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

Abstract

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.

Figure 1

The Wood-Ljungdahl pathway in T. kivui . The encoding genes are indicated (A) and the genetic organization is shown in (B). CoFeSP, corrinoid/FeS protein; THF, tetrahydrofolate; [H], reducing equivalent, corresponding to one electron; orf1, probably encoding a thymidylate synthase.

Figure 2

Sequence alignment of c subunits of Na ⁺ -dependent ATP synthases and the subunit c (TKV_c06420) of T. kivui . The Na⁺ binding motif is highlighted in bold.

Figure 3

Effect of Na ⁺ on autotrophic growth of T. kivui . Cultures grown on H₂ + CO₂ were transferred into Na⁺-enriched (●) and Na⁺-deficient (○) minimal medium. The curves shown are representative for three independent experiments. Precultures were grown for four transfers in the same medium.

Figure 4

Acetate formation from H ₂ + CO ₂ by resting cells of T. kivui is inhibited by TCS. Whole cells of T. kivui were incubated with H₂ + CO₂ in buffer containing 50 mM imidazole, 20 mM MgSO₄, 20 mM KCl, 50 mM KHCO₃ and 4 mM DTE (pH 7.0). A: 350 ± 20 μM (■) or 20 mM (▲) NaCl; B: 20 mM NaCl with (▼) or without (▲) 30 μM ETH2120; C: 350 ± 20 μM NaCl with (♦) or without (■) 30 μM TCS. The final protein concentration of the resting cells in the assay was 1 mg/ml. All values are mean from three replicates.

Figure 5

Arrangement of genes in the cluster (A) and model (B) of the Ech-type complex Ech1. Electron flow from reduced ferredoxin to H⁺ and the coupled export of protons is shown. FeS clusters are indicated. *, predicted transmembrane protein (number of transmembrane helices); Fd, ferredoxin; orf1/orf2, encoding small FeS containing proteins (TKV_c01280 and TKV_c01290) with similarity to the N-terminus of MetV.

Figure 6

Arrangement of genes in the gene cluster (A) encoding a second potential Ech-type complex Ech2 (B). FeS clusters are indicated. *, predicted transmembrane protein (number of transmembrane helices); Fd, ferredoxin.

Figure 7

Model of electron and carbon flow in T. kivui when growing autotrophically. Fd_red, reduced ferredoxin; a H⁺/ATP ratio of 12/3 was assumed for the F₁F_O ATP synthase. *, reduction of methylene-THF might occur using an electron donor with a similar redox potential as NADH. CoFeSP, corrinoid/FeS protein.