract:	Xanthomonas campestris pv. campestris ( Xcc ) is a Gram negative, rod-shaped bacterium, and is a causative agent of black rot disease in cruciferous plants. According to previously reports, bacterial flagella and the type III secretion systems (TTSS) are highly related. The TTSS is essential for the virulence in many pathogens, including Xcc. Based on sequence homology, our search in Xcc revealed that more than 40 genes might be involved in the flagellar biogenesis. Six genes, rpoN, fleQ, fliA, flgM, fleN and flhF, have been demonstrated in our laboratory to be involved in the regulation of expression of many flagellar genes. In this work, we report the function of FlhA and FlhB which are homologs of TTSS proteins InvA and SpaS reported to be involved in the exportation of flagellar structure in Salmonlla. Xcc flhA and flhB mutants were obtained by insertional mutagenesis and were used for phenotypical assays. The flhA and flhB mutants are virulent, but immotile. Both mutants expressed a normal quantity of FleQ and RpoN protein. The expression of FliA protein was slightly decreased in an flhA mutant but not in an flhB mutant. Both mutants cannot synthesis the major flagellin protein FliC. Induction of plasmid-born wild-type flhA or flhB genes can successfully complement the motility defect, and restore the biosynthesis of FliC protein in flhA and flhB mutants, respectively. The promoter of fliC gene was transcribed by RNA polymerases containing an alternative sigma factor encoded by fliA gene. The FlgM protein is an anti-sigma factor acts against FliA. A mature flagellar TTSS expels FlgM from the cytoplasm and recover the function of FliA. As FlhA and FlhB are essential components of flagellar TTSS, the expression of fliC in flhA and flhB mutants might be blocked by an accumulation of cytoplasmic FlgM protein. The second part of this work is to use a yeast two-hybrid system to detect the protein-protein interaction between RpoN, FleQ, FleN, FlhF, FlgM, FliA and NtrC. The results suggested that RpoN2 and FlhF; RpoN2 and FleQ; RpoN2 and NtrC can interact with each other.
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