Functional Analysis Of The Two-Component System EnvZ-OmpR In Acidithiobacillus Caldus

Acidithiobacillus caldus,a chemoautotrophic and gram-negative bacterium,belongs to the Acidithiobacillus genus.This bacterium lives in extremely acidic environments(pH 1 to 3)and can obtain energy from oxidation of sulfur or reduction inorganic sulfur compounds.Because of its important properties in producing sulfuric acid and mitigating passivation of sulfide mineral,A.caldus has received extensive attentions in the field of bioleaching.In the process of bioleaching,A.caldus will face osmotic pressure with the dissolution of metal minerals.Therefore,it is important to study the mechanism of osmolarity regulation in this microorganism.The osmotic pressure is one of the environmental factors during the growth of microorganism.Hyperosmolality usually has effects on cell membrane structure,cell cycle arrest,DNA damage,transcription and translation inhibition.Therefore,bacteria need some regulation mechanisms to reduce the adverse effects of osmotic stress.The EnvZ/OmpR two-component system is the key regulation pathway in response to osmotic stress.However,the research of EnvZ/OmpR has not been reported in autotrophic bacterium till now.Therefore,it is of great significance to reveal the osmolality regulation mechanism in A.caldus.To explore the EnvZ/OmpR two-component regulation system in A.caldus,research was carried out according to the following parts in the work.Firstly,based on the sequence alignment,phylogenetic analysis and protein structure analysis,we found that the protein structures of EnvZ/OmpR in different bacteria were relatively conservative.The EnvZ/OmpR in A.caldus had a highly similarity to EnvZ/OmpR in Acidithiobacillus ferrooxidans,followed by EnvZ/OmpR in Salmonella and Escherichia coli.The transcription factor OmpR binding sites in Escherichia coli and Salmonella were used as matrices to scan the genome of A.caldus,there were 584 total matches.Combined with the results of RNAseq of the ompR mutant,50 matches were screened out.Secondly,using the gene knock-out strategy of A.caldus developed by our laboratory,envZ(orf2589)is successfully markeless knocked-out.Based on the mutants ?envZ and AompR,we constructed the complemented strain?envZ(pJRD215-PtetH-envZ)and AompR(pJRD215-PtetH-ompR),over-expression strain A.caldus MTH-04(pJRD215-PtetH-envZ)and A.caldus MTH-04(pJRD215-PtetH-ompR).The successful construction of these strains provided the ideal materials for the following molecular biological research.Thirdly,the growthes of ?envZ,?ompR and the wild type under different osmotic pressure were analyzed.When there was low osmotic pressure,the deletion of ompR had an impact on the growth.When there was a hyperosmolality,the knockout of envZ or ompR both had effects on the growth.Results indicated that envZ played a more important role in tetrathionate metabolism,while the absence of OmpR brought obvious effect on the cultivation of elemental sulfur.Fourthly,the real-time quantitative PCR was used to explore the regulation mechanism of EnvZ/OmpR in A.caldus.The regulated genes predicted by bioinformatic analysis were further confirmed by transcriptional analysis.Results indicated that EnvZ/OmpR mainly regulated substrate and energy metabolism,channel and membrane proteins,restriction-modification system,nucleotide modification and metabolism.