【作者】 黄宁；

【导师】 霍明昕； 王小雨；

【作者基本信息】 东北师范大学 ， 环境科学， 2019， 博士

【摘要】 重金属污染是全球关注的环境问题,往往带来严重的生态后果和健康威胁。铜是生物生长必需元素,但超过一定浓度就会产生毒性。铜毒性导致生物体酶失活、蛋白质氧化以及DNA损伤。为了避免铜的毒性,细菌进化了铜耐受机制来维持适当游离铜的细胞质环境。研究细菌的铜抗性响应机制可以为微生物更好应用于铜污染治理与修复提供理论支持。但目前细菌的铜抗性响应机制是分散的、片段式研究,缺乏系统整体的抗性网络研究。本论文通过对两株铜抗性细菌贪铜菌Cupriavidus gilardii CR3和弗氏柠檬酸杆菌Citrobacter freundii JPG1的铜抗性能力比选,选取一株抗性能力较好的优势菌株C.gilardii CR3,利用生物化学、全基因组学、高通量转录组学及生物信息学技术,分别研究铜胁迫下该细菌的生理生化响应及累积能力;测定菌株C.gilardii CR3的全基因序列信息并注释分析相关重金属抗性的基因;测序不同铜浓度胁迫下C.gilardii CR3的转录本,寻找显著差异表达基因,深入分析其生物学功能和可能参与的代谢通路,从物理、化学和分子生物学多角度解析C.gilardii CR3对铜抗性的内在响应机制,研究结果为生物修复重金属污染提供重要理论参考。主要结果如下:C.gilardii CR3对Cu最小抑制浓度为3 mM。当铜浓度为0.5 mM时,生长量和生长速率与对照组相似无显著变化(p>0.05),暗示该浓度下其生长速率完全不受影响。当铜浓度增加到1.0 mM时,C.gilardii CR3的生长受到一定抑制。与之对比,C.freundii JPG1的最小抑制浓度为2 mM,C.freundii JPG1在0.5 mM下生长量和生长速率与对照组相比出现明显抑制。因此C.gilardii CR3比C.freundii JPG1表现出更强的铜抗性能力,后续研究选取C.gilardii CR3作为铜抗性机制研究对象。菌株C.gilardii CR3的全基因组测序分析结果显示C.gilardii CR3基因组包含2条完整没有任何内环洞的环状染色体,但不存在质粒。染色体1(CHR1)的大小为3,539,530 bp,染色体2(CHR2)为2,039,213 bp,通过全基因注释分析发现C.gilardii CR3包含cop和cus两大铜的抗性基因系统,共包含copSRABCD~1、copQLFGJIDCBARS ompC copKBA~1、copKHFIDCBARS~1和cusFAB~1四个抗铜基因簇,涉及35个抗铜基因。这些研究结果暗示了C.gilardii CR3包含丰富的抗铜基因,可能参与铜抗性响应。铜胁迫下不同转录本之间存在大量差异表达基因。0.5 mM铜浓度下,显著差异表达基因为310个;1.5 mM铜浓度下,显著差异表达基因达413个;暗示着铜浓度增加,细菌响应表达的基因更多。利用生物信息学分析显著差异表达基因的注释功能、GO功能和KEGG代谢通路富集,结果发现0.5 mM和1.5 mM铜胁迫下GO和KEGG富集结果相似,发现显著差异表达的基因主要富集在抗铜基因、硫代谢系统、Fe-S装配、ABC转运系统、蛋白质分泌系统、氨基酸代谢和谷胱甘肽代谢多个代谢途径上。细菌对铜产生较高的去除能力,在胞内和胞外都有累积。铜离子在胞内积累达92%,胞内累积占主要作用。微观结构表征和官能团位点分析揭示了细菌表面铜胁迫的响应变化。结果表明,铜直接影响细菌的细胞活性和胞外聚合物分泌,低浓度(0.1 mM)刺激胞外聚合物分泌,但高浓度(1.5 mM)抑制分泌。官能团分析发现铜胁迫后肽键-O-C=O(羧基)基团、肽聚糖中C-O-C和C-O-P和酰胺I和酰胺II的吸收峰发现明显位移,这些官能团是细胞表面蛋白质、氨基酸和多糖的特征谱带,说明这些物质参与铜的响应,可能与铜发生结合。生物膜激光共聚焦观察铜胁迫刺激了,细菌表面蛋白的分泌,这是对细胞分泌系统上调表达的有力证明。值得注意,本研究首次发现Ⅲ型蛋白质分泌系统的显著响应,它可能刺激蛋白质分泌并通过通道蛋白输出到细胞壁表面,这也可以解释铜胁迫细胞表面聚合物的增多想象。综上,研究结果揭示了铜胁迫下细菌的多种抗性响应机制:细胞壁表面分泌大量聚合物,胞外聚合物官能团与铜离子发生静电等作用结合,阻止铜离子进入细胞内;铜离子通过ABC转运蛋白进入到细胞内,细胞内的铜抗性基因响应将过量铜离子外排降低毒性;硫代谢系统高度表达,其半胱氨酸和谷胱甘肽的生物合成产生重金属螯合分子,促进CR3对铜的解毒作用;Fe-S装配系统显著响应,因为Cu(I)可以夺取Fe离子位点与硫结合形成胞内累积可以达到保护细胞的目的。更多还原

【Abstract】 Heavy metal pollution is a global environmental issue because of its serious ecological consequences and health threats.Copper,an essential element of living organisms,exceeds critical concentration will cause toxicity which can lead to biological enzyme inactivation,protein oxidation and DNA damage.To avoid the damage mentioned above,bacteria evolved copper tolerance mechanisms to maintain a proper free copper in cytoplasmic environment.Therefore,studying the copper resistance response mechanism of bacteria can provide theoretical support for the application of microorganisms in copper pollution control and restoration.But the current study of copper resistance response mechanism is decentralized and fragmented.In this study,a dominant strain was selected by the capability for copper resistance in two strains C.gilardii CR3 and C.freundii JPG1,and the biochemistry,genome-wide,high-throughput transcriptomics and bioinformatics were used to study the physiological and biochemical responses and accumulation ability of bacteria under copper stress,whole gene sequence information of strain C.gilardii CR3 and annotation analysis of heavy metal resistance genes,transcriptome sequencing and annotation of C.gilardii CR3 under copper stress,significant difference gene and enrichment metabolic pathway of C.gilardii CR3 under copper stress.The main results are as follows:The effects of copper ions on bacterial growth and resistance were studied on strains C.gilardii CR3 and C.freundii JPG1.And the results show:C.gilardii CR3 exhibits greater copper resistance and the minimum inhibitory concentration for Cu is 3 mM.When the copper concentration was 0.5 mM,the growth and growth rate were completely unaffected and similar to the control group(P>0.05).And then the copper concentration was increased to 1.0 mM,the growth of C.gilardii CR3 was inhibited.Compared to the C.gilardii CR3,the minimum inhibitory concentration of C.freundii JPG1 is 3 mM,and the growth was inhibited when the copper concentration was 0.5mM.So we chose the C.gilardii CR3 as the object for studying copper resistance response mechanism.Based on the whole genome sequencing analysis of strain C.gilardii CR3,the copper resistance genes of C.gilardii CR3 contain two major systems,cop and cus,and consisting of four copper-resistant gene clusters(copSRABCD~1;copQLFGJIDCBARSompCcopKBA~1;copKHFIDCBARS~1;cusFAB~1),and involved 35 anti-copper genes.The above results indicated that C.gilardii CR3 is rich in anti-copper genes responsible for copper resistance responses.There are a large number of significantly differentially expressed genes between different transcriptomes under copper stress,and the bacteria responded more genes with the concentration of copper increasing.For example,at 0.5 mM copper,there were 310significant differentially expressed genes and the number of differentially expressed genes reached 413 at 1.5 mM copper.Based on the bioinformatics analysis of significant differentially expressed genes,GO gene function,and KEGG metabolic pathway enrichment,significantly differentially expressed genes are mainly enriched in multiple metabolic pathways such as anti-copper gene,sulfur metabolism system,Fe-S assembly,ABC transport system,protein secretion system,amino acid metabolism and glutathione metabolism.Changes in response to copper stress on bacterial surfaces were revealed through the analysis of microstructural characterization and functional group.The results shows:copper concentration directly affects bacterial cell activity and extracellular polymer secretion,low concentration(0.1 mM)stimulates extracellular polymer secretion and high concentration(1.5 mM)inhibits secretion.Under copper stress,significant shift in the absorption peaks of peptide bond-O-C=O(carboxyl)group,C-O-C and C-O-P and amide I and amide II in peptidoglycan,which are characteristics band of cell surface proteins,amino acids and polysaccharides,were discovered by functional group analysis.This means these materials mentioned above participated in copper response and may be combined with copper.At the same time,bacteria generated a high removal capacity for copper accumulated both intracellularly and extracellularly.Especially intracellular,copper ion accumulation reached 92%.In summary,the results revealed a variety of resistance response mechanisms of bacteria under copper stress:the surface of the cell wall secretes a amount of polymer,and the extracellular polymer functional group combines with the electrostatic action of copper ions to prevent copper ions from entering the cell;copper ions enter the cell through the ABC transporter,and the copper resistance gene response in the cell reduces the toxicity by excess copper ion efflux;the sulfur metabolism system is highly expressed,and the biosynthesis of cysteine and glutathione produces heavy metal chelate molecules,which promotes the detoxification of copper by CR3.The Fe-S assembly system responds significantly because Cu(I)can capture the Fe ion site and combine with sulfur to form intracellular accumulation.It is worth noting that for the first time,a significant response of the protein secretion system was discovered,which may stimulate protein secretion and export to the cell wall surface through channel proteins,and may also explain the increase of surface polymer in copper stress cells.Using various research methods,the study systematically revealed the resistance mechanism of bacteria to copper and provides a theoretical reference for bioremediation of heavy metal pollution.更多还原