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. 2013 Oct;195(19):4466-75.
doi: 10.1128/JB.00679-13. Epub 2013 Aug 2.

New Family of Tungstate-Responsive Transcriptional Regulators in Sulfate-Reducing Bacteria

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Free PMC article

New Family of Tungstate-Responsive Transcriptional Regulators in Sulfate-Reducing Bacteria

Alexey E Kazakov et al. J Bacteriol. .
Free PMC article

Abstract

The trace elements molybdenum and tungsten are essential components of cofactors of many metalloenzymes. However, in sulfate-reducing bacteria, high concentrations of molybdate and tungstate oxyanions inhibit growth, thus requiring the tight regulation of their homeostasis. By a combination of bioinformatic and experimental techniques, we identified a novel regulator family, tungstate-responsive regulator (TunR), controlling the homeostasis of tungstate and molybdate in sulfate-reducing deltaproteobacteria. The effector-sensing domains of these regulators are similar to those of the known molybdate-responsive regulator ModE, while their DNA-binding domains are homologous to XerC/XerD site-specific recombinases. Using a comparative genomics approach, we identified DNA motifs and reconstructed regulons for 40 TunR family members. Positional analysis of TunR sites and putative promoters allowed us to classify most TunR proteins into two groups: (i) activators of modABC genes encoding a high-affinity molybdenum and tungsten transporting system and (ii) repressors of genes for toluene sulfonate uptake (TSUP) family transporters. The activation of modA and modBC genes by TunR in Desulfovibrio vulgaris Hildenborough was confirmed in vivo, and we discovered that the activation was diminished in the presence of tungstate. A predicted 30-bp TunR-binding motif was confirmed by in vitro binding assays. A novel TunR family of bacterial transcriptional factors controls tungstate and molybdate homeostasis in sulfate-reducing deltaproteobacteria. We proposed that TunR proteins participate in protection of the cells from the inhibition by these oxyanions. To our knowledge, this is a unique case of a family of bacterial transcriptional factors evolved from site-specific recombinases.

Figures

Fig 1
Fig 1
Motif logos of deltaproteobacterial dif and Desulfovibrio TunR- and TunR2-binding sites.
Fig 2
Fig 2
Genomic context of genes associated with TunR family members.
Fig 3
Fig 3
Transcription of modA, modB, and tunR is activated by tunR and repressed by tungstate. (A) Effect of addition of molybdate in wild type and GZ6027 (tunR::mini-Tn5); (B) effect of tunR knockout in the presence and absence of molybdate; (C) effect of addition of tungstate in wild type and GZ6027 (tunR::mini-Tn5); (D) effect of tunR knockout in the presence and absence of tungstate.
Fig 4
Fig 4
TunR binds to the predicted DNA motif. Electrophoretic mobility shift assay where TunR (33 pmol) was tested for binding to a DNA fragment (200 fmol) containing the predicted binding site motif. Lanes 1 and 2, wild-type-binding motif; lanes 3 and 4, modified motif; the sequences of the 30-nt motif (total DNA size of 46 bp) are shown with the 8-bp conserved half sites in bold, and the base changes in the modified substrate are underlined.
Fig 5
Fig 5
Domain structure of TunR, ModE, and TunR homologs. Rectangles represent protein domains, and white diamonds show approximate positions of active site residues in PFAM [29] annotations.
Fig 6
Fig 6
Phylogenetic distribution of the TunR family.

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