It is generally considered that sulphur reduction was one of the earliest forms of microbial respiration, because the known microorganisms that are most closely related to the last common ancestor of modern life are primarily anaerobic, sulphur-reducing hyperthermophiles. However, geochemical evidence indicates that Fe(III) is more likely than sulphur to have been the first external electron acceptor of global significance in microbial metabolism. Here we show that Archaea and Bacteria that are most closely related to the last common ancestor can reduce Fe(III) to Fe(II) and conserve energy to support growth from this respiration. Surprisingly, even Thermotoga maritima, previously considered to have only a fermentative metabolism, could grow as a respiratory organism when Fe(III) was provided as an electron acceptor. These results provide microbiological evidence that Fe(III) reduction could have been an important process on early Earth and suggest that microorganisms might contribute to Fe(III) reduction in modern hot biospheres. Furthermore, our discovery that hyperthermophiles that had previously been thought to require sulphur for cultivation can instead be grown without the production of toxic and corrosive sulphide, should aid biochemical investigations of these poorly understood organisms.

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An insertion of transposon Tn5-lac, omega 4519, generates a lacZ fusion with a Myxococcus xanthus promoter expressed during both vegetative growth and development. Sequence analysis of the junction of omega 4519 with M. xanthus DNA shows that the insertion is in frzF, a homologue of cheR from Salmonella typhimurium. When frzF- (or frzCD-) cells are starved for nutrients at modest densities, they aggregate to form a radial pattern and produce fewer than 1% of the wild-type complement of spores. At higher densities, frzF::omega 4519 cells form 'frizzy' aggregates and produce 80-90% of the wild-type complement of spores. In contrast, when cells with both a frzF- (or frzCD-) and an sglA1 mutation are allowed to develop at either low or high cell densities, they produce frizzy aggregates containing a near wild-type complement of heat-resistant spores. In addition to suppressing the density dependence of fruiting-body morphogenesis, the sglA1 mutation also suppresses the sporulation defect caused by two different frzF- mutations and a frzCD- mutation. In contrast, a mutation in a different S motility gene, sglG1, does not suppress the frz- mutations. Thus, the suppression of frz- mutations by sgl- mutations is allele-specific, and depends on the sgl allele, but not the frz allele. Because the phenotypes of frz- mutations have been determined in a (suppressing) sglA1 genetic background, the frz genes may play more central roles in development than initially recognized.

It has recently been noted that a diversity of hyperthermophilic microorganisms have the ability to reduce Fe(III) with hydrogen as the electron donor, but the reduction of Fe(III) or other metals by these organisms has not been previously examined in detail. When Pyrobaculum islandicum was grown at 100°C in a medium with hydrogen as the electron donor and Fe(III)-citrate as the electron acceptor, the increase in cell numbers of P. islandicum per mole of Fe(III) reduced was found to be ca. 10-fold higher than previously reported. Poorly crystalline Fe(III) oxide could also serve as the electron acceptor for growth on hydrogen. The stoichiometry of hydrogen uptake and Fe(III) oxide reduction was consistent with the oxidation of 1 mol of hydrogen resulting in the reduction of 2 mol of Fe(III). The poorly crystalline Fe(III) oxide was reduced to extracellular magnetite. P. islandicum could not effectively reduce the crystalline Fe(III) oxide minerals goethite and hematite. In addition to using hydrogen as an electron donor for Fe(III) reduction, P. islandicum grew via Fe(III) reduction in media in which peptone and yeast extract served as potential electron donors. The closely related species P. aerophilum grew via Fe(III) reduction in a similar complex medium. Cell suspensions of P. islandicum reduced the following metals with hydrogen as the electron donor: U(VI), Tc(VII), Cr(VI), Co(III), and Mn(IV). The reduction of these metals was dependent upon the presence of cells and hydrogen. The metalloids arsenate and selenate were not reduced. U(VI) was reduced to the insoluble U(IV) mineral uraninite, which was extracellular. Tc(VII) was reduced to insoluble Tc(IV) or Tc(V). Cr(VI) was reduced to the less toxic, less soluble Cr(III). Co(III) was reduced to Co(II). Mn(IV) was reduced to Mn(II) with the formation of manganese carbonate. These results demonstrate that biological reduction may contribute to the speciation of metals in hydrothermal environments and could account for such phenomena as magnetite accumulation and the formation of uranium deposits at ca. 100°C. Reduction of toxic metals with hyperthermophilic microorganisms or their enzymes might be applied to the remediation of metal-contaminated waters or waste streams.

Studies with a diversity of hyperthermophilic and mesophilic dissimilatory Fe(III)-reducing Bacteria andArchaea demonstrated that some of these organisms are capable of precipitating gold by reducing Au(III) to Au(0) with hydrogen as the electron donor. These studies suggest that models for the formation of gold deposits in both hydrothermal and cooler environments should consider the possibility that dissimilatory metalreducing microorganisms can reductively precipitate gold from solution.A wide diversity of both Bacteria and Archaea have the ability to transfer electrons to Fe(III) (11,12,14). Many of these Fe(III)-reducing microorganisms are also capable of transferring electrons to other metals and metalloids. Microbial reduction of Fe(III) and other metals can influence the fate of metals in aquatic sediments, submerged soils, and the subsurface (10,11,13). One of the more geologically significant impacts of microbial metal reduction is the formation of minerals that can be important geological signatures of the activity of metal-reducing microorganisms and, in some instances, that may represent economically important ore deposits. For example, the formation of magnetite during Fe(III) oxide reduction (21) has been considered to be an indication of the activity of Fe(III)-reducing microorganisms in aquatic sediments (4), in the deep, hot biosphere (5), and on Mars (22). The massive magnetite accumulations formed in the Precambrian period, presumably as the result of microbial activity (1,8,27), represent an important source of iron ore. It has also been suggested that microbial reduction of U(VI) to U(IV), which precipitates uranium from solution (19), might account for the formation of some uranium ores (6, 17).Like uranium, gold is soluble in the oxidized form, Au(III), but the reduced form of gold, Au(0), is insoluble (23). The finding that addition of Au(III) to cell suspensions of Geobacter metallireducens oxidized c-type cytochromes, which are thought to be involved in electron transport to metals (15), suggested that this dissimilatory metal reducer might be able to transfer electrons to Au(III) (9). Furthermore, the c 3 -cytochrome of Desulfovibrio vulgaris, known to be involved in the reduction of U(VI) (20) and Cr(VI) (18), can also transfer electrons to Au(III) (9). However, the possibility that whole cells of either G. metallireducens, D. vulgaris, or other dissimilatory Fe(III)-reducing microorganisms reduced Au(III) was not further evaluated.In order to determine the potential for dissimilatory Fe(III)-reducing microorganisms to precipitate gold from solution, the following organisms were cultured (800 ml) in 1-liter bottles, using strict anaerobic techniques as previously described (16 (ATCC 35115), "Desulfitobacterium metallireducens" (laboratory culture collection), and the acetate-oxidizing hyperthermophile strain 234 (laboratory culture collection). Cell suspensions were prepared as previously described (26). Briefly, cells were harvested anaerobically by centrifugation, resuspended in 8...