【作者】 黄东亮；

【导师】 唐纪良；

【作者基本信息】 广西大学 ， 微生物学， 2008， 博士

【摘要】 细菌中锌离子的平衡主要是通过调控Zn²⁺吸收系统和Zn²⁺排放系统的表达来维持的。长期以来一直认为,细菌的金属离子吸收系统和排放系统是由不同的调控蛋白分开调控的。众所周知,Zur蛋白的主要功能是负调控Zn²⁺吸收系统的表达。但是,在十字花科黑腐病菌（Xanthomonas campestrispv.campestris,简称Xcc）中,zur突变体对高浓度Zn²⁺敏感、EPS产量下降和致病力降低,表明在Xcc中,Zur不但调控Zn²⁺平衡,还与EPS产生和致病性相关（Tang et al,MPMI Vol.18,No.7,2005,pp.652-658.）。为了研究Xcc中Zur蛋白在调控锌平衡方面的真正功能,我们用DNA芯片分析的方法鉴定出在锌丰富条件下Zur的调控元。芯片结果表明,在锌丰富条件下,有38个基因受Zur负调控,而有29个基因受Zur正调控。通过构建基因启动子与gusA基因转录融合的报告质粒检测表明芯片结果是可靠的。对这67个受Zur调控的基因进行生物信息学分析,我们推测XC0266-XC0267,XC2471-XC2472-XC2473和XC3786-XC3787-XC3788可能分别编码三个Zn²⁺吸收系统,而XC2976可能编码一个Zn²⁺排放泵。对这些Zn²⁺平衡相关基因的启动子与gusA基因转录融合的报告质粒在野生型背景下的GUS活性检测表明,三个假定的Zn²⁺吸收系统的表达均受低Zn²⁺浓度诱导,受高Zn²⁺浓度抑制,而假定的Zn²⁺排放泵受低Zn²⁺浓度抑制,受高Zn²⁺浓度诱导。而所有这些Zn²⁺平衡相关基因的报告质粒在zur突变体背景下的GUS活性不受Zn²⁺浓度影响,说明Zn²⁺浓度对这些基因的诱导依赖于Zur蛋白。为了鉴定这些基因在Zn²⁺平衡方面的功能,我们构建了这些基因的突变体并检测假定的Zn²⁺吸收系统的基因突变体在低Zn²⁺浓度下生长情况,而检测假定的Zn²⁺排放泵的基因突变体在高Zn²⁺浓度下生长情况。结果表明,所有假定的Zn²⁺吸收系统的基因突变体在低Zn²⁺浓度培养基中的生长均比野生型菌株差,但加入一定浓度的Zn²⁺又使突变体的生长恢复到野生型水平,这些结果表明,XC0266-XC0267,XC2471-XC2472-XC2473和XC3786-XC3787-XC3788可能分别编码三个Zn²⁺吸收系统。XC2976基因突变体在高Zn²⁺浓度条件下生长比野生型菌株差,且XC2976的产物与已鉴定的一个Zn²⁺排放泵,Ralstonia的CzcD,在序列和结构上很相似,表明XC2976编码一个CDF型Zn²⁺排放泵。对基因启动子与gusA基因转录融合报告质粒的GUS活性检测表明,在Xcc中,Zur不但负调控三个假定的Zn²⁺吸收系统的表达,还正调控一个Zn²⁺排放泵的表达。为了检测Zur蛋白是否直接调控这些基因的表达,我们用EMSA检测Zur与这些基因的启动子在体外的结合情况。结果表明,Xcc的Zur蛋白不仅能与Zn²⁺吸收系统的启动子结合,而且能与Zn²⁺排放泵的启动子结合,且Zur与它们的结合需要Zn²⁺,说明Zur直接调控这些基因的表达。为了证明Zur确实直接激活Zn²⁺排放泵的转录和直接抑制Zn²⁺吸收系统的转录,我们进行体外转录实验。结果表明,XC0267、XC2471-2和XC3788的转录随Zur蛋白的增加而减少,而XC2976的转录随Zur蛋白的增加而增加,证明Zur蛋白直接抑制XC0267、XC2471-2和XC3788的转录而直接激活XC2976的转录。为了确定Zur结合在这些基因启动子区的序列,我们进行DNaseI足迹试验。结果表明,Xcc的Zur蛋白结合在假定的Zn²⁺吸收系统启动子区的序列是一段约30 bp的与Ecoli的Zur-box相似的保守序列,而结合在Zn²⁺排放泵的启动子区的序列是一段59 bp的富含GC的序列,该序列有一个20 bp的反向重复序列。序列比对表明Zur结合在Zn²⁺排放泵启动子区的序列与结合在假定的Zn²⁺吸收系统启动子区的序列相似性极低,表明在Xcc中,Zur能识别两个完全不同的序列。用5’-RACE方法确定Zn²⁺平衡相关基因的转录起始位点后发现,Xcc的Zur蛋白均结合在这些Zn²⁺平衡相关基因启动子的-10和-35区。zur突变体与Zn²⁺排放泵的突变体对高Zn²⁺浓度的敏感程度及细胞内聚集的锌含量基本一致,表明zur突变体对高浓度Zn²⁺敏感是由于Zn²⁺排放泵的功能缺失引起的。所有Zn²⁺平衡相关基因的致病性和EPS产量与野生型菌株一致,说明Xcc中Zur蛋白调控Zn²⁺敏感性是独立于致病性和EPS产生的。为了研究Xcc中,Zur蛋白在调控致病性方面的功能,我们用DNA芯片分析的方法鉴定出在基本培养基MMX（大多数致病相关基因受诱导表达）条件下Zur的调控元。芯片结果显示,在MMX培养条件下,有149个基因受Zur负调控,而有216个基因受Zur正调控。在216个受Zur正调控的基因中,几乎包括所有的hrp基因,表明zur突变体致病力的下降可能是由于hrp基因的表达下降引起的。RT-PCR检测进一步证实了zur突变体中hrp基因的表达是下降的。通过检测所有hrp基因的启动子与gusA基因转录融合的报告质粒在野生型菌株Xcc 8004和在zur突变体中的GUS活性,我们发现Zur蛋白出除了正调控hrp基因簇外,还调控hrpX,但不调控hrpG。说明Zur通过hrpX调控hrp基因簇的表达。Zur不能与hrpX的启动子结合,说明Zur可能间接调控hrpX的表达。为了进一步证实Zur通过hrpX调控hrp基因簇,我们构建了组成型表达的hrpX并导入到zur突变体中。HR和致病性检测结果表明,组成型表达的hrpX能使zur突变体延迟的HR恢复到野生型水平,且能使zur突变体的致病力得到部分恢复,说明Zur通过hrpX调控hrp基因簇的表达。组成型表达的hrpX仅部分恢复zur突变体的致病力说明除hrp基因外,Zur还调控其它致病相关基因的表达。更多还原

【Abstract】 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.更多还原