Reconstitution of membrane protein complexes involved in pneumococcal septal cell wall assembly

PLoS One. 2013 Sep 23;8(9):e75522. doi: 10.1371/journal.pone.0075522. eCollection 2013.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / isolation & purification
  • Bacterial Proteins / metabolism*
  • Cell Division*
  • Cell Wall / metabolism*
  • Hydrolysis
  • Kinetics
  • Membrane Proteins / genetics
  • Membrane Proteins / isolation & purification
  • Membrane Proteins / metabolism*
  • Proteolysis
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Streptococcus pneumoniae / genetics
  • Streptococcus pneumoniae / metabolism*

Substances

  • Bacterial Proteins
  • Membrane Proteins
  • Recombinant Proteins

Grant support

This work was partly funded by the “Coopol innovation France-Chine” program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study. This work used the platforms of the Grenoble Instruct centre (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) with support from FRISBI(ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB).