Functional Insights into the High-Molecular-Mass Penicillin-Binding Proteins of Streptococcus agalactiae Revealed by Gene Deletion and Transposon Mutagenesis Analysis.

High-molecular-mass penicillin-binding proteins (PBPs) are enzymes that catalyze the biosynthesis of bacterial cell wall peptidoglycan. The Gram-positive bacterial pathogen Streptococcus agalactiae (group B streptococcus [GBS]) produces five high-molecular-mass PBPs, namely, PBP1A, PBP1B, PBP2A, PBP2B, and PBP2X. Among these, only PBP2X is essential for cell viability, whereas the other four PBPs are individually dispensable. The biological function of the four nonessential PBPs is poorly characterized in GBS. We deleted the , , , and genes individually from a genetically well-characterized serotype V GBS strain and studied the phenotypes of the four isogenic mutant strains. Compared to the wild-type parental strain, (i) none of the isogenic mutant strains had a significant growth defect in Todd-Hewitt broth supplemented with 0.2% yeast extract (THY) rich medium, (ii) isogenic mutant Δ and Δ strains had significantly increased susceptibility to penicillin and ampicillin, and (iii) isogenic mutant Δ and Δ strains had significantly longer chain lengths. Using saturated transposon mutagenesis and transposon insertion site sequencing, we determined the genes essential for the viability of the wild-type GBS strain and each of the four isogenic deletion mutant strains in THY rich medium. The gene is essential for cell viability in the deletion background. Reciprocally, is essential in the deletion background. Moreover, the gene encoding RodA, a peptidoglycan polymerase that works in conjunction with PBP2B, is also essential in the deletion background. Together, our results suggest functional overlap between PBP1A and the PBP2B-RodA complex in GBS cell wall peptidoglycan biosynthesis. High-molecular-mass penicillin-binding proteins (HMM PBPs) are enzymes required for bacterial cell wall biosynthesis. Bacterial pathogen group B streptococcus (GBS) produces five distinct HMM PBPs. The biological functions of these proteins are not well characterized in GBS. In this study, we performed a comprehensive deletion analysis of genes encoding HMM PBPs in GBS. We found that deleting certain PBP-encoding genes altered bacterial susceptibility to beta-lactam antibiotics, cell morphology, and the essentiality of other enzymes involved in cell wall peptidoglycan synthesis. The results of our study shed new light on the biological functions of PBPs in GBS.