SwrA extends DegU over an UP element to activate flagellar gene expression in Bacillus subtilis

SwrA activates flagellar gene expression in Bacillus subtilis to increase the frequency of motile cells in liquid and further increase flagellar density to swarm over solid surfaces. Here we perform ChIP-seq and demonstrate that SwrA interacts with many sites on the chromosome indirectly through the response regulator DegU. We identify a DegU-specific inverted repeat and show that SwrA increased DegU DNA binding affinity in parallel to phosphorylation. Moreover, we show that SwrA increased the size of the DegU footprint expanding the region of protection towards the promoter. The remote location of the DegU inverted repeat was critical such that relocating the binding site by full turns of DNA impaired activation by SwrA/DegU and moving the binding site closer to the promoter severely impaired transcription. We conclude that SwrA/DegU forms a heteromeric complex that enables both remote binding and activator-RNA polymerase interaction in the context of an interceding UP element. We speculate that multimeric activators that resolve similar cis-element spatial conflicts are common in bacteria such as the diverse master activators of flagellar biosynthesis and those that activate long operons of other multi-subunit complexes. Overall design: Chromatin Immunoprecipitation Sequencing (ChIP-Seq): Bacillus subtilis cultures were grown to an OD600 of 1.0 at 37°C with constant rotation. 20 ml of cells were cross-linked for 30 minutes at room temperature using 3% formaldehyde (Sigma), quenched with 125 mM glycine, washed with PBS, and then lysed. DNA was sheared to an average fragment size of ~200 bp using Qsonica sonicator (Q8000R), and then incubated overnight at 4ºC with ?-SwrA or ??-DegU as indicated. Immunoprecipitation was performed using Protein A Magnetic Sepharose beads (Cytiva #45002511), washed, and DNA was eluted in TES (50mM Tris pH8, 10mM EDTA and 1% SDS). Crosslinks were reversed overnight at 65ºC. DNA samples were treated with a final concentration of 0.2mg/ml RNaseA (Promega #A7973) and 0.2mg/ml Proteinase K (NEB #P8107S) respectively, and subsequently extracted using phenol/chloroform/isoamyl (25:24:1). DNA samples were then used for library preparation using NEBNext UltraII DNA library prep kit (NEB #E7645L). Paired end sequencing of the libraries was performed using Illumina NextSeq 550 platform and atleast 3 million paired end reads were obtained for each sample. Two or three biological replicates were sequenced for each sample. Whole genome sequencing (WGS): Bacillus subtilis cultures were grown to an OD600 of 1.0 at 37°C with constant rotation and 5ml of cells were collected, pelleted and DNA was extracted using Qiagen DNeasy kit (#69504). Genomic DNA was sonicated using Qsonica sonicator (Q8000R) and the sonicated DNA was used to prepare libraries using the NEBNext UltraII DNA library prep kit (NEB #E7645L). Paired end sequencing of the libraries was performed using Illumina NextSeq 550 platform and atleast 3 million paired end reads were obtained for each sample. Data from WGS was used as input for the ChIP. Analysis of ChIP-Seq and WGS data: Sequencing reads for both ChIP and WGS were mapped individually to B. subtilis 3610 genome (CP020102) (Nye et al, 2017) using CLC Genomics Workbench software (Qiagen). The enrichment at ribosomal RNA locations were eliminated and the number of reads mapped to each base pair in the genome was exported into a .csv file. Data was normalized to the total number of reads and fold enrichment was calculated as the ratio of number of reads at each genome location in ChIP-Seq and WGS (ChIP/input). Analysis was performed and graphs were plotted in 1kb bins to show enrichment across the entire genome using custom R-scripts. When required, individual peaks were plotted in 10bp bins across a 4kb range centered around the peak summit. Detailed protocols and scripts are available upon request.