MarR-like transcriptional regulator involved in detoxification of aromatic compounds in Sulfolobus solfataricus

Abstract

A DNA binding protein, BldR, was identified in the crenarchaeon Sulfolobus solfataricus as a protein 5- to 10-fold more abundant in cells grown in the presence of toxic aldehydes; it binds to regulatory sequences located upstream of an alcohol dehydrogenase gene (Sso2536). BldR is homologous to bacterial representatives of the MarR (multiple antibiotic resistance) family of transcriptional regulators that mediate response to multiple environmental stresses. Transcriptional analysis revealed that the bldR gene was transcribed in a bicistronic unit composed of the genes encoding the transcriptional regulator (Sso1352) and a putative multidrug transporter (Sso1351) upstream. By homology to bacterial counterparts, the bicistron was named the mar-like operon. The level of mar-like operon expression was found to be increased at least 10-fold in response to chemical stress by aromatic aldehydes. Under the same growth conditions, similar enhanced in vivo levels of Sso2536 gene transcript were also measured. The gene encoding BldR was expressed in E. coli, and the recombinant protein was purified to homogeneity. DNA binding assays demonstrated that the protein is indeed a transcription factor able to recognize site specifically both the Sso2536 and mar-like promoters at sites containing palindromic consensus sequences. Benzaldehyde, the substrate of ADH(Ss), stimulates DNA binding of BldR at both promoters. The role of BldR in the auto-activation as well as in the regulation of the Sso2536 gene, together with results of increased operon and gene expression under conditions of exposure to aromatic aldehydes, indicates a novel coordinate regulatory mechanism in cell defense against stress by aromatic compounds.

FIG. 1.

Multiple alignment of BldR and characterized MarR family members. Proteins are BldR (Sso1352) from S. solfataricus, SlyA from Salmonella enterica serovar Typhimurium, PecS from Erwinia chrysantemi, MarR from E. coli, HucR from Deinococcus radiodurans, and MexR from Pseudomonas aeruginosa. The alignment was generated using Clustal W, and the numbering is based upon the HucR sequence. The winged helix-turn-helix DNA binding motif predicted for BldR is also represented: alfa-helices are gray bars, beta sheets are black arrows, and turns are black lines.

FIG. 2.

Northern blot analysis of the mar-like operon and Sso2536 mRNAs. Total RNA was prepared from S. solfataricus cells grown in the presence of different aromatic aldehydes and harvested in exponential phase. Lane 1, untreated control cells; lane 2, cells grown in the presence of 0.35 mM cynnamaldehyde; lane 3, 1 mM veratrylaldehyde; lane 4, 1 mM benzaldehyde. The filters were probed with the Sso1352 (A), Sso2536 (B), and Sso7d (C) genes. (D) Structure of the mar-like operon. BldR and the putative drug efflux permease are encoded by Sso1352 and Sso1351, respectively, and are transcribed in a single polycistronic mRNA as indicated. The stop/start codons of the ORFs are underlined in the sequence. The putative ribosome binding site(s) upstream of the Sso1352 gene is in bold. nt, nucleotides.

FIG. 3.

Primer extension and sequence analysis of the mar-like promoter region. Total RNA was prepared from cells grown in the presence or absence of benzaldehyde and harvested in exponential growth phase. (A) Primer extended products were separated by electrophoresis under denaturing conditions alongside sequencing reactions with the same primer. (B) Promoter sequence. The mapped transcription/translation start site (+1) is in bold. The TBP and TFB binding sites are indicated by boxed regions, and tandem inverted repeats are underlined.

FIG. 4.

Purification and gel filtration analysis of recombinant BldR. (A) Coomassie brilliant blue-stained SDS-PAGE gels of the protein samples from E. coli transformed with Sso1352 after each purification step. Lane 1, crude extract; lane 2, heat-treated cell extract; lane 3, fraction from Resource S chromatography; lane 4, fraction from Heparin chromatography. M, molecular mass markers. (B) Elution profile of the purified protein from gel filtration on a superdex PC75 column. Arrows indicate the elution volumes of the protein standards in the relative calibration of the column. MW, molecular mass (kDa).

FIG. 5.

Detection of BldR by Western blot analysis. S. solfataricus protein extracts were prepared from cells grown in the presence of different aromatic aldehydes and harvested in exponential phase, as for Northern analysis. (A) Untreated control cells (lane 1) and cells grown in the presence of 0.35 mM cynnamaldehyde (lane 2), 1 mM veratrylaldehyde (lane 3), and 1 mM benzaldehyde (lane 4). (B) Western blot analysis was performed on known amounts (indicated on the top) of purified recombinant protein (rBldR) as a reference for quantitative determination of the native BldR protein in S. solfataricus cell extracts.

FIG. 6.

Binding of recombinant BldR to the promoter regions of Sso2536 and the mar-like operon. EMSAs of the −152/+10 region of the Sso2536 gene (A) and of the −271/+21 region of the mar-like operon (C) were performed both in the absence (lanes 2) and in the presence (lanes 3 to 6) of two different excess amounts of unlabeled unspecific (Sso10b coding sequence; lanes 3 and 4) or specific (lanes 5 and 6) DNAs, using 2 μM protein. The specificity of the binding was tested with three different protein amounts (5, 10, and 20 μM) on the S. solfataricus superoxide dismutase gene promoter (B, lanes 2, 3, and 4). Shown are the effects of increasing concentrations of benzaldehyde (D) or benzoic acid (E) on the mobility of the mar-like promoter with BldR. Lanes 2 to 9, 0.8 μM BldR and 0, 0.08, 0.16, 0.2, 0.4, 1.6, and 10 μM aromatic compounds. Similar results were obtained with the Sso2536 promoter. Lanes 1 in all panels indicate free labeled DNAs.

FIG. 7.

BldR binding sites at the promoters of the Sso2536 gene and the mar-like operon. DNase I footprinting analyses of BldR were performed at the nontemplate strand of both the Sso2536 gene (A) and the mar-like operon (B). DNase I footprint analyses were performed using 0.0, 0.5, 1.0, and 2.0 μg of purified recombinant BldR. DNA fragments were analyzed in parallel with a sequencing reaction (relative lanes are indicated by the corresponding nucleotide positions on the top) by denaturing gel electrophoresis. The positions of the BldR footprints are indicated on the nucleotide sequences relative to the transcription start site.