Fig 1.
Lactate utilization operon in Desulfovibrio.
A. Lactate is transported inside the cell by a lactate permease (DVU3026, llp). D- (DVU3027-3028, dld-II) and L-lactate dehydrogenases (DVU3032-3033, lldGH) oxidize lactate to pyruvate [6]. Pyruvate is then oxidatively decarboxylated to acetyl-CoA via a pyruvate ferredoxin oxidoreductase (DVU3025, por) [7,8]. Acetyl-CoA is then oxidized to acetate in two steps by phosphotransacetylase (pta) and acetate kinase (ack) enzymes [8]. The operon also contains pta-N gene (DVU3031) encoding the N-terminal domain of phosphotransacetylase, whose function is unknown. B. The DVU3025-3033 lactate utilization operon and the associated lurSR two- component system are conserved across Desulfovibrio and related species (also see S1 Table). Gene numbers are indicated above the gene. Genes are color coded according to the key. Gut isolates such as D. piger have a highly reduced operon and lack lurSR. C. DAP-chip revealed a regulatory network where four response regulators–LurR, NrfR, PhoB, and DVU0539 –target the DVU3025-3033 genes (colored circles–see key) [9]. Other gene targets are shown in grey circles, and arrows indicate regulatory interactions between an RR and its target. Figure generated using Cytoscape [10].
Fig 2.
Deletion in lurR affects growth and lactate consumption.
A and C. Growth on lactate-sulfate (A) or pyruvate-sulfate (C) monitored as OD600 readings for WT, ΔlurR and ΔlurR complemented. Data are average for three biological replicates; error bars indicate standard deviation (For growth curves in log scale, please see S1 Fig). B and D. Lactate/pyruvate consumption and acetate production monitored by HPLC during growth on lactate-sulfate (B) and pyruvate-sulfate (D). Data are average for three biological replicates; error bars indicate standard deviation.
Fig 3.
LurR activates lactate utilization genes.
Fold changes in expression of select genes were measured by RT-qPCR during growth on lactate-sulfate (LS) or pyruvate-sulfate (PS) of ΔlurR and ΔlurR complemented strains relative to that of WT. Expression was normalized to that of two reference genes, rpoD and rpoH. Data are the average from three independent experiments, each with three biological replicates, and error indicates standard deviation.
Fig 4.
Electrophoretic mobility shift assays with promoter deletions of por.
The upstream region of the lactate utilization operon contains a σ54-dependent promoter (orange box, ATTGGCACATTTCTTGTTA), a predicted binding site for PhoB (green box, AGGTTACAGCATAGTTAC), and three 16 bp binding sites for LurR (red boxes, ATCCGCTTTTTCAGAC, GTCCGCTTTTCAAGAC, and GTCCACTTTTTCAGAC). Five biotin-labeled DNA promoter substrates (I to V) of decreasing lengths (426, 348, 283, 220 and 159 bp) were used in EMSAs with purified His-tagged protein. Protein concentrations used were LurR—2.5, 1, 0.5 pmol; NrfR– 10 and 5 pmol; PhoB– 250 and 125 pmol.
Fig 5.
Validation of predicted binding sites.
A. Comparison of NrfR and LurR binding motifs. Motif images were generated using Weblogo [20]. B. NrfR shifts LurR motif. The top strands of the DNA substrates used are shown on top (wt = wild-type; mut = modified). Bases in bold indicate the conserved motif positions, and bases in red indicate the modified bases in the mutated substrate. Lanes 1–5: por wt motif; lanes 6–10: por mut motif. Lanes 1, 6 –DNA only; lanes 2, 7–1 pmol of LurR; lanes 3, 8–0.5 pmol of LurR; lanes 4, 9–25 pmol of NrfR; and lanes 5, 10–10 pmol of NrfR. C. The motif on top indicates the 18 bp consensus PhoB binding sequence [9]. Gel-shift assays with purified His-tagged PhoB protein and the predicted PhoB binding site upstream of por. Lanes 1–3: WT substrate (conserved bases are shown in bold); lanes 4–6: mutated substrate (underlined bases indicate substitutions made in the conserved positions); lanes 1 and 4: No protein; lanes 2 and 5: 250 pmol PhoB; lanes 3 and 6: 125 pmol PhoB.