Identification Of Zinc Uptake Regulator In Xanthomonas Campestris Pv. Campestris

Zur (Zinc uptake regulator) is a metalloregulatory protein, which negatively regulates the high-affinity zinc uptake systems in many bacteria. When the concentration of Zn2+ in the cell is over the proper concentration, Zur is activated by bound to a Zn2+and then binds to the -35 region of the promoter of the high-affinity Zn2+ uptake systems and represses the expression of the target genes. No zur gene was annotated in Xcc8004 genome, but when we do the Blast Xcc 8004 genome with amino acid sequence of Zur of Escherichia coli, and found that a predicted product of ORF XC1430 (annotated as Fur family transcriptional regulator), shows 42% amino acid similarity with Escherichia coli Zur. To determine if XC 1430 function as a Zur protein, XC1430 was activated by using pK18mob homologous integration method. The XC1430 mutant NK1430 fails to grow in rich medium supplemented with Zn2+at the concentration of 400 uM and in non-rich medium supplemented with Zn2+ at the concentration of 110 uM, whereas the wild type strain 8004 and complementation strain CNK1430 grow well in the same conditions. In rich medium with 400 uM Zn2+, NK1430 accumulates 4.21μg/1010cells Zn2+ but 8004, CNK1430 accumulates only 1.67 and 1.83μg/10l0cells respectively. These results demonstrated that XC1430 encodes a Zur protein in Xcc. To determinate if XC 1430 is relate to the virulence of Xcc, the virulence of the mutant was tested on Chinese radish (Raphanus sativus L. var. radiculus Pers.) by the leaf clipping method. Lesion lengths were meansured 10 days post inoculation, as a result, the mutant NK1430 showed a reduction in virulence compared with the wild type strain 8004, which could be fully restored by complementation strain CNK1430. These results demonstrated that Zur is essential for the full virulence of Xcc. We further found that XC1430 may be involved in the functionning of the type III secretion systems because mutation in XC1430 led to adelated and weaken hypersensitive response (HR) in pepper. Finally, we tested the extracellular enzymes and extracellular polysaccharide (EPS) production in zur mutant. Result shows that the activities of extracellular protease, extracellular amylase, and extracellular endoglucanase are no affected by XC1430 mutation, but the EPS production of XC1430 mutant NK1430 is two fold less than that of wild type strain 8004. This indicates that XC1430 is not required for the activities of these extracellular enzymes, but required for EPS production of Xcc.