The Response Mechanism Of Cupriavidus Gilardii CR3 To Copper Stress

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.