The energy-converting hydrogenase Ech2 is important for the growth of the thermophilic acetogen Thermoanaerobacter kivui on ferredoxin-dependent substrates

Abstract

Thermoanaerobacter kivui is the thermophilic acetogenic bacterium with the highest temperature optimum (66°C) and with high growth rates on hydrogen (H₂) plus carbon dioxide (CO₂). The bioenergetic model suggests that its redox and energy metabolism depends on energy-converting hydrogenases (Ech). Its genome encodes two Echs, Ech1 and Ech2, as sole coupling sites for energy conservation during growth on H₂ + CO₂. During growth on other substrates, its redox activity, the (proton-gradient-coupled) oxidation of H₂ may be essential to provide reduced ferredoxin (Fd) to the cell. While Ech activity has been demonstrated biochemically, the physiological function of both Ech's is unclear. Toward that, we deleted the complete gene cluster encoding Ech2. Surprisingly, the ech2 mutant grew as fast as the wild type on sugar substrates and H₂ + CO₂. Hence, Ech1 may be the essential enzyme for energy conservation, and either Ech1 or another enzyme may substitute for H₂-dependent Fd reduction during growth on sugar substrates, putatively the H₂-dependent CO₂ reductase (HDCR). Growth on pyruvate and CO, substrates that are oxidized by Fd-dependent enzymes, was significantly impaired, but to a different extent. While ∆ech2 grew well on pyruvate after four transfers, ∆ech2 did not adapt to CO. Cell suspensions of ∆ech2 converted pyruvate to acetate, but no acetate was produced from CO. We analyzed the genome of five T. kivui strains adapted to CO. Strikingly, all strains carried mutations in the hycB3 subunit of HDCR. These mutations are obviously essential for the growth on CO but may inhibit its ability to utilize Fd as substrate.

Importance: Acetogens thrive by converting H₂+CO₂ to acetate. Under environmental conditions, this allows for only very little energy to be conserved (∆G'<-20 kJ mol^-1). CO₂ serves as a terminal electron acceptor in the ancient Wood-Ljungdahl pathway (WLP). Since the WLP is ATP neutral, energy conservation during growth on H₂ + CO₂ is dependent on the redox metabolism. Two types of acetogens can be distinguished, Rnf- and Ech-type. The function of both membrane-bound enzyme complexes is twofold-energy conversion and redox balancing. Ech couples the Fd-dependent reduction of protons to H₂ to the formation of a proton gradient in the thermophilic bacterium Thermoanaerobacter kivui. This bacterium may be utilized in gas fermentation at high temperatures, due to very high conversion rates and the availability of genetic tools. The physiological function of an Ech hydrogenase in T. kivui was studied to contribute an understanding of its energy and redox metabolism, a prerequisite for future industrial applications.

Keywords: Thermoanaerobacter kivui; acetogen; carbon monoxide; energy-converting hydrogenase; ferredoxin; pyruvate; thermophilic.

Fig 1

(A) Deletion of the entire operon encoding Ech2 via two independent homologous recombination events using plasmid pEch2TK02. Plasmid pEch2Tk02 contained homologous regions for integration at the 5′ or 3′ region flanking the ech2 operon (UFR, upstream flanking region; DFR, downstream flanking region) into the parent strain, T. kivui TKV002 (TKV_MB002) lacking pyrE; and ech2 operon replacement via homologous recombination. For methodological details, see Material and Methods. (B) 1% Agarose gel stained with midori green (Biozym, Hessisch Oldendorf, Germany). The loss of ech2 (6,770 bp) was verified by PCR using primers binding outside of the ech2 cluster. Shown is the electrophoretic separation of the DNA fragments from the PCRs using cells of T. kivui wild type (WT) (expected fragment size, 8.8 kbp) and ∆ech2 mutant (expected fragment size, 2.0 kbp).

Fig 2

Model of acetogenesis from glucose in Thermoanaerobacter kivui (A) wild type (3) and (B) ech2 mutant. (A) Glucose oxidation provides the required reducing equivalents for the reduction of CO₂ in the WLP. The electron-bifurcating transhydrogenase Nfn concomitantly oxidizes NADH and Fd_red from glucose and pyruvate oxidation to provide NADPH to the WLP and to the electron-bifurcating hydrogenase (HydABC) that oxidizes NADPH and Fd_red. The produced H₂ is used by HDCR in the WLP. To balance redox carriers, the energy-conserving hydrogenase (Ech complex) oxidizes H₂ to reduce ferredoxin (Fd_ox). (B) Hypothetical takeover of Ech2 function by the HDCR. Ech1-MetFV is assumed to translocate 1 + x H⁺ across the membrane. The generated proton motive force is used to synthesize ATP via ATP synthase.

Fig 3

Model of acetogenesis from H₂ + CO₂ in Thermoanaerobacter kivui (A) wild type (3) and (B) ech2 mutant. (A) H₂ oxidation provides the required reducing equivalents for the reduction of CO₂ in the WLP. The electron-bifurcating hydrogenase (HydABC) and the energy-conserving hydrogenase (Ech complex) oxidize H₂ to reduce ferredoxin (Fd_ox) and NADP⁺ to Fd_red and NADPH. (B) In the ech2 mutant, HDCR may catalyze the Fd-dependent H₂ oxidation. Ech1-MetFV is assumed to translocate 1 + x H⁺ across the membrane. The generated proton motive force is used to synthesize ATP via ATP synthase.

Fig 4

(A) Representative growth curve of the T. kivui Δech2 mutant (gray) and the wild type, strain DSM 2030 (black) with 25 mM glucose. A representative growth curve is shown. (B) Concentrations of glucose (continuous line) and acetate (dashed line). All experiments were performed on complex medium at 65°C and 160 rpm (n = 3).

Fig 5

Representative growth curve of the T. kivui Δech2 mutant (gray) and the wild type, strain DSM 2030 (black) with (A) 25 mM mannitol and (B) 25 mM fructose. All experiments were performed on complex medium at 65°C and 160 rpm (n = 3).

Fig 6

Specific growth rate (h⁻¹) of T. kivui Δech2 mutant (grey) and the wild type, strain DSM 2030 (black) grown on complex medium containing glucose (n = 8), fructose (n = 3), mannitol (n = 7) (25 mM each) pyruvate (50 mM; n = 3), H₂/CO₂ (3 atm; 66/33 vol/vol; n = 4) or H₂/CO₂/CO (3 atm; 44/22/33; vol/vol/vol; n = 3) or defined medium containing glucose or mannitol (25 mM each; n = 3). The P(T <= t) (n.s. = P > .05, * =P ≤ .05 or ** =P ≤ .01) was calculated by a t-test (one-tailed and two-sample unequal variance).

Fig 7

Representative growth curve of the T. kivui Δech2 mutant (gray) and the wild type, strain DSM 2030 (black) with (A) 25 mM glucose and (B) 25 mM mannitol. All experiments were performed on defined medium at 65°C and 160 rpm (n = 3).

Fig 8

(A) Representative growth curve of the T. kivui strain Δech2 mutant (gray) and the wild-type strain DSM 2030 (black) in the presence of 3 atm H₂/CO₂ (66/33 vol/vol). H₂/CO₂ was refilled every hour to a pressure of 3 atm. (B) Acetate concentration of wild type (black) and Δech2 mutant (gray) in media of growing cells or (C) resting cells. Resting cells were incubated for 24 h in a minimal medium and 3 atm H₂/CO₂ (66/33 vol/vol). All experiments were performed on a complex medium at 65°C and 160 rpm. Acetate was determined by gas chromatography (n = 4).

Fig 9

(A) Representative growth curve of ∆ech2 mutant on H₂/CO₂/CO. Growth of the wild type (black) and ∆ech2 mutant (gray) in the presence of 3 atm H₂/CO₂/CO. All experiments were performed on complex medium at 65°C and 160 rpm. Gas mix H₂/CO₂/CO (44/22/33; vol/vol/vol; [2 atm H₂/CO₂ (66/33; vol/vol) plus 1 atm pure CO]). (B) Increase of OD₆₀₀ of Δech2 mutant and the wild-type strain, DSM 2030 in the presence of CO/N₂ (3 atm; 14/86 vol/vol). The Δech2 mutant and the wild type were inoculated with an OD₆₀₀ of around 0.01 (passage 1; solid line), while passage 2 (dashed line) was inoculated with an OD₆₀₀ of 0.001 after 23 days from passage 1. All experiments were performed on complex medium at 65°C and 160 rpm (n = 3).

Fig 10

(A) First transfer of the Δech2 mutant and wild type from glucose to pyruvate. Growth of the T. kivui strain Δech2 mutant (gray) and the wild-type strain DSM 2030 (black) in the presence of 50 mM pyruvate (continuous line) or 25 mM glucose (dashed line). The experiment was performed in Hungate tubes with 5 mL complex medium at 65°C. (B) Growth of the T. kivui Δech2 mutant (gray) and the wild type, strain DSM 2030, (black) in the presence of 50 mM pyruvate (fourth transfer on pyruvate). All experiments were performed on a complex medium at 65°C and 160 rpm (n = 3±SD).

Fig 11

Resting cell experiment of the wild type (black) and ech2 mutant (gray). Incubated for 24 h in Tris-HCL buffer with ca. 100 mM pyruvate at 65 C. (A) Concentration changes in pyruvate (solid line), acetate (dashed line), and formate (dotted line) over 3 h. (B) Concentration at the beginning and after 24 h of pyruvate (filled column), acetate (striped column), and formate (tiled column). Acetate and formate concentrations at the beginning of the experiment were below 0.75 mM, while almost all pyruvate was consumed within 24 h.

Fig 12

Model of acetogenesis from pyruvate in Thermoanaerobacter kivui (A) wild type (modified from (3)) and (B) ech2 mutant. (A) pyruvate oxidation provides the required reducing equivalents for the reduction of CO₂ in the WLP. The electron-bifurcating hydrogenase (HydABC) oxidizes H₂ to provide Fd_red and NADPH and the energy-conserving hydrogenase (Ech complex) oxidize Fd_red to Fd_ox and reduce H⁺ to H₂. (B) Hypothetical replacement of Ech2 redox activities by HDCR. Ech1-MetFV is assumed to translocate 1 + x H⁺ across the membrane. The generated proton motive force is used to synthesize ATP via ATP synthase.