The function of the transmembrane and cytoplasmic domains of pneumococcal penicillin-binding proteins 2x and 2b extends beyond that of simple anchoring devices

Abstract

The biosynthesis of cell-wall peptidoglycan is a complex process that involves six different penicillin-binding proteins (PBPs) in Streptococcus pneumoniae. Two of these, PBP2x and PBP2b, are monofunctional transpeptidases that catalyse the formation of peptide cross-links between adjacent glycan strands. Both of them are bitopic membrane proteins with a small cytoplasmic and a large extracellular domain. PBP2x and PBP2b are essential for septal and peripheral peptidoglycan synthesis, respectively. Although several studies have investigated the properties of their extracellular catalytic domains, it is not known whether the role of their N-terminal non-catalytic domains extends beyond that of being simple anchoring devices. We therefore decided to use reciprocal domain swapping and mutational analysis to gain more information about the biological function of the membrane anchors and cytoplasmic tails of PBP2x and PBP2b. In the case of PBP2x both domains are essential, but neither the membrane anchor nor the cytoplasmic domain of PBP2x appear to serve as major localization signals. Instead, our results suggest that they are involved in interactions with other components of the divisome. Mutations of conserved amino acids in the cytoplasmic domain of PBP2x resulted in loss of function, underlining the importance of this region. The cytoplasmic domain of PBP2b could be swapped with the corresponding domain from PBP2x, whereas replacement of the PBP2b transmembrane domain with the corresponding PBP2x domain gave rise to slow-growing cells with grossly abnormal morphology. When both domains were exchanged simultaneously the cells were no longer viable.

Pbp2x localizes separately from Pbp2b and other peptidoglycan synthesis proteins during later stages of cell division of Streptococcus pneumoniae D39

“…Recent results establish that the extracellular C‐terminal PASTA domains, but not the transpeptidase domain or its activity, are required for normal localization of pneumococcal Pbp2x (Peters et al ., ). This conclusion was further supported in a new paper showing that the cytoplasmic and transmembrane domains of pneumococcal Pbp2x are essential for function, but not for normal localization (Berg et al ., ). In another recent study, the extracellular PASTA domains of the StkP Ser/Thr kinase were implicated in normal localization of Pbp2x, possibly through a direct interaction (Morlot et al ., ).…”

“…B; Results ; Berg et al ., ). These depletion experiments suggest that Pbp2x and Pbp2b are essential in S. pneumoniae ( Results ; Berg et al ., 2013; 2014; Fleurie et al ., ; Peters et al ., ).…”

The Pneumococcal Cell Wall

“…These data indicate a specific interaction between PBP2x and DivIB, perhaps mediated by the transmembrane regions of both proteins, and of PBP2x with the large extracellular loop between transmembrane segments 7 and 8 of the lipid II transporter FtsW [38]. Insights into the role of the N-terminal domain came from domain swapping and mutational analysis of PBP2x and PBP2b [39]. These results revealed that both the cytoplasmic and transmembrane domains of PBP2x and PBP2b are needed for correct functioning of these enzymes, suggesting possible interactions with divisome proteins such as DivIB and FtsW [39].…”

“…Insights into the role of the N-terminal domain came from domain swapping and mutational analysis of PBP2x and PBP2b [39]. These results revealed that both the cytoplasmic and transmembrane domains of PBP2x and PBP2b are needed for correct functioning of these enzymes, suggesting possible interactions with divisome proteins such as DivIB and FtsW [39].…”

Suppression of a deletion mutation in the gene encoding essential PBP2b reveals a new lytic transglycosylase involved in peripheral peptidoglycan synthesis in Streptococcus pneumoniae D39

“…Recent work shows that class B PBP2x and PBP2b are essential for growth and required for septal and peripheral PG synthesis, respectively, in dividing S. pneumoniae cells (Berg et al, 2013;Land et al, 2013;Morlot et al, 2013;Berg et al, 2014;Fleurie et al, 2014;Peters et al, 2014;Tsui et al, 2014). pbp1a and pbp2a, encoding PBP1a and PBP2a, respectively, are synthetically lethal and cannot both be inactivated (Hoskins et al, 1999;Paik et al, 1999).…”