Sulfur Attachment And Cytoplasmic Sulfur Trafficking In The Sulfur Metabolism Process Of Acidithiobacillus Caldus MTH-04

Acidithiobacillus caldus(A.caldus)is a Gram-negative,obligate chemolithoautotrophic,acidophilic sulfur-oxidizing bacterium and widely used in the bioleaching industry.A.caldus has the remarkable capability of oxidizing various reduced inorganic sulfur compounds(RISCs)to obtain electrons for carbon dioxide fixation.Sulfur metabolism is the core energy system of A.caldus and the basis of its industrial application.The sulfur metabolism process in A.caldus is a complex oxidation process from extracellular interaction to intracellular reaction.Firstly,cells need to move and attach on the surface of elemental sulfur.Second,elemental sulfur is activated,converted,and transferred into periplasmic space,at where sulfur is oxidized to different sulfur compounds.Third,some compounds in periplasm can be transferred into cytoplasm for further oxidation.Therfore,the metabolism of elemental sulfur in A.caldus is a gradual oxidation process involving various systems.The sulfur-oxidizing enzymes have been extensively identified and studied in A.caldus and other Acidithiobacillus spp.,however,the researches on the attachment to the elemental sulfur and the sulfur trafficking mechanism are still not well carried out in these sulfur-oxidizing bacteria.Here,the cell adsorption process and intracellular sulfur transport system were systematically studied in A.caldus.(I)Effects of flagellum-mediated motility on sulfur adsorption and metabolism in A.caldus.1.Distribution characteristics of flagellum gene clusters and the transcriptional analysis of rpoF gene.Bioinformatics analysis of flagellar gene cluster in A.caldus revealed all flagellar biosynthesis related genes,including flagellin genes,regulatory genes and chemotactic genes,are arranged together in one large gene cluster,involving nearly 40 genes and accounting for about 2%of the whole genome ORFs(Opening read frames).Comparative genomics is used to analyzing the synteny of flagellar gene clusters in Acidithibacillia and comparing with other heterotrophic microorganisms habor the monotrichous flagellum.In summarizing the taxonomic characters,it's also found that the sulfur oxidizers that harbor the Sox system tend to form the flagella.The coexistence of Sox system and flagella suggesting that flagella are closely related to sulfur metabolism in these bacteria.The promoter sequence of RpoF regulatory gene in A.caldus is different from heterotrophic bacteria.Phylogenetic analysis of A.caldus RpoF showed that it's different from heterotrophic bacteria in evolution,suggesting the species specificity of RpoF.2.The role of flagellum in energy metabolism and the regulatory effect of RpoF on flagellar biosynthesis in A.caldus.Based on homologous recombination,the flagellum synthesis regulator RpoF gene in A.caldus genome was knocked out by using the technique of markerless gene knockout.The overexpression and complementary strains of rpoF were constructed using the wide host plasmid pJRD215 as the skeleton.These plasmids were transformed into A.caldus cells.No flagellum was found in A.caldus ArpoF strain.On semisolid agar gel plates,the colony of ArpoF strain became smaller and the movement ability decreased.As a result,the absence of RpoF has no obvious influence on A.caldus growth under optimal conditions(1.2 g S0 and 150 rpm),but brings negative effects on its growth in unfavorable conditions(no shaking,low substrate concentration or non-optimal substrate).Results also indicate that the absence of RpoF results in the tendency of cells to attach to S0-coupons.In addition,the effects of rpoF on flagellum synthesis genes and other life activities were explored at the transcriptome level.RNA-seq results showed that rpoF also participated in energy metabolism,DNA modification,pili synthesis and c-di-GMP production(the second messenger),which had a global regulatory effect on cell life activities.The regulatory role of RpoF for flagellar assembly in A.caldus MTH-04 was determined by TEM observation and RNA-seq analysis.These findings indicate that A.caldus probably develops the similar flagellar transcription hierarchy as the reported pattern in P.aeruginosa.As a proposed model,the flagellar biosynthesis in A.caldus is probably controlled by a four-class hierarchy involving the regulatory proteins RpoN,FleQ,FleS,FleR,and RpoF(FliA).(II)The function of Dsr system in the sulfur trafficking of A.caldus.1.Analysis of the Dsr system in A.caldus:Bioinformatics was used to analyze the small molecule sulfur carrier proteins in A.caldus genome.The dsrE3A-tusA-rhd-hdr gene cluster is also found in A.caldus genome,which is an incomplete Dsr gene cluster.The characteristics of sulfur transport related genes in A.caldus were analyzed by BlastP and phylogenetic analysis.At the same time,the transcriptional changes of these genes during sulfur metabolism and the co-transcriptional relationship of gene clusters were studied by RT-q PCR.2.Studies on the function of sulfur transfer proteins in A.caldus.In order to investigate the sulfur trafficking in A.caldus,the knockout mutants of genes related to sulfur transfer,dsrE2A,tusA and 1004 were constructed by method of markerless gene knockout.Using different reduced sulfides as substrates,the effects of deletion of related genes on cell growth were studied.Overall,the deletion of dsrE2A and orf1004 had the limited effects on elemental sulfur oxidation,while the deletion of tusA had a significant inhibitory effect on elemental sulfur metabolism,leading to delayed cell growth and slow growth rate.When K2S4O6 was used as the energy source,the growth of AdsrE2A and AtusA were significantly different from that of wild type except ?1004.The deletion of dsrE2A promoted the metabolism of K2S4O6,while the deletion of tusA leads to the accumulation of extracellular sulfur,which seriously hindered the oxidation of 34O62-and lead to a considerable stagnation period,and the final cell density was much higher than that of wild-type cells.It is speculated that the oxidation of the accumulated extracellular sulfur could produce more energy for cell growth.In addition,two knockout strains of dsrE2A and tusA were found to promote the oxidation of Na2S2O3.As the sulfur carrier proteins in cells,the absence of these two genes result in trafficking block of thiosulfonate groups and hinder its further oxidation.With the growth of cells,the pH decreased in the medium,and the accumulated Na2S2O3 is naturally hydrolyzed to produce elemental sulfur and rapidly oxidized by Sox system to obtain energy.Overexpression plasmids of dsrE2A,tusA and other sulfur trafficking genes were constructed using pJRD215 as a vector and conjugated them into corresponding A.caldus MTH-04 cells.As results,a series of complementary strains of dsrE2A and tusA and overexpression strains of related genes were constructed.The growth of each strain under different energy conditions was studied.On the contrary to knockout strians,the overexpression of dsrE2A had a weak effect on sulfur oxidation,but a significant inhibition on the metabolism of S4O62-and slow growth rate.Although the overexpression of orf0418 has no obvious effect on sulfur oxidation,it significantly promoted sulfur oxidation whose cells begin to grow directly without growth stagnation.The overexpression of tusA also showed the opposite trend compare with its knockout strain.However,the overexpression of orf1004,in concert with its knockout strain,had no significant effect on the sulfur metabolism of cells under the experimental conditions.BlastP analysis showed that small molecule sulfur carrier proteins contained highly conservative amino acid Cys.In order to study the functions of DsrE2A and TusA,the key amino acid sites of Cys were mutated to Ser.The mutation vectors of corresponding genes were constructed using pJRD215 as the framework.The conjugation method was used to transform them into the corresponding mutants.The in vivo experiments were conducted to study their oxidation of RISCs.The results showed that Cys117 and Cys101 at the end of C-terminal of DsrE2A protein were the two key amino acid sites.The mutation had different effects on sulfur metabolism,especially the oxidation of S4O62-and the growth characteristics of the double mutants were consistent with the DsrE2A knockout strain.Cys 15 at the N-terminal of TusA protein is crucial for its function.After mutation,cell growth returned to the same growth with AtusA.In addition,the function of other genes in Dsr gene cluster were explored.A series of overexpressed strains were constructed and their effects on the oxidation of different sulfides were studied.The results showed that the overexpression of them had little effect on the oxidation of elemental sulfur.Most genes overexpression even showed almost the same growth as wild type,but had different effects on S4O62-oxidation.These results suggest that these genes are involved in the trafficking and oxidation of S4O62-in vivo.