Effect of the Deletion of qmoABC and the Promoter-Distal Gene Encoding a Hypothetical Protein on Sulfate Reduction in Desulfovibrio vulgaris Hildenborough

Abstract

The pathway of electrons required for the reduction of sulfate in sulfate-reducing bacteria (SRB) is not yet fully characterized. In order to determine the role of a transmembrane protein complex suggested to be involved in this process, a deletion in Desulfovibrio vulgaris Hildenborough was created by marker exchange mutagenesis that eliminated four genes putatively encoding the QmoABC complex and a hypothetical protein (DVU0851). The Qmo (quinone-interacting membrane-bound oxidoreductase) complex is proposed to be responsible for transporting electrons to the dissimilatory adenosine-5-phosphosulfate reductase in SRB. In support of the predicted role of this complex, the deletion mutant was unable to grow using sulfate as its sole electron acceptor with a range of electron donors. To explore a possible role for the hypothetical protein in sulfate reduction, a second mutant was constructed that had lost only the gene that codes for the DVU0851 protein. The second constructed mutant grew with sulfate as the sole electron acceptor; however, there was a lag that was not present with the wild-type or complemented strain. Neither deletion strain was significantly impaired for growth with sulfite or thiosulfate as the terminal electron acceptor. Complementation of the ⌬(qmoABC-DVU0851) mutant with all four genes or only the qmoABC genes restored its ability to grow by sulfate respiration. These results confirmed the prediction that the Qmo complex is in the electron pathway for sulfate reduction and revealed that no other transmembrane complex could compensate when Qmo was lacking.

Metabolic niche of a prominent sulfate-reducing human gut bacterium

“…Genes involved in H 2 consumption and sulfate reduction are required for fitness in vivo in both diet contexts; they include (i) a predicted periplasmic [NiFeSe] hydrogenase complex (Dpig-GOR1_1496-DpigGOR1_1497) important in other Desulfovibrio species for growth with H 2 (21); (ii) hydrogenase maturation genes (DpigGOR1_0739-DpigGOR1_0740); (iii) a predicted transport system for nickel, which functions as an important cofactor for the hydrogenase (DpigGOR1_1393-DpigGOR1_1398); (iv) two electron transport systems involved in sulfate reduction in other microbial species, a high molecular weight cytochrome complex, Hmc (DpigGOR1_0741-DpigGOR1_0744) and the QmoABC complex (DpigGOR1_0790-DpigGOR1_0792) (22,23); plus (v) components of sulfite reductase (DpigGOR1_0170-DpigGOR1_0174). These results emphasize the importance of hydrogen metabolism and respiration of sulfate and/or other oxidized sulfur compounds for survival of D. piger in the distal gut and underscore the restricted metabolic options that D. piger has to efficiently generate energy in this environment.…”

Methods for Engineering Sulfate Reducing Bacteria of the Genus Desulfovibrio

“…vulgaris cultures are grown in medium adapted from Postgate (Postgate, 1984) named MOYLS4 medium (Zane et al, 2010) [60 mM sodium lactate, 30mM Na 2 SO 4 , 8 mM MgCl 2 , 20 mM NH 4 Cl, 0.6 mM CaCl 2 , 2 mM phosphate (K 2 HPO 4 /NaH 2 PO4), 60 µM FeCl 2 , 120 mM EDTA, 30 mM Tris (pH 7.4), 0.1% wt/vol yeast extract, 1 ml Thauers vitamin solution per liter (Brandis and Thauer, 1981), and 6 ml trace elements solution per liter, with pH adjusted to 7.2]. The trace elements solution contains 2.5 mM MnCl 2 , 1.26 mM CoCl 2 , 1.47 mM ZnCl 2 , 210 µM Na 2 MoO 4 , 320 µM H 3 BO 3 , 380 µM NiSO 4 , 11.7 µM CuCl 2 , 35 µM Na 2 SeO 3 , and 24 µM Na 2 WO 4 .…”

“…Alternative electron donors or acceptors provide the opportunity of obtaining conditional lethal mutants in other pathways (Zane et al, 2010). Commonly used electron donors:acceptors for D. vulgaris and Desulfovibrio G20 are found in Table II. WARNING: To obtain and test mutants of genes in various metabolic pathways, it may be necessary to grow Desulfovibrio in fermenting conditions (pyruvate only) or dismutating fumarate (Desulfovibrio G20).…”

“…Protocols related to conjugal transfer have been described extensively elsewhere (van Dongen et al, 1994). In contrast, the introduction of DNA into the SRB with methods other than conjugation has been minimally utilized or described (Bender et al, 2007;Keller et al, 2009;Zane et al, 2010). We have successfully employed electroporation with D.…”

“…Once a gene deletion is verified, growth studies in minimal medium are required to determine the effect of the deletion. If a strain with a deletion has a phenotype different than that of the wild-type, the missing gene vulgaris; however, studies have shown that the constitutive kanamycin promoter is sufficient to get expression in cultures Zane et al, 2010). Therefore, complementation plasmids can readily be generated with the vector plasmid pMO9075 (Fig.…”