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The Calyptogena magnifica Chemoautotrophic Symbiont Genome

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Science  16 Feb 2007:
Vol. 315, Issue 5814, pp. 998-1000
DOI: 10.1126/science.1138438

Abstract

Chemoautotrophic endosymbionts are the metabolic cornerstone of hydrothermal vent communities, providing invertebrate hosts with nearly all of their nutrition. The Calyptogena magnifica (Bivalvia: Vesicomyidae) symbiont, Candidatus Ruthia magnifica, is the first intracellular sulfur-oxidizing endosymbiont to have its genome sequenced, revealing a suite of metabolic capabilities. The genome encodes major chemoautotrophic pathways as well as pathways for biosynthesis of vitamins, cofactors, and all 20 amino acids required by the clam.

Metazoans at deep-sea hydrothermal vents flourish with the support of symbiotic chemoautotrophic bacteria (1). Analogous to photosynthetic chloroplasts, which evolved from cyanobacterial ancestors and use light energy to fix carbon for their plant and algal hosts, chemoautotrophic endosymbionts use the chemical energy of reduced sulfur emanating from vents to provide their hosts with carbon and a large array of additional necessary nutrients. In return, host invertebrates bridge the oxic-anoxic interface to provide symbiotic bacteria with the inorganic substrates necessary for chemoautotrophy. The giant clam, Calyptogena magnifica Boss and Turner (Bivalvia: Vesicomyidae), was one of the first organisms described after the discovery of hydrothermal vents (2). It has a reduced gut and ciliary food groove and is nutritionally dependent on its γ-proteobacterial symbionts (here named Candidatus Ruthia magnifica) (3, 4). We present the complete genome of Ruthia magnifica (Fig. 1). Despite having a relatively small genome (1.2 Mb), R. magnifica is predicted to encode all the metabolic pathways typical of free-living chemoautotrophs, including carbon fixation, sulfur oxidation, nitrogen assimilation, and amino acid and cofactor/vitamin biosynthesis (fig. S1 and table S1).

Fig. 1.

A circular representation of the R. magnifica genome. The innermost circle highlights genes of special interest: cbb (Calvin-Benson-Bassham cycle, red), sox (sulfur oxidation, green), dsr (dissimilatory sulfite reductase, blue), and rnf (NADH dehydrogenase). The second and third circles show GC skew and %G+C, respectively. The distribution of genes is depicted on the two outer rings (fourth and fifth, forward and reverse, respectively) colored by role category.

The following sections outline the reconstruction of R. magnifica's chemoautotrophic metabolism and what this might mean for the biology of its host. Our analysis provides direct evidence that this symbiont fixes carbon via the Calvin cycle, the dominant form of carbon fixation in vent symbioses (5), by using energy derived from sulfur oxidation. The R. magnifica genome encodes enzymes specific to this cycle, including a form II ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO) and phosphoribulokinase (6). Interestingly, however, it appears that R. magnifica lacks homologs of sedoheptulose 1,7-bis phosphatase and fructose 1,6-bis-phosphatase and may regenerate ribulose 1,5-bisphosphate via an unconventional route, one that was a reversible pyrophosphate-dependent phosphofructokinase [supporting online material (SOM) text].

Energy for carbon fixation in R. magnifica appears to result from sulfur oxidation via the sox (sulfur oxidation) and dsr (dissimilatory sulfite reductase) genes (fig. S1). The symbiont may oxidize its sulfur granules via dsr homologs when external sulfide is lacking, as occurs in both Allochromatium vinosum and Chlorobium limicola (7, 8). Homologs encoding both a sulfide:quinone oxidoreductase and rhodanese are also present, and, with the dsr and sox proteins, these enzymes can oxidize sulfide or thiosulfate to sulfite (fig. S1). Sulfite can then be oxidized to sulfate by adenosine 5′-phosphosulfate (APS) reductase and adenosine triphosphate (ATP) sulfurylase before being exported from the cell via a sulfate transporter. Genomic evidence of the Calvin cycle and the sulfur oxidation pathways confirms the chemoautoautotrophic nature of this symbiosis. These data support prior reports showing RuBisCO and ATP sulfurylase activity in C. magnifica gill tissue (4, 9), carbon dioxide uptake by the clam in response to sulfide or thiosulfate (10), and sulfide-binding, zinc-containing lipoprotein in the host blood stream (11).

Energy conservation via the creation of a charge across a membrane proceeds in R. magnifica through a nicotinamide adenine dinucleotide (NADH) dehydrogenase, a sulfide: quinone oxidoreductase, and an rnf complex (12). The genome encodes a straightforward electron transport chain; thus, the reduced quinone in the symbiont membrane may transfer electrons to cytochrome c via a bc1 complex, and a terminal cytochrome c oxidase could then transfer these electrons to oxygen (fig. S1).

Our analysis shows that R. magnifica has the potential to produce 20 amino acids, 10 vitamins or cofactors, and all necessary biosynthetic intermediates, supporting an essential role of symbiont metabolism in the nutrition of this symbiosis. Two nitrogen assimilation pathways, essential to provisioning of amino acids in the symbiosis, occur in R. magnifica. Nitrate and ammonia, which enter the cell via a nitrate or nitrite transporter and two ammonium permeases, are reduced via nitrate and nitrite reductase and assimilated via glutamine synthetase and glutamate synthase, respectively (fig. S1). Although nitrate is the dominant nitrogen form at vents (13), the symbiont may also assimilate ammonia via recycling of the host's amino acid waste. Unlike any other sequenced endosymbiont genome, R. magnifica encodes complete pathways for the biosynthesis of 20 amino acids, including 9 essential amino acids or their precursors (fig. S3). R. magnifica also has complete biosynthetic pathways for the majority of vitamins and cofactors (SOM text). The genome encodes a complete glycolytic pathway and the nonoxidative branch of the pentose phosphate pathway and, interestingly, also encodes a tricarboxylic acid (TCA) cycle lacking α-ketoglutarate dehydrogenase. The lack of this enzyme has been suggested to indicate obligate autotrophy in other bacteria (14). Carbon fixed via the Calvin cycle can enter the TCA cycle through phosphoenolpyruvate and here could follow biosynthetic routes either to fumarate or to α-ketoglutarate.

As with other intracellular species, genes not found in the R. magnifica genome reveal important details about the interaction between host and symbiont. Few substrate-specific transporters were found, suggesting that the symbionts are leaky or that the host actively digests symbiont cells. Indeed, Ruthia's closest known relatives, the bathymodiolid mussel symbionts, are digested intracellularly by their host (15). Although the vesicomyids and the bathymodiolids are distantly related, putative degradative stages of symbionts also are observed within C. magnifica bacteriocytes (fig. S2b). The symbiont is also lacking the key cell division protein ftsZ suggesting that, like Chlamydia spp., intracellular division may not proceed through usual pathways (SOM text).

Intracellular endosymbionts often follow distinctive evolutionary routes, including genome reduction, skewed base compositions, and elevated mutation rates (16). Given the apparent defects in DNA repair in R. magnifica (SOM text) and the likely evolutionary forces pushing this genome toward degradation, it is particularly informative that Ruthia retains genes encoding a full suite of chemoautotrophic processes. Indeed, R. magnifica has the largest genome of any intracellular symbiont sequenced to date and may represent an early intermediate in the evolution toward a plastid-like chemoautotrophic organelle. The broad array of metabolic pathways revealed through sequencing of the R. magnifica genome confirms and extends prior knowledge of host nutritional dependency.

Supporting Online Material

www.sciencemag.org/cgi/content/full/315/5814/998/DC1

Materials and Methods

SOM Text

Figs. S1 to S3

Table S1

References

References and Notes

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