Structural analyses of the permease like protein SoxT: A member of the sulfur compound metabolizing sox operon

One of the oldest known gene clusters that are involved in biological oxidation processes is the sox operon. This operon is present in different microbial species. In the present study an attempt has been made to analyze the probable structural role of SoxT protein from Pseudaminobacter salicylatoxidans. This protein has been predicted to be a permease-like protein. A comparative model of the protein has been made and analyzed. The possible membrane spanning region of the protein has been detected by structural bioinformatics approach. The inducer of the sulfur oxidation process has been predicted. And thereby the plausible mechanism of the transport of the sulfur anion inside the bacterial cell has been elucidated. Since this is the first study regarding the structural aspect of the protein this study may shed light on the theory of the yet unknown molecular mechanism of the sulfur oxidation process by sox operon.     

The Sulfur Oxidation Operon Repressor Function is Influenced by the Product of its Adjacent Upstream ORF in <Emphasis Type="Italic">Pseudaminobacter salicylatoxidans</Emphasis> KCT001

The repressor of sulfur-oxidizing (sox) operon regulates expression of genes encoding a multienzyme complex that governs the chemolithotrophic sulfur oxidation in Pseudaminobacter salycylatoxidans KCT001. The inducer of sox operon viz., thiosulfate and other sulfur anions had no impact on in vitro repressor–operator interaction which indicates an atypical derepression mechanism. The reduced repressor has higher affinity for its operator DNA. The sulfur oxidation repressor binds with operator regions and led to efficient repression in trans, however, increased repressor concentration resulted in higher gene expression. Using a reporter system in E. coli, the present study established that the thioredoxin-like protein, encoded in immediate upstream ORF, could nullify the observed reversal of the repression at higher repressor concentration. In this context, the involvement of the upstream gene product in the regulation of the sulfur oxidation gene expression has been reported.     

Structural insight into the mode of interactions of SoxL from Allochromatium vinosum in the global sulfur oxidation cycle

Microbial redox reactions of inorganic sulfur compounds are one of the important reactions for the recycling of sulfur to maintain the environmental sulfur balance. These reactions are carried out by phylogenetically diverse microorganisms. The sulfur oxidizing gene cluster (sox) of α-proteobacteria, Allochromatium vinosum comprises two divergently transcribed units. The central players of this process are SoxY, SoxZ and SoxL. SoxY is sulfur compound binder which binds to sulfur anions with the help of SoxZ. SoxL is a rhodanese like protein, which then cleaves off the sulfur substrate from the SoxYZ complex to recycle the SoxY and SoxZ. In the present work, homology modeling has been employed to build the three dimensional structures of SoxY, SoxZ and SoxL. With the help of docking simulations the amino acid residues of these proteins involved in the interactions have been identified. The interactions between the SoxY, SoxZ and SoxL proteins are mediated mainly through hydrogen bonding. Strong positive fields created by the SoxZ and SoxL proteins are found to be responsible for the binding and removal of the sulfur anion. The probable biochemical mechanism of sulfur anion oxidation process has been identified.     

Molecular characterization of inorganic sulfur‐compound metabolism in the deep‐sea epsilonproteobacterium Sulfurovum sp. NBC37‐1

The molecular components involved in energy metabolism of deep‐sea Epsilonproteobacteria were characterized in the mesophilic hydrogen‐ and sulfur‐oxidizing chemolithoautotroph Sulfurovum sp. NBC37‐1. Previous whole‐genome analysis of strain NBC37‐1 identified key genes likely to be associated with both sulfur reduction (psr gene families) and oxidation (two sox gene clusters). However, the sox gene clusters showed unique organizations and low homologies to those in other bacteria. Therefore, the biochemical mechanism of inorganic sulfur metabolism has been uncertain. Enzymatic activity measurements and partial protein purification indicated that the Sox enzyme system was constitutively expressed, whereas the expression of sulfur‐reduction enzymes varied depending on the culture conditions. The operative Sox system in strain NBC37‐1 required membrane components. The molecular basis of energy metabolism reported in this study provides important insight into how deep‐sea Epsilonproteobacteria change their energy metabolism in response to variable physical and chemical conditions in mixing zones between hydrothermal fluid and ambient seawater.     

Thiosulfate oxidation by a moderately thermophilic hydrogen-oxidizing bacterium, <Emphasis Type="Italic">Hydrogenophilus thermoluteolus</Emphasis>

The moderately thermophilic Betaproteobacterium, Hydrogenophilus thermoluteolus, not only oxidizes hydrogen, the principal electron donor for growth, but also sulfur compounds including thiosulfate, a process enabled by sox genes. A periplasmic extract of H. thermoluteolus showed significant thiosulfate oxidation activity. Ten genes apparently involved in thiosulfate oxidation (soxEFCDYZAXBH) were found on a 9.7-kb DNA fragment of the H. thermoluteolus chromosome. The proteins SoxAX, which represent c-type cytochromes, were co-purified from the cells of H. thermoluteolus; they enhanced the thiosulfate oxidation activity of the periplasmic extract when added to the latter.     

Structural Interaction Between DsrE-DsrF-DsrH Proteins Involved in the Transport of Electrons in the dsr Operon

Abstract

Biological redox reactions of inorganic sulfur compounds are important for the proper maintenance of environmental sulfur balance. These reactions are mediated by phylogeneticaly diverse set of microorganisms. The protein complex that is involved in such redox reactions of sulfur compounds is the complex encoded by dsr operon. The ecological and industrial importance of these microorganisms led us to investigate the structural details of the mechanism of the process of electron transport during such redox reactions performed by the dsr operon. Among the gene products of the operon, the proteins DsrE, DsrF, and DsrH are small soluble cytoplasmic proteins acting as α₂β₂γ₂ heterohexamer and are involved in the process of electron transport in these ecologically as well as industrially important microorganisms.

Since no structural details of the proteins were available we employed homology modeling to construct the three-dimensional structures of the DsrE, DsrF, and DsrH from Chlorobium tepidum. The putative three dimensional structures of the proteins were predicted from the models. Since DsrE, DsrF, and DsrH proteins act as a hetero-hexameric complex, the modeled proteins were subjected to molecular docking analyses to generate the model of the biochemically active complex. This allowed us to predict the probable binding modes of the proteins as well as the biochemical and the structural basis of the mechanism of the electron transport process by this complex. The hexamerization of the proteins would help to bring the Cys residues in close proximity, which enables the complex to actively take part electron transport process.     

Expression of genes for sulfur oxidation in the intracellular chemoautotrophic symbiont of the deep-sea bivalve Calyptogena okutanii

To understand sulfur oxidation in thioautotrophic deep-sea clam symbionts, we analyzed the recently reported genomes of two chemoautotrophic symbionts of Calyptogena okutanii (Candidatus Vesicomyosocius okutanii strain HA: Vok) and C. magnifica (Candidatus Ruthia magnifica strain Cm: Rma), and examined the sulfur oxidation gene expressions in the Vok by RT-PCR. Both symbionts have genes for sulfide-quinone oxidoreductase (sqr), dissimilatory sulfite reductase (dsr), reversible dissimilatory sulfite reductase (rdsr), sulfur-oxidizing multienzyme system (sox) (soxXYZA and soxB but lacking soxCD), adenosine phosphosulfate reductase (apr), and ATP sulfurylase (sat). While these genomes share 29 orthologous genes for sulfur oxidation implying that both symbionts possess the same sulfur oxidation pathway, Rma has a rhodanese-related sulfurtransferase putative gene (Rmag0316) that has no corresponding ortholog in Vok, and Vok has one unique dsrR (COSY0782). We propose that Calyptogena symbionts oxidize sulfide and thiosulfate, and that sulfur oxidation proceeds as follows. Sulfide is oxidized to sulfite by rdsr. Sulfite is oxidized to sulfate by apr and sat. Thiosulfate is oxidized to zero-valence sulfur by sox, which is then reduced to sulfide by dsr. In addition, thiosulfate may also be oxidized into sulfate by another component of sox. The result of the RT-PCR showed that genes (dsrA, dsrB, dsrC, aprA, aprB, sat, soxB, and sqr) encoding key enzymes catalyzing sulfur oxidation were all equally expressed in the Vok under three different environmental conditions (aerobic, semioxic, and aerobic under high pressure at 9 MPa), indicating that all sulfur oxidation pathways function simultaneously to support intracellular symbiotic life.     

Mechanism of electron transport during thiosulfate oxidation in an obligately mixotrophic bacterium Thiomonas bhubaneswarensis strain S10 (DSM 18181T)

This study describes the thiosulfate-supported respiratory electron transport activity of Thiomonas bhubaneswarensis strain S10 (DSM 18181^T). Whole-genome sequence analysis revealed the presence of complete sox (sulfur oxidation) gene cluster (soxCDYZAXB) including the sulfur oxygenase reductase (SOR), sulfide quinone reductase (SQR), sulfide dehydrogenase (flavocytochrome c (fcc)), thiosulfate dehydrogenase (Tsd), sulfite dehydrogenase (SorAB), and intracellular sulfur oxidation protein (DsrE/DsrF). In addition, genes encoding respiratory electron transport chain components viz. complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (ubiquinone-cytochrome c reductase), and various types of terminal oxidases (cytochrome c and quinol oxidase) were identified in the genome. Using site-specific electron donors and inhibitors and by analyzing the cytochrome spectra, we identified the shortest thiosulfate-dependent electron transport chain in T. bhubaneswarensis DSM 18181^T. Our results showed that thiosulfate supports the electron transport activity in a bifurcated manner, donating electrons to quinol (bd) and cytochrome c (Caa ₃ ) oxidase; these two sites (quinol oxidase and cytochrome c oxidase) also showed differences in their phosphate esterification potential (oxidative phosphorylation efficiency (P/O)). Further, it was evidenced that the substrate-level phosphorylation is the major contributor to the total energy budget in this bacterium.

     

Dynamics of Metabolic Activities and Gene Expression in the Roseobacter Clade Bacterium Phaeobacter sp. Strain MED193 during Growth with Thiosulfate

Metagenomic analyses of surface seawater reveal that genes for sulfur oxidation are widespread in bacterioplankton communities. However, little is known about the metabolic processes used to exploit the energy potentially gained from inorganic sulfur oxidation in oxic seawater. We therefore studied the sox gene system containing Roseobacter clade isolate Phaeobacter sp. strain MED193 in acetate minimal medium with and without thiosulfate. The addition of thiosulfate enhanced the bacterial growth yields up to 40% in this strain. Concomitantly, soxB and soxY gene expression increased about 8-fold with thiosulfate and remained 11-fold higher than that in controls through stationary phase. At stationary phase, thiosulfate stimulated protein synthesis and anaplerotic CO₂ fixation rates up to 5- and 35-fold, respectively. Several genes involved in anaplerotic CO₂ fixation (i.e., pyruvate carboxylase, propionyl coenzyme A [CoA], and crotonyl-CoA carboxylase) were highly expressed during active growth, coinciding with high CO₂ fixation rates. The high expression of key genes in the ethylmalonyl-CoA pathway suggests that this is an important pathway for the utilization of two-carbon compounds in Phaeobacter sp. MED193. Overall, our findings imply that Roseobacter clade bacteria carrying sox genes can use their lithotrophic potential to gain additional energy from sulfur oxidation for both increasing their growth capacity and improving their long-term survival.     

Expression,purification and characterization of a cysteine desulfurase,IscS, from <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis>

Iron–sulfur clusters are one of the most common types of redox center in nature. Three proteins of IscS (a cysteine desulfurase), IscU (a scaffold protein) and IscA (an iron chaperon) encoded by the operon iscSUA are involved in the iron–sulfur cluster assembly in Acidithiobacillus ferrooxidans. In this study the gene of IscS from A. ferrooxidans ATCC 23270 was cloned and expressed in Escherichia coli, the protein was purified by one-step affinity chromatography to homogeneity. The molecular mass of recombinant IscS was 46 kDa by SDS-PAGE. The IscS was a pyridoxal phosphate-containing protein, that catalyzed the elimination of S from l-cysteine to yield l-alanine and elemental sulfur or H₂S, depending on whether or not a reducing agent was added to the reaction mixture. Jia Zeng and Yanfei Zhang contributed equally to this work.     

Cold adaptation of the mononuclear molybdoenzyme periplasmic nitrate reductase from the Antarctic bacterium Shewanella gelidimarina

The reduction of nitrate to nitrite is catalysed in bacteria by periplasmic nitrate reductase (Nap) which describes a system of variable protein subunits encoded by the nap operon. Nitrate reduction occurs in the NapA subunit, which contains a bis-molybdopterin guanine dinucleotide (Mo–MGD) cofactor and one [4Fe–4S] iron–sulfur cluster. The activity of periplasmic nitrate reductase (Nap) isolated as native protein from the cold-adapted (psychrophilic) Antarctic bacterium Shewanella gelidimarina (Nap_Sgel) and middle-temperature adapted (mesophilic) Shewanella putrefaciens (Nap_Sput) was examined at varied temperature. Irreversible deactivation of Nap_Sgel and Nap_Sput occurred at 54.5 and 65 °C, respectively. When Nap_Sgel was preincubated at 21–70 °C for 30 min, the room-temperature nitrate reductase activity was maximal and invariant between 21 and 54 °C, which suggested that Nap_Sgel was poised for optimal catalysis at modest temperatures and, unlike Nap_Sput, did not benefit from thermally-induced refolding. At 20 °C, Nap_Sgel reduced selenate at 16% of the rate of nitrate reduction. Nap_Sput did not reduce selenate. Sequence alignment showed 46 amino acid residue substitutions in Nap_Sgel that were conserved in NapA from mesophilic Shewanella, Rhodobacter and Escherichia species and could be associated with the Nap_Sgel cold-adapted phenotype. Protein homology modeling of Nap_Sgel using a mesophilic template with 66% amino acid identity showed the majority of substitutions occurred at the protein surface distal to the Mo–MGD cofactor. Two mesophilic ↔ psychrophilic substitutions (Asn ↔ His, Val ↔ Trp) occurred in a region close to the surface of the NapA substrate funnel resulting in potential interdomain π–π and/or cation–π interactions. Three mesophilic ↔ psychrophilic substitutions occurred within 4.5 Å of the Mo–MGD cofactor (Phe ↔ Met, Ala ↔ Ser, Ser ↔ Thr) resulting in local regions that varied in hydrophobicity and hydrogen bonding networks. These results contribute to the understanding of thermal protein adaptation in a redox-active mononuclear molybdenum enzyme and have implications in optimizing the design of low-temperature environmental biosensors.     

Genome-wide analysis of the Chinese sturgeon sox gene family: identification,characterisation and expression profiles of different tissues

The sox family is assumed to be responsible for a number of developmental systems. Genome sequencing technology makes it possible to scan sox genes and conduct characteristic analyses of different species. In fish, full characterisation of sox genes at the genome-wide level has been reported for pufferfish Takifugu rubripes, medaka Oryzias latipes, tilapia Oreochromis niloticus and channel catfish Ictalurus punctatus. However, no systematic investigation of the sox family in sturgeons (Acipenseridae) has been reported to date. This study conducted genome-wide identification of the sox genes in the Chinese sturgeon Acipenser sinensis and profiled their tissue distribution between male and female individuals. In total, 19 sox genes were identified, including soxb1, b2, c, d, e, f and h, in the Chinese sturgeon. Genomic structure analysis indicated relatively conserved exon–intron structures in each sox group and phylogenetic analysis supported the previous classification of the sox family. Most of the sox genes showed a tissue-specific expression pattern, indicating the possible involvement of Chinese sturgeon sox genes at different developmental processes such as cardiac and gonadal development. This study provides a comprehensive resource of Chinese sturgeon sox genes and enables a better understanding of the evolution and function of the sox family.     

Effects of inhibitors and NaCl on the oxidation of reduced inorganic sulfur compounds by a marine acidophilic, sulfur-oxidizing bacterium, Acidithiobacillus thiooxidans strain SH

The effect of NaCl and the pathways of the oxidation of reduced inorganic sulfur compounds were studied using resting cells and cell-free extracts of Acidithiobacillus thiooxidans strain SH. This isolate specifically requires NaCl for growth. The oxidation of sulfur and sulfite by resting cells was strongly inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide. Carbonylcyanide m-chlorophenyl-hydrazone and monensin were also relatively strong inhibitors. Thiosulfate-oxidizing activity was not inhibited by these uncouplers. Valinomycin did not inhibit the oxidation of sulfur compounds. NaCl stimulated the sulfur- and sulfite-oxidizing activities in resting cells but not in cell-free extracts. The tetrathionate-oxidizing activity in resting cells was slightly stimulated by NaCl, whereas it did not influence the thiosulfate-oxidizing activity. Sulfide oxidation was biphasic, suggesting the formation of intermediate sulfur. The initial phase of sulfide oxidation was not affected by NaCl, whereas the subsequent oxidation of sulfur in the second phase was Na⁺-dependent. A model is proposed for the role of NaCl in the metabolism of reduced sulfur compounds in A. thiooxidans strain SH.     

Insight into the molecular mechanism of the sulfur oxidation process by reverse sulfite reductase (rSiR) from sulfur oxidizer <Emphasis Type="Italic">Allochromatium vinosum</Emphasis>

Sulfur metabolism is one of the oldest known biochemical processes. Chemotrophic or phototrophic proteobacteria, through the dissimilatory pathway, use sulfate, sulfide, sulfite, thiosulfate or elementary sulfur by either reductive or oxidative mechanisms. During anoxygenic photosynthesis, anaerobic sulfur oxidizer Allochromatium vinosum forms sulfur globules that are further oxidized by dsr operon. One of the key redox enzymes in reductive or oxidative sulfur metabolic pathways is the DsrAB protein complex. However, there are practically no reports to elucidate the molecular mechanism of the sulfur oxidation process by the DsrAB protein complex from sulfur oxidizer Allochromatium vinosum. In the present context, we tried to analyze the structural details of the DsrAB protein complex from sulfur oxidizer Allochromatium vinosum by molecular dynamics simulations. The molecular dynamics simulation results revealed the various types of molecular interactions between DsrA and DsrB proteins during the formation of DsrAB protein complex. We, for the first time, predicted the mode of binding interactions between the co-factor and DsrAB protein complex from Allochromatium vinosum. We also compared the binding interfaces of DsrAB from sulfur oxidizer Allochromatium vinosum and sulfate reducer Desulfovibrio vulgaris. This study is the first to provide a comparative aspect of binding modes of sulfur oxidizer Allochromatium vinosum and sulfate reducer Desulfovibrio vulgaris.     

The effects of ATP and sodium chloride on the cytochrome c-cardiolipin interaction: the contrasting behavior of the horse heart and yeast proteins

In cells a portion of cytochrome c (cyt c) (15–20%) is tightly bound to cardiolipin (CL), one of the phospholipids constituting the mitochondrial membrane. The CL-bound protein, which has nonnative tertiary structure, altered heme pocket, and disrupted Fe(III)-M80 axial bond, is thought to play a role in the apoptotic process. This has attracted considerable interest in order to clarify the mechanisms governing the cyt c–CL interaction. Herein we have investigated the binding reaction of CL with the c-type cytochromes from horse heart and yeast. Although the two proteins possess a similar tertiary architecture, yeast cyt c displays lower stability and, contrary to the equine protein, it does not bind ATP and lacks pro-apoptotic activity. The study has been performed in the absence and in the presence of ATP and NaCl, two compounds that influence the (horse cyt c)-CL binding process and, thus, the pro-apoptotic activity of the protein. The two proteins behave differently: while CL interaction with horse cyt c is strongly influenced by the two effectors, no effect is observed for yeast cyt c. It is noteworthy that NaCl induces dissociation of the (horse cyt c)–CL complex but has no influence on that of yeast cyt c. The differences found for the two proteins highlight that specific structural factors, such as the different local structure conformation of the regions involved in the interactions with either CL or ATP, can significantly affect the behavior of cyt c in its reaction with liposomes and the subsequent pro-apoptotic action of the protein.