Osmolarity Response Regulator OmpR Gene Markerless Knockout And Its Functional Analysis In Acidithiobacillus Caldus

Acidithiobacillus caldus is a kind of obligated autotrophic sulfur-oxidizing bacterium.It through oxidizing inorganic reduced sulfur compounds to obtain energy and reducing power,and fixes CO2 from atmosphere through Calvin cycle as the carbon source.It usually distributed in acidic mine water,sulfide deposit,is the most important bacterium in bio leaching,the treatment of waste water and coal desulfurization.It has been subject to widespread attention due to its special way of life and the important role it played in sulfur cycle in nature.However,its chemoautotrophic features,long generation time and lower cell yield,brought difficult to our study.So further study of its metabolism and adaptability to the environment,increase the growth rate is the current primary subject.Osmolarity response regulator(ompR)plays an important role in the process of bacteria perceive and rapid response high osmotic pressure environment,related research are most deeply in Escherichia.coli.More and more studies show that in addition to osmotic adjustment,OmpR plays many other regulatory functions role.But in Acidithiobacillus caldus,how OmpR deal with high osmotic stress,which genes it directly regulate expression,there has not been reported.Discovered by genome sequencing,A.caldus MTH-04 has two ompR genes of orf1207 and orf2590 in the genome as the genome sequences reveal.Therefore,to explore the regulatory mechanism of ompR will not only contribute to explore how A.caldus MTH-04 adapt to changes in the environment of the osmotic pressure,but also to improving the sulfur metabolism system model.This article is the first report on the function analysis of OmpR in A.caldus,which contains four parts as the following:Firstly,the two ompR genes in A.caldus MTH-04 genome are successfully markeless knocked-out and three mutants are obtained which are A.caldus(?orfl207),A.caldus(?orf2590)and A.caldus(?orf1207?orf2590).Secondly,ompR mutants phenotypic has been analysised in A.caldus.Determined the curve of three mutant strains and wild-type strain grown under different osmotic pressure,compare the strain sensitivity to high osmotic pressure,speculate ompR2590 plays a greater role in the regulation of the osmotic pressure;using flow cytometry to test the physiological state of bacteria under high osmotic,we found that under high osmotic stimulate for a long time,permeability of cell membranes has increased and membrane strains been destroyed.Thirdly,OmpR regulon and its regulatory mechanism has been explored in A.caldus.Using chain-specific transcriptome sequencing technology,compared the difference expression beteen the wild-type strain and the ompR mutant gene at the level of mRNA at high osmolarity stimulate.Then,using the real-time quantitative PCR(Real time RT-PCR)to verification the results of transcription sequencing.Fourthly,OmpR gene are analyzed and identified in A.caldus,OmpR(orf1207)and OmpR(orf2590)are expressed in E.coli BL21(DE3)and purified,using MicroCal iTC200 initially measured ompR2590 can interact with tetH.