Subtle sequence differences between two interacting σ⁵⁴ -dependent regulators lead to different activation...

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Aromatoleum anaerobium anaerobically degrades 1,3-dihydroxybenzene (1,3-DHB) using a pathway regulated by two bacterial enhancer-binding proteins (bEBPs), RedR1 and RedR2.
• These proteins control transcription of σ⁵⁴-dependent promoters, crucial for pathway gene expression.
• RedR1 and RedR2 share structural similarities but differ in their N-terminal tails, N-terminal domains (NTDs), and C-terminal domains (CTDs), suggesting distinct regulatory functions.

Purpose of the Study:

• To elucidate the distinct regulatory mechanisms of RedR1 and RedR2 in 1,3-DHB degradation.
• To investigate the role of protein-protein interactions, specifically between RedR1, RedR2, and BtdS, in pathway regulation.
• To understand the assembly dynamics of RedR1 and RedR2, including homo- and hetero-oligomerization, and the influence of DNA binding on their activity.

Main Methods:

• Comparative analysis of RedR1 and RedR2 structures and sequences.
• Investigating protein-protein interactions using biochemical assays.
• Studying the effect of effector molecules (1,3-DHB) on regulator activity.
• Analyzing the role of specific protein domains (NTD, CTD, PAS domain) in regulation and assembly.
• Assessing promoter activation by different regulator forms and oligomeric states.

Main Results:

• RedR1 is regulated by classical NTD-mediated negative control released by the effector, while RedR2 is constitutively active and membrane-associated via BtdS.
• BtdS sequesters RedR2 to the membrane through its NTD, involving specific residues in the PAS domain and N-terminal tail; 1,3-DHB metabolism releases RedR2.
• Hetero-oligomer formation between RedR1 and RedR2 is favored over homo-oligomers, although truncated RedR1 or full-length RedR2 can activate promoters independently.
• Promoter DNA acts as an allosteric effector, binding the CTD to modulate ΔNTD-RedR1 multimerization and activity.

Conclusions:

• A novel mode of bEBP activation and assembly governs the anaerobic 1,3-DHB degradation pathway.
• The distinct regulatory strategies of RedR1 and RedR2, coupled with their preferential hetero-oligomerization, provide sophisticated control over gene expression.
• Understanding these mechanisms offers insights into bacterial adaptation and metabolic regulation in anaerobic environments.