Functional Aspects

The phylogenetic diversity of the oligochaete symbionts is mirrored in their physiological diversity. Before the discovery that gutless oligochaetes harbor multiple symbionts, it was assumed that these associations are driven solely by chemoautotrophy. Evidence for a symbiosis fueled by sulfur-oxidizing, CO2-fixing bacteria was based on rapid uptake and incorporation of radiolabeled bicarbonate as well as the presence of characteristic enzymes for the fixation of CO2, such as ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) and the oxidation of sulfur (Felbeck et al. 1983). However, these studies were conducted on whole animal homogenates so that it remained unresolved whether all or only some of the bacteria that coexist in gutless oligochaetes are thiotrophic.

It is now clear that the Gamma 1 symbionts, which occur in all host species and are the dominant symbionts in the worms in terms of biomass, are responsible for most if not all of the observed thiotrophic activity. The thiotrophic nature of these symbionts is suggested by their close evolutionary relationship to a clade of purple sulfur bacteria that includes Allochromatium vinosum and Thiococcus pfennigii (Fig. 2). Immuno-cytochemistry analyses with an antiserum directed against Form I RubisCO showed consistent labeling of the Gamma 1 symbionts, and large deposits of sulfur in intracellular globules of these symbionts were shown using electron microscopic spectroscopy (Krieger 2000; Dubilier et al. 2001). It is assumed that the Gamma 1 symbionts provide the oligochaetes with a source of nutrition, either through regular transfer of carbon compounds, or through their lysis and digestion in host vacuoles (Giere and Langheld 1987). However, this has not been confirmed experimentally.

The physiological nature of the Gamma 2 symbionts is not currently known. They do not appear to be essential to the hosts, as they only occur in some, but not in all species (Fig. 2). The Gamma 2 symbionts are related to phylogenetic groups that include bacteria from cold seeps and a thiotrophic vent isolate (Sect. 5.2.1), suggesting that these symbionts might also participate in chemosynthethic pathways. It is not clear if the Gamma 2 bacteria express RubisCO, as the immunocytochemistry analyses described above were conducted prior to the discovery of these symbionts. We are currently using immunofluorescence studies with antisera against Form I and II RubisCO in combination with 16S rRNA FISH to understand more about the metabolism of these symbionts.

The metabolism of the alpha proteobacterial symbionts is another as yet unsolved puzzle. The Alpha 1 symbionts are most closely related to Rhodovibrio salinarum and R. sodomensis, halophilic bacteria that are photoheterotrophic under anoxic conditions but can also grow in the dark heterotrophically under aerobic conditions. It is intriguing that in all gutless oligochaetes, either Alpha 1 or Delta 1 symbionts coexist with the Gamma 1 symbionts, and it is tempting to speculate that the Alpha 1 symbionts might also play a role in recycling of anaerobic waste products of the worms as suggested for the delta proteobacterial symbionts (see below).

The Alpha 2 symbiont of I. leukodermatus is most closely related to nitrogen-fixing symbionts of leguminous plants (Fig. 2), indicating that these symbionts might also fix N2. However, numerous attempts to amplify a gene characteristic of nitrogen-fixing bacteria, the nifH gene (Zehr and Ward 2002), were unsuccessful (N. Dubilier and J.P. Zehr, unpubl. data). Since this symbiont is only present in I. leukodermatus, it does not appear to be essential for nitrogen uptake, as other oligochaete hosts are clearly able to acquire nitrogen without Alpha 2 symbionts.

The spirochete symbionts fall on a neighboring branch with a sequence from the tubes of the hydrothermal vent polychaete, Alvinella pompejana, obtained from an enrichment culture grown on a very rich medium (M.A.Cambon-Bonavita, unpubl. data). The free-living, marine spirochetes Spirochaeta isovalerica and S. litoralis consistently form a neighboring clade of the oligochaete spirochetes. These bacteria were isolated from sulfidic muddy sediments and are obligate anaerobes that ferment carbohydrates mainly to acetate, ethanol, CO2, and H2 (Hespell and Canale-Parola 1973; Harwood and Canale-Parola 1983). While fermentation is one possible metabolic pathway of the oligochaete spirochetes, they could also have a completely different metabolism, just as the spirochete symbionts in termites do not possess properties common to their closest free-living relatives within the genera Treponema. Instead, termite spirochetes were recently discovered to be chemoautotrophic, using H2 and CO2 to produce acetate (Leadbetter et al. 1999), and were also shown to be able to fix nitrogen (Lilburn et al. 2001). These types of metabolism would clearly be beneficial to the oligochaete hosts, providing them with additional sources of reduced carbon and nitrogen.

In contrast to the uncertainties in the function of the alpha proteobacterial and spirochete symbionts, the metabolism of the delta proteobacterial symbionts is clear. These symbionts are sulfate-reducing bacteria based on their close phylogenetic relationship to free-living sulfate-reducers, the presence of a gene (afcrAB) encoding an enzyme used for dissimilatory sulfate reduction, and the detection of sulfate reduction in the worms at rates comparable to those of free-living sulfate reducers (Dubilier et al. 2001). The coexistence of sulfate-reducing and sulfide-oxidizing bacteria as endosymbionts in oligochaete hosts suggests that these are engaged in a syntrophic sulfur cycle in which oxidized and reduced sulfur compounds are cycled between the two symbionts (Fig. 4e). The sulfate reducer produces reduced sulfur compounds (sulfide or sulfur) using organic carbon (or H2 if it is autotrophic) from the environment. The reduced sulfur compounds are used by the sulfide-oxidizer as an electron donor for the autotrophic fixation of CO2.

This syntrophic association provides several benefits for the hosts. The cycling of reduced and oxidized sulfur compounds between the symbionts increases their energy yields, as shown for continuous co-cultures of sulfate-reducing and sulfide-oxidizing bacteria (van den Ende et al. 1997). Another advantage for the worm is that the sulfate-reducers could take up anaerobic metabolites from the worm such as succinate and fatty acids that are produced when oxygen becomes limiting and are normally excreted. This would allow the worms to recycle these energy rich fermentation products. Finally, the presence of an internal sulfide producer allows these hosts to colonize habitats in which sulfide is present at very low concentrations as in Elba, or only intermittently available, or even completely absent. However, the presence of sulfate-reducing symbionts in O. crassitunicatus that occurs in sediments that appear to be well supplied with sulfide (see Sect. 3.1), shows that the role of these bacteria is not restricted to supplying sulfide. For these worms, the benefits of internal sulfur cycling and the reuse of fermentation products appears to provide a sufficient selective advantage.

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