Detoxification of aromatic aldehydes in the archaeon Sulfolobus solfataricus is regulated by a Mar-like transcription factor.

INTRODUCTION: Investigation of mechanisms underlying transcriptional regulation of Sso2536, encoding for an alcohol dehydrogenase gene (adh) in the crenarchaeon S. solfataricus has shown an active 5’ flanking region responsive to physiologically relevant DNA binding proteins. In particular, one DNA binding protein, Bald16 (Sso1352), has been identified whose levels are higher when cells are grown in the presence of the toxic benzaldehyde, substrate of the ADH enzyme; it has been proposed that this protein could act as a transcriptional activator triggering adh expression to protect cells from an environmental stress due to phenolic-derived aldehydes. Bald16 encodes for a putative transcriptional regulator, which has a bacterial homologue belonging to the Mar (Multiple Antibiotic Resistance) family of regulators involved in the control of gene expression of aromatic compound metabolism and antibiotic resistance. To better investigate the molecular mechanisms underlying transcriptional regulation in S.solfataricus, with greater attention with respect to defense response upon chemical stress, we analyzed the expression of the bald16 gene in the presence of aromatic aldehydes. Furthermore, recombinant Bald16 has been obtained in E. coli and characterized for its DNA binding activity. MATERIALS AND METHODS S. solfataricus cultures were grown in different conditions among which DSM182 medium supplemented with 1 mM benzaldehyde, 1mM veratrylaldehyde, 0,35 mM cynnamaldehyde and harvested at exponential or stationary phase. mRNA and crude extracts were prepared according to conventional procedures to perform Northern and Western blot analyses, respectively. Recombinant Bald16 was obtained by PCR amplification of the Sso1352 ORF and cloning in the pTrc99A vector and purified by thermal precipitation and three chromatographic steps. Functional analysis has been performed essentially by band shift analysis and DNAseI footprinting using synthetic double stranded oligonucleotides designed on the sequences of the adh or bald16 promoter and DNA fragments prepared by endonuclease restriction of the 5’ flanking regions upstream of the adh or bald16 gene. RESULTS Transcriptional analysis of the Bald16 gene allowed the identification of a new a mar-like locus in S. solfataricus composed of a putative multidrug transporter and the transcriptional regulator downstream (Sso1351, Sso1352). The genes are transcribed as a polycistronic unit whose expression is sensitive to the addition to the cell growth medium of different aromatic aldehydes. The gene encoding for the transcriptional regulator, has been overexpressed in E. coli and the recombinant protein purified to homogeneity. The protein is indeed a DNA binding protein, which binds site-specifically to both the adh and Bald16 promoters, reasonably strengthening the hypothesis of a coordinate expression in response to stress determined by phenolic-derived materials.