Reconstitution of Membrane Protein Complexes Involved in Pneumococcal Septal Cell Wall Assembly

Abstract

The synthesis of peptidoglycan, the major component of the bacterial cell wall, is essential to cell survival, yet its mechanism remains poorly understood. In the present work, we have isolated several membrane protein complexes consisting of the late division proteins of Streptococcus pneumoniae: DivIB, DivIC, FtsL, PBP2x and FtsW, or subsets thereof. We have co-expressed membrane proteins from S. pneumoniae in Escherichia coli. By combining two successive affinity chromatography steps, we obtained membrane protein complexes with a very good purity. These complexes are functional, as indicated by the retained activity of PBP2x to bind a fluorescent derivative of penicillin and to hydrolyze the substrate analogue S2d. Moreover, we have evidenced the stabilizing role of protein-protein interactions within each complex. This work paves the way for a complete reconstitution of peptidoglycan synthesis in vitro, which will be critical to the elucidation of its intricate regulation mechanisms.

Substitutions in PBP2b from β-Lactam-resistant Streptococcus pneumoniae Have Different Effects on Enzymatic Activity and Drug Reactivity

“…In any case, the in vitro transpeptidase activities reported here and previously are extremely weak and far from realistic physiologic rates. In the future, it will be necessary to study monofunctional PBPs in a membrane environment and/or in the presence of their protein partners (36), in particular SEDS proteins that act as transglycosylases (37).…”

Identification of pneumococcal proteins that are functionally linked to penicillin‐binding protein 2b (PBP2b)

“…FtsW and PBP3 are both essential for septal peptidoglycan synthesis during cell division in E. coli (Boyle et al, 1997;Pastoret et al, 2004). Localization of FtsW to the divisome has also been demonstrated in B. subtilis and S. pneumoniae (Morlot et al, 2004;Gamba et al, 2009;Noirclerc-Savoye et al, 2013). Due to their sequence homology and topological equivalence, it is likely that FtsW and RodA have the same or similar functions in the bacterial cell (Ikeda et al, 1989;G erard et al, 2002).…”

The Pneumococcal Cell Wall

“…Another recent study reported the formation of stable membrane protein complexes consisting of late division proteins DivIB, DivIC, FtsL, PBP2x, and FtsW [38]. 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].…”