Exploring ?⁵⁴ Regulated Genes And Tetrahydrosulfate Metabolic Pathway In Acidithiobacillus Caldus

Acidithiobacillus caldus is a kind of moderately acidophilic thermophilic autotrophic sulfur-oxidizing bacteria.It is Gram-negative and can grow on elemental sulfur,thiosulfate,tetrasulfate,sulfite and sulfide.The optimum growth temperature is 45 ?,and the optimum pH is 2-2.5.A.caldus plays an important role in bioleaching,biological desulfurization of coal,and sewage treatment.Through the study of its a54 regulated genes and S4I pathway,we can further understand the sulfur metabolism system and transcriptional regulation network in A.caldus,improve the sulfur oxidation model,and realize the efficient utilization of A.caldus in biological leaching and sewage treatment.The sigma factor is the key to the transcriptional regulation mechanism of bacteria.It is usually responsible for some genes necessary for non-cell growth,such as nitrogen metabolism,dicarboxylic acid transport,and heat shock response.In the A.caldus MTH-04,?54 specifically regulates which genes are not clear,and the transcriptional regulatory network is still not perfect.Therefore,we use bioinformatics analysis to apply direct molecular and indirect regulation of a54 by molecular biology techniques.Gene research is of great significance for understanding the transcriptional regulation mechanism of a54 and constructing the a54 regulatory network.The S4I pathway is an important sulphur metabolic pathway in the acidophilic Thiobacillus thermophilum,by which the interconversion of thiosulfate and tetrathionate can be carried out.Based on the tetH knockout strains obtained in our laboratory,molecular techniques were used to measure the growth of tetH knockout strains on tetrahydrosulfate,analyze tetrahydrosulfate consumption and transcription levels.In addition to tetrasulfate hydrolase(TetH),is there any other tetrathionate metabolic pathway,and what is the relationship between this pathway and the S4I pathway need to be answered.The aim of this study is to understand the sulfur metabolism pathway,explore the tetrahydrosulfate metabolic pathway,and provide a theoretical basis for the targeted genetic modification of bioleaching.This paper mainly carries out research work in the following aspects:1.Bioinformatics analysis of ?54 regulated genes in A.caldus MTH-04.Because A.caldus MTH-04 genome annotation is incomplete,the bioinformatics analysis of a54-regulated genes was performed in two ways.(1)Due to the high similarity between the genomic sequences of A.caldus MTH-04 and A.caldus SM-1,and the genomic annotation of A.caldus SM-1 being more complete,the genes regulated by ?54 were searched in A.caldus SM-1,and the searched gene sequences were aligned in the A.caldus MTH-04 genome.Seven possible a54-regulated genes and promoters in A.caldus MTH-04 were identified and predicted.(2)According to high conservation of the RpoN protein-specific recognition promoter sequence-12(GC)/24(GG)during the start of transcription,possible ?54 in the A.caldus MTH-04 genome are predicted using Virtual Footprint online analysis software.The regulated promoter sequences were analyzed and 13 possible RpoN-binding promoter sequences were found and the 13 promoter-corresponding genes were identified.2.Gel retardation analysis of RpoN protein and predictor regulatory genepromoter sequences.The RpoN protein was purified and expressed.The binding of RpoN protein to the predicted promoter sequence was carried out by gel retardation analysis,and four promoter sequences for RpoN protein binding were determined,and the genes directly regulated by ?54 were determined to be orf2328,orf2805,orf1640,orf2486.3.Transcriptional level analysis of ?54 regulated genes.RT-qPCR was used to analyze the effect ?54 overexpression on gene transcription level.It was found that rpoN overexpression resulted in orf1479,orf1638,orf2129,orf0213,orf0969,orf0019,orf2237,orf1076,orf0414,orf2330,orf0021,orf0211 down-regulation,orf2803,orf2139,orf1074,orf2486 up-regulation,which implied a close relationship between a54 and these genes.4.The study of metabolic pathways for tetrahydrosulfate.On the basis of the tetH knockout strain in the laboratory,the tetH knockout strain was cultured using Starkey-K2S4O6 medium supplemented with sulfur powder,and the growth curve of the tetH knockout strain was measured,and the consumption of tetrahydrosulfate was measured.Observation of sulfur consumption and analysis of transcription levels were made.When measuring the growth curve,it was found that the TetH knockout strain could be grown in the Starkey-K2S4O6 medium supplemented with sulfur powder.Under the same culture conditions,it showed a superiority compared with the wild type strain,and the growth trend of the late stage was inferior.High-performance liquid chromatography was used to determine the consumption of tetrahydrosulfate.It was found for the first time that the tetH knockout strain would consume the tetrasulfate in the Starkey-K2S4O6 medium supplemented with sulfur powder.It can be confirmed that this consumption is related to the tetrahydrosulfuric acid.Regardless of the salt hydrolase(TetH),it is speculated that there may be an unknown tetrahydrosulfate metabolism or transport pathway in A.caldus MTH-04,and a transcription level analysis is performed in order to give the answer.