The RemB structural paralogue is a co-activator for the DNA-binding biofilm regulator RemA

Biofilm formation in Bacillus subtilis depends on the transcriptional activator RemA, an unusual DNA-binding protein that assembles into ring-shaped octamers and activates expression of the major biofilm matrix operons. Although RemB was identified together with RemA as an important regulator of biofilm formation, its molecular function has remained unknown. Here, we show that RemB is a structural paralogue of RemA despite sharing only limited sequence identity. Structural modeling revealed that RemB adopts the characteristic RemA fold, but lacks the conserved three arginine residues required for DNA binding. Biochemical analyses showed that RemA and RemB form a stable hetero-oligomeric complex consistent with a 7:1 RemA/RemB stoichiometry, supporting a model in which a single RemB subunit replaces one RemA protomer within the octameric ring. Electrophoretic mobility shift assays further showed that incorporation of RemB into the RemA ring enhances DNA binding of the RemA complex to the eps promoter, whereas mutation of the RemA DNA-binding surface abolishes DNA binding irrespective of the presence of RemB. In vivo, both depletion and overproduction of RemB impaired matrix gene expression and biofilm formation, indicating that RemB activity is concentration dependent. Together, these findings identify RemB as a structural and functional modulator of RemA and suggest that the composition of the RemA oligomer provides an additional layer of control over biofilm gene regulation in B. subtilis. More generally, our work shows how the activity of an oligomeric transcription factor can be tuned through incorporation of a structurally related, DNA-binding-deficient paralogue into its assembly.