Regulation of the essential peptidoglycan hydrolytic complex FtsEX-PcsB during Streptococcus pneumoniae cell division

In the human commensal Gram-positive bacterial pathogen Streptococcus pneumoniae, the essential extracellular cell-division-associated peptidoglycan (PG) hydrolase PcsB interacts directly with the cytoplasmic-membrane-bound complex between FtsE and FtsX (1-3). PcsB contains a cysteine, hishdine-dependent amidohydrolase/pephdase (CHAP) domain responsible for PG hydrolysis, as well as a coiled-coil domain required for interaction with FtsEX (1,4). ATP hydrolysis of FtsE in the cytoplasm drives conformational changes in FtsX in the cytoplasmic membrane, which ultimately regulates the PG hydrolase on the outside of the cell (5). In this work we show using in vitro and in vivo approaches, that the CHAP domain of PcsB predominately functions as an iso -D-Glutaminyl-Lysyl D,L-endopeptidase, with particular substrate specificity for Lys-containing, amidated PG, cleaving between the second and third amino acids of the peptidoglycan stem peptide. The catalytic activity of PcsB is regulated and activated by conformation changes of the coiled-coil region of PcsB and in part by a short helical region immediately adjacent to the CHAP domain to guard against PcsB hydrolytic activation outside of its cell division specific functional requirement. This work supports a model for the overall biological activity of the FtsEX-PcsB complex, in which ATP hydrolysis by FtsE in the cytoplasm, drives conformational changes in FtsX and PcsB resulting in the liberation of the hydrolytic CHAP domain of PcsB from its regulatory helix to allow PG stem peptide cleavage that splits the septal disk and marks a region of the peptidoglycan sacculus for subsequent cell division remodelling.

Bacterial cell division requires coordinated assembly of specific proteins acting on the cell envelope to induce division. The cell division complex FtsEX is recruited early in divisome assembly and controls extracellular hydrolase function, which modifies the peptidoglycan layer. We demonstrate in Streptococcus pneumoniae , where these proteins are essential, connections between ATP hydrolysis in cytosolic FtsE, mechanotransduction through FtsX in the membrane, activation of PcsB in the intracellular space and molecular details of the chemical modification of the peptidoglycan layer. Here we present a model whereby the FtsEX-PcsB complex is assembled early in order to produce a hydrolase-modified form of peptidoglycan during septal splitting, marking the site of later division events; thus providing a rationale for its biological and temporal function.

‒ Major - Biological Sciences‒ Minor - Biochemistry.