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

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

Author information

1
Institut de Biologie Structurale, Université Grenoble Alpes, Grenoble, France ; Institut de Biologie Structurale, Direction des Sciences du Vivant, Commissariat à l'energie atomique et aux Energies Alternatives, Grenoble, France ; Institut de Biologie Structurale, Centre National de la Recherche Scientifique, Grenoble, France.

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.

PMID:
24147156
PMCID:
PMC3798694
DOI:
10.1371/journal.pone.0075522
[Indexed for MEDLINE]
Free PMC Article

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