Rex (encoded by DVU_0916) in Desulfovibrio vulgaris Hildenborough is a repressor of sulfate adenylyl transferase and is regulated by NADH

Abstract

Although the enzymes for dissimilatory sulfate reduction by microbes have been studied, the mechanisms for transcriptional regulation of the encoding genes remain unknown. In a number of bacteria the transcriptional regulator Rex has been shown to play a key role as a repressor of genes producing proteins involved in energy conversion. In the model sulfate-reducing microbe Desulfovibrio vulgaris Hildenborough, the gene DVU_0916 was observed to resemble other known Rex proteins. Therefore, the DVU_0916 protein has been predicted to be a transcriptional repressor of genes encoding proteins that function in the process of sulfate reduction in D. vulgaris Hildenborough. Examination of the deduced DVU_0916 protein identified two domains, one a winged helix DNA-binding domain common for transcription factors, and the other a Rossman fold that could potentially interact with pyridine nucleotides. A deletion of the putative rex gene was made in D. vulgaris Hildenborough, and transcript expression studies of sat, encoding sulfate adenylyl transferase, showed increased levels in the D. vulgaris Hildenborough Rex (RexDvH) mutant relative to the parental strain. The RexDvH-binding site upstream of sat was identified, confirming RexDvH to be a repressor of sat. We established in vitro that the presence of elevated NADH disrupted the interaction between RexDvH and DNA. Examination of the 5' transcriptional start site for the sat mRNA revealed two unique start sites, one for respiring cells that correlated with the RexDvH-binding site and a second for fermenting cells. Collectively, these data support the role of RexDvH as a transcription repressor for sat that senses the redox status of the cell.

FIG 1

EMSA demonstrating specific interaction between Rex_DvH and the predicted Rex_DvH-binding site within the sat promoter. (A) Schematic representation of the sat promoter region drawn approximately to scale. The predicted Rex_DvH-binding site is annotated by an oval. Fragments (A, B, C, and D) used in EMSA are shown with their positions noted. Fragments A (121 bp), B (130 bp), and D (271 bp) were PCR amplified, while fragment C (40 bp) was generated by annealing two oligonucleotides. (B) Native polyacrylamide gel of individual DNA fragments (A, B, C, and D; 1 nM stock prior to column purification) without (−) or with (+) Rex_DvH. An equal concentration of DNA was labeled and then passed over a column to separate the fragments from the rest of the components, i.e., unlabeled nucleotides. Fragment C, the smallest fragment, is below the size cutoff for the column (∼100 bp), and so only a small amount of this fragment is actually recovered compared to the other three larger fragments. Each fragment was eluted in the same volume of buffer, and so the concentration of this smaller fragment is considerably lower than the rest. Therefore, the band intensity for fragment C is less than the others. The lowest band common in all lanes is the dye front.

FIG 2

Two transcription start sites (TSSs) for sat identified dependent on growth substrates. Parental and Rex_DvH mutant strains were grown in medium that would allow for sulfate respiration or pyruvate fermentation. Samples at the early-exponential-growth phase were analyzed for the TSS of sat by 5′-RACE. (A) Schematic representation of sat promoter. The predicted Rex_DvH-binding site is annotated with a yellow oval with nucleotide positions listed relative to the assumed ATG translational start codon of sat in D. vulgaris Hildenborough. Horizontal arrows denote the half-sites (inverted repeats) within the Rex_DvH-binding site. The predicted −35 site is indicated by a green box. TSSs are identified with vertical arrows (and positions) for each sample tested. (B) The Rex-binding site (underlined, highlighted in yellow) and the surrounding region is shown for the promoter sequence of sat of Desulfovibrio strains, with the predicted −35 site displayed (TTGACA, highlighted in green). A TSS (respiration) for D. vulgaris Hildenborough is highlighted in blue.

FIG 3

NADH disrupts interaction between Rex_DvH and the Rex_DvH-binding site. The results of an electrophoretic assay demonstrate the effect of the pyridine nucleotide on Rex_DvH binding. Fragment C (40 bp, including the Rex_DvH-binding site upstream of sat) at 0.1 nM (1 nM prior to column purification) was incubated with Rex_DvH in the presence of a low or high concentration of the specified pyridine nucleotide. The location of the DNA or protein-DNA complex is indicated.

FIG 4

Characterization of alterations to the Rex_DvH-binding site within the sat promoter. (A) List of strains and alterations made to the Rex_DvH-binding site upstream of sat, with a sequence logo of the predicted Rex_DvH-binding site shown above. The Rex_DvH-binding site is underlined, with the alterations made shown in red. An alignment of sequences is shown relative to the assumed translational start codon for sat. The fragments used for EMSA are displayed, along with the estimated K_d (nM) for each 40-bp fragment. N.A., not assessed. The strains examined included the parental strain (JW710), the sat promoter exchange deletion strain (ΔP_sat), the restored promoter strain (P_sat), the conserved “G−147” altered to “A” strain (G−147A), the distal inverted repeat “GTA” altered to “ACG” strain (IR1), the proximal inverted repeat “CAC” altered to “TGT” strain (IR2), and the strain with alterations to both inverted repeat sites (IR1and2). The relative growth of three replicates of mutants and the parental strain by sulfate respiration (B) or pyruvate fermentation (C) was examined. (D) An electrophoretic assay demonstrated Rex_DvH binding to native (fragment C) and altered (C^I, C^II, C^III, and C^IV) Rex_DvH-binding sites. Rex_DvH was added with increasing concentrations (0, 10, 100, 250, 500, 750, 1,000, and 2,000 nM) to each DNA fragment (0.1 nM; 1 nM prior to column purification). The estimated K_d is shown. The location of the DNA or protein-DNA complex is noted. Triangles represent increasing Rex_DvH concentration across the lanes.