| The process of using microorganisms to decompose metallic sulfide minerals is called as bioleaching,it' s a friendly environmental bioleaching technique.Acidithiobacillus caldus(A.caldus)is a kind of obligated autotrophic gram negative bacteria,which grow at low pH(<3),belonging to the extreme acidophilus,and usually widely distribute in the Acidic hot springs,sulfide deposit and other extreme acidic environments.A.caldus is one of the dominant bacteria in the bioleaching.It has received the widespread attentions for its high-efficiency sulfur metabolism and significant application value in industry.There is many difficulties in the industry application of A.caldus,because of its long generation time and lower growth yield,especially the strong inhibitory effects for high iron and low pH conditions in the later bioleaching period.Therefore,it becomes more and more important to research the environmental adaption of A.caldus for its theoretical value and practical applicationThe RpoS protein encoded by the rpoS gene,also referred as ?38 or aS,is a kind of a-factor that is a subunit of RNA polymerase in prokaryotes.It is a regulator of stimuli response,such as nutrient depletion,oxidative stress,high temperature,high osmotic pressure and acidic environment.It mainly plays a role in the late log phase and stationary phase.RpoS functions varys in different strains.In A.caldus MTH-04,there are no reports on which metabolic pathways RpoS regulates and how it exerts regulatory effects.Therefore,the study of the regulatory mechanism of RpoS is of great significance for improving the metabolic regulatory network of A.caldus and elucidating its mechanism for adapting to an extremely acidic metallurgical environment.This article studies the function of rpoS gene in stress-resistance mechanism of A caldus MTH-04,and mainly carries out the following work:Firstly,the amino acid sequence of RpoS protein was compared with other acidophilic bacteria and other model bacteria which RpoS is widely-studied to analyze their evolutionary relationship.The results showed that the RpoS protein of the genus Acidothiobacillus was closely related and the protein sequence was more conservative.However,with a relatively long-standing genetic relationship with model strains such as E.coli and Pseudomonas.In addition,according to the RpoS amino acid sequence,the spatial structure and transmembrane structure of RpoS protein were predicted by online software.It was found that RpoS protein is a hydrophilic protein with no signal peptide and no transmembrane structure.Second,an over expression engineered strain WT(pSDUl-PropS-rpoS)of the rpoS gene was constructed.The growth characteristics of the over-expressed strains were analyzed by measuring the growth curves of the strains at different initial pH values,0.25 M osmotic pressure stimulation and initial addition of 1%glucose,respectively,and it was found that over-expression strain grew better than the control strain.Third,RNA-seq and quantitative quantification qPCR were used to analyze the transcriptional changes of wild-type,knockout strains of rpoS(constructed in our laboratory pre-studies)and overexpression strains using S0 as an energy source.The study found that the regulation of RpoS protein involves not only flagellar assembly,bacterial chemotaxis,and osmotic pressure-regulatory two-component systems ompR/envZ and related stress-resistant genes,but also enzymes activity related to sulfur metabolism,pyruvate metabolism,and glycolysis pathways.It shows that RpoS plays a role in the environmental adaptation and material metabolism of A.caldus MTH-04This work analyzes the biological function of RpoS protein(?38)for the first time,and provided basic data for further identification of RpoS-regulon,and elucidate its regulatory mechanism and regulatory network,and has the theoretical value and practical application. |
PDF Full Text Request