Direct interactions of early and late assembling division proteins in Escherichia coli cells resolved by FRET

Abstract

SummaryThe 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.

Bacterial SPOR domains are recruited to septal peptidoglycan by binding to glycan strands that lack stem peptides

“…Understanding how these proteins are targeted to that site is important for understanding how division is spatially and temporally regulated. The division apparatus is generally thought of as a multiprotein complex (or a collection of smaller complexes) whose assembly is driven, for the most part, by protein-protein interactions (23,(31)(32)(33)(34)(35)(36). However proteins that contain a small PG-binding domain known as a "SPOR domain" are thought to be recruited by binding to septal PG (15)(16)(17).…”

Positive control of cell division: FtsZ is recruited by SsgB during sporulation ofStreptomyces

“…To quantitatively assess the in vivo proximity and direct interactions between SsgA, SsgB, and FtsZ, Fö rster resonance energy transfer (FRET) combined with FLIM was applied (Cremazy et al 2005). Its application was successfully applied recently to study the interaction between members of the Escherichia coli divisome (Alexeeva et al 2010). FRET is the capacity of a low-wavelength fluorophore to transfer its excited state energy to a longerwavelength counterpart given a sufficient overlap of the corresponding emission and excitation spectra, if the proximity between the two fluorescent proteins is between 2 and 10 nm.…”

In Vivo Bacterial Morphogenetic Protein Interactions

“…More than twenty proteins have been identified to be involved in the division process of which at least twelve are essential. Between the latter proteins many interactions are observed and it is therefore hypothesized that a big protein complex is formed referred to as the Divisome (Alexeeva et al, 2010;Di lallo et al, 2003;Karimova et al, 1998;Maggi et al, 2008).…”

“…The contribution of each component can be quantified by 'linear unmixing' of the spectra (Clegg, 1992;Clegg et al, 1992;Murchie et al, 1989;Wlodarczyk et al, 2008). An elaborate unmixing description fitting to our setup can be found in the supplemental information of the corresponding publication and will be explained in less detail (Alexeeva et al, 2010).…”

“…Real-time measurements will then be difficult especially when emission spectra over a broad range of wavelengths are required. To minimize the level of auto-fluorescence originating from the bacteria, redshifted fluorescent proteins are more suitable for these studies (Alexeeva et al, 2010). The Fig.…”