苜蓿中华根瘤菌P-type ATPase介导的铜抗性机制及其调控机理研究

Copper resistance genes and their regulation vary among microbes. These genes provide a selective advantage for microbes to survive in contaminated environment. Sinorhizobium meliloti CCNWSX0020 was isolated from the nodules of Medicago lupulina L. which grew on a gold mine tailing in Shanxi province in northwestern China. Two genes SM0020_05727 and SM0020_05862 encoding P-type ATPase were identified in our previous studies, and these genes conferring Zn2+, Pb2+ and Cd2+ as well as Cu2+ resistance to S. meliloti CCNWSX0020. Moreover, Compared to the typical copper efflux P-type ATPase which was only responsible for Cu (I) ef?ux, the primary structure of the two P-type ATPase from S. meliloti CCNWSX0020 were completely different from other P-type ATPase. This project will research the copper resistance and regulation mechanism, the effection of rhizobium-legume symbiosis process and copper absorption ability of the novel P-type ATPase. Based on the results of our previous studies, this study will help us understand the mechanism of copper resistance in bacteria.

长期铜污染环境中存在的微生物对铜形成了一定的抗性，种类繁多的微生物对铜的抗性机制不尽相同。本课题组从生长于矿区的天蓝苜蓿（Medicago lupulina L.）根瘤中分离到一株根瘤菌CCNWSX0020经鉴定命名为Sinorhizobium meliloti，在全基因组测序和突变体筛选的基础上，发现两个基因SM0020_05727和SM0020_05862编码阳离子外排P-type ATPase，它们对铜、锌、铅和镉多种重金属均有输出作用，与已报道的只能专一性输出铜的P-type ATPase相比，在蛋白质一级结构上存在明显差异。该项目将对这种新型P-type ATPase结合与转运铜离子的机理以及对共生结瘤和共生体吸收铜的影响，SM0020_05727和SM0020_05862这两个基因的转录调节机制进行研究，其结果将为揭示细菌抗铜分子机理提供依据。

苜蓿中华根瘤CCNWSX0020分离自陕西凤县重金属尾矿的天蓝苜蓿根瘤内，具有多种重金属抗性。经全基因测序及分析发现，CCNWSX0020中存在五个P1B-type ATPase。通过基因敲除和功能互补实验证实其中CopA1b和ZntA两个P1B-type ATPase与Zn/Cd/Pb抗性相关。根据序列同源性分析，CopA1b属于P1B-1 ATPase，而ZntA属于P1B-2 ATPase。P1B-1 ATPase转运底物为Cu+/Ag+，而P1B-2 ATPase转运底物为Zn2+/Cd2+/Pb2+。CCNWSX0020中zntA基因的表达受Zn2+/Cd2+/Pb2+诱导，且zntA基因缺失后造成细胞对Zn/Cd/Pb高度敏感，菌体内大量Zn/Cd/Pb积累，说明CCNWSX0020中的ZntA是一个典型的Zn2+-ATPase，参与细胞内Zn2+/Cd2+/Pb2+的外排。而CCNWSX0020中copA1b基因的表达受Cu+/Ag+诱导，但copA1b基因缺失后不影响细胞的Cu耐受性，反而对高浓度的Zn/Cd/Pb敏感，造成菌体内Zn/Cd/Pb积累，说明CCNWSX0020中的CopA1b虽然属于P1B-1 ATPase，但也可能参与细胞内的Zn2+/Cd2+/Pb2+的稳态调节。.通过比较转录组学方法分析揭示了CCNWSX0020野生型菌株和∆copA1b突变体对Cu和Zn胁迫的响应机制。在Cu或Zn胁迫下，野生型菌株中编码P1B-type ATPase、多铜氧化酶和外膜蛋白等基因的表达量显著上调。另外，一些碳水化合物及氨基酸代谢相关基因表达量也提高。而在∆copA1b突变株中，编码一些氨基酸代谢、离子转运以及呼吸链电子传递过程相关的基因表达量下调了，表明copA1b缺失会扰乱细胞内多种离子稳态平衡，从而进一步影响酶促反应和产能以及物质代谢等过程。.共生结瘤实验表明，CCNWSX0020野生型菌株和5个P1B-type ATPase基因敲除突变体均可与天蓝苜蓿共生结瘤；而∆copA3突变体由于缺失CopA3，形成的根瘤多为小而黑的坏死根瘤；而∆fixI1突变体由于缺失FixI1，形成的根瘤多为小而圆的无效根瘤。这些结果表明CCNWSX0020中的多个P1B-type ATPase不仅与细菌的重金属抗性有关，还与共生结瘤过程密切相关。