Periplasmic sulfide and thiosulfate oxidation

In the periplasm of the S. velum symbiont, sulfide, thiosulfate, and, possibly, elemental sulfur, may be oxidized for energy by the Sox system, which is represented in the genome (Figure 4). The encoded SoxYZAXB, flavocytochrome c dehydrogenase (FccAB), and type I and IV sulfide-quinone reductases (Sqr) potentially reduce cytochromes c and quinones, which along the course of the electron-transport chain translate into membrane-ion gradients, NADH, and ATP, ultimately fueling biosynthetic and other energy-requiring cellular processes, including autotrophy (Figure 1). In A. vinosum and the green non-sulfur bacterium, Chlorobium tepidum, SoxYZ, SoxAX, and SoxB proteins participate in the formation of transient sulfur deposits as intermediates during sulfur oxidation [43]. In fact, sulfur deposits are common to all known sulfur-oxidizing bacteria (SOB) which, like the S. velum symbiont, lack SoxCD sulfur dehydrogenase (Figure 4) [44], including the symbionts of the hydrothermal vent tubeworm, R. pachyptila[31, 45], and the clam, C. magnifica[22, 46]. Microscopically-detectable intracellular or extracellular sulfur has not been observed either in the symbiont-containing gills of S. velum or directly within the symbionts (Cavanaugh, unpublished observation). Absence of sulfur deposits may be attributed to a very rapid consumption of any available reduced sulfur substrate. This agrees with the fact that the S. velum symbiont have the highest known carbon fixation rate, and, hence, demand for energy, of all the studied chemosynthetic symbionts, i.e., 65 μmol min^-1 g of protein^-1[13] compared to 0.45 μmol min^-1 g of protein^-1 of the next highest rate measured in the symbionts of R. pachyptila[47]. Alternatively, in the S. velum symbiont intermediate sulfur may be stored in a chemical form that is not easily observed microscopically.

Cytoplasmic sulfide oxidation

Energy generating oxidation of sulfide to sulfite may be catalyzed in the cytoplasm of the S. velum symbiont by the reverse-acting dissimilatory sulfite reductase (rDsr) pathway (Figure 1). All of the enzymes and accessory proteins required for this pathway are encoded in a dsrABEFHCMKLJOPNRS operon (Figure 4). While multiple homologues of dsrC were identified outside the dsr operon, these genes did not encode the two conserved C-terminal cysteines required for the protein to function [48, 49]. The DsrC enzyme likely mediates transfer of electrons from sulfide reductase, DsrAB, to a transmembrane electron transport complex DsrKMJOP, an entry point for electrons derived from cytoplasmic oxidation of sulfur into the electron transport chain [50]. rDsr may be the key energy-generating pathway in the symbiont, as sulfide has a six-fold higher effect on carbon fixation in the S. velum symbiosis [13] compared to thiosulfate oxidized by the Sox pathway.

Sulfite oxidation

Sulfite generated by rDsr may be further oxidized to sulfate in the cytoplasm by a sequential action of APS reductase (AprABM) and an ATP-generating ATP sulfurylase (Sat) (Figures 1 and 4). Identification of the respective genes agrees with measured Apr and Sat activity in the symbiont-containing S. velum tissue [51]. Sulfate generated in this pathway may be exported from the cytoplasm via a sulfate-bicarbonate antiporter SulP (Figure 1). While electrons obtained from the oxidation of sulfide, thiosulfate, and, possibly, elemental sulfur by Sox and rDsr are shuttled into the electron transport chain, energy obtained from the oxidation of sulfite is immediately available in the form of ATP.

Rnf complexes

The versatile electron transport chain of the S. velum symbiont may utilize electron donors like ferrodoxins, which have a redox potential as negative as -500 mV [52], compared, for example, to -400 mV of S₂O₃^2- and -270 mV of H₂S. The reversible oxidation of ferrodoxins coupled to the reduction of NAD⁺ in the S. velum symbiont may be catalyzed by the H⁺ or/and Na⁺-motive Rnf complexes (Figure 1) encoded in the genome by two complete rnfABCDGE (rnf1) and rnfBCDGEA (rnf2) operons. The organization of these genes in the operons is conserved with other bacteria, suggesting that these clusters did not arise from duplication. Previously, only Axotobacter vinelandii and Desulfobacterium autotrophicum were known to harbor two rnf operons [52]. Based on the presence of genes for pyruvate:ferredoxin oxidoreductase located between rnfB2 and rnfC2, pyruvate may serve as an electron donor for at least one of the Rnf complexes. In general, rnf genes are distributed mainly among obligate and facultative anaerobes, including many pathogens that colonize oxygen-limited host tissues [52]. Together with the fact that ferrodoxins play a key role in anaerobic metabolism [53], this suggests that the S. velum symbiont, as well as other sequenced chemosynthetic symbionts, which all contain rnf genes, may be capable of facultative anaerobiosis.

Hydrogenases

Hydrogen is another highly electron negative reductant (-420 mV) that the S. velum symbiont may harness for the reduction of the quinone and the NAD⁺ cellular pools (Figure 1). Hydrogen oxidation is suggested by the presence in the symbiont genome of hup and hox2 operons encoding an uptake and a bidirectional hydrogenase, respectively. The two subunits of the symbiont [Ni-Fe]-uptake hydrogenase, HupSL, are most similar in amino acid sequence to HupS and HupL proteins from the symbionts of the tubeworms, R. pachyptila and the T. jerichona, (73% and 78% identity for the S and L subunits respectively), the sulfur bacterium, Thiocapsa roseopersicina, (68 and 74%), and the symbionts of the scaly-foot snail, C. squamiferum, (50 and 53%). In T. roseopersicina, HupSL has been experimentally demonstrated to reduce quinones of the respiratory chain with H₂[54, 55]. Unlike all the other γ-proteobacteria containing HupSL, the hup operon in the S. velum symbiont does not encode the di-heme cytochrome b, which is necessary to link H₂ oxidation to quinone reduction in the cellular membrane [56]. However, a [Ni-Fe] hydrogenase cytochrome b homolog was found on a different genomic scaffold. Though this discordant gene organization is unlike that in other H₂ oxidizers, it is possible that the identified cytochrome b may act in tandem with HupSL to enable H₂ oxidation.

Apart from potentially reducing the respiratory quinone pool with H₂, the symbiont, by means of a bidirectional hydrogenase, may produce H₂ by oxidizing NAD⁺. The S. velum symbiont Hox2FUYH enzyme complex is most similar in amino acid sequence (63-66%) to the recently-characterized NAD⁺-reducing [Ni-Fe]-hydrogenase from T. roseopersicina, which can operate in reverse, generating H₂ when the high reduction state of the dinucleotide pool is growth-limiting [57]. As H₂ concentrations available to the S. velum symbiont have not been measured, it is unknown whether the H₂ oxidation contributes to primary production to the degree that has been recently demonstrated in a hydrothermal vent symbiosis [58].

Primary ion pumps

NADH (-320 mV), potentially derived from oxidation of H₂ or heterotrophic metabolism (see Heterotrophy) in the S. velum symbiont, could be converted into an electrochemical gradient by two NADH:quinone oxidoreductases. The genome of the symbiont encodes the conventional H⁺-translocating quinone-reducing NADH dehydrogenase (NdhABCDEFGHIJKLMN), a homolog of the mitochondrial Complex I, as well as an alternative Na⁺-translocating NADH dehydrogenase (NqrABCDEF) (Figure 1). While Complex I is ubiquitous in bacteria, Nqr is found mainly in pathogenic and marine species [59]. Among symbiotic bacteria, nqr genes have so far been described only in Buchnera spp., an obligate endosymbiont of aphids [60]. The S. velum symbiont may be able to switch between Complex I and Nqr, preferentially generating either H⁺ or Na⁺ electrochemical gradients. Thus, depending on the cellular requirements, the symbiont may synthesize ATP (see ATP synthases) and regulate pH (see Ion gradient driven transporters) independently from each other.

Quinone reductases

Apart from the electron donors such as sulfur and NADH, the S. velum symbiont may be able to directly reduce its quinone pool with a number of other substrates. This is suggested by the presence of genes encoding malate:quinone oxidoreductase (Mqo), succinate dehydrogenase (ShdCDAB), homologous to Complex II in mitochondria, and formate dehydrogenase-O (FdoGHI) (Figure 1). This is the first report of FdoGHI in a chemosynthetic symbiont genome. In E. coli this enzyme, which is common to facultative anaerobes [61], is used in formate-dependent oxygen respiration, allowing the bacteria to rapidly adapt to shifts from aerobiosis to anaerobiosis [62]. The presence of FdoGHI is additional evidence that the S. velum symbiont may be capable of facultative anaerobiosis (see Rnf complexes).

The genome-encoded quinol-cytochrome-c oxidoreductase (bc ₁ , Complex III homologue) potentially links oxidation of quinols to the generation of a proton membrane gradient and the reduction of terminal electron acceptors (Figure 1), discussed next.

Terminal oxygen reductases

Similar to most aerobic and microaerophilic bacteria, the genome of the S. velum symbiont encodes three types of H⁺-motive terminal oxygen reductases (Figure 1), which suggest a capacity to respire O₂ over a wide range of concentrations. The genome contains a ccoNOQP operon encoding a cbb ₃ cytochrome oxidase, which is known to function at nanomolar O₂ concentrations in the nitrogen-fixing plant symbionts, Bradyrhizobium japonicum[63], and in the microaerophilic human pathogens, Campylobacter jejuni, Helicobacter pylori, and Neisseria meningitidis[64]. The genome also encodes a aa ₃ cytochrome oxidase (CoxAB), which is thought to function primarily under atmospheric oxygen concentrations [65] and is the only terminal oxidase in the symbionts of the bivalves C. magnifica[22] and C. okutanii[20]. The third terminal oxidase identified in the symbiont genome is a cydAB-encoded quinol oxidase, which is thought to oxidize quinols instead of cytochromes. CydAB may operate when an excess of reductants, potentially coming from the host, limits metabolic turnover and a redox balance needs to be achieved [66]. The observed diversity of terminal oxygen reductases indicates that the supply of oxygen to the symbionts may fluctuate over time or between free-living and symbiotic stages, necessitating adjustments in respiratory metabolism.

Alternative terminal reductases

When oxygen is limited or unavailable, potentially either through competition for oxygen with the host or if the symbionts find themselves in the anoxic sediment that surrounds the burrow, the S. velum symbiont may be capable of using terminal electron acceptors other than oxygen. Although it is unknown whether the symbiont-containing gill bacteriocytes ever become anaerobic, the presence of genes for periplasmic NO₃^- reductase (napFDAGHBC) suggests that symbiont energy generation may involve electron transfer to nitrate, which is available in the porewater surrounding S. velum at concentrations of ~1-10 μM ([67], in preparation). The structure of the symbiont napFDAGHBC operon is consistent with that of enteric bacteria that are thought to use Nap for effectively scavenging nitrate during anaerobic growth under nitrate-limited conditions (5 μM) [68]. The symbiont genome also encodes a DMSO reductase (dmsABC), which suggests an ability to respire dimethyl sulfoxide (DMSO), a breakdown product of dimethylsulfoniopropionate (DMSP) produced, for example, by marine algae. DMSO is available at nanomolar concentrations in the coastal eutrophic environments inhabited by S. velum[69], and Dms genes are common to many marine sediment-dwelling bacteria, e.g., Beggiatoa and Shewanella[70, 71].

ATP synthases

Based on the genome data, both H⁺ and Na⁺ membrane gradients, established along the course of the electron transport chain during respiration, may drive ATP synthesis in the S. velum symbiont via either H⁺- or Na⁺-dependent ATP synthases (Figure 1). The H⁺-specificity of the F₀F₁-type ATP synthase is suggested by the presence of two characteristic transmembrane helixes within the c subunit. In contrast, an A₀A₁-type ATP synthase detected in the genome contains the characteristic Na⁺-binding PXXXQ motif I and ES motif II in the rotor subunit K. While proton-translocating ATP synthases are predominant in bacteria, Na⁺-coupled ATP synthesis driven by respiration has recently been recognized in some marine and pathogenic species [72, 73]. To our knowledge, this is the first report of a Na⁺-translocating ATP synthase in a chemosynthetic symbiont.

Ion gradient driven transporters

Cellular roles of the H⁺ and Na⁺ gradients in the S. velum symbiont appear to extend beyond ATP synthesis. Besides ATP synthases, the genome encodes diverse Na⁺:substrate symporters and numerous Na⁺:H⁺ antiporters, including the multi-subunit MrpEFGBBCDD complex (Figure 1). These transporters, together with ATP synthases and respiratory ion pumps, may establish and consume simultaneous transmembrane gradients of protons and sodium ions in the symbiont [72]. These parallel cycles of H⁺ and Na⁺ would allow the S. velum symbiont to synthesize ATP and maintain pH homeostasis via two separate mechanisms.

Autotrophy

The genome of the S. velum symbiont encodes a version of the Calvin cycle which appears to be prevalent in chemosynthetic symbionts but may also operate in a few free-living bacteria [75–77]. In these organisms genes for fructose 1,6-bisphosphatase and sedoheptulose 1,7-bisphosphatase, which process obligate intermediates in the cycle, are absent. Instead, the role of the missing enzymes may be performed by a single reversible pyrophosphate-dependent phosphofructokinase (PPi-PFK), the gene for which was identified in the genome of the S. velum symbiont (Figure 1). The ability of this enzyme to dephosphorylate fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate in vitro was demonstrated for the PPi-PFK from Methylococcus capsulatus[75], which shares 73% amino acid sequence identity with the homologue from the S. velum symbiont. Notably, during dephosphorylation this enzyme generates pyrophosphate, which bears a high-energy phosphate bond unlike the orthophosphate liberated by fructose 1,6-bisphosphatase and sedoheptulose 1,7-bisphosphatase. In M. capsulatus[75] and in the chemosynthetic symbionts of R. pachyptila[76] and the oligochete, O. algarvensis[77], it was proposed that the pyrophosphate produced this way could be converted into a proton gradient by a membrane-bound proton-pumping pyrophosphatase (V-type H⁺-PPase) co-encoded with the PPi-PFK. This proton gradient could then be used for ATP synthesis. Compared to the classical Calvin cycle [78], this mechanism may allow bacteria to spend up to 9.25% less energy on CO₂ fixation [77]. Judging from the similar gene content, this version of the cycle may also be at work in the symbionts of the vent clams, C. magnifica[22] and C. okutanii[20]. Apart from the membrane-bound V-type H⁺-PPase, the S. velum symbiont genome also encodes a soluble pyrophosphatase (PPase) immediately upstream of the PPi-PFK gene. The PPase cannot convert the energy of pyrophosphate into a proton gradient but, by controlling the availability of pyrophosphate, may serve to regulate the catalytic direction of the PPi-PFK, which may also participate in glycolysis as a kinase. This additional PPase suggests that it may be important for the S. velum symbiont to control the direction of its carbon flux to a higher degree than what has been seen in other chemosynthetic symbionts.

Carbon Flux

Carbon fixed by the S. velum symbiont may be stored as polyglucose or fed into catabolic and anabolic reactions (Figure 1). The overall direction of the metabolic carbon flux in the symbiont can be controlled by at least two putative mechanisms. First, the reversible PPi-PFK, participating in the Calvin cycle as discussed above, may also phosphorylate fructose 6-phosphate during glycolysis. PPi-PFK appears to be the only enzyme encoded in the genome that could catalyze both the forward and the reverse reactions. The directionality of the catalysis may depend on the concentration of pyrophosphate and the other substrates of the enzyme in the cytoplasm [79], since this PPi-PFK is likely nonallosteric [75]. Second, the two encoded glyceraldehyde 3-phosphate dehydrogenases, GapA and GapB, may be specific to glycolysis and the Calvin cycle/gluconeogenesis, respectively, by analogy to the homologous enzymes in Staphylococcus aureus[80]. In the symbiont genome, gapB is adjacent to the gene for transketolase, an enzyme in the Calvin cycle, further suggesting that these two Gap proteins may play a role in regulating the direction of the carbon flux either in the direction of glycolysis or the Calvin cycle and gluconeogenesis. The symbionts of C. magnifica, C. okutanii, R. pachyptila, T. jerichona, and the scaly snail possess just a single gap gene, which has a much higher amino acid sequence identity to gapB than to gapA from the S. velum symbiont. In line of the above evidence the symbiont of S. velum appears to be distinct from other chemosynthetic symbionts in placing a stronger emphasis on controlling the direction of its carbon flux.

Heterotrophy

The S. velum symbiont is the third chemosynthetic symbiont, along with the γ3-symbiont of O. algarvensis[34] and the intracellular γ-proteobacterial symbionts of the scaly-foot snail [33], known to encode all of the enzymes required for the complete TCA cycle, and, therefore, could oxidize organic carbon for energy (Figure 1). All of the other sequenced chemosynthetic symbionts lack genes for α-ketoglutarate dehydrogenase and citrate synthase, which suggests their obligate autotrophy [81].

Furthermore, genes for the glyoxylate bypass of the TCA cycle, encoding isocitrate lyase and malate synthase, were also found in the genome of the S. velum symbiont (Figure 1). These enzymes could allow the symbiont to grow on various carbon sources, including acetate and other two-carbon compounds, [82] or rapidly replenish intermediates of biosynthetic reactions. The presence of the glyoxylate bypass and the TCA cycle suggests that the symbiont may be a facultative mixo- or heterotroph. The adaptive role of having both heterotrophic pathways, however, is unclear, and may relate either to the intracellular conditions specific to this particular symbiosis or to the yet unconfirmed host-free existence of the symbiont.

Nitrogen assimilation

Extracellular ammonia may be imported by the symbiont via specific AmtB transporters and incorporated into glutamate and glutamine, which serve as amino group donors for the other nitrogen-containing compounds in the cell (Figure 1). The S. velum symbiosis comes in contact with 20–100 μM concentration of ammonia in its coastal environment ([67] in preparation). Thus, it is not surprising that, unlike the chemosynthetic symbionts found at nitrate-rich (40 μM) hydrothermal vents [85, 86], the S. velum symbiont lacks nar genes for nitrate reductases capable of assimilatory nitrate reduction [32, 87–89]. Assimilation of ammonia has been previously demonstrated in the gills of S. velum, but was initially ascribed to the activity of the host glutamine synthetase (GS) [88]. The present analysis identified glnA, the gene that encodes GS, in the genome of the symbiont. A preliminary transcriptional study showed glnA to be one of the fifty most highly transcribed genes in the symbiont [40]. The biosynthetic pathways reconstructed on the basis of gene content suggest that the symbiont has the ability to make all of the 20 proteinogenic amino acids. The amino acid prototrophy of the symbiont is in keeping with its proposed role in providing most, if not all, of the host’s nutrition [14, 15].

Urea metabolism

Host urea may serve as an additional source of assimilatory nitrogen for the S. velum symbiont. The identified ureHABCEFG operon encodes a cytoplasmic urease UreABC, which can hydrolyze urea, releasing ammonia that may be re-utilized by the symbiont. Urea can enter the bacterial cell by passive diffusion [90], but under nitrogen starvation the symbiont may be able to take it up more rapidly via an ABC-transporter UrtABCDE, encoded directly upstream of the ure genes. Among chemosynthetic symbionts, urease genes have been previously described only in the γ-symbionts from the marine oligochaete worm O. algarvensis[34, 77], which, like S. velum, lives in coastal sediments. The sequenced chemosynthetic symbionts from hydrothermal vents lack urease genes, even though some of their host organisms, for instance R. pachyptila[91], are known to produce urea. This discrepancy may be accounted for by the fact that in coastal sediments urea is also present outside the host in the pore water ([67] in preparation).

Taurine synthesis

The S. velum symbiont may also provide its host with nitrogenous osmoregulants, such as the non-proteinogenic amino acid taurine [92]. In the host tissues, taurine accounts for up to 70% of the total free amino acids and shows an isotopic composition (δ¹³C, δ¹⁴N, δ³⁴S) suggestive of symbiont origin [93]. Synthesis of taurine may be accomplished by the two homologs of the reversible taurine dioxygenase (TauD) encoded in the symbiont genome. Taurine could be actively secreted to the host by the TauABCD ABC transporter, the genes for which were found to contain a conserved binding domain for sulfonate, characteristic of the taurine molecule. Since taurine synthesis requires sulfite [94], one of the final intermediates in sulfur oxidation, this pathway could serve to dispose of SO₃^2-, and, thus, to drive forward sulfur oxidation in the S. velum symbiont, benefiting both the host and the symbiont.

Transporters

Multi-drug efflux pumps

The S. velum symbiont genome contains at least five sets of genes encoding multi-drug efflux pumps (AcrAB-TolC), suggesting the ability to expel host-derived antimicrobial agents. A comparable genetic capacity for the AcrAB-TolC efflux system has been found in bacteria, such as the plant symbiont Rhizobium leguminosarum, that have a free-living stage, but not in bacteria that are obligately intracellular (Table 2, ATP-dependent transporters). The plant host of R. leguminosarum manipulates the cellular fate of its symbionts using antimicrobial-like peptide factors [102]. As a result, R. leguminosarum undergoes cell elongation and genome replication but looses its ability to divide. Only a small number of R. leguminosarum cells remain vegetative [103]. A very similar morphological differentiation of the symbiont has been observed in S. velum[104]. Assuming the bivalve host also uses peptide factors to control its symbiont, the S. velum symbiont may rely on the efflux pumps to maintain a small undifferentiated population in the bacteriocytes for transmission to future host generations.