Respiration and growth of Shewanella oneidensis MR-1 using vanadate as the sole electron acceptor

Abstract

Shewanella oneidensis MR-1 is a free-living gram-negative gamma-proteobacterium that is able to use a large number of oxidizing molecules, including fumarate, nitrate, dimethyl sulfoxide, trimethylamine N-oxide, nitrite, and insoluble iron and manganese oxides, to drive anaerobic respiration. Here we show that S. oneidensis MR-1 is able to grow on vanadate as the sole electron acceptor. Oxidant pulse experiments demonstrated that proton translocation across the cytoplasmic membrane occurs during vanadate reduction. Proton translocation is abolished in the presence of protonophores and the inhibitors 2-heptyl-4-hydroxyquinoline N-oxide and antimycin A. Redox difference spectra indicated the involvement of membrane-bound menaquinone and cytochromes c, which was confirmed by transposon mutagenesis and screening for a vanadate reduction-deficient phenotype. Two mutants which are deficient in menaquinone synthesis were isolated. Another mutant with disruption in the cytochrome c maturation gene ccmA was unable to produce any cytochrome c and to grow on vanadate. This phenotype could be restored by complementation with the pEC86 plasmid expressing ccm genes from Escherichia coli. To our knowledge, this is the first report of E. coli ccm genes being functional in another organism. Analysis of an mtrB-deficient mutant confirmed the results of a previous paper indicating that OmcB may function as a vanadate reductase or may be part of a vanadate reductase complex.

FIG. 1.

Vanadate reduction capacity of S. oneidensis MR-1 cells grown in SM medium either aerobically or anaerobically on fumarate, KNO₃, Fe(III) citrate, TMAO, or V(V) (in VM medium). Values were obtained from the initial reduction rates, as described previously (9) and are expressed as mM V(V) reduced per gram (wet weight) of cells per hour.

FIG. 2.

(A) Proton pulse traces obtained with fumarate-grown S. oneidensis MR-1 cells in response to 100 nmol ferric citrate, fumarate, and vanadate. (B) Pulse traces from 100 nmol vanadate in the presence of dinitrophenol (DNP), CCCP, HOQNO, antimycin A, rotenone, and cyanide at the concentrations indicated.

FIG. 3.

(A) Anaerobic growth in VM medium under an N₂ atmosphere (▴) and concomitant reduction of 5 mM V(V) (⧫); V(V) reduction with cells omitted (□); and growth with vanadate omitted (▵). (B) Cellular protein formed using 0.5 (▪), 1 (▴), 2.5 (▾), and 5 (♦) mM of vanadate as electron acceptor in VM medium under an N₂ atmosphere. All measurements at each time were obtained by calculating the average values from at least three independent incubations.

FIG. 4.

Redox difference spectra [V(V) oxidized minus reduced] of the membrane fraction of S. oneidensis MR-1. The difference spectra were recorded in the quinone (A) and cytochrome (B) absorption ranges. AU, absorption units.

FIG. 5.

(A and B) Anaerobic growth in VM medium (A) and concomitant reduction of vanadate (B) for VRD1 (□), VRD2 (▴), MR-1R (▪), VRD1 (▿) amended with 50 μM vitamin K₂, and VRD2 (▵) amended with 50 μM vitamin K₂. (C and D) Growth of (C) and concomitant reduction of vanadate by (D) mutant VRD3 (♦) and VRD3/pEC86 (▵), compared to MR-1R (▪). (E and F) Growth of (E) and concomitant reduction of vanadate by (F) mutant VRD4 (▾), compared to MR-1R (▴). All measurements at each time were obtained by calculating the average values for at least three independent incubations.

FIG. 6.

Thin-layer chromatogram of quinone standards and quinones isolated from S. oneidensis cells grown anaerobically with fumarate as the electron acceptor. Lanes 1 and 2 were loaded with ubiquinone-6 and menaquinone-4, respectively. The other lanes were loaded with quinone extracts isolated from cells of the following strains: lane 3, MR-1R; lane 4, VRD1; lane 5, MR-1R; lane 6, VRD2. Slight differences in migration of quinone standards compared to the quinones from MR-1R were due to differences in the length and composition of the isoprenyl side chain. Spots I (methylmenaquinone), II (menaquinone), and III to V (ubiquinones) were identified by relative migration (27).

FIG. 7.

(A) Heme-stained SDS-PAGE analysis of loosely attached proteins from cells grown anaerobically on fumarate. Equal amounts (∼30 μg protein) of sample were loaded. Lane 1, molecular weight reference sample; lane 2, S. oneidensis MR1-R protein extract; lane 3, mutant VRD4 protein extract. X indicates differences at ∼65 and ∼90 kDa. (B) Heme-stained SDS-PAGE analysis of total cellular protein of cells grown aerobically in LB broth. Lane 1, molecular weight reference sample; lane 2, S. oneidensis MR1-R; lane 3, VRD3; lane 4, VRD3/pEC86.