Study On The Molecular Mechanism Of Gene Regulation Of Zur In Xanthomonas Campestris Pv.Campestris

Zur (Zinc uptake regulator) exists in many bacteria. The primary function of Zur is repressing the high affinity zinc uptake system when the zinc concentration in the cell reaches a threshold. It has been reported that Zur is encoded by ORF XC1430 in Xanthomonas campestris pv. campestris strain 8004 (Xcc 8004), and that, in addition to involving in regulation of zinc uptake, Zur plays an important role in the EPS production and virulence to the host plant ( Dong-Jie Tang, et al.2005.MPMI.) . In this work, Xcc Zur protein was purified using the E. coli M15/pEQ30 system, and the binding ability of Zur to the promoter of its target genes, XC0376, XC2386 and XC3668, was tested using electrophoresis mobility shift assay (EMSA) . Results showed that Xcc Zur can specifically bind to the promoters in vitro. Sequence analysis result showed that a putative Zur-box that shows sequence similarity to the E. coli Zur-box (5'-AAGTGTGATATTAAACATTTCATGAC TA-3') were present in the -35 region of the promoters. DNase I foot-printing assay showed there exist a protected region by Zur in the promoter of XC3668. All these results indicated that the mechanism of gene regulation of Xcc Zur may be similar to that of E. coli Zur. In order to investigate the mechanism of Zur involved in zinc uptake regulation, EPS production and virulence, the 23 highly conserved amino acid residues of Xcc Zur were mutated by site-directed mutagenesis, and the function of the mutated Zur were assay by complementation the zur mutant NK1430. Results showed among the 23 amino acids substitutions of Xcc Zur, Ala88 →Thr88, GlyI52→Alal52 and Phel53→Tyrl53 resulted in the lose of zinc uptake regulatory function of Zur but the EPS production and virulence is not affected; Glul34→Lysl34 and Glul61→Lysl61 resulted in the decline of EPS production but thezinc uptake regulatory and virulence is not affected; Thr45→Met45, AIa64→Gly64, Pro82→Ser82,Cys110→Ala110, Cysl26→Vall26, Glyl64→Alal64 and Cysl69→Trp169 affected both zinc uptake regulatory and EPS production; Val51→Gly51, Tyr65→Lue65, Tyr86→Leu86, Arg87→Gly87, Leu89→Ser89, Cysl29→Leul29, Cysl66→Ilel66 affected all three functions; Leu92→Ser92, Gly96→Ala96, His99→Asp99 and Asn105→Ile105 have no effects on all the three functions. These results indicate that the role of Zur in zinc uptake regulation, EPS production and virulence to the host of Xcc can be functionally and structurally separated. Thus, we proposed that Xcc Zur might control the three processes through different metabolism pathways. Finally, we purified mutant Zur proteins with a amino acid substitution in Cysl26→Vall26, Cysl29→Leul29, Cysl66→Ilel66 and Cysl69→Trpl69, respectively, and the binding ability of these protein to their target promoters were tested by EMSA. Result showed that Cysl26→Vall26 and Cysl29→Leul29 substitution lost the binding ability of Zur with their target promoters, while Cysl66→llel66 and Cys169→Trpl69 substitution did not affect their binding ability. This indicated that Cysl26 and Cysl29 are essential for the binding of Arc Zur with its target promoters.