Cytochrome bd oxidase, oxidative stress, and dioxygen tolerance of the strictly anaerobic bacterium Moorella thermoacetica

Abstract

The gram-positive, thermophilic, acetogenic bacterium Moorella thermoacetica can reduce CO2 to acetate via the Wood-Ljungdahl (acetyl coenzyme A synthesis) pathway. This report demonstrates that, despite its classification as a strict anaerobe, M. thermoacetica contains a membrane-bound cytochrome bd oxidase that can catalyze reduction of low levels of dioxygen. Whole-cell suspensions of M. thermoacetica had significant endogenous O2 uptake activity, and this activity was increased in the presence of methanol or CO, which are substrates in the Wood-Ljungdahl pathway. Cyanide and azide strongly (approximately 70%) inhibited both the endogenous and CO/methanol-dependent O2 uptake. UV-visible light absorption and electron paramagnetic resonance spectra of n-dodecyl-beta-maltoside extracts of M. thermoacetica membranes showed the presence of a cytochrome bd oxidase complex containing cytochrome b561, cytochrome b595, and cytochrome d (chlorin). Subunits I and II of the bd oxidase were identified by N-terminal amino acid sequencing. The M. thermoacetica cytochrome bd oxidase exhibited cyanide-sensitive quinol oxidase activity. The M. thermoacetica cytochrome bd (cyd) operon consists of four genes, encoding subunits I and II along with two ABC-type transporter proteins, homologs of which in other bacteria are required for assembly of the bd complex. The level of this cyd operon transcript was significantly increased when M. thermoacetica was grown in the absence of added reducing agent (cysteine + H2S). Expression of a 35-kDa cytosolic protein, identified as a cysteine synthase (CysK), was also induced by the nonreducing growth conditions. The combined evidence indicates that cytochrome bd oxidase and cysteine synthase protect against oxidative stress and contribute to the limited dioxygen tolerance of M. thermoacetica.

FIG. 1.

Composition of the M. thermoacetica cytochrome bd oxidase operon (cydABDC) and the relationship between the genes and their products. The direction of transcription is from left to right. AA, amino acid; mol. wt., molecular weight.

FIG. 2.

Alignment of the deduced amino acid sequences at the COOH-terminal end of CydAs from M. thermoacetica (MtCydA), B. subtilis (BsCydA; accession no. A69611), M. tuberculosis (MyCydA; accession no. E70558), E. coli (EcCydA; accession no. S17958), A. vinelandii (AvCydA; accession no. A38170), and V. cholerae (VcCydA; accession no. E82149).

FIG. 3.

(A) Northern blot of total RNA (5 μg in each lane) isolated from M. thermoacetica grown on methanol in the presence (+R) or absence (−R) of reducing agent after hybridization with a DIG-labeled DNA fragments amplified from cydA (A) of the M. thermoacetica cyd operon. The positions of RNA size standards are indicated on the left. The probe also hybridized nonspecifically to 23S (2.928 kb) and 16S (1.553 kb) rRNA. (B) RNA dot blots of M. thermoacetica total RNA (each spot loaded with 5 μg of DNase-treated RNA) after hybridization with the DIG-labeled DNA fragment amplified from cydA, the F₁-ATPase β-subunit gene (atpD), and the CO dehydrogenase β-subunit gene.

FIG. 4.

PAGE and SDS-PAGE of M. thermoacetica cytochrome bd oxidase. (A) Native PAGE (8% acrylamide in resolving gel) of DM extracts (40 μg of protein) after heme staining with TMBZ. (B) SDS-PAGE of the heme-positive band excised from the native gel shown in panel A after staining with Coomassie brilliant blue. (C) SDS-PAGE of DM extracts (lane 2, 40 μg of protein) and partially purified cytochrome bd oxidase (lane 1, 20 μg of protein).

FIG. 5.

UV-visible absorption spectra of M. thermoacetica cytochrome bd oxidase. The spectra were recorded on DM extracts (4.5 mg of protein/ml) of cholate-washed M. thermoacetica membranes in 0.1 M potassium phosphate (pH 7.5) and 1 mM EDTA. Trace 1 is air oxidized, trace 2 is dithionite reduced, and trace 3 is dithionite reduced minus air oxidized.

FIG. 6.

EPR spectra of as-isolated cytochrome bd oxidases from M. thermoacetica and E. coli. DM extracts of M. thermoacetica or E. coli membranes (20 mg of protein/ml) in 50 mM Tris-HCl (pH 8.0) and 100 mM EDTA. EPR conditions were as follows: temperature, 4.0 K; microwave frequency, 9.602 GHz; microwave power, 2 mW; and modulation amplitude, 6.34 G.

FIG. 7.

Dithionite-reduced + CO − dithionite-reduced UV-visible absorption difference spectra of cytochrome bd oxidase in DM extracts (5.5 mg of protein/ml) of cholate-washed membranes from M. thermoacetica (solid trace) or E. coli (dashed trace).

FIG. 8.

UV-visible absorption difference spectra of M. thermoacetica cytochrome bd oxidase in DM extracts (6 mg of protein/ml). Traces: 1, DQH₂ reduced − air oxidized; 2, air oxidized + cyanide + DQH₂ − air oxidized + cyanide; 3, air oxidized + cyanide − air oxidized; 4, dithionite reduced − air oxidized.

FIG. 9.

Effect of oxidative stress on growth of M. thermoacetica. Cultures were grown on methanol (200 mM) in Drake's medium (65) (80 ml) under 100% CO₂ in anoxic bottles (125-ml capacity) in the presence (trace 1) or absence (traces 2 through 5) of reducing agent (cysteine + H₂S). After 48 h, either cysteine (2.8 mM) or azide (2 mM) was injected (shown by arrow) into replicate cultures grown in the absence of reducing agent followed immediately by injection of air into the headspace to give a final dioxygen concentration of 1.5 vol% (based on 21 vol% dioxygen in air). The cultures were shaken vigorously for 20 s (to equilibrate dioxygen) and then incubated at 58°C. Traces: 2, cysteine + dioxygen; 3, no treatment; 4, dioxygen only; 5, azide (2 mM) + dioxygen. At the times indicated, 1-ml aliquots of the cultures were withdrawn via syringe and centrifuged, and the cell pellets were suspended in equal volumes of 50 mM Tris-HCl, pH 8.0, prior to measurement of the OD₆₀₀. The results are average of three replicate experiments with standard deviations between 0 and 8%.

FIG. 10.

(A) SDS-PAGE (12% acrylamide in resolving gel) of cytosolic extracts (40 μg of protein) of M. thermoacetica grown in the presence (+R) or absence (−R) of reducing agent. Proteins overexpressed in the absence of reducing agent were indicated by arrows. (B) Northern blot of total RNA (5 μg in each lane) isolated from M. thermoacetica grown on methanol in the presence (+R) or absence (−R) of reducing agent after hybridization with DIG-labeled DNA fragments amplified from cysK.