Phenylacetyl coenzyme A is an effector molecule of the TetR family transcriptional repressor PaaR from Thermus thermophilus HB8

Abstract

Phenylacetic acid (PAA) is a common intermediate in the catabolic pathways of several structurally related aromatic compounds. It is converted into phenylacetyl coenzyme A (PA-CoA), which is degraded to general metabolites by a set of enzymes. Within the genome of the extremely thermophilic bacterium Thermus thermophilus HB8, a cluster of genes, including a TetR family transcriptional regulator, may be involved in PAA degradation. The gene product, which we named T. thermophilus PaaR, negatively regulated the expression of the two operons composing the gene cluster in vitro. T. thermophilus PaaR repressed the target gene expression by binding pseudopalindromic sequences, with a consensus sequence of 5'-CNAACGNNCGTTNG-3', surrounding the promoters. PA-CoA is a ligand of PaaR, with a proposed binding stoichiometry of 1:1 protein monomer, and was effective for transcriptional derepression. Thus, PaaR is a functional homolog of PaaX, a GntR transcriptional repressor found in Escherichia coli and Pseudomonas strains. A three-dimensional structure of T. thermophilus PaaR was predicted by homology modeling. In the putative structure, PaaR adopts the typical three-dimensional structure of the TetR family proteins, with 10 α-helices. A positively charged surface at the center of the molecule is similar to the acyl-CoA-binding site of another TetR family transcriptional regulator, T. thermophilus FadR, which is involved in fatty acid degradation. The CoA moiety of PA-CoA may bind to the center of the PaaR molecule, in a manner similar to the binding of the CoA moiety of acyl-CoA to FadR.

Fig. 1.

Organization of the putative paagene cluster in T. thermophilusHB8. (A) The locus tags (numerals preceded by TTHA) are denoted above the genes. The locations of the genes on the chromosomal DNA are shown. The putative paagenes, besides paaR, are named according to the consensus names defined by Luengo et al. (17); i.e., paaD, paaF, paaN, paaJ, paaI, paaH, and paaGencode a putative thioesterase, PA-CoA ligase, ring opening enzyme, ring-hydroxylating complex protein 4, ring-hydroxylating complex protein 3, ring-hydroxylating complex protein 2, and ring-hydroxylating complex protein 1, respectively. paaRand hypencode a TetR family transcriptional regulator and a hypothetical protein, respectively. TTHA0963and TTHA0964encode ABC transporter ATP-binding protein-related proteins. The nucleotide sequences upstream of TTHA0963and TTHA0973are shown with their locations on the chromosomal DNA. Probable PaaR-binding pseudopalindromic sequences are indicated by bold capital letters. Possible −10 and −35 hexamer sequences of the promoters are underlined. The representative transcriptional start site (+1) of each gene, as determined by the 5′-RACE experiment, is indicated. In the case of the start site of TTHA0963, G (+1) was converted to C in all seven sequenced clones. As for TTHA0973, two of the seven clones were identified at the A (−2) site, and one was converted to C. The positions of the primers used for RT-PCR (see below) are indicated by open triangles and filled triangles for the TTHA0963to TTHA0967and TTHA0973 to TTHA0968operons, respectively. (B) RT-PCR analysis to confirm the operon composed of TTHA0963to TTHA0967(lane 2) and that composed of TTHA0973to TTHA0968(lane 4). The positions of the primers are shown in panel A. PCR was also performed with no RT, as control using the same primers (lanes 3 and 5 are the controls for lanes 2 and 4, respectively). The samples were fractionated on a 1% agarose gel, followed by staining with ethidium bromide and photography. The expected bands are indicated with asterisks. Lane 1, 500-bp DNA ladder markers.

Fig. 2.

Sequence alignment of T. thermophilusPaaR (Tt_PaaR) with representative homologous proteins. Strictly conserved residues are represented by white letters on a black background, and similar residues are depicted by boxed bold letters. The homologs are from D. maricopensisDSM 21211 (Deima_1092, YP_004170410), Thermobifida fuscaYX (Tfu_2799, YP_290855), A. evansii(Ae_PaaR, CAC10611), and T. thermophilusFadR (Tt_FadR, YP_143367). The sequences were aligned using Clustal W2 (16). The secondary structure of Tt_PaaR was predicted with DSSP (13), and the figure was generated with ESPript 2.2 (9). α and η represent α-helix and 3₁₀-helix, respectively. The percent identities [id(%)] and E values relative to Tt_PaaR, determined by BLAST, are indicated on the right.

Fig. 3.

BIAcore biosensor analysis of the T. thermophilusPaaR-DNA interaction. (A) A dsDNA corresponding to the upstream region of the TTHA0963gene (TTHA0963p) (see text) was immobilized on the sensor chip, and then PaaR was injected over the DNA surface at concentrations of 0.4, 0.3, 0.2, and 0.1 μM dimer, in buffer B containing 0.3 M NaCl. (B) A dsDNA corresponding to the upstream region of the TTHA0973gene (TTHA0973p) (see text) was used under the same experimental conditions as those of panel A. (C) The PaaR dimer (0.4 μM), along with 0, 0.2, 0.4, 0.6, 1, or 2 μM PA-CoA, was injected over the TTHA0963p-immobilized surface, in buffer B containing 0.3 M NaCl. (D) The PaaR dimer (0.4 μM) plus nothing (middle line), 2 μM Bz-CoA (bottom line), or 2 μM Ac-CoA (top line) was injected over the TTHA0963p-immobilized surface, as in panel C. Representative sensorgrams, minus the bulk refractive index background, were recorded and normalized to the baseline of 0 RU.

Fig. 4.

ITC profiles of the titrations of T. thermophilusPaaR with PA-CoA (A), Bz-CoA (B), and Ac-CoA (C) at 30°C. The raw thermogram of each experiment is shown. The lower panel in panel A indicates the titration curve fitted to the one-site model.

Fig. 5.

Effects of T. thermophilusPaaR on transcription in vitro. (A) Runoff transcription assays were performed with templates containing the upstream sequences of TTHA0890, TTHA0963, and TTHA0973genes, in the absence or presence of 0.25, 0.5, or 1 μM PaaR as a dimer and in the absence or presence of 2, 5, 10, or 20 μM PA-CoA. In lanes 2 to 11, equivalent volumes were fractionated on the polyacrylamide gel, followed by autoradiography. In lanes 12 to 19, 4-fold-higher sample volumes were applied on the gel, which was exposed six times longer than in lanes 2 to 11. Lane 1, [α-³²P]dCTP-labeled MspI fragments of pBR322. (B) Runoff transcription assays were performed with a template containing the upstream sequence of the TTHA0973gene, in the absence or presence of 1 μM dimer PaaR, and in the absence or presence of 20 μM Bz-CoA or Ac-CoA. After the reaction, equivalent volumes of samples were fractionated on the polyacrylamide gel, followed by autoradiography.