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5.10C: Oxidation of Reduced Sulfur Compounds - Biology

5.10C: Oxidation of Reduced Sulfur Compounds - Biology



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Sulfur oxidation involves the oxidation of reduced sulfur compounds, inorganic sulfur, and thiosulfate to form sulfuric acid.

Learning Objectives

  • Describe the process of sulfur oxidation

Key Points

  • The oxidation of sulfide occurs in stages, with inorganic sulfur being stored either inside or outside of the cell until needed.
  • This two step process occurs because sulfide is a better electron donor than inorganic sulfur or thiosulfate; this allows a greater number of protons to be translocated across the membrane.
  • Sulfur-oxidizing organisms generate reducing power for carbon dioxide fixation via the Calvin cycle using reverse electron flow—an energy-requiring process that pushes the electrons against their thermodynamic gradient to produce NADH.

Key Terms

  • calvin cycle: A series of biochemical reactions that take place in the stroma of chloroplasts in photosynthetic organisms.
  • thiosulfate: Any salt or ester of thiosulfuric acid.
  • chemolithoautotrophic: The characteristic of a microorganism that obtains energy from the oxidation of inorganic compounds and carbon from the fixation of carbon dioxide.

Sulfur is an essential element for all life, and it is widely used in biochemical processes. In metabolic reactions, sulfur compounds serve as both fuels and respiratory (oxygen-alternative) materials for simple organisms. Sulfur is an important part of many enzymes and antioxidant molecules such as glutathione and thioredoxin.

Sulfur Oxidation

Sulfur oxidation involves the oxidation of reduced sulfur compounds such as sulfide (H2S), inorganic sulfur (S0), and thiosulfate (S2O2−3) to form sulfuric acid (H2SO4). An example of a sulfur-oxidizing bacterium is Paracoccus.

Generally, the oxidation of sulfide occurs in stages, with inorganic sulfur being stored either inside or outside of the cell until needed. The two step process occurs because sulfide is a better electron donor than inorganic sulfur or thiosulfate; this allows a greater number of protons to be translocated across the membrane. Sulfur-oxidizing organisms generate reducing power for carbon dioxide fixation via the Calvin cycle using reverse electron flow—an energy-requiring process that pushes the electrons against their thermodynamic gradient to produce NADH. Biochemically, reduced sulfur compounds are converted to sulfite (SO2−3) and, subsequently, sulfate (SO2−4) by the enzyme sulfite oxidase. Some organisms, however, accomplish the same oxidation using a reversal of the APS reductase system used by sulfate-reducing bacteria (see above). In all cases the energy liberated is transferred to the electron transport chain for ATP and NADH production. In addition to aerobic sulfur oxidation, some organisms (e.g. Thiobacillus denitrificans) use nitrate (NO−3) as a terminal electron acceptor and therefore grow anaerobically.

Beggiatoa

A classic example of a sulfur-oxidizing bacterium is Beggiatoa, a microbe originally described by Sergei Winogradsky, one of the founders of environmental microbiology. Beggiatoa can be found in marine or freshwater environments. They can usually be found in habitats that have high levels of hydrogen sulfide. These environments include cold seeps, sulfur springs, sewage contaminated water, mud layers of lakes, and near deep hydrothermal vents. Beggiatoa can also be found in the rhizosphere of swamp plants. During his research in Anton de Bary’s laboratory of botany in 1887, Russian botanist Winogradsky found that Beggiatoa oxidized hydrogen sulfide (H2S) as an energy source, forming intracellular sulfur droplets. Winogradsky referred to this form of metabolism as inorgoxidation (oxidation of inorganic compounds). The finding represented the first discovery of lithotrophy.

Beggiatoa can grow chemoorgano-heterotrophically by oxidizing organic compounds to carbon dioxide in the presence of oxygen, though high concentrations of oxygen can be a limiting factor. Organic compounds are also the carbon source for biosynthesis. Some species may oxidize hydrogen sulfide to elemental sulfur as a supplemental source of energy (facultatively litho-heterotroph). This sulfur is stored intracellularly. Some species have the ability of chemolithoautotrophic growth, using sulfide oxidation for energy and carbon dioxide as a source of carbon for biosynthesis. In this metabolic process, internal stored nitrate is the electron acceptor and reduced to ammonia.

Sulfide oxidation: 2H2S + O2 → 2S + 2H2O

Marine autotrophic Beggiatoa species are able to oxidize intracellular sulfur to sulfate. The reduction of elemental sulfur frequently occurs when oxygen is lacking. Sulfur is reduced to sulfide at the cost of stored carbon or by added hydrogen gas. This may be a survival strategy to bridge periods without oxygen


Oxidation of Inorganic Sulfur Compounds by Obligately Organotrophic Bacteria

New data obtained by the author and other researchers on two different groups of obligately heterotrophic bacteria capable of inorganic sulfur oxidation are reviewed. Among culturable marine and (halo)alkaliphilic heterotrophs oxidizing sulfur compounds (thiosulfate and, much less actively, elemental sulfur and sulfide) incompletely to tetrathionate, representatives of the gammaproteobacteria, especially from the Halomonas group, dominate. Some denitrifying species from this group are able to carry out anaerobic oxidation of thiosulfate and sulfide using nitrogen oxides as electron acceptors. Despite the low energy output of the reaction of thiosulfate oxidation to tetrathionate, it can be utilized for ATP synthesis by some tetrathionate-producing heterotrophs however, this potential is not always realized during their growth. Another group of marine and (halo)alkaliphilic heterotrophic bacteria capable of complete oxidation of sulfur compounds to sulfate mostly includes representatives of the alphaproteobacteria which are most closely related to nonsulfur purple bacteria. They can oxidize sulfide (polysulfide), thiosulfate, and elemental sulfur via sulfite to sulfate but neither produce nor oxidize tetrathionate. All of the investigated sulfate-forming heterotrophic bacteria belong to lithoheterotrophs, being able to gain additional energy from the oxidation of sulfur compounds during heterotrophic growth on organic substrates. Some doubtful cases of heterotrophic sulfur oxidation described in the literature are also discussed.


Abstract

The feasibility of reusing wood ash as an inexpensive catalyst in a catalytic ozonation process has been demonstrated. Catalytic ozonation was demonstrated to oxidize H2S, methanethiol (MT), dimethyl sulfide (DMS), and dimethyl disulfide (DMDS) at low temperatures (23−25 °C). The process oxidized 25−50% of an inlet MT stream at 70 ppmv without the formation of DMDS (contrary to ash plus oxygen in air), oxidized 90−95% of an 85 ppmv stream of DMS, and oxidized 50% of a 100 ppmv DMDS stream using 2 g of wood ash at a space velocity of 720 h - 1 using ozone concentrations ranging from 100 to 300 ppmv. Similarly, 60−70% conversion of a 70 ppmv H2S stream was achieved with 2 g of ash in 1.1 s without catalytic deactivation (∼44 h). The overall oxidation rate of H2S, DMS, and DMDS increased with increasing ozone concentration contrary to the oxidation rate of MT, which was independent of ozone concentration. Dimethyl sulfoxide and dimethyl sulfone were identified as the primary end products of DMS oxidation, and SO2 was the end product of H2S and MT oxidation.

Corresponding author phone: (706)583-0155 fax: (706)542-8806 e-mail: [email protected]


Reduced inorganic sulfur oxidation supports autotrophic and mixotrophic growth of Magnetospirillum strain J10 and Magnetospirillum gryphiswaldense

N2 - Magnetotactic bacteria are present at the oxic-anoxic transition zone where opposing gradients of oxygen and reduced sulfur and iron exist. Growth of non-magnetotactic lithoautotrophic Magnetospirillum strain J10 and its close relative magnetotactic Magnetospirillum gryphiswaldense was characterized in microaerobic continuous culture. Both strains were able to grow in mixotrophic (acetate + sulfide) and autotrophic (sulfide or thiosulfate) conditions. Autotrophically growing cells completely converted sulfide or thiosulfate to sulfate and produced 7.5 g dry weight per mol substrate at a maximum observed growth rate of 0.09 h(-1) for strain J10 and 0.07 h(-1) for M. gryphiswaldense. The respiratory activity for acetate was repressed in autotrophic and also in mixotrophic cultures, suggesting acetate was used as C-source in the latter. We have estimated the proportions of substrate used for assimilatory processes and evaluated the biomass yields per mol dissimilated substrate. The yield for lithoheterotrophic growth using acetate as the C-source was approximately twice the autotrophic growth yield and very similar to the heterotrophic yield, showing the importance of reduced sulfur compounds for growth. In the draft genome sequence of M. gryphiswaldense homologues of genes encoding a partial sulfur-oxidizing (Sox) enzyme system and reverse dissimilatory sulfite reductase (Dsr) were identified, which may be involved in the oxidation of sulfide and thiosulfate. Magnetospirillum gryphiswaldense is the first freshwater magnetotactic species for which autotrophic growth is shown

AB - Magnetotactic bacteria are present at the oxic-anoxic transition zone where opposing gradients of oxygen and reduced sulfur and iron exist. Growth of non-magnetotactic lithoautotrophic Magnetospirillum strain J10 and its close relative magnetotactic Magnetospirillum gryphiswaldense was characterized in microaerobic continuous culture. Both strains were able to grow in mixotrophic (acetate + sulfide) and autotrophic (sulfide or thiosulfate) conditions. Autotrophically growing cells completely converted sulfide or thiosulfate to sulfate and produced 7.5 g dry weight per mol substrate at a maximum observed growth rate of 0.09 h(-1) for strain J10 and 0.07 h(-1) for M. gryphiswaldense. The respiratory activity for acetate was repressed in autotrophic and also in mixotrophic cultures, suggesting acetate was used as C-source in the latter. We have estimated the proportions of substrate used for assimilatory processes and evaluated the biomass yields per mol dissimilated substrate. The yield for lithoheterotrophic growth using acetate as the C-source was approximately twice the autotrophic growth yield and very similar to the heterotrophic yield, showing the importance of reduced sulfur compounds for growth. In the draft genome sequence of M. gryphiswaldense homologues of genes encoding a partial sulfur-oxidizing (Sox) enzyme system and reverse dissimilatory sulfite reductase (Dsr) were identified, which may be involved in the oxidation of sulfide and thiosulfate. Magnetospirillum gryphiswaldense is the first freshwater magnetotactic species for which autotrophic growth is shown


Iron Oxidation

Ferric iron is an anaerobic terminal electron acceptor, with the final enzyme a ferric iron reductase.

Learning Objectives

Outline the purpose of iron oxidation and the three types of ferrous iron-oxidizing microbes (acidophiles, microaerophiles and anaerobic photosynthetic bacteria)

Key Takeaways

Key Points

  • Ferrous iron is a soluble form of iron that is stable at extremely low pHs or under anaerobic conditions. Under aerobic, moderate pH conditions ferrous iron is oxidized spontaneously to the ferric (Fe3+) form and is hydrolyzed abiotically to insoluble ferric hydroxide (Fe(OH)3).
  • Three distinct types of ferrous iron-oxidizing microbes: acidophiles, microaerophiles that oxidize ferrous iron at cirum-neutral pH, anaerobic photosynthetic bacteria which use ferrous iron to produce NADH for autotrophic carbon dioxide fixation.
  • Although ferric iron is the most prevalent inorganic electron acceptor, a number of organisms can use other inorganic ions in anaerobic respiration.

Key Terms

  • autotroph: Any organism that can synthesize its food from inorganic substances, using heat or light as a source of energy.
  • heterotroph: An organism that requires an external supply of energy in the form of food as it cannot synthesize its own.

Ferric iron (Fe 3+ ) is a widespread anaerobic terminal electron acceptor both for autotrophic and heterotrophic organisms. Electron flow in these organisms is similar to those in electron transport, ending in oxygen or nitrate, except that in ferric iron-reducing organisms the final enzyme in this system is a ferric iron reductase. Model organisms include Shewanella putrefaciens and Geobacter metallireducens. Since some ferric iron-reducing bacteria (e.g. G. metallireducens) can use toxic hydrocarbons such as toluene as a carbon source, there is significant interest in using these organisms as bioremediation agents in ferric iron-rich contaminated aquifers.

Iron Bacteria: Common effects of excess iron in water are a reddish-brown color and stained laundry. Iron bacteria are a natural part of the environment in most parts of the world. These microorganisms combine dissolved iron or manganese with oxygen and use it to form rust-colored deposits. In the process, the bacteria produce a brown slime that builds up on well screens, pipes, and plumbing fixtures. Bacteria known to feed on iron are Thiobacillus ferrooxidans and Leptospirillum ferrooxidans.

Ferrous iron is a soluble form of iron that is stable at extremely low pHs or under anaerobic conditions. Under aerobic, moderate pH conditions ferrous iron is oxidized spontaneously to the ferric (Fe 3+ ) form and is hydrolyzed abiotically to insoluble ferric hydroxide (Fe(OH)3). There are three distinct types of ferrous iron-oxidizing microbes. The first are acidophiles, such as the bacteria Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans, as well as the archaeon Ferroplasma. These microbes oxidize iron in environments that have a very low pH and are important in acid mine drainage. The second type of microbes oxidizes ferrous iron at cirum-neutral pH. These micro-organisms (for example Gallionella ferruginea or Leptothrix ochracea) live at the oxic-anoxic interfaces and are microaerophiles. The third type of iron-oxidizing microbes is anaerobic photosynthetic bacteria such as Rhodopseudomonas, which use ferrous iron to produce NADH for autotrophic carbon dioxide fixation. Biochemically, aerobic iron oxidation is a very energetically poor process which therefore requires large amounts of iron to be oxidized by the enzyme rusticyanin to facilitate the formation of proton motive force. Like sulfur oxidation, reverse electron flow must be used to form the NADH used for carbon dioxide fixation via the Calvin cycle.

Although ferric iron is the most prevalent inorganic electron acceptor, a number of organisms (including the iron-reducing bacteria mentioned above) can use other inorganic ions in anaerobic respiration. While these processes may often be less significant ecologically, they are of considerable interest for bioremediation, especially when heavy metals or radionuclides are used as electron acceptors. Examples include:

  • Manganic ion (Mn 4+ ) reduction to manganous ion]] (Mn 2+ )
  • Selenate (SeO2 −4 ) reduction to selenite (SeO2 −3 ) and selenite reduction to inorganic selenium (Se0)
  • Arsenate (AsO3 −4 ) reduction to arsenite (AsO3 −3 )
  • Uranyl ion ion (UO2 +2 ) reduction to uranium dioxide (UO2)

5.10C: Oxidation of Reduced Sulfur Compounds - Biology

Aerobic respiration—the reduction of molecular oxygen (O 2 ) coupled to the oxidation of reduced compounds such as organic carbon, ferrous iron, reduced sulfur compounds, or molecular hydrogen while conserving energy to drive cellular processes—is the most widespread and bioenergetically favorable metabolism on Earth today. Aerobic respiration is essential for the development of complex multicellular life thus the presence of abundant O 2 is an important metric for planetary habitability. O 2 on Earth is supplied by oxygenic photosynthesis, but it is becoming more widely understood that abiotic processes may supply meaningful amounts of O 2 on other worlds. The modern atmosphere and rock record of Mars suggest a history of relatively high O 2 as a result of photochemical processes, potentially overlapping with the range of O 2 concentrations used by biology. Europa may have accumulated high O 2 concentrations in its subsurface ocean due to the radiolysis of water ice at its surface. Recent modeling efforts suggest that coexisting water and O 2 may be common on exoplanets, with confirmation from measurements of exoplanet atmospheres potentially coming soon. In all these cases, O 2 accumulates through abiotic processes—independent of water-oxidizing photosynthesis. We hypothesize that abiogenic O 2 may enhance the habitability of some planetary environments, allowing highly energetic aerobic respiration and potentially even the development of complex multicellular life which depends on it, without the need to first evolve oxygenic photosynthesis. This hypothesis is testable with further exploration and life-detection efforts on O 2 -rich worlds such as Mars and Europa, and comparison to O 2 -poor worlds such as Enceladus. This hypothesis further suggests a new dimension to planetary habitability: "Follow the Oxygen," in which environments with opportunities for energy-rich metabolisms such as aerobic respiration are preferentially targeted for investigation and life detection.


The TusA Protein

TusA (cd00291) is a highly conserved, widely distributed ∼8-kDa protein that may be found as a single protein or fused to other proteins such as cysteine desulfurases or rhodaneses 4, 24, 25 . TusA has been extensively studied both structurally and functionally 25, 26 . All TusA proteins contain a common Cys-Pro-X-Pro sequence motif in the N-terminal region with the sulfane sulfur-binding cysteine as the central element 4, 11, 25 . In E. coli, the motif contains a glutamate residue at position “X,” which is replaced by a hydrophobic residue (most often leucine, sometimes isoleucine or methionine) or glycine in the proteins from sulfur oxidizers 4 . Evidence is emerging that the nonconserved amino acid in the motif is an element decisive for interaction of TusA with its various possible protein partners 4, 24 . TusA alone is incapable of mobilizing sulfur from thiosulfate or low-molecular-weight organic persulfides like glutathione persulfide (GSSH ref. 4).

For E. coli, it is well established that TusA functions as a sulfur mediator for the synthesis of 2-thiouridine of the modified wobble base 5-methyl-aminomethyl-2-thiouridine [(mnm) 5 s 2 U] in tRNA 27 . Over the last years, several additional roles have been assigned to TusA. A tusA-deficient E. coli strain was found to form filamentous cells due to a severe impairment of FtsZ ring formation 28, 29 . Furthermore, TusA in conjunction with TusBCD and TusE appears to play a role in the maintenance of the intracellular redox state 30 , and it has been suggested that TusA is involved in sulfur transfer for the synthesis of molybdopterin and also in the balanced regulation of the availability of IscS to various biomolecules in E. coli 24 . Taken together, all these findings indicate a pleiotropic role for TusA in E. coli that involves specific interaction with several different protein partners 24 .

Bioinformatic analyses and transcriptomic profiling provided first hints that the role of TusA in bacteria other than E. coli is not limited to biosynthetic processes. In several sulfur oxidizers including Acidithiobacillus ferrooxidans, Metallosphaera sedula, and the purple sulfur bacterium Allochromatium vinosum, relative mRNA levels for tusA were significantly higher under sulfur-oxidizing conditions than in the absence of reduced sulfur compounds 22, 31-33 . An A. vinosum strain deficient of the rhd-tusA-dsrE2 genes was impaired in its ability to degrade zero-valent sulfur formed during the oxidation of sulfide and thiosulfate 4 . The eminently important role of TusA is further highlighted by the finding that TusA is among the most abundant proteins in A. vinosum cells grown photolithoautotrophically on reduced sulfur compounds (Fig. 2 ref. 34). In these cells, enzymes involved in carbon dioxide fixation (RubisCo, phosphoribulokinase), photosynthesis (light-harvesting complexes), and energy conservation (subunits of ATP synthase) are very abundant. Furthermore, enzymes of oxidative sulfur metabolism like flavocytochrome c sulfide dehydrogenase (FccAB), DsrA, and DsrC and components of the Sox system dominate among the highest ranking proteins. It is intriguing that even SoxYZ and DsrC, the sulfur substrate donor proteins in the Sox and Dsr systems, respectively, are outnumbered by TusA (Fig. 2).

The 150 most abundant proteins in A. vinosum grown with sulfur as electron donor. Proteins are ranked according to the ratio of their peptide spectrum matches and the number of amino acids thereby avoiding an underestimation of small and overestimation of large proteins. Proteins belonging to five functional groups were distinguished: energy conservation (blue) C-metabolism (green) gene expression, replication, and chaperones (gray) photosynthesis (red) and sulfur metabolism (yellow). Data taken from ref. 34.


Discussion

This study provides the first molecular evidence that diverse genes of sulfur energy metabolism are present and actively transcribed in the S. velum symbiont population under in situ conditions. The metatranscriptome contained Bacteria transcripts encoding genes of several primary sulfur oxidation pathways, including the reverse dissimilatory sulfite reductase (Dsr) complex for sulfur oxidation to sulfite, the adenosine-5 ′ -phosphate (APS) reductase pathway for sulfite oxidation, and the sulfur oxidation (Sox) pathway mediating thiosulfate oxidation. In total, the sulfur oxidation genes identified here (n = 28 Figure 4) represented 6.6% of the Bacteria protein-coding reads in the dataset (1 in 15 sequences). Several genes encoding enzymes for sulfide and thiosulfate oxidation, including the dsr genes and SAT (ATP sulfurylase), were among the top most abundant reference genes (i.e., unique accession numbers) detected in the study (Figure 3). Many of these were most closely related to homologs from known sulfur-oxidizing Gammaproteobacteria (Figures 1𠄳), in particular A. vinosum and Endoriftia persephone, the symbiont of the tubeworm Riftia pachyptila, consistent with the phylogenetic placement of the Solemya symbiont within this group (Eisen et al., 1992 Distel, 1998). Strikingly, a single reference gene, the previously characterized cbbL gene encoding the large subunit of the CO2 fixation enzyme RubisCO (ribulose 1,5-bisphosphate carboxylase–oxygenase) of the S. velum symbiont, accounted for over 4% of all protein-coding reads (Figure 3). The high abundance of transcripts for enzymes of sulfur compound oxidation and carbon fixation suggests that thioautotrophy dominated symbiont community metabolism at the time of collection in early winter.

Genes of the reverse Dsr pathway were among the most highly expressed in the symbiont metatranscriptome (Figures 3 and 4). In sulfate-reducing Bacteria and Archaea, dissimilatory sulfite reductase (DsrAB) mediates sulfite reduction to sulfide (Klein et al., 2001 Wagner et al., 2005). DsrAB homologs have been isolated from a wide variety of sulfur-oxidizing taxa (Loy et al., 2009), in which the enzymes are thought to operate in the oxidative direction in conjunction with a diverse set of accessory proteins, including the transmembrane complex DsrMKJOP, the sulfur shuttle molecules DsrEFH and DsrC, and the cytoplasmic flavoprotein DsrL (Dahl et al., 2005 Ghosh and Dam, 2009), all of which were detected in the Solemya symbiont transcriptome (Figure 4). It has been suggested that sulfide for oxidation by the DsrAB complex is released via the reduction of a polysulfur carrier molecule, as catalyzed by the NADH: (acceptor) oxidoreductase activity of DsrL (Dahl et al., 2005), which has been shown to be critical for sulfur oxidation in A. vinosum (Lubbe et al., 2006). In this species, the carrier molecule may be an organic perthiol involved with bringing stored elemental sulfur (sulfur globules) from the periplasm to the cytoplasm (Frigaard and Dahl, 2009). Stored sulfur globules in A. vinosum are produced during growth on sulfide by enzymes including FccAB and Sqr or via the activity of the Sox pathway during growth on thiosulfate (Frigaard and Dahl, 2009 Ghosh and Dam, 2009). Interestingly, the deposition of sulfur storage globules, while common in other thioautotrophic symbionts (e.g., Endoriftia persephone), has not been observed in Solemya, suggesting a potentially rapid turnover of elemental sulfur compounds by an active Dsr system. While the exact roles of Dsr enzymes remain to be confirmed in the Solemya symbiont, our data indicate a prominent role for sulfur oxidation to sulfite in this symbiosis.

Consistent with this finding, transcripts encoding the APS pathway for sulfite oxidation were abundant in the dataset. Use of the APS pathway by Solemya symbionts was established previously by enzyme assays showing the activity of APS reductase (AprAB) and ATP sulfurylase (SAT Chen et al., 1987). Together, these enzymes catalyze the AMP-dependent oxidation of sulfite to APS and the subsequent ATP-generating conversion of APS to sulfate (Figure 4). Here, AprAB and SAT-encoding transcripts represented a combined 1.5% of all protein-coding Bacteria reads. Additionally, transcripts encoding the transmembrane anchor AprM were detected at low abundance. In other Bacteria taxa, electron transport from the cytoplasmic APS reductase to the membrane quinol/quinone pool is mediated via interactions with either AprM, which is more common in Gammaproteobacterial sulfur-oxidizers (e.g., A. vinosum), or the functionally associated membrane-bound redox complex QmoABC, common in green sulfur bacteria of the Order Chlorobiales (Meyer and Kuever, 2007). Here, detection of aprM but not qmoABC transcripts is consistent with the placement of the Solemya symbiont among the Gammaproteobacteria. Together, these data suggest a functional sulfite oxidation complex in this symbiosis.

The detection of transcripts matching genes of the Sox pathway also confirmed an active role for thiosulfate oxidation in S. velum symbionts. The Sox (sulfur oxidation) pathway mediates the oxidation of thiosulfate to sulfate in a variety of both non-photosynthetic and photosynthetic sulfur-oxidizing bacteria (Frigaard and Dahl, 2009 Ghosh and Dam, 2009 Sakurai et al., 2010). The pathway has been extensively studied in the Alphaproteobacterium Paracoccus pantotrophus, in which 15 genes soxRSVWXYZABCDEFGH comprise the sox operon (Friedrich et al., 2001, 2005 Rother et al., 2001). Of these, seven genes (soxABCDXYZ) encode the core periplasmic sox proteins, SoxB, SoxXA, SoxYZ, and SoxCD, with SoxYZ acting as the primary carrier molecule at every step in the pathway (Friedrich et al., 2001 Figure 4). Analysis of the S. velum symbiont metatranscriptome revealed transcripts matching two peripheral sox genes (soxHW) at low abundance and five of the seven core sox genes (soxABXYZ) at higher abundances (Figure 4). Transcripts encoding the sulfur dehydrogenase SoxCD were not detected in our analysis, consistent with the absence of these genes from the genomes of a variety of green and purple sulfur bacteria, including A. vinosum and the endosymbionts of deep-sea clams and tubeworms (Frigaard and Dahl, 2009 Harada et al., 2009). Alternatively, it is possible that soxC and soxD are present in the symbiont genome, but were not transcribed (above the level of detection) at the time of collection. In organisms lacking soxCD, the sulfur atom of the sulfane intermediate (SoxYZ-S-SH, Figure 4) is transferred to other acceptor substrates (RSnH − and − HSn − in Figure 4) and eventually into sulfur storage globules, or enters other sulfur oxidation pathways (Sauve et al., 2007 Frigaard and Dahl, 2009). The deposition of elemental sulfur globules has not yet been demonstrated for S. velum. However, the potential for globule storage under varying concentrations of thiosulfate or sulfide has not yet been exhaustively explored.

Implications

Our metatranscriptome data indicates the use of diverse pathways for both sulfide and thiosulfate oxidation by S. velum symbionts under environmental conditions, consistent with prior experiments showing that both substrates stimulate symbiont carbon fixation (Cavanaugh, 1983 Scott and Cavanaugh, 2007). The utilization of multiple sulfur oxidation pathways is not unusual among thioautotrophic bacteria and is likely related to substrate availability (Ghosh and Dam, 2009). For the endosymbionts of deep-sea clams, which share a complement of sulfur oxidation genes similar to that in S. velum symbionts (Kuwahara et al., 2007 Newton et al., 2007), it has been argued that the ability to oxidize both thiosulfate (Sox system) and sulfide (Dsr, Sqr/Fcc) is linked to the relative abundance of these compounds in the clam microhabitat (basaltic cracks and fissures in zones of diffuse-flow venting Harada et al., 2009). In contrast, the endosymbiont of the tubeworm Riftia pachyptila putatively lacks the Sox pathway (Markert et al., 2007 Robidart et al., 2008), which may reflect an adaptation to a greater reliance on sulfide in the zones surrounding black smokers where this symbiosis occurs (Nelson and Fisher, 1995 Harada et al., 2009).

For S. velum, the relative abundance of reduced sulfur species available for symbiont oxidation has not been directly characterized. The host bivalve lives in coastal muds, positioning itself at the convergence of a Y-shaped burrow (Levinton, 1977) where it can access anoxic water from lower in the sediment. This water is likely enriched in sulfide but may also contain thiosulfate, perhaps resulting from chemical sulfide oxidation during burrow ventilation (Howarth et al., 1983 Elsgaard and Jorgensen, 1992). Alternatively, thiosulfate may also be available via sulfide oxidation by host mitochondria, as has been demonstrated in the Pacific congener S. reidi (Obrien and Vetter, 1990). Though both sulfide and thiosulfate can fuel symbiont autotrophy, sulfide has a much stronger effect (sevenfold) on carbon fixation rates in S. velum symbionts (Scott and Cavanaugh, 2007), suggesting that the symbiosis is adapted to use sulfide most efficiently.

Our transcriptome data, showing the relative abundance of transcripts in the Dsr and Sqr/Fcc pathways (Figure 4), potentially indicates a dominant role for sulfide metabolism in the S. velum symbiosis. However, transcript abundance may not be tightly coupled to protein abundance or enzyme activity (Taniguchi et al., 2010) and should be interpreted as a proxy for metabolic activity without supporting biochemical data. In other endosymbioses, namely those involving deep-sea hydrothermal vent clams, diverse sulfur pathways (Sox, Dsr, APS) have been shown to be constitutively expressed under variable environmental conditions (e.g., oxygen concentration (Harada et al., 2009). However, compared to gene expression in the relatively stable environment of the deep sea, expression of S. velum symbiont genes, and the relative reliance on diverse reduced sulfur sources, may be more stochastic given the potential for diel and seasonal variation in the coastal environment.

Metatranscriptomics can now provide a comprehensive molecular framework for interpreting physiochemical measurements of metabolism in uncultured symbionts. Such analyses can be easily integrated into studies exploring the links between sulfur availability, environmental conditions, and symbiont gene expression. Our study confirms that genes of sulfur energy metabolism are a dominant component of the transcriptionally active symbiont gene pool in S. velum. Future experiments will help determine the extent to which genes of thioautotrophy co-vary with other metabolic pathways in the transcriptome (Dmytrenko et al., in preparation) and with the biochemical transformations of sulfur mediated by this model symbiosis.


Author information

These authors contributed equally: Kristopher Kieft, Zhichao Zhou.

Affiliations

Department of Bacteriology, University of Wisconsin–Madison, Madison, WI, USA

Kristopher Kieft, Zhichao Zhou & Karthik Anantharaman

Biology Department, Carleton College, Northfield, MN, USA

Department of Microbiology, University of Tennessee, Knoxville, TN, USA

Department of Biological Sciences, Life Science Facility, Clemson University, Clemson, SC, USA

Department of Microbiology & Immunology, University of British Columbia, Vancouver, BC, Canada

Graduate Program in Bioinformatics, University of British Columbia, Genome Sciences Centre, Vancouver, BC, Canada

Genome Science and Technology Program, University of British Columbia, Vancouver, BC, Canada

Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada

ECOSCOPE Training Program, University of British Columbia, Vancouver, BC, Canada

Department of Animal Science, University of California Davis, Davis, CA, USA

Department of Microbiology, The Ohio State University, Columbus, OH, USA

Groupe de recherche interuniversitaire en limnologie, Department of Biology, Concordia University, Montréal, QC, Canada

DOE Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

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Contributions

K.K., Z.Z., S.R., and K.A. designed the study. K.K. and S.R. identified the genomes. K.K., Z.Z., and K.A. conducted the analyses. K.K., Z.Z., and K.A. drafted the manuscript. All authors (K.K., Z.Z., R.E.A., A.B., B.J.C., S.J.H., M.H., M.B.S., D.A.W., S.R., and K.A.) reviewed the results, revised, and approved the manuscript.

Corresponding author


Results and discussion

The architecture of the SoxAX complex

The crystal structure of the SoxAX heterodimer with four molecules in the asymmetric unit of a P21 cell was solved at 2.5 Å resolution by Fe-multiple wavelength anomalous dispersion (MAD). The structure subsequently was refined at 1.75 Å resolution using data collected from a crystal of space group P21212 with a single molecular copy in the asymmetric unit (Table I). The heterodimer seen in the crystal corresponds to the oligomeric state observed in solution by analytical ultracentrifugation studies ( Appia-Ayme et al., 2001 ). SoxAX is an elongated molecule of approximate dimensions 34 × 36 × 75 Å 3 (Figure 3A and B). The SoxA subunit is organized into two domains each binding a c-type haem with histidine–cysteine axial coordination. These are designated haems 1 and 2, labelled in the order in which the corresponding CXXCH ligand motifs appear in the SoxA sequence. Post-translational modification of the cysteine ligand to haem 2 is apparent and is discussed further below. The single domain SoxX subunit binds a c-type haem with histidine–methionine axial ligands (haem 3) and forms the interface with the C-terminal domain of SoxA. The haem ligands observed in the structure are in agreement with those inferred from previous spectroscopic characterization of oxidized protein samples ( Cheesman et al., 2001 ). The geometrical arrangement of the haem groups is presented in Table II. The use of cysteinate as a haem iron ligand is unusual, with only cytochromes P450, the CO sensor protein CooA and cystathionine β-synthase previously having been reported to have such a ligand ( Ojha et al., 2000 ). Indeed, SoxA is the first c-type cytochrome with cysteine haem iron coordination and only the second structurally characterized haemoprotein containing a haem with a cysteine–histidine ligand set, cystathionine β-synthase being the other ( Meier et al., 2001 ).

Data collection MAD data collection space group P21a = 81.2 Åb = 102.9 Åc = 114.4 Åβ = 110.5° Oxidized, space group P21212 a = 66.4 Å b = 87.3 Å c = 71.7Å Reduced, space group P21212 a = 65.8 Å b = 87.2 Å c = 71.5 Å
λ1 = 1.729 Å λ2 = 1.741 Å λ3 = 1.000 Å λ = 0.933 Å λ = 0.98 Å
Resolution (Å) 2.5 2.5 2.55 1.75 1.5
Completeness (%) 95.0 (92.2) 94.8 (89.6) 98.2 (88.7) 93.4 (91.4) 99.3 (98.4)
Rsym a a Rsym = ΣΣ|Ii − <I>|/Ii, where <I> is the average of symmetry equivalent reflections and the summation extends over all observations for all unique reflections.
(%)
4.9 (9.1) 5.1 (10.4) 3.1 (5.0) 7.0 (16.8) 4.1 (28.4)
Ranom b b Ranom = Σ|<I+> − <I−>|/Σ|<I+> + <I−>|.
(%)
4.4 (7.1) 3.5 (8.7) 1.8 (5.4)
(<I>/<σI>) >3 (%) 92.9 (82.4) 90.6 (73.9) 96.6 (89.2) 78.7 (44.2) 78.6 (48.4)
Independent reflections 61 072 61 950 58 706 42 647 66 141
Total reflections 903 590 901 411 101 074 262 534 670 264
Overall temperature factor (Å 2 ) 34.7 36.7 36.7 17.8 13.2
SOLVE mean FOM c c Average figure of merit (FOM) calculated by SOLVE ( Terwilliger and Berendzen, 1999 ).
0.74
Refinement statistics P21 P21212 oxidized P21212 reduced
Wavelength (Å) 1.000 0.933 0.98
Resolution range (Å) 20–2.55 20–1.75 25–1.5
Refined structure d d The final structural model for the P21 crystal form contained four copies of the indicated residues of SoxA and SoxX in the asymmetric unit
SoxA 1–261, SoxX 1–138, + 691 water SoxA 1–261, SoxX 1–51, 55–137, + 353 water SoxA 1–261, SoxX 1–51, 55–137, + 667 water, + 2 ethylene glycol
Rcryst e e Rcryst = Σ‖F| − |Fc‖/Σ|F|.
21.0 (29.6) 17.7 (21.3) 19.9 (23.5)
Rfree f f For Rfree, the summations extends over a subset (5%) of reflections excluded from all stages of refinement.
26.4 (36.8) 21.4 (23.0) 23.0 (27.4)
C–N 0.005 0.009 0.005
N–Cα 0.009 0.014 0.008
Cα–C 0.012 0.016 0.009
N–Cα–C 0.88 1.08 1.36
C–N–Cα 1.30 1.77 2.16
Main chain atoms 18.5 16.5 14.4
Side chain atoms 19.1 21.9 17.3
  • Figures in parentheses refer to data in the highest resolution bin.
  • a Rsym = ΣΣ|Ii − <I>|/Ii, where <I> is the average of symmetry equivalent reflections and the summation extends over all observations for all unique reflections.
  • b Ranom = Σ|<I+> − <I−>|/Σ|<I+> + <I−>|.
  • c Average figure of merit (FOM) calculated by SOLVE ( Terwilliger and Berendzen, 1999 ).
  • d The final structural model for the P21 crystal form contained four copies of the indicated residues of SoxA and SoxX in the asymmetric unit
  • e Rcryst = Σ‖F| − |Fc‖/Σ|F|.
  • f For Rfree, the summations extends over a subset (5%) of reflections excluded from all stages of refinement.
Haems 1–2 Haems 2–3
Distance (Fe–Fe) (Å) 31.4 (31.0) 19.0 (18.8)
Distance (closest approach) (Å) 23.9 (23.4) 10.7 (10.6)
Interplanar angle (°) 56.1 (55.1) 80.95 (78.3)
Haem 1 Haem 2 Haem 3
Ligand distances (Å)
Fe–S 2.45 (2.37) 2.41 (2.35) 2.34 (2.43)
Fe–N 2.08 (2.11) 2.08 (2.08) 2.05 (2.07)
Haem solvent accessibility (Å 2 ) 179.2 (179.7) 56.7 (53.3) 80.8 (82.9)
  • The figures in parentheses refer to the average calculated over the four independent molecules of the P21 cell.

The haem 2–haem 3 interplanar angle and distance of closest approach together with the haem solvent-accessible areas are consistent with haem 3 being the route for electron transfer away from the complex (Table II). The solvent-exposed edge of haem 3, the presumed locus for cytochrome c2 binding, lies on the opposite face of SoxX from haem 2. The edge–edge separation of haems 1 and 2, in contrast to that between haems 2 and 3, is sufficiently long to preclude efficient electron transfer in the absence of a chain of cofactors ( Page et al., 1999 ). An extended groove runs across the molecule at the subunit interface. This leads to a solvent-filled channel lined with basic residues, at the bottom of which the haem 2 iron atom and modified cysteine ligand lie exposed to solvent (Figure 3C). These observations, together with the post-translational modification to haem 2, led us to identify this haem as the catalytic active site.

The folds of the SoxA and SoxX subunits

The SoxX subunit has a c-type cytochrome fold as defined in the SCOP database ( Holm and Sander, 1993 ). Yeast mitochondrial cytochrome c (PDB entry 1ycc, 108 residues) was found to be most similar, with 82 structurally aligned residues and an r.m.s.d. of 2.3 Å. The SoxX subunit can thus be classified as a member of the mono-domain cytochrome c family ( Murzin et al., 1995 ). Inspection of the SoxA subunit reveals a pronounced internal pseudo-dyad encompassing residues 51–150 and 151–250 for domains that we term SoxAN and SoxAC, respectively. Alignment of these domains gave an overall r.m.s.d. of 1.8 Å for 93 structurally conserved residues for which the percentage residue identity was 18%. The haem groups in each domain are also well conserved, having an r.m.s.d. of 1.2 Å in this alignment (excluding propionate groups). The folds of these domains are clearly highly homologous and, as was the case for SoxX, each of these A-subunit domains has a c-type cytochrome fold. Again taking the mitochondrial cytochrome c for comparison, the r.m.s.ds are 2.7 and 3.1 Å for the SoxAN and SoxAC domains (both 69 aligned residues), respectively. The structure, in conjunction with sequence alignment, suggests that SoxA, apart from the N-terminal 50 amino acids, has evolved from duplication of a cytochrome c gene.

The di-haem SoxA subunit

Like SoxA, proteins of the two-domain cytochrome c family possess a pseudo 2-fold symmetry axis relating the two haem domains ( Murzin et al., 1995 ). However, fold homology searches failed to align the SoxA subunit with members of this family ( Chen et al., 1994 Fülöp et al., 1995 Kadziola and Larsen, 1997 ). As such, SoxA represents the first example of a new family of two-domain cytochromes c in which the packing of the two domains differs radically from the previously characterized examples (Figure 3D). The novel domain arrangement seen in SoxA may arise because a combination of the C-terminal (residues 251–261) and N-terminal polypeptides (residues 1–50) of SoxA block the face of the C-terminal domain that forms the domain interface in the other two-domain cytochrome c proteins. The result is that in SoxA the two domains pack with their C-terminal α4 helices arranged about the local pseudo 2-fold axis relating the SoxAN and SoxAC domains. A major consequence of this unusual packing is to give a distance of closest approach of the haems in SoxA (24 Å) that is much greater than that seen in the other two-domain cytochrome c proteins (<14 Å). The result is that effective electron transfer between the SoxA haems is precluded.

It is notable that the haem 1 consensus Cys-Xaa-Xaa-Cys-His haem-binding motif is absent from the SoxA proteins of Rhodopseudomonas palustris and of Chlorobium species (Figure 2A). In place of haem 1, an intradomain disulfide bridge is found between the remaining (second) cysteine of the haem-binding motif and the conserved cysteine residue that is a ligand to that haem in R.sulfidophilum SoxA ( Klarskov et al., 1998 ). However, the α-carbon atoms of these cysteines in the SoxAN domain are >10 Å apart, a distance greater than the maximum for a disulfide bridge ( Sowdhamini et al., 1989 ). The question then naturally arises as to whether, nevertheless, the AN domain of the R.palustris and Chlorobium sp. SoxA proteins can adopt the cytochrome c fold but substitute a disulfide bridge for the c haem to form a different superfamily. An important observation in this respect is that the haem-less SoxA domains preserve the cluster of hydrophobic residues at positions A61 (6), A65 (10), A134 (94) and A137 (97) (tuna cytochrome c residue numbering in parentheses see Figure 2A) in the N- and C-terminal helices of the fold. These four residues are not involved in haem binding and are conserved in otherwise highly divergent cytochromes c. This, together with the absence of an apparent functional role for these residues, has led to the suggestion that they are involved in a common folding nucleus of all subfamilies of c-type cytochromes ( Ptitsyn, 1998 ). It therefore appears reasonable to conclude that the haem-less SoxAN domain in these proteins may adopt a modified cytochrome c fold.

The nature of the subunit interface

The SoxAX complex is purified readily from the periplasm of R.sulfidophilum, suggesting a subunit interaction that is both specific and of relatively high affinity ( Appia-Ayme et al., 2001 ). At present, the complex of yeast cytochrome bc1 with cytochrome c ( Lange and Hunte, 2002 ) is the sole example of a crystal structure of a productive electron transfer complex involving two cytochrome c proteins. Furthermore, there are few examples of stable, structurally characterized homo-oligomeric proteins with the cytochrome c fold. Under these circumstances, the nature of the subunit interface in the SoxAX complex is of some interest. In an attempt to determine whether the SoxAC: SoxX packing is structurally unique, the PQS server ( Henrick and Thornton, 1998 ) was used to generate an independent set of stable cytochrome c dimer packings. Adding to these the domain packings observed in the two-domain cytochromes c, a total of 11 independent cytochrome c:cytochrome c domain packings were found. Comparison reveals that the SoxAC:SoxX domain packing bears a passing resemblance to that observed between monomers of the cytochrome c6 from the green alga Chlamydomonas reinhardtii although the relative orientation of the haems is significantly different. It is appropriate to note that there is some evidence that the dimerization of the latter has functional significance ( Kerfeld et al., 1995 ). Notably, there is no similarity between the packing of cytochrome c domains at the SoxAC:SoxX interface and that observed in the yeast cytochrome bc1:cytochrome c complex ( Lange and Hunte, 2002 ). Thus, the packings of the c-type cytochrome domains both within SoxA and at the subunit interface are essentially unique. Cytochrome c domains are limited in their packing arrangements if the haem groups are to fall within suitable minimum edge–edge distances for electron transfer without the requirement for other cofactors. This requirement is clearly illustrated in the packing of domains within the SoxA subunit.

Analysis of the high resolution structure reveals a subunit interface that is well packed. This is evidenced by the shape complementarity statistic (Sc) ( Lawrence and Colman, 1993 ), which has a value of 0.74 and is consistent with previously compiled statistics on permanent heterodimeric complexes ( Jones and Thornton, 1996 ). Despite being well packed, the interface is also apparently fairly plastic. The largest r.m.s.d. between pairs of high and low resolution structures of the complex is ∼0.6 Å (based on all α-carbon atoms), and this difference can be accounted for in the most part by a rigid body screw domain displacement of SoxX relative to SoxAC of 4° and a translation of 0.2 Å. As is often the case, the root cause of this difference in the SoxAX structures may be attributable to crystal lattice packing contacts. However, whilst the haem edge–edge distances and interplanar angles are not affected greatly by the excursions of the SoxX subunit relative to SoxA (Table II), the observed plasticity at least suggests that conformational changes may occur on binding of SoxYZ. The increased mobility of SoxX is also reflected in the average atomic temperature factor for atoms in this subunit (25.5 Å 2 ) being greater than that for the SoxA subunit (15.1 Å 2 ).

The interface of the complex is predominantly hydrophobic in nature, in keeping with that observed in other stable electron transfer complexes ( Mathews and White, 1993 ). Approximately 2500 Å 2 is buried on complex formation (6.8% of the total accessible surface area), of which apolar and aromatic residues contribute 42%. This buried area is consistent with that observed in other heterodimeric complexes ( Jones and Thornton, 1996 ). The interface is composed of two distinct, non-overlapping contact regions which we label A and B. Four short polypeptide stretches in SoxA form the majority of the interaction surface with SoxX (Figure 2A). Those contributing to region A are 183–184 and 187–191, whilst 168–175 and 224–232 contribute to region B. In SoxX, a number of short polypeptides are involved in forming the interface: residues 37–44 and 55–60 (region A), and residues 89–93 (region B) (Figure 2B). These three polypeptides contribute ∼50% of the total buried surface area of the SoxX subunit in the complex. The remainder comes from residues 101–110 (region B) which form part of a loop extending from the core domain structure to form an intimate association with residues of SoxA. Notably, this interface loop (residues 99–118) constitutes a major insertion in SoxX relative to other members of the cytochrome c superfamily. Interestingly, this loop is also missing in the SoxX proteins from Chlorobium tepidum, Aquifex aeolicus and R.palustris (Figure 2B). In addition, the overall sequence homology of the other contact peptides in SoxX relative to the corresponding regions in the loop-less proteins appears to be low (Figure 2B) and this may have important consequences in terms of the stability of their complexes. It should be noted that the absence of a permanent SoxAX complex in these organisms may imply generation of a means of stabilizing one-electron-oxidized intermediates.

A single midpoint potential using NADH as reductant has been observed for R.sulfidophilum SoxAX with a value of +180 mV that has been assigned to two redox-linked haems ( Little, 2000 ). The packing of SoxAC to SoxX at the subunit interface provides a haem–haem distance of closest approach of 10.7 Å. This distance is marginally shorter than that seen in the di-haem cytochrome c peroxidase (14.5 Å) and the cytochrome c4 protein (11.2 Å) and involves transversing a region of the interface predicted to be solvent accessible. However, electron transfer should be rapid and essentially independent of the environment between the haems for a separation of 14 Å or less ( Page et al., 1999 ). We therefore assume that haems 2 and 3 constitute the redox-linked pair. The haem groups arranged closest by the SoxAC:SoxX packing are the C-pyrrole rings. Several lines of evidence have implicated the exposed edge of the haem pyrrole ring C in electron transfer in c-type cytochromes ( Pelletier and Kraut, 1992 ).

Post-translational modification to catalytic haem ligand

Fourier maps calculated using a fully refined structural model based on the predicted amino acid sequence of SoxA reveal regions of unexplained difference electron density adjacent to the cysteine axial haem ligand to haem 2 in the vicinity of its terminal sulfur atom. The |FoFc| difference electron density peaks are at >5.2σ and 11.5σ from the mean electron density calculated over the volume of the molecule (where σ is the standard deviation of the distribution), indicating the presence of at least one additional atom (Figure 4A). The refined cysteine sulfur to ferric haem iron distance is 2.57 Å in this model. Given the likely geometry of the modified amino acid as indicated by the electron density, cysteine persulfide and cysteine sulfenic acid are the most probable derivatives. There is precedent for both modifications in other protein crystal structures ( Claiborne et al., 1999 Bordo et al., 2000 ). We attempted to refine both possibilities against our X-ray data with the result that both the cysteine persulfide sulfane sulfur to haem iron distance and the alternative cysteine sulfenic acid terminal oxygen to haem iron distance refined to 2.41 Å. However, whilst the cysteine persulfide form showed only minor residual difference electron density after the modification, the cysteine sulfenic acid residue showed a +7.0σ difference electron density peak in the vicinity of the sulfenic oxygen atom. This strongly suggests that cysteine persulfide is the post-translational modification observed for CysA222. Unfortunately, anomalous difference Fourier maps calculated using the iron K-edge wavelength data set (λ1 in Table I), which could positively identify a terminal sulfane sulfur atom on this residue, were inconclusive due to the proximity of the additional atom to the iron centre. So, in the absence of an unambiguous sulfur anomalous scattering signal, further supportive evidence was sought for this interpretation.