SwrA-mediated Multimerization of DegU and an Upstream Activation Sequence Enhance Flagellar Gene Expression in Bacillus subtilis

[Display omitted] •SwrA is a small protein that causes oligomerization of the response regulator DegU to increase DNA binding both in vivo and in vitro.•DegU binds upstream of the Pflache promoter but requires SwrA-induced oligomerization to activate transcription.•The Pflache promoter has an upstream activating sequence (UAS) between the -35 box and the DegU binding site that is required for basal transcription.•The SwrA-DegU complex heteromeric activator appears to be an adaptation to further enhance flagellar transcription in the context of the UAS. The earliest genes in bacterial flagellar assembly are activated by narrowly-conserved proteins called master regulators that often act as heteromeric complexes. A complex of SwrA and the response-regulator transcription factor DegU is thought to form the master flagellar regulator in Bacillus subtilis but how the two proteins co-operate to activate gene expression is poorly-understood. Here we find using ChIP-Seq that SwrA interacts with a subset of DegU binding sites in the chromosome and does so in a DegU-dependent manner. Using this information, we identify a DegU-specific inverted repeat DNA sequence in the Pflache promoter region and show that SwrA synergizes with DegU phosphorylation to increase binding affinity. We further demonstrate that the SwrA/DegU footprint extends from the DegU binding site towards the promoter, likely through SwrA-induced DegU multimerization. The location of the DegU inverted repeat was critical and moving the binding site closer to the promoter impaired transcription by disrupting a previously-unrecognized upstream activation sequence (UAS). Thus, the SwrA-DegU heteromeric complex likely enables both remote binding and interaction between the activator and RNA polymerase. Small co-activator proteins like SwrA may allow selective activation of subsets of genes where activator multimerization is needed. Why some promoters require activator multimerization and some require UAS sequences is unknown.