Study On Key Enzymes Of Sulfur Reduction Metabolism And Redox Balance In Thermotoga Maritima

Thermotoga maritima is a strictly anaerobic,extremophilic microorganism that lives in submarine sediments at around 80℃ and grows heterotrophically by fermenting a variety of carbohydrates.The ability of T.maritima to ferment carbohydrates and produce hydrogen with high efficiency has good potential for biohydrogen energy development.Early studies found that T.maritima was able to reduce elemental sulfur and thiosulfate to produce H2S,and that sulfur reduction metabolism has a significant effect on T.maritima growth.Howe the mechanism of sulfur reduction metabolism has remained unclear.The studies on the mechanism of T.maritima sulfur reduction metabolism and intracellular redox homeostasis were conducted as follows:Ⅰ.The study of the effects of S0 and Na2S2O3 on the growth and metabolism of T.maritima revealed that T.maritima may have a novel bacterial sulfur-reducing metabolic mechanism.Growth experiments revealed that S0 and Na2S2O3 were able to promote the growth of T.maritima.It was also found that Na2Sn promoted the growth of T.maritima more significantly than S0.The growth promoting effects of S0 and Na2S2O3 on T.maritima were mainly reflected in the late stage of T.maritima exponential growth and did not have significant effects in the early stage of growth.Combined with the fact that genome of T.maritima lacks sulfur respiration-related genes,it can be concluded that the reduction of S0 and Na2S2O3 by T.maritima does not take place through the traditional sulfur respiration chain.Further experiments revealed that growth was inhibited by H2.Addition of S0 or Na2S2O3 to the medium reduced the inhibition of growth by H2 and promoted the growth of T.maritima,and the promotion effect of S0 and Na2S2O3 was also more obvious under the inhibition of higher concentration of H2.In the absence of H2 inhibition,the growth promoting effect of S0 and Na2S2O3 was minimal.Study on the transcription level of the hydrogenase gene hydA and the activity of the hydrogenase HydABC enzyme revealed that the reduction of S0 and Na2S2O3 affects the expression of the hydrogenase gene and ultimately affects the hydrogen production,while producing a large amount of H2S.Studies of enzyme activity in cytoplasmic and membrane fractions of T.maritima revealed the presence of Fdreddependent sulfane sulfur reductase activity in the cell membrane,and genomic investigations revealed that this may be the activity of MBX,a membrane-bound sulfane sulfur reductase.Transcriptional level studies revealed that mbx is transcriptionally up-regulated in the presence of S0,indicating that it is involved in sulfur reduction metabolism.NADH-dependent elemental sulfur reductase activity and thiosulfate reductase activity were also detected in the cytoplasmic fractions of T.maritima,suggesting that novel bacterial sulfur reductases and thiosulfate reductases may be present in T.maritima.Ⅱ.The polysulfide reductase NSR was obtained by heterologous expression and purification,which was also found to possess thiosulfate reductase activity,and its characteristics and functions were investigated.Based on the sulfur reductase activity detected in cytoplasmic fractions,the genome of T.maritima was investigated to find possible sulfur reductase genes.The protein NSR encoded by TM0379,a flavoprotein with a molecular weight of about 50 kDa that binds to a molecule of FAD,was obtained by heterologous expression and purification.NSR has NAD(P)H-dependent elemental sulfur reduction activity of 1.3 U/mg.Polysulfides as its possible direct electron acceptor with an acticities of 0.35 U/mg.CoA has a significant facilitation effect on the NSR reaction and increase the activity to about 4 times,which may be due to the ability of CoA to react with S0 to generate CoA disulfide or persulfide,promoting the solubilization of S0 and its binding to the enzyme molecule.Mutation of its conserved amino acid residue Cys43 causes the loss of NSR activity,suggesting that this cysteine residue plays a key role in sulfur reduction.The polysulfide reductase NSR of T.maritima has many similarities in properties and functions with its archaeal homologs,but also has significant differences,for example,the NSR of T.maritima does not possess NAD(P)H oxidase activity.One of the most obvious differences is that the NSR of T.maritima also has thiosulfate reductase activity of about 6.8 U/mg.Transcription of nsr gene was regulated by the global redox-sensing transcriptional regulator Rex,indicating that NSR has a direct effect on the redox balance of T.maritima.Transcriptional level studies showed that nsr gene was up regulated by 5-6 times in the presence of S0 and Na2S2O3,suggesting that the NSR may be involved in both elemental sulfur and thiosulfate sulfur reduction metabolism in T.maritima.In thiosulfate reduction metabolism,the excess Fdred can transfer electrons to NAD+through Rnf protein on cell membrane to generate NADH for thiosulfate reduction,rnf gene was up regulated by around 10 times in the presence of Na2S2O3.The yeeE gene was up regulated by 42 times and it may be involved in the thiosulfate transport.Ⅲ.The NADH oxidase Nox and electron-bifurcating transhydrogenase NfnAB were heterologously expressed.Their characteristics and functions were investigated through enzyme activity and transcriptional studies,and mutations of key amino acids were performed on NfnAB to investigate the function of these amino acids.Heterologous expression and purification of TM0395 did not reveal that it exhibited sulfur reduction as well as NADH oxidation related activities,and its specific biochemical functions need to be further investigated.The Nox protein was obtained by heterologous expression of TM0754,which has a molecular weight of about 45 kDa and binds a molecule of FMN.Nox does not have the activity of reducing elemental sulfur or thiosulfate,but exhibits N AD(P)H oxidase activity,and NADH is its optimal electron donor,which can directly reduce O2 to generate.H2O.FprA encoded by TM0755,which is located upstream of TM0754,also has NADH oxidase activity.also has NADH oxidase activity.and both are located on the same operon.Transcription levels are influenced by the redox potential of the growth environment and are higher in normal anaerobic environments and lower when exposed to higher concentrations of oxygen,indicating that these two NADH oxidases are mainly used for the removal of small amounts of O2 under normal growth conditions.NfnAB is a two-subunit electronbifurcating transhydrogenase that catalyzes the reduction ofNAD+ and Fdox by NADPH.It was expressed under different growth conditions,indicating that it plays an important role in the regulation of redox homeostasis under different conditions.The binding of the b-FAD molecule was found to be essential for the activity of NfnAB,and the N5 atom of its isoalloxazine ring was the site of electron transfer,and its interaction with Arg residues played a key role in the electron disproportionation reaction.The Nfn homologs are widely distributed in anaerobic archaea and bacteria.Based on their protein sequence and domain arrangement,they can be divided into four types.Studies on the fusion-type Nfn from Clostridium ljungdahlii have found that it has consistent electron bifurcation enzyme activity.After knocking out the gene,this bacterium cannot grow.These results indicate that Nfn plays an important role in maintaining the intracellular redox balance of anaerobic microorganisms.Taken together,a model of the metabolic mechanism of T.maritima sulfur reduction metabolism and redox homeostasis can be derived:T.maritima degrades carbohydrates through the EMP and ED pathways to produce three reducing equivalents,Fdred,NADH and NADPH.NADPH can transfer electrons to NADH and Fdred through NfnAB.In the absence of S0 and Na2S2O3,NADH and Fdred release reducing power mainly through hydrogen production by HydABC,a hydrogenase for electron disproportionation.When S0 was present,HydABC expression decreased.at which time NADH released reducing power mainly through NSR reduction of sulfur.while Fdred released reducing power through MBX reduction of sulfur.In the presence of Na2S2O3,the hydrogenase still functions,while NSR uses NADH to reduce thiosulfate to release part of the reducing power,and the excess Fdred transfers electrons to NADH through Rnf to be further released.In the presence of a small amount of O2 in the system,it can be scavenged by the NADH oxidase encoded by two genes.TM0754 and TM0755.For the investigation of the distribution of NSR in microorganisms,it can be concluded that this sulfur reduction mechanism may be widely distributed in heterotrophic sulfur-reducing bacteria and thiosulfate-reducing bacteria in geothermal environments and play an important role for sulfur cycle in geothermal environments such as hot springs.