Prophage-encoded small protein YqaH counteracts the activities of the replication initiator DnaA in Bacillus subtilis.

Bacterial genomes harbor cryptic prophages that are mostly transcriptionally silent with many unannotated genes. Still, cryptic prophages may contribute to their host fitness and phenotypes. In B. subtilis, the yqaF-yqaN operon belongs to the prophage element skin, and is tightly repressed by the Xre-like repressor sknR. This operon contains several short open reading frames (smORFs) potentially encoding small-sized proteins. The smORF-encoded peptide YqaH was previously reported to bind to the replication initiator DnaA. Here, using a yeast two-hybrid assay, we found that YqaH binds to the DNA binding domain IV of DnaA and interacts with Spo0A, a master regulator of sporulation. We isolated single amino acid substitutions in YqaH that abolished interaction with DnaA but not with Spo0A. Then, we studied in B. subtilis the phenotypes associated with the specific loss-of-interaction with DnaA (DnaA-LOI). We found that expression of yqaH carrying DnaA-LOI mutations abolished the deleterious effects of yqaH WT expression on chromosome segregation, replication initiation and DnaA-regulated transcription. When YqaH was induced after vegetative growth, DnaA-LOI mutations abolished the deleterious effects of YqaH WT on sporulation and biofilm formation. Thus, YqaH inhibits replication, sporulation and biofilm formation mainly by antagonizing DnaA in a manner that is independent of the cell cycle checkpoint Sda.