RND驱动重金属外排泵介导的细菌性果斑病菌抗铜机制及其调控机理研究

Bacterial fruit blotch (BFB), caused by Acidovorax citrulli, is a devastating, quarantine disease of cucurbits. Due to the existence of copper-resistant strains in A. citrulli, very limited antibacterial chemicals, and short of resistant cultivars, BFB has been a serious threat to the cucurbit industry worldwide. Understanding the molecular and genetic basis of A. citrulli copper resistance will be helpful to develop effective disease control measures. By screening a transposon tagged A. citrulli mutant library, a mutant deficient in copper resistance was obtained in our previous research. Sequence alignment and gene functional complementation assays confirmed that the predicted resistance-nodulation-cell division family efflux transporter MFP subunit was essential for copper resistance in A. citrulli. This result shows that a RND-driven heavy-metal-efflux pump (i.e. HME-RND, named Cus system in Escherichia coli ) mediated copper resistance exists in A. citrulli. There is only one amino acid residue difference between MFP proteins from copper resistance strain FC440 and copper sensitive strain AAC00-1. Genomic DNA scanning shows the homolog of CusCFBA found in copper sensitive strain of A. citrulli, except CusF. In this application we aim at characteristics and regulation mechanism analysis of the RND-driven HME pump in A. citrulli, by gene knockout, genetic complementation, genetic cross-complementation, bacterial two-hybrid assay and site-directed mutagenesis. This study will finally provide a module on the mechanism of copper resistance driven by HME-RND pump in A. citrulli.

细菌性果斑病（BFB）是一种能够对葫芦科植物造成毁灭性为害的检疫性病害。BFB病菌群体中出现抗铜菌株，使得现有杀菌剂难以防控。本研究小组前期以抗铜菌株为材料发现：注释编码RND家族外排泵MFP亚基的基因表达产物是菌株抗铜必需因子，表明BFB菌中存在RND家族重金属外排泵（即HME-RND，大肠杆菌为Cus系统）介导的抗铜途径。MFP基因产物与铜敏感菌株的仅有一个氨基酸的差异；基因组序列扫描显示铜敏感菌株中存在Cus系统主要组分的同源基因。本研究拟利用同源重组基因敲除、基因功能互补和交叉互补实验、定点突变技术、细菌双杂交体系蛋白质互作分析等方法和手段，辅助生物信息学分析手段，对BFB菌中类Cus系统各组分特征、抗铜机制中的作用、蛋白质功能域特点、功能性HME-RND的调控因子及其互作关系等进行解析。通过本研究，将初步揭示出BFB病菌的HME-RND系统外排铜的机制及其功能的调控模式。

细菌性果斑病（Bacterial Fruit Blotch）是为害葫芦科植物的检疫性病害，其病原菌--西瓜食酸菌（Acidovorax citrulli）群体中存在铜抗性菌株导致病害难以防控，认识该病菌的抗铜系统对全面解析其抗铜机制有重要意义。本项目围绕类RND驱动重金属外排泵介导的抗铜系统（Cus系统）各组分候选基因的发掘、基因鉴定和功能分析、抗铜系统各组分间作用关系解析开展研究。本研究获得以下重要结果和关键数据：.1. 获得西瓜食酸菌FC440菌株的基因组和铜胁迫下的病菌转录组数据信息，显示FC440菌株含4,386个基因，其与已测序的pstlb65菌株的遗传距离较近，与AAC00-1的距离较远；铜胁迫下，FC440菌株表现266个基因上调表达，200个基因表达下调；本研究筛选了31个候选基因，鉴定并验证了4个铜抗性相关基因的功能。.2. 两种常用基因功能表达载体pBBR1MCS-5和pHC60对宿主菌生物学特性的影响不同，pBBR1MCS-5载体对宿主的影响较小。.3. 本研究验证了多个铜抗性相关的基因的功能：其中类cusC基因的发现，进一步确认了类Cus抗铜途径的存在；类cueR基因被验证，其产物作为转录调控因子启动copA、cueO基因的转录表达，完善了对类Cue系统的认识；首次发现葡萄糖酸转录阻遏蛋白操纵子gntR和类copZ基因对细菌抗铜性有贡献。上述基因均未参与菌的致病性、游动性、胞外酶或多糖的分泌等活动。.本研究结果揭示出西瓜食酸菌中至少存在Cus 、Cue等多条抗铜途径，首次发现了GntR、CopZ参与细菌对重金属外排，反映出该菌抗铜机制的复杂性和多样性，研究结果对全面认识该菌的抗铜机理有重要意义。