Microorganisms growing near and above 100C have recently been discovered near shaflow and deep sea hydrothermal vents. Most are obligately dependent upon the reduction of elemental sulfur (S°) to hydrogen sulfide (H2S) for optimal growth, even though SO reduction readily occurs abioticafly at their growth temperatures. The sulfur reductase activity of the anaerobic archaeon Pyrococcus furiosus, which grows optimally at 100°C by a metabolism that produces H2S if S0 is present, was found in the cytoplasm. It was purified anaerobically and was shown to be identical to the hydrogenase that had been previously purified from this organism. Both S0 and polysulfide served as substrates for H2S production, and the SO reduction activity but not the H2-oxidation activity was enhanced by the redox protein rubredoxin. The H2-oxidizing and SO-reduction activities of the enzyme also showed different responses to pH, temperature, and inhibitors. This bifunctional "sulfhydrogenase" enzyme can, therefore, dispose of the excess reductant generated during fermentation using either protons or polysulfides as the electron acceptor. In addition, purified hydrogenases from both hyperthermophilic and mesophilic representatives of the archaeal and bacterial domains were shown to reduce SO to H2S. It is suggested that the function of some form of ancestral hydrogenase was SO reduction rather than, or in addition to, the reduction of protons.

The bioenergetic role of the reduction of elemental sulfur (SO) in the hyperthermophilic archaeon (formerly archaebacterium) Pyrococcusfuriosus was investigated with chemostat cultures with maltose as the limiting carbon source. The maximal yield coefficient was 99.8 g (dry weight) of cells (cdw) per mol of maltose in the presence of So but only 51.3 g (cdw) per mol of maltose if So was omitted. However, the corresponding maintenance coefficients were not found to be significantly different. The primary fermentation products detected were H2, C02, and acetate, together with H2S, when So was also added to the growth medium. If H2S was summed with H2 to represent total reducing equivalents released during fermentation, the presence of So had no significant effect on the pattern of fermentation products. In addition, the presence of So did not significantly affect the specific activities in cell extracts of hydrogenase, sulfur reductase, a-glucosidase, or protease. These results suggest either that So reduction is an energy-conserving reaction, i.e., So respiration, or that So has a stimulatory effect on or helps overcome a process that is yield limiting. A modification of the Entner-Doudoroff glycolytic pathway has been proposed as the primary route of glucose catabolism in P.furiosus (S. Mukund and M. W. W. Adams, J. Biol. Chem. 266:14208-14216, 1991). Operation of this pathway should yield 4 mol of ATP per mol of maltose oxidized, from which one can calculate a value of 12.9 g (cdw)per mol of ATP for non-So growth. Comparison of this value to the yield data for growth in the presence of S0 indicates that So reduction is equivalent to an ATP yield of 0.5 mol of ATP per mol of So reduced. Possible mechanisms to account for this apparent energy conservation are discussed.Microorganisms that are able to grow optimally near the normal boiling point of water are a very recent discovery (48). To date, all have been classified as archaea (formerly archaebacteria [57]). The existence of such organisms has raised many fundamental biological questions. For example, one of the distinguishing growth characteristics of these so-called hyperthermophiles is their ability to reduce elemental sulfur (S) to H2S, and there has been much speculation as to So's physiological role (4, 22, 44) (for reviews, see references 1, 26, 42, and 48). On the other hand, the reduction of S to H2S is extremely limited in nonhyperthermophilic organisms. For example, only the mesophilic (eu) bacterium Desulfuromonas acetoxidans (39) and the moderate thermophile Desulfurella acetivorans (6) are obligately dependent upon the anaerobic respiration of So for growth, and only a few organisms which facultatively reduce So are known (28, 30a, 33, 57a, 58). In contrast, all known hyperthermophiles metabolize SO; in fact, the majority are obligately dependent upon So reduction and use either H2 or organic compounds as electron donors. The best studied is the autotroph Pyrodictium brockii (49), from which several components of its membrane-bound electron tra...

Pyrodictium brockii is a hyperthermophilic archaebacterium with an optimal growth temperature of 1050C. P. brockii is also a chemolithotroph, requiring H2 and CO2 for growth. We have characterized P. brockii hydrogenuptake activity with regard to temperature, ability to couple hydrogen oxidation to artificial electron acceptor reduction, sensitivity to 02, and cellular localization. The hydrogenuptake activity was localized predominantly in a particulate fraction, was reversibly inhibited by 02, and coupled H2 uptake to the reduction of positive potential artificial electron acceptors. Comparisons between these results and those of the well-studied hydrogen-uptake hydrogenase from the mesophile Bradyrhizobium japonicum showed the two enzymes to be similar despite the very different natural environments of the organisms. However, the optimum temperature for activity differed greatly in the two organisms. We have also used immunological and genetic probes specific to the 65-kDa subunit of B. japonicum hydrogenase to assay crude extracts and genomic DNA, respectively, from P. brockii and found the enzymes to be similar in these respects as well. In addition, we report a formulation for artificial seawater capable of sustaining the growth of P. brockii.The discovery of microorganisms capable of growth at extreme thermal conditions has fostered an expanding interest in the effects of temperature on biological systems (1). In addition to several important applications that have been considered for organisms and their associated biomolecules at elevated temperatures (1, 2), a variety of fundamental scientific questions have arisen concerning the biochemical basis for life under extreme conditions. Understanding the intrinsic nature of enzyme thermostability at high temperatures, for example, may be a key to gaining further insight into several issues related to protein structure and function. By comparing enzymes obtained from organisms growing at high temperatures with their mesophilic counterparts, the subtleties of protein thermostability should become clearer.Pyrodictium brockii is a hyperthermophilic (grows above 100'C) sulfur-metabolizing archaebacterium with a reported optimal growth temperature of 105'C (3, 4). To date, this is the highest reported optimal growth temperature for an organism grown in pure culture. P. brockii is a chemolithotroph, fixing CO2 for cell mass and oxidizing H2 for energy generation (5). The necessity of H2 for growth and metabolism suggests that a hydrogen-uptake hydrogenase is present in P. brockii.The work described here involved a comparison between the hydrogen-uptake system of P. brockii with that of the mesophilic bacterium Bradyrhizobium japonicum. Among the issues addressed were optimal temperature for hydrogen uptake, ability to use artificial electron acceptors, sensitivity to oxygen, and cellular localization. Furthermore, the hydrogenases from the two bacteria were compared on a structural (immunological) and genetic (DNA) level. In addition, we describe a formula...

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The effect of hydrogen and carbon dioxide partial pressure on the growth of the extremely thermophilic archaebacterium Pyrodictium brockii at 98 degrees C was investigated. Previous work with this bacterium has been done using an 80:20 hydrogen-carbon dioxide gas phase with a total pressure of 4 atm; no attempt has been made to determine if this mixture is optimal. It was found in this study that reduced hydrogen partial pressures affected cell yield, growth rate, and sulfide production. The effect of hydrogen partial pressure on cell yield and growth rate was less dramatic when compared to the effect on sulfide production, which was not found to be growth-associated. Carbon dioxide was also found to affect growth but only at very low partial pressures. The relationship between growth rate and substrate concentration could be correlated with a Monod-type expression for either carbon dioxide or hydrogen as the limiting substrate. The results from this study indicate that a balance must be struck between cell yields and sulfide production in choosing an optimal hydrogen partial pressure for the growth of P. brockii.