Effects of elemental sulfur on the metabolism of the deep-sea hyperthermophilic archaeon Thermococcus strain ES-1: characterization of a sulfur-regulated, non-heme iron alcohol dehydrogenase

Abstract

The strictly anaerobic archaeon Thermococcus strain ES-1 was recently isolated from near a deep-sea hydrothermal vent. It grows at temperatures up to 91؇C by the fermentation of peptides and reduces elemental sulfur (S 0 ) to H 2 S. It is shown here that the growth rates and cell yields of strain ES-1 are dependent upon the concentration of S 0 in the medium, and no growth was observed in the absence of S 0 . The activities of various catabolic enzymes in cells grown under conditions of sufficient and limiting S 0 concentrations were investigated. These enzymes included alcohol dehydrogenase (ADH); formate benzyl viologen oxidoreductase; hydrogenase; glutamate dehydrogenase; alanine dehydrogenase; aldehyde ferredoxin (Fd) oxidoreductase; formaldehyde Fd oxidoreductase; and coenzyme A-dependent, Fd-linked oxidoreductases specific for pyruvate, indolepyruvate, 2-ketoglutarate, and 2-ketoisovalerate. Of these, changes were observed only with ADH, formate benzyl viologen oxidoreductase, and hydrogenase, the specific activities of which all dramatically increased in cells grown under S 0 limitation. This was accompanied by increased amounts of H 2 and alcohol (ethanol and butanol) from cultures grown with limiting S 0 . Such cells were used to purify ADH to electrophoretic homogeneity. ADH is a homotetramer with a subunit M r of 46,000 and contains 1 g-atom of Fe per subunit, which, as determined by electron paramagnetic resonance analyses, is present as a mixture of ferrous and ferric forms. No other metals or acid-labile sulfide was detected by colorimetric and elemental analyses. ADH utilized NADP(H) as a cofactor and preferentially catalyzed aldehyde reduction. It is proposed that, under S 0 limitation, ADH reduces to alcohols the aldehydes that are generated by fermentation, thereby serving to dispose of excess reductant.Hyperthermophiles are a recently discovered group of microorganisms that have the remarkable property of growing at temperatures of 90ЊC and above (1, 2, 64, 65). They have been isolated from a variety of geothermally heated environments, and almost all are classified as Archaea (formerly Archaeobacteria [75]). The majority are strict anaerobes, and most are obligately dependent upon the reduction of elemental sulfur (S 0 ) to H 2 S for optimal growth. Various organic compounds and molecular H 2 serve as electron donors for the apparent respiration of S 0 . The mechanism of S 0 reduction in one autotrophic hyperthermophile, Pyrodictium brockii, an obligate S 0 -reducing species which grows optimally at 105ЊC, has been investigated. This organism was shown to have a primitive membrane-bound electron transport chain for coupling H 2 oxidation and H 2 S production (52), although the S 0 -reducing entity was not purified.On the other hand, some of the heterotrophic hyperthermophiles are able to grow well in the absence of S 0 . These have fermentative-type metabolisms and produce H 2 during growth, although they also produce H 2 S if S 0 is added to the growth medium. S 0 reduction was thought to be...

Dethiosulfovibrio russensis sp. nov., Dethosulfovibrio marinus sp. nov. and Dethosulfovibrio acidaminovorans sp. nov., novel anaerobic, thiosulfate- and sulfur-reducing bacteria isolated from 'Thiodendron' sulfur mats in different saline environments.

“…Since sulfur and thiosulfate increased both the cell yield and the growth rate in the cultures, the organisms must derive some advantage from the reduction process. As has been found for some Bacteria (Janssen & Morgan, 1992 ;Childers et al, 1992) and Archaea (Adams, 1990 ;Ma et al, 1995 ;Stetter, 1996), there was a significant stimulation of growth by the removal of high concentrations of hydrogen (one of the final products of fermentation). A possible explanation would be that these compounds are used as alternatives to protons by an enzyme similar to sulfhydrogenase, as described for Pyrococcus furiosus (Kelly & Adams, 1994).…”

“…It was demonstrated that all isolates were strictly anaerobic, obligately moderately halophilic, Gram-negative bacteria with a fermentative type of metabolism. Elemental sulfur and thiosulfate were reduced to sulfide during fermentation, but growth of the new isolates was not dependent upon sulfur reduction, as has been shown for some sulfurrespiring mesophilic and thermophilic Bacteria and Archaea (Widdel & Hansen, 1992 ;Schauder & Kro$ ger, 1993 ;Kelly & Adams, 1994 ;Bonch-Osmolovskaya, 1994 ;Ma et al, 1995 ;Stetter, 1996). As has been demonstrated already for members of the genera Dethiosulfovibrio, Thermoanaerobacter, Thermosipho and Haloanaerobium, sulfidogenesis was accompanied by a decrease in the hydrogen concentration in the growth medium and by a shift of the fermentation products to more oxidized compounds, e.g.…”

Purification, characterization, and metabolic function of tungsten-containing aldehyde ferredoxin oxidoreductase from the hyperthermophilic and proteolytic archaeon Thermococcus strain ES-1

“…To address the question of the physiological function of AOR, in this paper we have focused on the novel deep-sea hyperthermophile isolate ES-1 (36), which, by 16S rRNA analysis, has been determined to be a member of the genus Thermococcus (27). Thermococcus strain ES-1 grows at temperatures up to 91ЊC and is an obligately anaerobic heterotroph that reduces S 0 and utilizes proteinaceous materials (peptone, yeast extract, and casein) as a carbon source (36).…”

Two novel metal-independent long-chain alkyl alcohol dehydrogenases from Geobacillus thermodenitrificans NG80-2

“…M-1, which exhibited mainly reductive activity on medium-chain alkyl aldehydes (C 2 to C 14 ), with little oxidative activity detected, and was assumed not to participate in the degradation of long-chain alkanes (Tani et al, 2000). On the other hand, both ADH1 and ADH2 are expressed at similar levels (less than 50 % difference) in NG80-2 cells grown with hexadecane and with sucrose, as indicated by 2-DE analysis (Feng et al, 2007); thus they are are expected to be involved in other cellular processes such as aldehyde detoxication and alcohol fermentation, as for other characterized ADHs (Antoine et al, 1999;Daniel et al, 1995;Hirakawa et al, 2004;Hosaka et al, 2001;Larroy et al, 2003;Ma et al, 1994Ma et al, , 1995Ma & Adams, 1999;Scopes, 1983;Tani et al, 2000;Ying et al, 2007). It is likely that the major activity of ADH1 and ADH2 is diverted to oxidation of long-chain alcohols when long-chain alkanes are utilized as carbon and energy sources.…”

“…Fe-containing ADHs still contain Fe after purification, such as the ADHs from E. coli ECL1 (Montella et al, 2005), Thermotoga hypogea DSM 11164 (Ying et al, 2007), Thermotoga maritima strain MSB8 (Schwarzenbacher et al, 2004) and Thermococcus strain ES-1 (Ma et al, 1995), as well as the recombinant ADH from Thermococcus hydrothermalis strain AL662T (Antoine et al, 1999). Fe-activated ADHs do not contain Fe after purification, but the ion must be present in the assay mixture to obtain ADH activity, as seen for the ADH2 from Z. mobilis ZM4 (Bakshi et al, 1989;Scopes, 1983).…”