The morphogenic protein CopD controls the spatio-temporal dynamics of PBP1a and PBP2b in Streptococcus pneumoniae

Abstract

ABSTRACT Penicillin-binding proteins (PBPs) are key to the assembly of peptidoglycan, the major component of the bacterial cell wall. Although several PBP-specific regulatory proteins have been identified in different species, little is known about how the activity of PBPs is controlled and coordinated during the cell cycle. In this study, we characterize the unknown function protein Spr1400 and demonstrate its regulatory function on two PBPs in Streptococcus pneumoniae. For that, we use a combination of technics ranging from bacterial genetics and protein biochemistry to microscopy imaging. First, we show that pneumococcal Spr1400 localizes late to the cell division septum. Furthermore, deletion of spr1400 results in wider cells. Using co-immunoprecipitation and bacterial two hybrid (B2H), we observe that Spr1400 interacts with two PBPs, the class A PBP PBP1a and the class B PBP PBP2b, which are required for cell elongation. Microscale thermophoresis combined with B2H further reveals that these interactions occur through their transmembrane domains. We also show that Spr1400 co-localizes with PBP1a and PBP2b throughout the cell cycle. Strikingly, deletion of spr1400 alters the dynamics of PBP1a and PBP2b. Indeed, the two PBPs persist longer at the division site and localize later at the division site of daughter cells. Collectively, these data demonstrate that Spr1400, thus named CopD for coordinator of PBP1a and 2b dynamics, is a spatio-temporal regulator of PBP1a and PBP2b required for pneumococcal morphogenesis. IMPORTANCE Penicillin-binding proteins (PBPs) are essential for proper bacterial cell division and morphogenesis. The genome of Streptococcus pneumoniae encodes for two class B PBPs (PBP2x and 2b), which are required for the assembly of the peptidoglycan framework and three class A PBPs (PBP1a, 1b and 2a), which remodel the peptidoglycan mesh during cell division. Therefore, their activities should be finely regulated in space and time to generate the pneumococcal ovoid cell shape. To date, two proteins, CozE and MacP, are known to regulate the function of PBP1a and PBP2a, respectively. In this study, we describe a novel regulator (CopD) that acts on both PBP1a and PBP2b. These findings provide valuable information for understanding bacterial cell division. Furthermore, knowing that ß-lactam antibiotic resistance often arises from PBP mutations, the characterization of such a regulator represents a promising opportunity to develop new strategies to resensitize resistant strains.

Keywords

• peptidoglycan
• penicillin-binding proteins
• cell division
• cell morphogenesis
• Streptococcus pneumoniae