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Evidence of a chemiosmotic model for halorespiration in desulfomonile tiedjei DCB-1

University of British Columbia

Desulfomonile tiedjeiDCB-1, a sulfate-reducing bacterium, conserves energy for growth from reductive dehalogenation of 3-chlorobenzoate by an uncharacterized anaerobic respiratory process. Different electron carriers and respiratory enzymes of D. tiedjei cells grown under conditions for reductive dehalogenation, pyruvate fermentation and sulfate respiration were therefore examined quantitatively. Only cytochromes c were detected in the soluble and membrane fractions of cells grown under the three conditions. These cytochromes include a constitutively expressed 17-kDa cytochrome c, which was detected in cells grown under all three conditions, and a unique diheme cytochrome c with an apparent molecular mass of 50 kDa, which was present only in the membrane fractions of dehalogenatingcells. This inducible cytochrome c had a very negative midpoint potential of --342 mV. Absorption spectra and the putative gene sequence suggest that the inducible cytochrome c is substantially different from previously characterized cytochromes. Reductive dehalogenation activity of D. tiedjei-was shown to be dependent on 1,4-naphthoquinone, a possible precursor for a respiratory quinone. Moreover, cell suspension experiments indicated that reductive dehalogenation of D. tiedjei was inhibited by the respiratory quinone inhibitor, 2-heptyl-4-hydroxyquinoline JV-oxide, suggesting a respiratory quinone is involved in the electron transport chain coupled to reductive dehalogenation. However, no ubiquinone or menaquinone could be extracted from D. tiedjei. Rather, an UV-absorbing, quinone-like molecule or quinoid, was extracted. The oxidized and reduced UV-absorption spectra of the quinoid were similar in some ways to those of ubiquinones and pyrrolo-quinoline quinones, respectively. But the quinoid was different from these common respiratory quinones in chemical structure according to mass spectrometric analysis. ATP sulfurylase, APS reductase and desulfoviridin, the enzymes involved in sulfate-reduction, appeared to be constitutively expressed in the cytoplasm of D. tiedjei cells grown under the three metabolic conditions. An inducible, periplasmic hydrogenase was detected in cells grown under reductive-dehalogenating and pyruvate-fermenting conditions. An inducible, membrane-bound, periplasm-oriented formate dehydrogenase was active only in cells grown with formate as electron donor; while, a cytoplasmic formate dehydrogenase was detected in cells grown under reductive-dehalogenating and pyruvate-fermenting conditions. Results from dehalogenation assays with D. tiedjei cell suspensions suggest the membrane-bound reductive dehalogenaseis facing the cytoplasm . The inducible cytochrome c, or the quinoid, alone or in combination, failed to replace reduced methyl viologen as the electron donor for the reductive dehalogenase in vitro. The putative gene sequence of the reductive dehalogenase small subunit was determined from inverse PCR products amplified from genomic DNA, but the sequence did not have substantial similarity to any sequences in GenBank. These data clearly demonstrate that D. tiedjei possesses elements necessary for producing protons directly in the periplasm, generating a proton-motive force across the cytoplasmic membrane. However, the data did not exclude the existence of additional transmembrane proton translocation mechanisms, which would further enhance the proton-motive force.

Thesis/Dissertation

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2009-06-24

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http://hdl.handle.net/2429/9564

Microbiology and Immunology

University of British Columbia

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