Heide N. Schulz is a postdoctoral researcher in the Section of Microbiology, University of California, Davis.

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Thiomargarita namibiensis: Giant Microbe Holding Its Breath

The largest known bacterium survives in a highly sulfidic environment with only occasional access to electron acceptors

Heide N. Schulz

Thiomargarita namibiensis, the largest known bacterium, was discovered in sediments off the west coast of Africa in April 1997. Single spherical cells typically are 100-300 µm in diameter, but may be as large as 750 µm, exceeding the volume of all other known prokaryotes by several orders of magnitude. In terms of size, a T. namibiensis cell is to an Escherichia coli cell what blue whale is compared to a newly born mouse.

Figure 1

The single spherical cells, held together in a chain by a slime sheath, shine white due to intracellular sulfur inclusions (Fig. 1). This particular morphology inspired the selection of the genus and species names, which mean "Namibian sulfur pearl." Although these bacteria were discovered recently, in their natural environment they are by no means rare and may occur at biomass densities of up to 180 g per square meter, accounting for up to 0.8% of the sediment volume and making Thiomargarita namibiensis the dominant benthic organism in Namibian shelf sediments.

A New Genus within the Old Group of Colorless Sulfur Bacteria

Most members of the group of colorless sulfur bacteria were described from the middle of the 19th until the beginning of the 20th century, when numerous genera where defined based on morphology. For these sulfur bacteria this approach to nomenclature has survived remarkably well, since the numerous diverse morphologies are often directly linked to their physiology. All members of the group are unpigmented and carry internal sulfur inclusions—reflecting their characteristic physiology, namely the lithotrophic, nonphotosynthetic oxidation of sulfide. Almost everywhere in nature where free sulfide occurs, either in fresh or salt water, some member of the colorless sulfur bacteria can be found.

The best-known, and possibly also the most widespread, colorless sulfur bacteria belong to the genus Beggiatoa, whose members form fast-gliding filaments that accumulate in a thin zone where sulfide diffusing from the sediment contacts oxygen diffusing into the sediment. In 1887, the Russian microbiologist Sergei Winogradsky reasoned, based on his observations on accumulation and disappearance of sulfur inclusions, that Beggiatoa filaments gain energy by the oxidation of sulfide. This was the first proposal of a lithotrophic physiology. Despite many efforts by Winogradsky and other microbiologists, a lithoautotrophic Beggiatoa strain was not isolated into pure culture until 1980. This difficulty of isolating strains into pure cultures remains a common feature for many members belonging to the group of colorless sulfur bacteria.

A Giant in a Family of Large Bacteria

Thiomargarita namibiensis shares many traits with members of the marine sulfide-oxidizing genera Beggiatoa and Thioploca. These three genera include bacteria that are unusually large, with diameters ranging from 10 to more than 100 ?m. These giant cells contain a liquid vacuole that accounts for much of the internal space, while the cytoplasm is confined to a thin 1- to 2-?m layer surrounding the vacuole. Despite their overall large size, the active cytoplasm of such cells is not much thicker than that of an ordinary-sized bacterium. Hence, like other prokaryotes, these giant cells can take up nutrients by means of diffusion without needing special transport systems.

Figure 2

Like all colorless sulfur bacteria, Thiomargarita as well as Beggiatoa and Thioploca cells contain numerous sulfur inclusions (Fig. 2). The sulfur, which is stored in the periplasm, apparently represents conversion of the otherwise toxic electron donor sulfide into a nontoxic, highly condensed form that can be safely stored. Under sulfide starvation conditions, the sulfur inclusions slowly disappear.

In 1995, Osvaldo Ulloa and collaborators at the University of Concepcion in Chile found that the vacuole stores nitrate in very high concentrations (>0.1 M), maintaining this internal concentration against a steep gradient. Nitrate in the ambient bottom water typically occurs in concentrations below 50 µM, and often falls below detection limits within the sediment. The mechanism by which Thiomargarita and the larger Beggiatoa and Thioploca cells concentrate nitrate and maintain such high internal concentrations has not been studied. It appears that the ability to accumulate nitrate has only evolved once, since, according to the few 16S rDNA sequences that have been obtained so far, nitrate-storing species of Beggiatoa, Thioploca, and Thiomargarita seem to form a monophyletic group.

The large nitrate-storing sulfur bacteria typically occur in sediments characterized by high sulfide fluxes, due to either high sulfate reduction rates or to transport of sulfide from deeper layers to the sediment surface as is found at thermal vents or hydrocarbon seeps. In spite of the close phylogenetic relationship of these bacteria and their many similarities, each of these sulfide oxidizers seems to occupy a slightly different ecological niche.

Apart from relative differences in size, the most notable morphological difference between Thiomargarita on the one side and Beggiatoa and Thioploca on the other is that the latter two form filaments, whereas Thiomargarita cells do not. Each individual Thiomargarita cell is surrounded by a slime layer that attaches neighboring cells to one another after cell division (Fig. 1). Motility has not been observed and seems unlikely within this viscous sheath enclosing each cell.

Internal Stored Nitrate Used for Oxidation of Sulfide

Ulloa and his collaborators, studying two dominant marine species of Thioploca, were the first to recognize this vacuole-based capacity to store nitrate in high concentrations. These marine species dominate sediments underlying the highly productive upwelling areas off the coast of Peru and Chile.

Thioploca filaments are specialists in bringing together nitrate, which is available only in the bottom water overlying the sediment, with sulfide, which is found several centimeters below the surface of the sediment. These filaments occur in bundles held together by mucus sheaths that penetrate many centimeters into the sediment. The sheaths connect the deeper sulfidic zones with the bottom water that contains nitrate. By shuttling up and down in these conduits, Thioploca filaments oxidize sulfide at a sediment depth where no other electron acceptor, apart from their internally stored nitrate, is available. The end product of the nitrate reduction seems to be ammonia, whereas nitrite may be formed under environmental stress. So far, there is no indication that Thioploca filaments can use oxygen as an electron acceptor. The dense populations of Thioploca off the west coast of South America seem to be very sensitive to either oxygen or sulfide in higher concentrations.

Beggiatoa of the narrow, nonvacuolate variety occur as individual filaments at the surface of sediments where sulfide, diffusing from below, creates a steep sulfide gradient that overlaps with oxygen diffusing into the sediment from the water above. The filaments avoid high concentrations of sulfide and oxygen, accumulating in a small band directly in the narrow zone where their electron donor and acceptor overlap. Larger, vacuolated Beggiatoa filaments also can store nitrate. Nevertheless, the larger forms of Beggiatoa filaments tend to populate distinct horizontal zones in the sediment rather than spreading deep into the sediments, as is typical for Thioploca filaments. Because Beggiatoa spp. are usually not found in environments that are completely anoxic, it seems that the larger, nitrate-storing Beggiatoa spp. might use nitrate as an alternative electron acceptor only during anoxic periods.

Namibian Shelf Sediments: a Highly Changeable Environment

Beggiatoa and Thioploca spp. both require motility to oxidize sulfide. Thus, the discovery of a third genus of nitrate-storing sulfur bacteria apparently lacking motility points to a previously unrecognized strategy for using internally stored nitrate. Thiomargarita namibiensis occurs off the Namibian coast in sediments that are unusually fluid, consisting predominantly of empty diatom shells.

These high levels of organic material in Namibian coastal sediments lead to very high sulfide concentrations of up to 10 mM. Oxygen is usually absent in the bottom water covering these sediments, while nitrate may be found in low concentrations. Because they are not motile, Thiomargarita cells are trapped within the sediment without access to nitrate and are subject to sulfide concentrations that would be toxic for their relatives.

Figure 3

Occasionally, the loose sulfidic mud may be resuspended—for example, by methane eruptions that occur regularly in the area off Walvis Bay in Namibia and may be so dramatic that they raise "islands of mud" to the sea surface. Only such resuspension events would allow Thiomargarita cells to come into contact with nitrate for storage needed for later survival in the sulfidic sediment. During such events, the bacteria are also likely to encounter high oxygen concentrations that would be toxic for related microorganisms, including Thioploca filaments (Fig. 3).

Like the cytoplasm of the larger Beggiatoa and Thioploca cells, the active cytoplasm of Thiomargarita cells is typically 1-2 ?m. Nevertheless, because such cells are spherical rather than cylindrical and because of their larger diameter, their vacuole-to-cytoplasm ratio is considerably greater than is that for either Beggiatoa or Thioploca cells. Thus, even though they have similarly high internal concentrations of nitrate, this nitrate storage can sustain them through a longer period than a Thioploca or Beggiatoa filament can withstand, assuming all of them have similar metabolic activity.

Therefore, the large size of Thiomargarita cells can be interpreted as an adaptation for surviving long periods without access to a suitable electron acceptor. In addition, Thiomargarita cells seem capable of surviving by reducing their metabolic activity under conditions of nitrate starvation. For example, Namibian sediments kept in the cold for more than four years without added nitrate still contained a few living cells of T. namibiensis.

In contrast to Beggiatoa and Thioploca, Thiomargarita cells can survive exposure to atmospheric oxygen and to sulfide in millimolar concentrations. This tolerance of a broad spectrum of environmental conditions from very reduced to very oxidized chemical milieus seems to be another adaptation to the fluid, sulfidic sediment off Walvis Bay. This, together with the capacity to store both nitrate and sulfide, and the ability to survive long intervals without access to nitrate, enables Thiomargarita cells to continue to oxidize sulfide, even though electron acceptors are seldom available in the sediment. Thus, unlike Thioploca filaments, whose electron acceptor and donor are spatially separated, the electron donors and acceptors used by Thiomargarita are separated in time.

Additional Use of Oxygen

Single cells of T. namibiensis are so large that their metabolic activity produces measurable gradients of substrates. Such measurements indicate that Thiomargarita cells do not simply survive exposure to atmospheric oxygen but also consume it. Sulfide enhances this uptake, and oxygen enhances sulfide uptake, suggesting that Thiomargarita cells use oxygen as an electron acceptor in addition to nitrate for oxidizing sulfide or internal sulfur. Thus, during periods of sediment suspension, when the sulfidic sediments mix with oxygen-containing seawater, Thiomargarita cells take up nitrate for later survival in the highly sulfidic mud and also may gain energy by rapidly oxidizing sulfide or stored sulfur globules with oxygen (Fig. 3).

In addition to passively enduring a highly reduced and occasionally very oxidized environment, Thiomargarita cells may actively switch between a slow metabolism during the long phases that the cells are buried in the sulfidic sediment and a very rapid metabolism during resuspension from that sediment when oxygen and sulfide are simultaneously available.

Anaerobic Oxidation of Sulfide

Nitrate-storing sulfur bacteria have been discovered in a broad range of marine sulfidic environments. Each of the genera of nitrate-storing sulfur bacteria can occur in enormously high biomass if they are sustained by a high flux of sulfide, as is often the case in coastal sediments. Thus, apart from showing exotic adaptations to sulfidic environments, these bacteria may play a more important role in the oxidative part of the sulfur cycle than was previously thought.

Although microbiologists long ago realized that bacteria may oxidize sulfide with nitrate, they considered anaerobic oxidation of sulfide to be of little importance, and it receives no mention in textbook illustrations of the sulfur cycle. Because the large marine Beggiatoa and Thioploca species use their vacuoles for storing nitrate, this view of the sulfur cycle now seems outdated. Furthermore, the discovery of Thiomargarita, a third genus of sulfur bacteria that can use nitrate as an electron acceptor for the oxidation of sulfide, stresses the underlying ecological importance of this process.

Acknowledgments

This work was financed by the Max-Planck-Society. I thank Douglas Nelson and Sherry Huston for editing the manuscript and Bo Barker Jørgensen for his continuous support.

Suggested reading

Fossing, H., V. A. Gallardo, B. B. Jørgensen, M. Huttel, L. P. Nielsen, H. Schulz, D. E. Canfield, S. Forster, R. N. Glud, J. K. Gundersen, J. Kuver, N. B. Ramsing, A. Teske, B. Thamdrup, and O. Ulloa. 1995. Concentration and transport of nitrate by the mat-forming sulphur bacterium Thioploca. Nature 374:713-715.

Jannasch, H. W., D. C. Nelson, and C. O. Wirsen. 1989. Massive natural occurrence of unusually large bacteria (Beggiatoa sp.) at a hydrothermal deep-sea vent site. Nature 342: 834-836.

Jørgensen, B. B., and V. A. Gallardo. 1999. Thioploca spp: filamentous sulfur bacteria with nitrate vacuoles. FEMS Microbiol. Ecol. 28:301-313.

McHatton, S. C., J. P. Barry, H. W. Jannasch, and D. C. Nelson. 1996. High nitrate concentrations in vacuolated, autotrophic marine Beggiatoa spp. Appl. Environ. Microbiol. 62:954-958.

Møller, M. M., L. P. Nielsen, and B. B. Jørgensen. 1985. Oxygen responses and mat formation by Beggiatoa spp. Appl. Environ. Microbiol. 50:373-382.

Nelson, D. C., and H. W. Jannasch. 1983. Chemoautotrophic growth of a marine Beggiatoa in sulfide-gradient cultures. Arch. Microbiol. 136:262-269.

Nelson, D. C., B. B. Jørgensen, and N. P. Revsbech. 1986. Growth-pattern and yield of a chemoautotrophic Beggiatoa sp. in oxygen-sulfide microgradients. Appl. Environ. Microbiol. 52: 225-233.

Otte, S., J. G. Kuenen, L. P. Nielsen, H. W. Paerl, J. Zopfi, H. N. Schulz, A. Teske, B. Strotmann, V. A. Gallardo, and B. B. Jørgensen. 1999. Nitrogen, carbon, and sulfur metabolism in natural Thioploca samples. Appl. Environ. Microbiol. 65:3148-3157.

Schulz, H. N., T. Brinkhoff, T. G. Ferdelman, M. Hernandez Marine, A. Teske, and B. B. Jørgensen. 1999. Dense populations of a giant sulfur bacterium in Namibian shelf sediments. Science 284:493-495.

Schulz, H. N., and B. B. Jørgensen. 2001. Big bacteria. Annu. Rev. Microbiol. 55:105-137.