The <i>Bacillus subtilis</i> class A penicillin-binding protein 4 (PBP4) requires an accessory protein RpdA

Peptidoglycan (PG) synthesis is essential to maintain cell integrity during bacterial growth and division. In <i>Bacillus subtilis,</i> PG synthesis involves class A PBPs that act independently and class B PBPs that function in complexes for cell elongation (the elongasome) and division (the divisome). Here, we identify RpdA (formerly YufK) as a specific regulator of PBP4. Inactivation of either vegetative class A PBP (PBP1 or PBP4) by the β-lactam antibiotic cefuroxime (CEF) is toxic and their loss confers CEF resistance. Similarly, loss of RpdA increases CEF resistance and genetic epistasis studies reveal that RpdA functions in a pathway with PBP4. In the absence of RpdA, PBP4 is no longer membrane localized. Analysis of a predicted RpdA-PBP4 protein complex suggests that RpdA has a second function in addition to serving as a membrane scaffold. Induction of RpdA reduces sensitivity to an undecaprenyl-phosphate binding antibiotic, consistent with a role in recycling of this important lipid carrier. We conclude that RpdA is a PBP4 accessory protein critical for its localization and activity.