DbdR, a New Member of the LysR Family of Transcriptional Regulators, Coordinately Controls Four Promoters in the Thauera aromatica AR-1 3,5-Dihydroxybenzoate Anaerobic Degradation Pathway

Abstract

The facultative anaerobe Thauera aromatica strain AR-1 uses 3,5-dihydroxybenzoate (3,5-DHB) as a sole carbon and energy source under anoxic conditions using an unusual oxidative strategy to overcome aromatic ring stability. A 25-kb gene cluster organized in four main operons encodes the anaerobic degradation pathway for this aromatic. The dbdR gene coding for a LysR-type transcriptional regulator (LTTR), which is present at the foremost end of the cluster, is required for anaerobic growth on 3,5-DHB and for the expression of the main pathway operons. A model structure of DbdR showed conserved key residues for effector binding with its closest relative TsaR for p-toluenesulfonate degradation. We found that DbdR controlled expression of three promoters upstream from the operons coding for the three main steps of the pathway. While one of them (Porf20) was only active in the presence of 3,5-DHB, the other two (PdbhL and Porf18) showed moderate basal levels that were further induced in the presence of the pathway substrate, which needed be converted to hydroxyhydroquinone to activate transcription. Both basal and induced activities were strictly dependent on DbdR, which was also required for transcription from its own promoter. DbdR basal expression was moderately high and, unlike most LTTR, increased 2-fold in response to the presence of the effector. DbdR was found to be a tetramer in solution, producing a single retardation complex in binding assays with the three enzymatic promoters, consistent with its tetrameric structure. The three promoters had a conserved organization with a clear putative primary (regulatory) binding site and a putative secondary (activating) binding site positioned at the expected distances from the transcription start site. In contrast, two protein-DNA complexes were observed for the PdbdR promoter, which also showed significant sequence divergence from those of the three other promoters. Taken together, our results show that a single LTTR coordinately controls expression of the entire 3,5-DHB anaerobic degradation pathway in Thauera aromatica AR-1, allowing a fast and optimized response to the presence of the aromatic. IMPORTANCE Thauera aromatica AR-1 is a facultative anaerobe that is able to use 3,5-dihydroxybenzoat (3,5-DHB) as the sole carbon and energy source in a process that is dependent on nitrate respiration. We have shown that a single LysR-type regulator with unusual properties, DbdR, controls the expression of the pathway in response to the presence of the substrate; unlike other regulators of the family, DbdR does not repress but activates its own synthesis and is able to bind and activate three promoters directing the synthesis of the pathway enzymes. The promoter architecture is conserved among the three promoters but deviates from that of typical LTTR-dependent promoters. The substrate must be metabolized to an intermediate compound to activate transcription, which requires basal enzyme levels to always be present. The regulatory network present in this strain is designed to allow basal expression of the enzymatic machinery, which would rapidly metabolize the substrate when exposed to it, thus rendering the effector molecule. Once activated, the regulator induces the synthesis of the entire pathway through a positive feedback, increasing expression from all the target promoters to allow maximum growth.

A widely distributed metalloenzyme class enables gut microbial metabolism of host- and diet-derived catechols

“…5B). Strains lacking this enzyme exhibit impaired 447 growth on 3,5-DHB as a sole carbon source, suggesting a possible role for this enzyme in 448 metabolizing the one of the two catecholic intermediates involved in this pathway (58,(60)(61). 449…”

PsrA is a novel regulator contributes to antibiotic synthesis, bacterial virulence, cell motility and extracellular polysaccharides production in Serratia marcescens

“…The binding activity of LTTRs is generally influenced by one or more effectors ( 29 , 45–48 ), and the effectors of the LTTRs is usually the substrate or the intermediate in the metabolism of the products ( 29 , 48 ). To identify the possible effectors of PsrA, different concentrations of l -proline (0, 1.0, 2.0 and 3.0 mM), 2-Octenal (0, 1.0, 2.0 and 3.0 mM), l -serine (0, 1.0, 2.0 and 3.0 mM) and S -adenosylmethionine (0, 1.0, 2.0 and 3.0 mM) were added to LB medium, respectively, and the β-galactosidase assays were determined with S. marcescens JNB5-1 carrying the P pigA 281 - lacZ .…”

“…To efficiently activate the transcription of corresponding target genes and hence influence cellular processes in bacterial, the binding activity of LTTRs to its target gene is generally influenced by one or more effectors. For example, acetate is the effector of AlsR and activates the transcription of the alsSD operon for acetoin formation in Bacillus subtilis ( 26 , 38 ), glutarate is the effector of GcdR and controls glutarate catabolism in Pseudomonas putida KT2440 ( 29 ), hydroxyhydroquinone or possibly 2,3,5-trihydroxybenzoate is the effector of DbdR and influences expression of the entire 3,5-DHB anaerobic degradation pathway in Thauera aromatica ( 48 ), benzoate and cis,cis-muconate are the effectors of BenM and control aromatic compound degradation in Acinetobacter sp. ADP1 ( 45 ), 1-Naphthol is the effector of McbG to activate mcbBCDEF cluster transcription and involved in the upstream pathway of carbaryl degradation in Pseudomonas sp.…”

Transcriptional organization and regulation of the Pseudomonas putida K1 type VI secretion system gene cluster

“…Primer extension analyses were performed as previously described by Pacheco-Sánchez et al . [52]. An oligonucleotide complementary to the coding strand of the tagB1 gene (P45, Table S3) was 32 P labelled at its 5′ ends in a 10 µl final volume that contained 1 µl 10× buffer, 10 pmol oligonucleotides, 1 µl [γ- 32 P]ATP (6000 mCi mmol −1 ), and 1 U phage T4 polynucleotide kinase.…”