Direct interactions of early and late assembling division proteins in Escherichia coli cells resolved by FRET.
The bacterial cell division machinery is organized in the so-called divisome composed of highly dynamic but low abundant interacting (membrane-bound) proteins. In order to elucidate the molecular interactions between these proteins, we developed a robust background-insensitive quantitative spectral unmixing method for estimating FRET efficiencies at near endogenous protein levels using fluorescent protein fusions. The assembly of the division machinery of Escherichia coli occurs in two steps that are discrete in time: first the FtsZ-ring and the so-called early localizing proteins that together seem to prepare the division assembly at midcell. Subsequently, the late localizing protein complexes that contain the peptidoglycan-synthesizing proteins PBP1B and FtsI (PBP3) are recruited to the division site, which initiates septation. Physical interactions were observed between members within each group but also between the early and late localizing proteins strongly suggesting that these proteins despite their differential localization in time are linked at the molecular and functional level. Interestingly, we find FtsN, one of the latest proteins in the divisome assembly, interacting with late assembling proteins FtsI and FtsW, but also with early (proto-ring) protein ZapA. This is in line with the recently described role of FtsN in divisome stabilization including the proto-ring elements.
Alexeeva
Svetlana
S
Molecular Cytology, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Amsterdam, the Netherlands.
Gadella
Theodorus W J
TW
Verheul
Jolanda
J
Verhoeven
Gertjan S
GS
den Blaauwen
Tanneke
T
D001426:Bacterial Proteins / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002455:Cell Division; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D008565:Membrane Proteins / Q000378:metabolism; D046915:Penicillin-Binding Proteins / Q000378:metabolism*; D046928:Peptidoglycan Glycosyltransferase / Q000378:metabolism*; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D029968:Escherichia coli Proteins; C478704:FtsI protein, E coli; C098711:FtsN protein, E coli; C071525:FtsZ protein, Bacteria; D008565:Membrane Proteins; D046915:Penicillin-Binding Proteins; C520744:ZapA protein, E coli; D046928:Peptidoglycan Glycosyltransferase; C478681:penicillin-binding protein 1B, E coli; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase