Genomic And Transcriptomic Analyses Jointly Reveal The Mechanism Of Copper Stress To Klebsiella Michiganensis Under Acid Conditions

Heavy metal copper?Cu? is a trace element necessary for organisms,in spite of low concentration,long-term accumulation will also have a toxic effect on the organisms.Excessive copper will induce the change of DNA,the integrity of cell membranes,respiration,and enzyme activity,leading to threats to the survival of organisms.Various organisms represented by bacteria are evolving constantly anti-copper strategies in order to cope with copper toxicity,however,copper-contaminated areas are generally in an acidic environment,therefore,bacteria will be exposed simultaneously to copper stress and the effect of pH value.Based on the above,the analysis of the copper resistance response mechanism of bacteria in different pH conditions not only provides theoretical support for a comprehensive understanding of the anti-copper performance of bacteria,but also provides corresponding references for the research on the use of bioremediation to control copper pollution.In this paper,Klebsiella michiganensis was a research object,and pH values?3,4 and 5?were study variables,the growth curve of the bacteria were separately measured with or without supplemented copper.In order to study the copper resistance mechanism of K.michiganensis after copper stress at different pH levels?4 and 5?,the whole genome and transcriptome sequencing technology were applied.The gene sequence of K.michiganensis was obtained by whole genome sequencing technology,the genes related to copper resistance were annotated and analyzed.The transcripts were obtained under copper stress at different pH levels by transcriptome sequencing technology,and the genes and metabolic pathways that are significantly differentially expressed in relation to copper resistance were screened,therefore,the resistive response mechanism to copper of K.michiganensis were comprehensively illuminated.The specific results are as follows:?1?The growth curves of K.michiganensis at different pH levels?3,4 and 5? with or without supplemented copper?0.5 mM CuCl₂? were detected.The results show that the strains had no growth phenomenon with and without supplemented copper at pH 3,suggesting that strain growth was inhibited under this condition.The growth curve characteristics of the strains were very similar with and without supplemented copper at pH 5,suggesting that there was relatively little role on the growth of strains after copper stress under this condition.In contrast,the growth curves of the strains were more different with and without supplemented copper at pH 4,suggesting that pH 4 had a greater effect on the growth of the strain after copper stress.Therefore,the copper stress transcription mechanism of K.michiganensis was measured at pH 4 and 5,respectively.?2?Based on the Oxford Nanopore PromethION sequencing platform,and the complete gene sequence of the bacteria was obtained through processes such as filtering and assembly.The Genome-encoded Genes of K.michiganensis were 5,409,which located on ring chromosomes?chromosome length was 5,159,508 bp?,and no plasmid.In addition,the copper resistance genes were observed in K.michiganensis strain,such as cusFAB,cueO,copA ect.?3?Transcriptome sequencing results showed that a large number of genes were significantly differentially expressed with and without supplemented copper at pH 4 and 5.There were 583 differential expression genes at pH 4,whereas,there were 203 differential expression genes at pH 5,suggesting that the stronger the acidity,the more genes the bacteria respond to copper stress.K.michiganensis cells were exposed to 0.5 mM CuCl₂ at pH 4 and 5.Lower pH?pH < 4?strongly inhibited K.michiganensis growth,while copper stress and higher pH?pH > 5?induced copper precipitation in the medium.Therefore,this study focuses on the analysis of copper stress resistance genes and metabolic pathways under pH 4 conditions.Transcriptomic analyses indicated that two groups of genes related to quorum sensing?QS??lsrABCDFGKR?and ? Type secretion system?T2SS??gspCDEFGHIJKLM?were significantly up-regulated at pH 4 only.These results suggest that ? Type secretion system may be induced and controlled by quorum sensing,thereby contributing to the formation of extracellular polymeric substances?EPS?and the secretion of proteins to prevent copper ions from entering cells.Six copper resistance genes?cusABF,copA,cueO,and gene05308?were more significantly up-regulated at pH 4 than at pH 5.In addition,the relative expression?log₂|FC|? of the sulfur assimilation genes cysHJIK was relatively higher at pH 4 than at pH 5,while the gene encoding organic sulfur metabolism,tauB,was also significantly up-regulated at only pH 4.These results indicate that the copper efflux system can remove intracellular copper ions from cells,and that the sulfur assimilation system is related to the detoxification of copper ions.Furthermore,increased free copper ions at lower pH?4?could induce communication signals among cells,thereby stimulating the response of T2SS-related genes in K.michiganensis to tolerate copper stress.Consequently,the resistance of K.michiganensis to copper stress is a multisystem collaborative process composed of intracellular and extracellular components.