Study On Copper Resistance Mechanism Of Two Component System Gene CopSR In Cupriavidus Gilardii CR3

Two component signal system?TCS?is the most common signal transduction system in bacteria.TCS can sense the external environmental signals,and regulate the corresponding gene expression in the cell,which play an important role in the process of bacteria resistance to stress in the environment.Cupriavidus gilardii CR3 is a representative bacterium of heavy metal resistance gene on the chromosome.C.gilardii CR3 has high resistance to copper,of which minimum inhibitory concentration is 3 mM.The two-component signal transduction system of Cupriavidus metallidurans CH34,Cupriavidus necator N-1 and Cupriavidus taiwanensis LMG 19424 are various and involved in the regulation mechanism of heavy metal resistance.So far,there is no report on the two component signal system for Cupriavidus gilardii CR3.In this study,the distribution of two component signal system in C.gilardii CR3 genome was studied for the first time,and the predicted copSR was identified by cloning.Real-time fluorescence quantitative PCR was used to determine the expression of copSR and their regulated copB gene at mRNA level.In addition,the structure and function of protein CopSR were predicted by bioinformatics analysis,and the KEGG metabolic pathway was analyzed.Based on above research,we analyzed the complex and diverse signal transduction mechanism of Cupriavidus sp.,revealed copper resistance mechanism of C.gilardii CR3 and provided important theoretical reference for C.gilardii CR3'potential application in copper pollution remediation of water and soil.The main findings are as follows:?1?Through comparing the genomic analysis of the C.gilardii CR3 genome,we find that C.gilardii CR3 has 96 genes encoding two component signal proteins.C.gilardii CR3 has 22 two component signal systems,which copper resistance-related two component signal system has the largest proportion.The two component signaling system of strain CR3 was compared with C.metallidurans CH34,C.necator N-1 and C.taiwanensis LMG 19424.Because of the Cupriavidus sp'conservative nature,they share 10 high homologous HK protein functional domains and 7 high homologous RR protein domains.At the same time,C.gilardii CR3 contains four HK-specific protein functional domains and one RR-specific protein domain,specifically adapting to the unique habitat of C.gilardii CR3.?2?The clonal results indicated that the two component signal system copSR does exist in C.gilardii CR3.Sequencing and NCBI sequence comparison showed that the copSR gene sequence in C.gilardii CR3 was identical with the standard copSR gene sequence.copS was 1404 bp and copR was 687 bp.Smart database analysis showed that copS was sensing protein CopS?WP₀53820945.1?in C.gilardii CR3 and CopR was regulatory protein CopR?NP₂51499.1?in C.gilardii CR3.Protein CopS was homologous to sensing kinase in Cupriavidus sp.HPC?L?,copR was homologous to regulatory protein Cupriavidus sp.HPC?L?.?3?The expression of target gene in C.gilardii CR3 under Cu2+ stimulation was studied by real-time fluorescence quantitative PCR.The results showed that the expression of copSR was up-regulated under copper ion stress.Under 0.5 mM,1 mM and 2 mM Cu2+ stress for 2 h,the expression of copSR increased with the increasion of copper ion concentration.The expression of copSR reached the maximum at 2 mM.Different gene expression pattern of C.gilardii CR3 was found under different copper concentrations during growth stage.The copSR reached the maximum expression at 7 h under 0.5 mM Cu2+stress.Gene copSR reached the maximum expression at 4 h under 1 mM Cu2+stress.The expression of copSR reached maximum at 2 h under Cu2+stress.Obviously,the higher concentration of copper,the time for copSR to achieve the maximum expression was shorter.Gene copB expression is consistent with the expression of copSR,which was regulated by copSR.?4?Bioinformatics analysis showed that CopS is transmembrane histidine kinase with functional domain HATPase_c,HisKA,and HAMP signal domains.The 255 th amino acid residue is a conserved histidine residue and undergoes autophosphorylation.CopR is cytoplasmic regulatory protein with functional domain REC and trans_reg_C,and at position 51 is an aspartic acid residue,where received phosphate group from CopS.CopS tertiary structure is similar to the DrrD structure in Thermotoga maritima.The structure of the CopR protein is similar to structure of Caulobacter crescentus DivL.In summary,C.gilardii CR3 contains a rich and diverse two component signaling system.The copSR in C.gilardii CR3 is a copper-resistant two component signaling system.Copper-resistant two-component protein CopS and CopR co-induction and conduction external environment of copper ion stimulation in C.gilardii CR3.Then CopR regulate the expression of downstream copper resistance structure gene copB,and maintain the intracellular copper ion balance,which allow C.gilardii CR3 resist high concentrations of copper ion stress.