The nucleotide messenger (p)ppGpp is an anti-inducer of the purine synthesis transcription regulator PurR in Bacillus

The nucleotide messenger (p)ppGpp allows bacteria to adapt to fluctuating environments by reprogramming the transcriptome. Despite its well-recognized role in gene regulation, (p)ppGpp is only known to directly affect transcription in Proteobacteria by binding to the RNA polymerase. Here we reveal a different mechanism of gene regulation by (p)ppGpp in Firmicutes from soil bacteria to pathogens: (p)ppGpp directly binds to the transcription factor PurR to downregulate purine biosynthesis. We identified PurR as a receptor of (p)ppGpp in Bacillus anthracis and revealed that (p)ppGpp strongly enhances PurR binding to its regulon in the Bacillus subtilis genome. A co-structure reveals that (p)ppGpp binds to a PurR pocket reminiscent of the active site of PRT enzymes that has been repurposed to serve a purely regulatory role, where the effectors (p)ppGpp and PRPP compete to allosterically control transcription. PRPP inhibits PurR DNA binding to induce transcription of purine synthesis genes, whereas (p)ppGpp antagonizes PRPP to enhance PurR DNA binding and repress transcription. A (p)ppGpp-refractory purR mutant fails to downregulate purine synthesis genes upon starvation. Our work establishes the precedent of (p)ppGpp as a classical effector of transcription repressors and reveals the key function of (p)ppGpp in regulating nucleotide synthesis through gene regulation, from the human intestinal tract to host-pathogen interfaces. Overall design: Comparing PurR ChIP enrichment in the Bacillus subtilis genome before and after treatment with arginine hydroxamate (RHX) to induce (p)ppGpp. This study used 25 total samples. Five samples (3 ChIP replicates and 2 input replicates) were from B. subtilis grown in a medium with nucleobases to enhance PurR repression and identify PurR binding sites genome-wide. Five samples (3 ChIP replicates and 2 input) were from B. subtilis before RHX treatment and five samples (3 ChIP replicates and 2 input) were from B. subtilis after RHX treatment. Lastly, the same RHX treatment was performed on a (p)ppGpp-null B. subtilis strain, producing ten samples (five before RHX, 3 ChIP replicates and 2 input; five after RHX, 3 ChIP replicates and 2 input).