Subtle sequence differences between two interacting σ54‐dependent regulators lead to different activation mechanisms

In the strictly anaerobic nitrate reducing bacterium Aromatoleum anaerobium, degradation of 1,3‐dihydroxybenzene (1,3‐DHB, resorcinol) is controlled by two bacterial enhancer‐binding proteins (bEBPs), RedR1 and RedR2, which regulate the transcription of three σ54‐dependent promoters controlling expression of the pathway. RedR1 and RedR2 are identical over their length except for their N‐terminal tail which differ in sequence and length (six and eight residues, respectively), a single change in their N‐terminal domain (NTD), and nine non‐identical residues in their C‐terminal domain (CTD). Their NTD is composed of a GAF and a PAS domain connected by a linker helix. We show that each regulator is controlled by a different mechanism: whilst RedR1 responds to the classical NTD‐mediated negative regulation that is released by the presence of its effector, RedR2 activity is constitutive and controlled through interaction with BtdS, an integral membrane subunit of hydroxyhydroquinone dehydrogenase carrying out the second step in 1,3‐DHB degradation. BtdS sequesters the RedR2 regulator to the membrane through its NTD, where a four‐Ile track in the PAS domain, interrupted by a Thr in RedR1, and the N‐terminal tail are involved. The presence of 1,3‐DHB, which is metabolized to hydroxybenzoquinone, releases RedR2 from the membrane. Most bEBPs assemble into homohexamers to activate transcription; we show that hetero‐oligomer formation between RedR1 and RedR2 is favoured over homo‐oligomers. However, either an NTD‐truncated version of RedR1 or a full‐length RedR2 are capable of promoter activation on their own, suggesting they should assemble into homohexamers in vivo. We show that promoter DNA behaves as an allosteric effector through binding the CTD to control ΔNTD‐RedR1 multimerization and activity. Overall, the regulation of the 1,3‐DHB anaerobic degradation pathway can be described as a novel mode of bEBP activation and assembly. RedR1 and RedR2 are bacterial enhancer‐binding proteins than only differ in 17 residues (labelled orange and lilac) but are controlled through opposite mechanisms: RedR2 is constitutive and kept inactive by binding the membrane protein BtdS. RedR1 is negatively controlled by its N‐terminal domain. In the presence of 1,3‐DHB, metabolized to hydroxybenzoquinone (HBQ) by BtdSL, active RedR2 is released from BtdS and RedR1 is activated by HBQ. Both proteins assemble into heterodimers, which oligomerize to heterohexamers and initiate transcrip