Cytochrome c maturation and the physiological role of c-type cytochromes in Vibrio cholerae

Abstract

Vibrio cholerae lives in different habitats, varying from aquatic ecosystems to the human intestinal tract. The organism has acquired a set of electron transport pathways for aerobic and anaerobic respiration that enable adaptation to the various environmental conditions. We have inactivated the V. cholerae ccmE gene, which is required for cytochrome c biogenesis. The resulting strain is deficient of all c-type cytochromes and allows us to characterize the physiological role of these proteins. Under aerobic conditions in rich medium, V. cholerae produces at least six c-type cytochromes, none of which is required for growth. Wild-type V. cholerae produces active fumarate reductase, trimethylamine N-oxide reductase, cbb3 oxidase, and nitrate reductase, of which only the fumarate reductase does not require maturation of c-type cytochromes. The reduction of nitrate in the medium resulted in the accumulation of nitrite, which is toxic for the cells. This suggests that V. cholerae is able to scavenge nitrate from the environment only in the presence of other nitrite-reducing organisms. The phenotypes of cytochrome c-deficient V. cholerae were used in a transposon mutagenesis screening to search for additional genes required for cytochrome c maturation. Over 55,000 mutants were analyzed for nitrate reductase and cbb3 oxidase activity. No transposon insertions other than those within the ccm genes for cytochrome c maturation and the dsbD gene, which encodes a disulphide bond reductase, were found. In addition, the role of a novel CcdA-like protein in cbb3 oxidase assembly is discussed.

FIG. 1.

Predicted respiratory electron transport chains of V. cholerea. Electrons are transported from the quinol pool (QH₂) to the terminal electron acceptors indicated. Arrows with dashed lines show electron transport chains with c-type cytochrome subunits. BSO, biotin sulfoxide.

FIG. 2.

CcmE is required for cytochrome c maturation in V. cholerae. (A) Heme stain of membrane (lanes 1 to 3) and soluble (lanes 4 to 6) proteins (100 μg per lane) of wild-type strain VC593 (lanes 1 and 4) and ccmE-mutant strains VC595 (lanes 2 and 5) and VC596 (lanes 3 and 6) separated by SDS-15% PAGE. Molecular mass marker bands (in kilodaltons) are indicated on the left. (B) Optical difference spectra of membrane proteins (5 mg ml⁻¹) in 50 mM Tris, pH 7.5. Dithionite-reduced minus air-oxidized spectra of the wild-type strain VC593 and the ccmE-mutant VC595 are shown. The difference spectrum of VC595 was subtracted from that of VC593, resulting in the double difference spectrum (dd). Inset, the α/β region at a higher magnification.

FIG. 3.

CcmE of V. cholerae binds heme in the presence of CcmA-D. E. coli EC06 (ΔccmA-H) was transformed with plasmids encoding the V. cholerae (Vc) proteins CcmE (pVC11, lane 1), CcmA-D (pVC60, lane 2), CcmA-E (pVC70, lane 3), and CcmC-E (pVC5, lane 4) or the E. coli (Ec) proteins CcmC-E (pEC406, lane 5) as the control.

FIG. 4.

Respiratory chains present in V. cholerae. (A) Anaerobic growth of the wild-type strain VC593 (open bars) and ccmE-mutant strain VC596 (black bars) in M63 containing 0.2% glycerol and the indicated final electron acceptors (blank, no electron acceptor; Fum, fumarate). Growth of the wild-type strain in the presence of fumarate was taken as 100%. The average growth of three independent cultures is shown, and standard deviations are indicated. (B) Nitrite accumulation. Wild-type VC593 (open bars) and ccmE-mutant VC596 (black bars) strains were grown aerobically in M63 medium containing 0.2% glucose and 0.2% glycerol in the presence of the KNO₃ concentrations indicated. Nitrite accumulation was determined after overnight growth at 37°C.

FIG. 5.

Type c cytochromes of mutant V. cholerae strains. Membrane extracts containing 200 μg protein of the indicated strains were separated by SDS-PAGE in a 15% polyacrylamide gel. The gel was treated with o-dianisidine and H₂O₂ to identify polypeptides possessing covalently bound heme groups. The electrophoretic mobility of marker proteins is indicated on the left. The positions of CcoP and CcoO are indicated on the right.