The Mechanism By Which The Zinc Uptake Regulator Zur Regulats Zinc Homeostasis And Pathogenesis Of Xanthomonas Campestris Pv. Campestris

In bacteria,the balance between Zn²⁺import and export,known as Zn²⁺ homeostasis,is maintained mainly through the coordinated expression of export and uptake systems,and it has been long considered that metal ion export and uptake systems are separately controlled by their own regulators.Although it is well-known that the Zn²⁺uptake regulator Zur typically acts as a repressor of Zn²⁺uptake systems,our previous work showed that,in addition to plays an important role in zinc homeostasis,the Zur of Xanthomonas campestris pv. campestris(Xcc)is involved in the extracellular polysaccharide(EPS) production and pathogenesis of the pathogen(Tang et al,MPMI Vol.18,No.7, 2005,pp.652-658.).To investigate the exact role of Zur in the zinc homeostasis of Xcc,in this study,we identified the genes that are regulated by Zur(Zur regulon)in zinc-rich condition using DNA microarray hybridization analysis.The results showed that 38 genes were negatively regulated and 29 genes were positively regulated by Zur in zinc-rich condition.The reliability of the microarray data was confirmed by promoter-gusA transcriptional fusion reporter analysis.By using bioinformatics analysis,of the 67 Zur-regulated genes,we found that XC0266-XC0267,XC2471-XC2472-XC2473 and XC3786-XC3787-XC3788 may encode zinc uptake systems,and XC2976 may be a zinc effiux gene.The promoter-gusA transcriptional fusion reporter analysis revealed that the expression of putative zinc uptake systems was induced by low Zn²⁺and repressed by high Zn²⁺concentration,in contrast,the expression of the putative zinc efflux gene was induced by high Zn²⁺and repressed by low Zn²⁺ concentration.We further found that the expression of the putative zinc homeostasis genes was not response to Zn²⁺in the zur mutant background, demonstrating that the induction of zinc homeostasis genes by Zn²⁺is mediated by Zur.To determine these putative zinc uptake systems really play a role in zinc uptake and the putative zinc efflux gene play a role in zinc export,the disruption mutants of these genes were constructed and the growths of the mutants of the putative zinc uptake systems were tested in Zn²⁺deficient conditions,while the mutants of the zinc export gene were tested in high Zn²⁺conditions.The results showed that mutants of the putative zinc uptake systems grew weakly than the wild type strains in zinc-deficient condition and addition of Zn²⁺restore the growth to the wild type level,indicating that XC0266-XC0267, XC2471-XC2472-XC247 and XC3786-XC3787-XC3788 may encode zinc uptake systems.And the mutant of XC2976 mutant showed more sensitivity to Zn²⁺ than the wild type strain.This result combined with the fact that XC2976 showed sequence and structural similarity to CzcD,a characterized CDF type Zn²⁺efflux protein,we concluded that XC2976 encoded a CDF-type Zn²⁺efflux pump.The promoter-gusA transcriptional fusion reporter analysis showed that Zur negatively regulated the expression of these putative Zn²⁺uptake systems and positively regulated the expression of a Zn²⁺efflux pump.To determine Zur regulates theses genes directly or indirectly,electrophoretic mobility shift assays (EMSAs)were performed to analysis the in vitro binding of Zur with the promoter DNA fragment of these genes.EMSA results showed that Zur binds specifically to the promoter DNA fragments of both Zn²⁺uptake systems and Zn²⁺efflux pump and the binding required Zn²⁺,indicating that Zur regulates these genes directly.To completely demonstrate that Zur indeed directly activates the transcription of the Zn²⁺efflux pump XC2976 and represses Zn²⁺ uptake systems XC0267,XC2471-2 and XC3788,in vitro transcription assays were performed.The results showed that addition of Zur to the in vitro reactions decreased the transcription from the promoters of XC0267,XC2471-2 and XC3788,but increased the transcription from the XC2976 promoter.These demonstrate that Zur represses the transcription of XC0267,XC2471-2 and XC3788,and activates the transcription ofXC2976,directly.To determine the Zur binding site in its target promoter,DNase I footprinting analysis was performed,and the results show that Zur bound to an～30bp E.coli "Zur-box" like sequence in the promoter of there putative Zn²⁺ uptake systems,but to a 59bp GC rich sequence containing a 20bp imperfect invert repeat in the promoter of the Zn²⁺efflux pump.The Zur-binding sequence in the promoter of Zn²⁺efflux pump show no sequence similarity to that in the promoter of putative Zn²⁺uptake systems,indicating that Zur can recognize at least two distinct target sequences in Xcc.After determining the transcription start site by 5'-RACE,we found that Zur bound to the -10 and -35 regions in all the promoters of zinc homeostasis genes.The Zn²⁺sensitivity and the zinc content in the cell of zur mutant were identical to those of the mutant of the Zn²⁺efflux pump,indicating that the Zn²⁺ sensitivity of zur mutant was due to the absence of the Zn²⁺efflux pump.All the mutants of zinc homeostasis genes showed identical virulence and EPS yield to wild type strain,revealing that the regulatory roles of Zur in zinc homeostasis was independent to that in virulence and EPS production.To investigate the exact role of Zur in the pathogenesis of Xcc,the genes that are regulated by Zur in minimal media MMX(a condition that most virulence genes were induced)were determined by DNA microarray analysis. The results showed that 149 genes were negatively regulated and 216 genes were positively regulated by Zur when grown in MMX.Of these 216 genes positively regulated by Zur,nearly all hrp genes were included,suggested that the reduced virulence of the zur mutant was due to the reduced expression of hrp genes.The reduced expression of hrp genes in the zur mutant was further confirmed by RT-PCR.Importantly,by construction of a promoter-gusA transcriptional reporter,we demonstrated that,in addition to the hrp cluster,Zur also positively regulates hrpX but not hrpG,indicating that Zur regulates the hrp cluster through controlling hrpX.Zur failed binding to the promoter of hrpX in EMSA indicating that Zur may regulate hrpX indirectly.To further confirm that Zur regulates hrp cluster via hrpX,a plasmid containing constitutively expressing hrpX was constructed and was induced into the zur mutant.Results of HR and virulence tests showed that a constitutive expressing hrpX fully restore the HR and partially restore the virulence of the zur mutant.These results demonstrated that Zur regulated hrp gene via hrpX.A constitutive expression hrpX conly partially restore the virulence of the zur mutant,indicating virulence gene(s)outside the HrpX regulon is regulated by Zur.