Discovery of a unique symbiosis between bacteria  and a marine worm (2001)

Sulfide is toxic for most animals. The symbiotic sulfur bacteria of the gutless oligochaetes take up sulfide and oxidize it to non-toxic products. The energy they gain during the oxidation of sulfide is used to fix CO2 into organic compounds. The animals use the bacteria for their nutrition, either by taking up organic compounds from the symbionts or by digesting them. The metabolic process of these bacteria is called chemosynthesis and is comparable to photosynthesis, in which CO2 is also fixed into organic compounds, but with light instead of sulfide as an energy source. Chemosynthetic symbionts also occur in other marine animals such as mussels, clams, and snails, and were discovered approximately 20 years ago at hydrothermal vents in the deep sea.

One of the essential requirements for chemoautotrophic symbioses is the availability of reduced sulfur compounds, such as sulfide. Without this energy source, the bacteria would not be able to feed their hosts, and these would eventually starve. Dr. Dubilier and her colleagues were therefore very surprised when they discovered an oligochaete worm with chemoautotrophic symbionts thriving in an environment in which, at first, they were not able to detect any sulfide. The worm, with the Latin name of Olavius algarvensis, was discovered in a small Mediterranean bay off the island of Elba (Italy) (Fig. 1 and 2). The worms live at 8 - 10 meters water depth near sea grass beds in coarse sediments 5 - 15 cm below the sediment surface. Directly under the skin of these worms the scientists observed, next to the already known primary sulfide-oxidizing symbionts, unique sulfide producing bacteria as secondary symbionts. This discovery immediately gave the scientists a clue as to how the symbiosis could survive without sulfide in the environment: the secondary symbionts produce sulfide, which is used by the primary symbionts as an energy source for the fixation of CO2. Using a wide array of modern molecular and biogeochemical methods, Dr. Dubilier was able to confirm this hypothesis in an intensive interdisciplinary collaboration with colleagues at the Max Planck Institute of Marine Microbiology and other institutes.

Caroline Mülders, a Ph. D. student in the laboratory of Dr. Rudolf Amann at the MPI, determined the identity of the bacterial symbionts by sequencing a gene commonly used in bacterial systematics, 16S ribosomal RNA (rRNA), and developing specific 16S rRNA gene probes to locate the bacteria in the worm. This method for identifying microorganisms in environment samples has been used and optimized for years in the laboratory of Dr. Amann, and is called FISH (fluorescence in situ hybridization). The 16S rRNA molecules of different bacteria vary in their RNA sequences and are therefore ideal for their identification. These variable regions are used for developing probes that are specific to the bacterial species of interest. In a sample with many different bacterial species, a specific probe will bind only to its target rRNA sequence. The probes are marked with a fluorescent dye so the bacerial cells that are labeled by the probe become clearly visible under fluorescent light. This method was decisive both in identifying the symbiotic bacteria and in showing where they occur in the worm, namely in close association with one another immediately below the outer skin (cuticle) of the animals (Fig. 3).

The close relationship of the secondary symbionts to known free-living sulfate-reducing bacteria suggested to the scientists that these were also sulfate reducers - that is, bacteria that produce sulfide. A close relationship between two bacteria, however, is not necessarily proof that they share the same metabolism. Even very closely related bacteria can have completely different metabolic pathways. The scientists had to therefore make sure that the newly discovered symbionts were indeed sulfate reducers, in particular because an endosymbiosis between a sulfate-reducer and a marine invertebrate seemed very unlikely. Such symbioses have not been previously described, and it has been assumed that they do not occur because the sulfide produced by the bacteria is toxic to most animals. Furthermore, most sulfate reducers are heterotrophic, and it does not seem advantageous for animals, which are also heterotrophs, to establish a symbiosis with another heterotrophic organism. When Dr. Dubilier and Ms. Mülders, in cooperation with Dr. Michael Wagner and Michael Klein (both Technical University Munich), discovered an essential and key gene for bacterial sulfate reduction in the symbiotic genome, they were able to prove that the secondary symbiont is in fact a sulfate reducer. 

But were the sulfate reducers active in the worm? Did they produce sulfide in their host? To answer this question Dr. Dubilier and two of her colleagues, Dr. Dirk de Beer and Dr. Tim Ferdelman from the Max Planck Institute in Bremen, developed a clever experiment. The scientists incubated the worms in radioactive sulfate and inserted an extremely thin silver needle into the worms. When they exposed the needles to an x-ray film after the experiment, a black signal appeared. This blackening occurred because the bacteria reduced the radioactive sulfate to sulfide, which precipitated on the needles. The experiment only worked under low oxygen concentrations. At higher oxygen concentrations, the symbionts no longer reduced sulfate to sulfide, a behavior common to almost all free-living, non-symbiotic sulfate reducers. These results were crucial in proving that the sulfate-reducing symbionts are active inside the worm and able to produce sulfide.

A final unresolved question remained: do the sulfide-oxidizing symbionts really need the sulfide produced by the sulfate-reducing symbionts? In their first experiments, the scientists had not been able to measure sulfide in the wormís environment, but they were worried that the methods they used might not be sensitive enough. They knew that free-living sulfide-oxidizing bacteria can gain energy even at very low sulfide concentrations. It was therefore possible that the sulfide oxidizers did not need the sulfide produced by the sulfate-reducers. To answer this question, the scientists developed a more sensitive method for measuring sulfide in the wormís environment. They discovered that the sediments contained only minute amounts of sulfide, comparable to concentrations in open sea waters, which are considered free of sulfide. The comparison of sulfide flux from the sediment (that is, the amount of sulfide that diffuses into the worm within a defined time period) with the internal production of sulfide from the symbionts showed that internally produced sulfide generally exceeded sulfide flux from the environment by at least 7 times, and sometimes by as much as 30 times. These results clearly demonstrated that the sulfate reducers are the main source of sulfide in this system.

How does this symbiosis work (Figure 4)? The sulfate-reducing symbionts produce sulfide, which is taken up by the neighbouring sulfide-oxidizing symbionts and converted to oxidized sulfur compounds such as sulfate. These oxidized sulfur compounds are in turn taken up by the sulfate-reducing symbionts and converted to reduced sulfur compounds such as sulfide. At first glance this cyclic or syntrophic sulfur cycle may seem to be a "Perpetuum mobile". However, for the worm and its symbionts to grow, energy sources must be taken up from the environment, such as dissolved organic compounds that occur in the pore waters of marine sediments.

What does the worm gain from this arrangement? The cycling of metabolic products between the two symbionts increases their energy yield, which in turn would be passed on to their host. Another advantage for the worm is that the sulfate reducers could take up metabolic byproducts that would otherwise be excreted. These waste products, called anaerobic metabolites (for example, succinate), are produced by marine animals as soon as environmental oxygen concentrations become limiting. Instead of excreting these waste products, the worms can reuse them in a kind of internal "symbiotic recycling" and thereby save additional energy. There is a further advantage to having a symbiotic sulfate reducer: these supply the worms and their primary sulfide-oxdizing symbionts with a continuous source of internal