A Role for the Class A Penicillin-Binding Protein PonA2 in the Survival of Mycobacterium smegmatis under Conditions of Nonreplication

Class A penicillin-binding proteins (PBPs) are large, bifunctional proteins that are responsible for glycan chain assembly and peptide cross-linking of bacterial peptidoglycan. Bacteria in the genus Mycobacterium have been reported to have only two class A PBPs, PonA1 and PonA2, that are encoded in their genomes. We report here that the genomes of Mycobacterium smegmatis and other soil mycobacteria contain an additional gene encoding a third class A penicillin-binding protein, PonA3, which is a paralog of PonA2. Both the PonA2 and PonA3 proteins contain a penicillin-binding protein and serine/threonine protein kinase-associated (PASTA) domain that we propose may be involved in sensing the cell cycle and a C-terminal proline-rich region (PRR) that may have a role in protein-protein or protein-carbohydrate interactions. We show here that an M. smegmatis Delta ponA2 mutant has an unusual antibiotic susceptibility profile, exhibits a spherical morphology and an altered cell surface in stationary phase, and is defective for stationary-phase survival and recovery from anaerobic culture. In contrast, a Delta ponA3 mutant has no discernible phenotype under laboratory conditions. We demonstrate that PonA2 and PonA3 can bind penicillin and that PonA3 can partially substitute for PonA2 when ponA3 is expressed from a constitutive promoter on a multicopy plasmid. Our studies suggest that PonA2 is involved in adaptation to periods of nonreplication in response to starvation or anaerobiosis and that PonA3 may have a similar role. However, the regulation of PonA3 is likely different, suggesting that its importance could be related to stresses encountered in the environmental niches occupied by M. smegmatis and other soil-dwelling mycobacteria.