The Azoarcus anaerobius 1,3-Dihydroxybenzene (Resorcinol) Anaerobic Degradation Pathway Is Controlled by the Coordinated Activity of Two Enhancer-Binding Proteins

Abstract

The anaerobic resorcinol degradation pathway in is unique in that it uses an oxidative rather than a reductive strategy to overcome the aromatic ring stability in degradation of this compound, in a process that is dependent on nitrate respiration. We show that the pathway is organized in five transcriptional units, three of which are inducible by the presence of the substrate. Three σ-dependent promoters located upstream from the three operons coding for the main pathway enzymes were identified, which shared a similar structure with conserved upstream activating sequences (UASs) located at 103 to 111 bp from the transcription start site. Expression of the pathway is controlled by the bacterial enhancer-binding proteins (bEBPs) RedR1 and RedR2, two homologous regulators that, despite their high sequence identity (97%), have nonredundant functions: RedR2, the master regulator which also controls RedR1 expression, is itself able to promote transcription from two of the promoters, while RedR1 activity is strictly dependent on the presence of RedR2. The two regulators were shown to interact with each other, suggesting that the natural mode of activation is by forming heterodimers, which become active in the presence of the substrate after its metabolization to hydroxybenzoquinone through the pathway enzymes. The model structure of the N-terminal domain of the proteins is composed of tandem GAF and PAS motifs; the possible mechanisms controlling the activity of the regulators are discussed. is a strict anaerobe that is able to use 1,3-dihydroxybenzene as the sole carbon source in a process that is dependent on nitrate respiration. We have shown that expression of the pathway is controlled by two regulators of almost identical sequences: the bEBPs RedR1 and RedR2, which share 97% identity. These regulators control three promoters with similar structure. Despite their sequence identity, the two bEBPs are not redundant and are both required for maximum pathway expression. In fact, the two proteins function as heterodimers and require activation by the pathway intermediate hydroxyhydroquinone. The structure of the domain sensing the activation signal resembles that of regulators that are known to interact with other proteins.

Occurrence and diversity of the oxidative hydroxyhydroquinone pathway for the anaerobic degradation of aromatic compounds in nitrate‐reducing bacteria

“…Interestingly, S. selenatireducens AKOH1 rehLS cluster included a regulatory gene belonging to NtrC family showing 44% identity with the RedR regulators present in A. anaerobius cluster (Pacheco‐Sánchez et al, ). It is worth mentioning that the clusters in C. bauzanensis BZ59 and T. chlorobenzoica 3CB‐1 also contained at least one regulatory gene of different families (Fig.…”

“…In A. anaerobius, a gene for a transposase was found 4 Kb upstream from the rehLS cluster, further supporting the idea of horizontal transfer events in the dissemination of the pathway. Interestingly, S. selenatireducens AKOH1 rehLS cluster included a regulatory gene belonging to NtrC family showing 44% identity with the RedR regulators present in A. anaerobius cluster (Pacheco-Sánchez et al, 2017). It is worth mentioning that the clusters in C. bauzanensis BZ59 and T. chlorobenzoica 3CB-1 also contained at least one regulatory gene of different families (Fig.…”

“…Analysis of the gene cluster coding for this pathway shows that genes homologous to A. anaerobius genes for the three first steps in the pathway, rehLS , btdL and bqdLMS , are involved in 3,5‐DHB degradation in this strain (Molina‐Fuentes et al, ). Additional genes of unknown function are also conserved in the two pathways, but the regulatory proteins controlling their expression belong to different families, suggesting that these regulatory elements were acquired independently (Molina‐Fuentes et al, ; Pacheco‐Sánchez et al, ). It is worth noting that none of the strains were able to grow aerobically on the corresponding dihydroxylated aromatic substrate.…”

Bacterial nanocellulose production from naphthalene

“…N1B used in this study was isolated from a hydrocarbon‐polluted aquifer after enrichment with naphthalene crystals as sole carbon source under microaerophilic conditions (Martirani‐Von Abercron et al ., ). The strain was routinely grown under microaerophilic conditions a 28°C in 50 ml static bottles filled with 40 ml of a modified non‐reduced Widdel Mineral Medium (WMM) prepared as described (Pacheco‐Sánchez et al ., ). The final WMM medium contained 30 mM 3‐(N‐morpholino) propanesulfonic acid (MOPS) as buffer, 8 mM potassium nitrate, 17.1 mM NaCl, 6.7 mM KCl, 5.9 mM CaCl 2 , 4.7 mM NH 4 Cl, 2 mM MgCl 2 , 1.44 mM KH 2 PO 4 , 1 mM Na 2 SO 4 and was supplemented with 12 μl of a vitamin solution (Widdel and Bak, ), 40 μl of the SL10 trace element solution (Widdel et al ., ) and 40 μl of a selenium‐tungsten solution (Widdel, ) per 40 ml medium.…”

“…The Δ bcsA deletion mutant was generated by double homologous recombination using a pKNG101 (Kaniga et al ., ) plasmid derivative as described (Pacheco‐Sánchez et al ., ). Briefly, a 2.3 Kb fragment encompassing the 5′ 851 first nucleotides of bcsA and the preceding 1449 bp sequence was amplified from strain N1B genome DNA and cloned in pCR2.1‐TOPO R to render plasmid pTOPO:bcsN; a 2.04 Kb fragment encompassing the last 3657 nucleotides of bcsA and the following 1382 bp sequence was cloned in pMBL‐T™ to render pMBL:bcsB.…”