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, 7 (11), e48015

Crystal Structures of Two Transcriptional Regulators From Bacillus Cereus Define the Conserved Structural Features of a PadR Subfamily

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Crystal Structures of Two Transcriptional Regulators From Bacillus Cereus Define the Conserved Structural Features of a PadR Subfamily

Guntur Fibriansah et al. PLoS One.

Abstract

PadR-like transcriptional regulators form a structurally-related family of proteins that control the expression of genes associated with detoxification, virulence and multi-drug resistance in bacteria. Only a few members of this family have been studied by genetic, biochemical and biophysical methods, and their structure/function relationships are still largely undefined. Here, we report the crystal structures of two PadR-like proteins from Bacillus cereus, which we named bcPadR1 and bcPadR2 (products of gene loci BC4206 and BCE3449 in strains ATCC 14579 and ATCC 10987, respectively). BC4206, together with its neighboring gene BC4207, was previously shown to become significantly upregulated in presence of the bacteriocin AS-48. DNA mobility shift assays reveal that bcPadR1 binds to a 250 bp intergenic region containing the putative BC4206-BC4207 promoter sequence, while in-situ expression of bcPadR1 decreases bacteriocin tolerance, together suggesting a role for bcPadR1 as repressor of BC4206-BC4207 transcription. The function of bcPadR2 (48% identical in sequence to bcPadR1) is unknown, but the location of its gene just upstream from genes encoding a putative antibiotic ABC efflux pump, suggests a role in regulating antibiotic resistance. The bcPadR proteins are structurally similar to LmrR, a PadR-like transcription regulator in Lactococcus lactis that controls expression of a multidrug ABC transporter via a mechanism of multidrug binding and induction. Together these proteins define a subfamily of conserved, relatively small PadR proteins characterized by a single C-terminal helix for dimerization. Unlike LmrR, bcPadR1 and bcPadR2 lack a central pore for ligand binding, making it unclear whether the transcriptional regulatory roles of bcPadR1 and bcPadR2 involve direct ligand recognition and induction.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Genomic neighborhood of the bcPadR1 and bcPadr2 encoding genes and analysis of promoter binding.
(A) Organization of the BC4206(bcPadR1)-BC4207 operon of B. cereus ATCC 14579, the putative BCE3449(bcPadR2)-BCE3448-BCE3447-BCE3446 regulon of B. cereus ATCC 10987 and the lmrR-lmrCD regulon of L. lactis. The relative scale of the genes and intergenic regions is proportional to nucleotide length. Boxes indicate the operon/regulon boundaries. The PadR-encoding genes are colored in yellow, while their (putative) target genes, encoding resistance-associated membrane proteins, are in green. (B) The sequence of the intergenic region between BC4205 and BC4206 that is used for the EMSA experiments. Putative -35 and -10 promoter sequences of BC4206 are underlined, the start codons of BC4205 and BC4206 are highlighted in bold, and the amino acids of the coded proteins are indicated above the DNA sequence. The putative binding site of bcPadR1 (homologous to the canonical ATGT/ACAT inverted sequence motif) is highlighted in blue. (C) EMSA experiments with bcPadR1. DNA fragments encompassing the promoter regions of BC4206 (lanes 1–10) and BC4029 (lanes 11–13, negative control) were prepared by PCR and end labeled with 33P. DNA binding was assayed as described in the Materials and Methods. Lanes 1, 10 and 11 contain the free DNA probe. Samples run on lanes 2 and 12 contain 0.5 µM; lane 3 1 µM; lanes 4–9 and 13 2 µM of purified bcPadR1 protein. Lanes 5 to 9 contains samples including increasing concentrations of AS-48 from 0.5 pM to 0.69 µM.
Figure 2
Figure 2. Multiple sequence alignment of bcPadR1, bcPadR2, and other members of the PadR-s2 subfamily.
Only sequences for which structures are available in the PDB are shown in the alignment. The PadR-s2 proteins with unpublished structures are addressed by their PDB entry names: 1XMA, a putative transcriptional regulator from Clostridium thermocellum; 3HHH, a putative transcriptional regulator from Enterococcus faecalis V583; 3L7W, uncharacterized protein SMU.1704 from Streptococcus mutans UA159; 3RI2, a putative transcriptional regulator from Eggerthella lenta DSM 2243. Residues that participate in dimerization (for bcPadR1, bcPadR2, and LmrR) and/or have a role in drug binding (only for LmrR) are indicated by small spheres below the sequences. The consensus sequence is derived from a multiple sequence alignment of 2156 PadR-s2 proteins using as criteria that the conserved residue(s) should be present in at least 50% of the sequences.
Figure 3
Figure 3. Ribbon representations of the bcPadR dimers and selected structural homolog.
(A) bcPadR1, (B) bcPadR2, (C) LmrR (PDB code 3F8B), and (D) RTP, a replication terminator protein from Bacillus subtilis (PDB code 1F4K). For each dimer, one of the subunits is shown in a rainbow color gradient from the N-terminus (blue) to the C-terminus (red), whereas the other subunit is colored grey. Helices involved in dimerization are indicated. Residues Trp91 of bcPadR1, Trp93 of bcPadR2, and Trp96 of LmrR are drawn in stick representation.
Figure 4
Figure 4. Structural comparison between bcPadR1, bcPadR2 and LmrR.
(A) Superpositions of the wHTH domains of bcPadR1 (light-green), bcPadR2 (red), LmrR (blue), and the following homologs: MexR (green), SmtB (magenta), BlaI (cyan), RTP (orange), and Pex (gray) with the secondary structure elements indicated and labeled. (B) Superposition of the single bcPadR1, bcPadR2 and LmR subunits. (D) Superposition of the bcPadR1, bcPadR2 and LmR dimers.
Figure 5
Figure 5. Modeling of the DNA-bound bcPadR complexes.
(A) Superposition of the bcPadR1 (light-green) and bcPadR2 (red) dimers onto DNA-bound RTP (orange, PDB code 1F4K). (B) Superposition of the bcPadR1 (light-green) and bcPadR2 (red) dimers onto DNA-bound BlaI (cyan, PDB code 1XSD). Below the superpositions are structure-based sequence alignments including secondary structures. Residues of RTP and BlaI which participate in DNA binding are indicated with small spheres using the following color scheme: green, interacting with the phosphate backbone; blue, interacting with the base moiety and yellow, interacting with the ribose backbone).
Figure 6
Figure 6. The bound sulfate anion in the bcPadR1 structure.
(A) Interactions between bcPadR1 residues and the sulfate ion. Also shown is the DNA-bound RTP structure (PDB entry 1F4K) onto which the bcPadR1 dimer was superimposed. The bcPadR1 residues and the sulfate ion are shown in ball-and-stick representation and labeled, while the DNA strand of the RTP-DNA complex is drawn using lines. (B) Interactions between the RTP residues and the DNA phosphate backbone adjacent to the sulfate anion in the superimposed bcPadR1 structure. The RTP residues and DNA strands are shown in ball-and-stick representation and labeled, while the bcPadR1 residues and sulfate are drawn using lines.

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References

    1. Gury J, Barthelmebs L, Tran NP, Divies C, Cavin JF (2004) Cloning, deletion, and characterization of PadR, the transcriptional repressor of the phenolic acid decarboxylase-encoding padA gene of Lactobacillus plantarum . Appl Environ Microbiol 70: 2146–2153. - PMC - PubMed
    1. Tran NP, Gury J, Dartois V, Nguyen TKC, Seraut H, et al. (2008) Phenolic acid-mediated regulation of the padC gene, encoding the phenolic acid decarboxylase of Bacillus subtilis . J Bacteriol 190: 3213–3224. - PMC - PubMed
    1. Kovacikova G, Lin W, Skorupski K (2003) The virulence activator AphA links quorum sensing to pathogenesis and physiology in Vibrio cholerae by repressing the expression of a penicillin amidase gene on the small chromosome. J Bacteriol 185: 4825–4836. - PMC - PubMed
    1. Huillet E, Velge P, Vallaeys T, Pardon P (2006) LadR, a new PadR-related transcriptional regulator from Listeria monocytogenes, negatively regulates the expression of the multidrug efflux pump MdrL. FEMS Microbiol Lett 254: 87–94. - PubMed
    1. Agustiandari H, Lubelski J, van den Berg van Saparoea HB, Kuipers OP, Driessen AJM (2008) LmrR is a transcriptional repressor of expression of the multidrug ABC transporter LmrCD in Lactococcus lactis . J Bacteriol 190: 759–763. - PMC - PubMed

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Funding was provided by the University of Groningen. Work was supported in part by an Ubbo Emmius Bursary (University of Groningen) awarded to GF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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