The Characterization Of Sulfur Dioxygenase And Its Functional Analysis In The Sulfur Metabolic Network Of Acidithtobacillus Caldus

The development and utilization of mineral resources play an important role in the economic and social development of our country.However,our country's mineral resources have the characteristics of more lean ore and less rich ore.The existing ore development technology can not make full use of resources,and it is easy to cause a series of problems like ecological damage and resource waste.Bio-hydrometallurgy technology?mainly bioleaching technology?refers to the method extracting the precious metals in the ore by using microbial physiological and biochemical effects directly or indirectly.Compared with the traditional metallurgical technology,it has the advantages of low energy consumption,high recovery,clean,simple and so on.It has a broad application prospects in the mining of our country's common low-grade ore,especially the sulfur mine.At present,the widely distributed leaching microbes can be divided into mesophile,moderate thermophile and thermophile.It was found that the moderate thermophile gradually replaced the mesophile and occupy the dominant position in the case of higher leaching temperature?45?60 ??.Acidithiobacillus caldus?A.caldus?is an acidophilic,moderately thermophilic,chemolithoautotrophic gram-negative bacterium that relies on the oxidation state of inorganic sulfides?including S4O62-,S2O32-,S0,S2-,etc.?to obtain energy and reducing power required for growth,and fixing air CO2 as the main carbon source through the Calvin cycle.Due to its moderately thermophilic properties and unique sulfur metabolism mechanism,it is concerned by the researchers once identified.In addition to bioleaching,A.caldus also has important application values in the field of resources and environment like the desulfurization of coal and sulfur-containing waste water treatment.However,A.caldus has the characteristics of slow growth,long generation time and low cell yield,which affect its working efficiency,thus the study of its sulfur metabolic metabolism has both theoretical and practical significances.Because of the complex properties of sulfur from-2 to+6 valence,the oxidative metabolism of sulfur is complicated,plus the genetic operation is difficult in A.caldus,at present,the study of its mechanism of sulfur metabolism is mainly based on the models established by bioinformatics and transcriptomics.Analysis of the sequencing genome of A.caldus MTH-04 revealed that the sulfur metabolic system composed of a number of sulfur oxidation related genes of different sources,in which incomplete Sox system-related genes and sulfur oxidation system related genes similar to Acidithiobacillus ferrooxidans?A.ferrooxidans?as the representative.In 2012,our group proposed the sulfur oxidation system model in A.caldus based on the gene expression profile and growth characteristics of A.caldus MTH-04 and its sor gene mutant in different sulfur sources.The model proposed the regional division of sulfur oxidation,that is,sulfur was metabolized in different regions with different sulfur oxidation system.In this sulfur oxidation model,sulfur dioxygenase?SDO?is presumed to be a key enzyme for the oxidation of elemental sulfur in the periplasmic space,which can oxidize elemental sulfur in the presence of reduced glutathione?GSH?to sulfite.It is speculated that SDO may play a role in two aspects:oxidizing the elemental sulfur transported to the periplasmic space after activation of the outer membrane protein,producing sulfite;or replacing the missing Sox?CD?2 subunit in the incomplete Sox system,oxidizing the sulfur atoms in the sulfur-containing substrate bound by the SoxY subunit to form a complete cycle of the Sox system.Although SDO was first identified in A.ferrooxidans,its coding gene was not reported for decades thereafter.In 2009,Tiranti et al.found a mammalian mitochondrial sulfur dioxygenase encoding gene ethel.The enzyme is a metalloproteinase which catalytic center binds to a Fe2+ and is functionally related to sulfide quinone reductase?SQR?and rhodanese?Rhd?,which facilitate the catabolism of sulfide and energy generation.Since then,sulfur dioxygenase has been found in plants,invertebrates and heterotrophic bacteria,and their functions are involved in the decomposition of hydrogen sulfide relating to SQR and Rhd,and whether sulfur dioxygenase can participate in other sulfur metabolism pathway has not been reported.In 2014,the ETHE1 homologous protein encoding genes AFE₀269 and A5904₀790 were found for the first time in the genome of two Acidithiobacillus strains,A.ferrooxidans ATCC 23270 and A.caldus MTH-04.In vitro expression and enzyme activity assay showed that the proteins encoded by AFE₀269 and A5904₀790 had SDO activity,both of which could encode SDO in A.ferrooxidans ATCC 23270 and A.caldus MTH-04.However,the specific function of A5904₀790 encoded protein in A.caldus MTH-04 and the presence of other genes encoding sulfur dioxygenase in A.caldus MTH-04 were not reported.Therefore,analyzed the genome of A.caldus MTH-04 by bioinformatics,and studied the function and regulation mechanism of sulfur dioxygenase in A.caldus by techniques such as gene markless knockout to study the sulfur metabolism mechanism of A.caldus and its regulatory network is of great significance.The study is conducted mainly through the following aspects:1.Identification of sulfur dioxygenase in A.caldus.First,searching for putative sulfur dioxygenase encoding gene in A.caldus MTH-04 genome based on A5904₀790.Two genes A5904₀421 and A5904₁112 encoding A5904₀790 homologous proteins were found in the A.caldus MTH-04 genome by homologous alignment.The functional predictions of these two proteins were carried out by domain alignment and metal binding site analysis.The results showed that A5904₀421 protein could have similar function of SDO,and A5904₁112 protein may not have SDO-like function due to the lack of key amino acid residues of metal-binding sites.Genes A5904₀421,A5904₀790 and A5904₁112 were successfully expressed in Escherichia coli BL21?DE3?.The purified recombinant proteins were detected by enzyme activity in vitro.The results showed that A5904₀421 and A5904₀790 had the activity of sulfur dioxygenase,while the A5904₁112 protein did not have the activity of sulfur dioxygenase,which was consistent with the predicted results.The coding genes for SDO in A.caldus MTH-04 were identified as A5904₀421?named sdol?and A5904₀790?named sdo2?Further studies on the enzymatic properties of purified SDO recombinant proteins were carried out.The results showed that the optimal temperature of A5904 0421 recombinant protein?SDO1?was about 45 ?,the optimum pH value was pH 8.0,which was consistent with the results of the corresponding reaction condition of A5904 0790 recombinant protein?SD02?.Effect of inhibitors on the activity of SDO1 are basically the same with the results in SD02,that is,common divalent metal ions?except Mg2+?,EDTA and NEM strongly inhibited the activity of SDO1,while DTT showed moderate inhibition of SDO1 enzyme activity.Using GSSH as the substrate,the kinetic analysis of SDO1 and SD02 showed that there was no significant difference in Km between SDO1 and SD02,but the kcat of SDO1 was significantly smaller than that of SD02,kcat/Km ratio showed that SD02 had higher catalytic efficiency.The metal content of SDO protein was analyzed by ICP-MS.It was found that SDO protein was mainly bound to Fe and Mn,and SDO1 bound more Fe than SD02.Along with the results of sequence analysis,it can be seen that the difference in enzyme activity between the two proteins may be due to the absence of conserved amino acid residues in the GSH binding site of SDO1.Homology analysis was done in the sequenced genome on NCBI using the sulfur dioxygenase in A.caldus as the query sequence,the results showed that the homologous proteins of SDO1 were mainly concentrated in the autotrophic bacteria,and the homologous proteins of SD02 were found in animals,plants and heterotrophic bacteria.Phylogenetic analysis showed that SDO2 belongs to the ETHE1 subclass of SDO,but the evolutionary relationship of SDO1 and its homologous proteins are far away from the known three SDO subclasses?ETHE1,SdoA,Blh?,so they are classified as a new subclass,named SdoS.Analysis of the Acidithiobacillus strains revealed that there were two to three copies of SDO in different strains of Acidithiobacillus spp.,and genes encoding ETHE1 and SdoS were usually present at the same time.Sequence analysis showed that the GSH binding sites of SdoS were different from those of the other three subclasses.It is consistent with the result reported in the SDO structure study that the GSH binding site can be used as a basis for distinguishing these subclasses.The discovery of the new subclass of SDO shed light on how SDO in different subclasses may be involved in sulfur oxidation through similar or complementary mechanisms.2.Study on the function of sulfur dioxygenase in A.caldus sulfur metabolism system.In order to study the specific function of SDO in the A.caldus sulfur metabolism system,the sdol and sdo2 genes in A.caldus were knocked out based on homologous recombination principle.The single knockout strains ?sdo1,?sdo2 were constructed based on A.caldus MTH-04,and the double knockout strain?sd01&2 was constructed based on ?sdo1.At the same time,the sdo overexpression strains OE-sdo1,OE-sdo2 based on A.caldus MTH-04 and the sdo complemented strains ?sdo1/sdo1',?sdo2/sdo2' based on sdo gene knockout strains were successfully constructed.The successful construction of this series of mutants laid the foundation for the study of the function of sdo in A.caldus.The series sdo mutants constructed in this paper were studied systematically from the aspects of growth curve,substrate consumption,metabolic end product detection and the level of transcription of genes related to sulfur metabolism under different energy culture conditions.In the growth curve,the growth of the knockout strains were weaker than that of the wild type when sulfur powder was used as the energy source.When tetrathionate was used as the energy source,?sdo1 and ?sdo1&2 could not grow,?sdo2 got into the logarithmic growth phase earlier than the wild type,and ?sdol/sdol' restored the ability to use tetrathionate for growth.When thiosulfate was used as energy source,?sdo1 and ?sdo1&2 got into the logarithmic growth phase later than the wild type,while Asdo2 still got into the logarithmic growth phase earlier than the wild type.The overexpression of sdo did not affect the growth curve when sulfur powder,tetrathionate and thiosulfate were used as energy source.High performance liquid chromatography was used to detect the consumption of tetrathionate.After entering the stable period,tetrathionate in Asdo2 and wild type were depleted,and tetrathionate in ?sdol and ?sdo1&2 was not consumed,which was consistent with the results of the growth curve.The metabolic end products were detected by high performance liquid chromatography and ion chromatography.The metabolic end products of each strain x under different energy culture conditions were found to be sulfate,but the yield was different.Under the condition of sulfur culture,the knockout strains produced more sulfate than the wild type.In the presence of tetrathionate,?sdo2 and wild type produced equal amounts of sulfate,and Asdol and ?sdo1&2 were unable to grow and had no sulfate formation.Under the conditions of thiosulfate culture,there was no significant difference in the sulfate yield.The sulfur oxidation capacity of each strain was determined by adding GSH as the cofactor.It was found that knockout sdol could significantly increase the sulfur oxidation activity compared with the wild type,and the knockout of sdo2 had little effect on the oxidation capacity.The transcriptional changes of sdol and sdo2 in different growth stages of wild type under different energy culture conditions and transcriptional changes of sulfur metabolism related genes in knockout strains ?sdol,?sdo2,?sdo1 ?sdo1&2,overexpression strains OE-sdol and OE-sdo2 were analyzed by RT-qPCR.The results showed that the transcription level of sdol and sdo2 were higher in the late stage of logarithm,and when sulfur powder was used as the energy source,the transcription level of sdol was significantly higher than that of tetrathionate.The knockout or overexpression of sdo could cause changes in the transcription levels of other sulfur metabolic gene,for instance,tetH,tqo and partial sox-? genes were upregulated in sdo knockout strains and downregulated in sdo overexpressing strains in different energy source.The above results indicate that SDO plays an important role in the metabolism of elemental sulfur formated in the cytoplasmic metabolism processes instead of the extracellular elemental sulfur;SDO can not replace the missing Sox?CD?2 subunit;SDO1 and SD02 play a different role in A.caldus metabolic process,and they can not completely replace each other:sdol is closely related to S4I pathway,it is necessary for the growth of A.caldus on tetrathionate,and sdo2 is closely related to the sqr-sdo-rhd pathway,sdo2 may also play an important role in sulfide detoxification in addition to sulfur metabolism.3.Study on regulatory mechanism of sulfur dioxygenase.The results of bioinformatics analysis showed that gene A5904₀789 in the upstream of sdo2 could be involved in the transcriptional regulation of sdo in A.caldus.The gene A5904 0789 was successfully expressed in Escherichia coli BL21?DE3?.The sdo gene promoter was analyzed by EMSA using purified A5904 0789 recombinant protein,the results showed that the CRP transcriptional regulator encoding by A5904 0789 could bind to the promoter region of sdo2?A5904₀790?,but it could not bind to the promoter region of sdol?A5904 0421?.The gene knockout strain of A5904 0789??crp?were constructed successfully,and the growth characteristics of Acrp were studied.The growth characteristics were similar to those of Asdo2 under different energy culture conditions.These results suggest that the transcriptional regulator of CRP family encoded by A5904 0789 might involve in the transcriptional regulation of sdo2,and sdo1-related transcription mechanism needs further study.In this paper,an encoding gene of sulfur dioxygenase?A5904₀421?was newly found in the A.caldus MTH-04 genome,and the enzymatic properties of the proteins?A5904₀421 and A5904₀790?were studied.Based on phylogenetic analysis,the evolutionary relationship of known sulfur dioxygenases and sulfur dioxygenases from A.caldus was analyzed.A new subclass?SdoS?of sulfur dioxygenase was found.The mutants of sdo genes were constructed and their growth characteristics,substrate consumption,product formation and the level of transcription of genes related to sulfur metabolism under different energy culture conditions were studied,and the function of SDO in A.caldus sulfur metabolism system were showed:SDO plays an important role in the metabolism of elemental sulfur formated in the cytoplasmic metabolism processes;sdol is necessary for the growth of A.caldus on tetrathionate,and sdo2 may also play an important role in sulfide detoxification in addition to sulfur metabolism;A5904 0789 encodes CRP family transcriptional regulator that might involve in sdo2-related transcriptional regulation.The identification of SDO and its function and regulatory mechanism study in the A.caldus are of great significance to further understand the sulfur metabolism of A.caldus and its regulatory network,and provides important information for constructing improved sulfur oxidation models for Acidithiobacillus spp.,which are important for optimizing bioleaching.