Membrane-anchored HDCR nanowires drive hydrogen-powered CO2 fixation.

Filamentous enzymes have been found in all domains of life, but the advantage of filamentation is often elusive1. Some anaerobic, autotrophic bacteria have an unusual filamentous enzyme for CO2 fixation-hydrogen-dependent CO2 reductase (HDCR)2,3-which directly converts H2 and CO2 into formic acid. HDCR reduces CO2 with a higher activity than any other known biological or chemical catalyst4,5, and it has therefore gained considerable interest in two areas of global relevance: hydrogen storage and combating climate change by capturing atmospheric CO2. However, the mechanistic basis of the high catalytic turnover rate of HDCR has remained unknown. Here we use cryo-electron microscopy to reveal the structure of a short HDCR filament from the acetogenic bacterium Thermoanaerobacter kivui. The minimum repeating unit is a hexamer that consists of a formate dehydrogenase (FdhF) and two hydrogenases (HydA2) bound around a central core of hydrogenase Fe-S subunits, one HycB3 and two HycB4. These small bacterial polyferredoxin-like proteins oligomerize through their C-terminal helices to form the backbone of the filament. By combining structure-directed mutagenesis with enzymatic analysis, we show that filamentation and rapid electron transfer through the filament enhance the activity of HDCR. To investigate the structure of HDCR in situ, we imaged T. kivui cells with cryo-electron tomography and found that HDCR filaments bundle into large ring-shaped superstructures attached to the plasma membrane. This supramolecular organization may further enhance the stability and connectivity of HDCR to form a specialized metabolic subcompartment within the cell.

A genome-guided analysis of energy conservation in the thermophilic, cytochrome-free acetogenic bacterium Thermoanaerobacter kivui.

BACKGROUND: Acetogenic bacteria are able to use CO2 as terminal electron acceptor of an anaerobic respiration, thereby producing acetate with electrons coming from H2. Due to this feature, acetogens came into focus as platforms to produce biocommodities from waste gases such as H2+CO2 and/or CO. A prerequisite for metabolic engineering is a detailed understanding of the mechanisms of ATP synthesis and electron-transfer reactions to ensure redox homeostasis. Acetogenesis involves the reduction of CO2 to acetate via soluble enzymes and is coupled to energy conservation by a chemiosmotic mechanism. The membrane-bound module, acting as an ion pump, was of special interest for decades and recently, an Rnf complex was shown to couple electron flow from reduced ferredoxin to NAD+ with the export of Na+ in Acetobacterium woodii. However, not all acetogens have rnf genes in their genome. In order to gain further insights into energy conservation of non-Rnf-containing, thermophilic acetogens, we sequenced the genome of Thermoanaerobacter kivui. RESULTS: The genome of Thermoanaerobacter kivui comprises 2.9 Mbp with a G+C content of 35% and 2,378 protein encoding orfs. Neither autotrophic growth nor acetate formation from H2+CO2 was dependent on Na+ and acetate formation was inhibited by a protonophore, indicating that H+ is used as coupling ion for primary bioenergetics. This is consistent with the finding that the c subunit of the F1FO ATP synthase does not have the conserved Na+ binding motif. A search for potential H+-translocating, membrane-bound protein complexes revealed genes potentially encoding two different proton-reducing, energy-conserving hydrogenases (Ech). CONCLUSIONS: The thermophilic acetogen T. kivui does not use Na+ but H+ for chemiosmotic ATP synthesis. It does not contain cytochromes and the electrochemical proton gradient is most likely established by an energy-conserving hydrogenase (Ech). Its thermophilic nature and the efficient conversion of H2+CO2 make T. kivui an interesting acetogen to be used for the production of biocommodities in industrial micobiology. Furthermore, our experimental data as well as the increasing number of sequenced genomes of acetogenic bacteria supported the new classification of acetogens into two groups: Rnf- and Ech-containing acetogens.

Thermoanaerobacter pseudethanolicus sp. nov., a thermophilic heterotrophic anaerobe from Yellowstone National Park.

Strain 39E(T), originally characterized as Clostridium thermohydrosulfuricum strain 39E and later renamed as Thermoanaerobacter ethanolicus strain 39E, shows less than 97 % 16S rRNA gene sequence similarity with the type strain of the type species of the genus Thermoanaerobacter, T. ethanolicus strain JW 200(T). On the basis of a polyphasic analysis that included DNA-DNA hybridization studies with the subspecies of Thermoanaerobacter brockii, its closest phylogenetic relatives, strain 39E(T) represents a novel species of the genus Thermoanaerobacter, for which the name Thermoanaerobacter pseudethanolicus sp. nov. is proposed. The type strain is 39E(T) (=DSM 2355(T)=ATCC 33223(T)).

Evaluation of continuous ethanol fermentation of dilute-acid corn stover hydrolysate using thermophilic anaerobic bacterium Thermoanaerobacter BG1L1.

Dilute sulfuric acid pretreated corn stover is potential feedstock of industrial interest for second generation fuel ethanol production. However, the toxicity of corn stover hydrolysate (PCS) has been a challenge for fermentation by recombinant xylose fermenting organisms. In this work, the thermophilic anaerobic bacterial strain Thermoanaerobacter BG1L1 was assessed for its ability to ferment undetoxified PCS hydrolysate in a continuous immobilized reactor system at 70 degrees C. The tested strain showed significant resistance to PCS, and substrate concentrations up to 15% total solids (TS) were fermented yielding ethanol of 0.39-0.42 g/g-sugars consumed. Xylose was nearly completely utilized (89-98%) for PCS up to 10% TS, whereas at 15% TS, xylose conversion was lowered to 67%. The reactor was operated continuously for 135 days, and no contamination was seen without the use of any agent for preventing bacterial infections. This study demonstrated that the use of immobilized thermophilic anaerobic bacteria for continuous ethanol fermentation could be promising in a commercial ethanol process in terms of system stability to process hardiness and reactor contamination. The tested microorganism has considerable potential to be a novel candidate for lignocellulose bioconversion into ethanol.

Thermosediminibacter oceani gen. nov., sp. nov. and Thermosediminibacter litoriperuensis sp. nov., new anaerobic thermophilic bacteria isolated from Peru Margin.

A new group of anaerobic thermophilic bacteria was isolated from enrichment cultures obtained from deep sea sediments of Peru Margin collected during Leg 201 of the Ocean Drilling Program. A total of ten isolates were obtained from cores of 1-2 m below seafloor (mbsf) incubated at 60 degrees C: three isolates came from the sediment 426 m below sea level with a surface temperature of 9 degrees C (Site 1227), one from 252 m below sea level with a temperature of 12 degrees C (Site 1228), and six isolates under sulfate-reducing condition from the lower slope of the Peru Trench (Site 1230). Strain JW/IW-1228P from the Site 1228 and strain JW/YJL-1230-7/2 from the Site 1230 were chosen as representatives of the two identified clades. Based on the 16S rDNA sequence analysis, these isolates represent a novel group with Thermovenabulum and Caldanaerobacter as their closest relatives. The temperature range for growth was 52-76 degrees C with an optimum at around 68 degrees C for JW/IW-1228P and 43-76 degrees C with an optimum at around 64 degrees C for JW/YJL-1230-7/2. The pH(25C) range for growth was from 6.3 to 9.3 with an optimum at 7.5 for JW/IW-1228P and from 5 to 9.5 with an optimum at 7.9-8.4 for JW/YJL-1230-7/2. The salinity range for growth was from 0% to 6% (w/v) for JW/IW-1228P and from 0% to 4.5% (w/v) for JW/YJL-1230-7/2. The G+C [corrected] mol% of the genomic DNA was 46.3 +/- 0.7% (n = 4) for Thermosediminibacter oceani [corrected] JW/IW-1228PT [corrected] and 45.2 +/- 0.7 (n = 6) for Thermosediminibacter litoriperuensis [corrected] JW/YJL-1230-7/2T [corrected] DNA-DNA hybridization yielded 52% similarity between the two strains. According to 16S rRNA gene sequence analysis, the isolates are located within the family, Thermoanaerobacteriaceae. Based on their morphological and physiological properties and phylogenetic analysis, it is proposed that strain JW/IW-1228P(T) is placed into a novel taxa, Thermosediminibacter oceani, gen. nov., sp. nov. (DSM 16646(T)=ATCC BAA-1034(T)), and JW/YJL-1230-7/2(T) into Thermosediminibacter litoriperuensis sp. nov. (DSM 16647(T) =ATCC BAA-1035(T)).