T. den Blaauwen

Escherichia coli Minicell Membranes Are Enriched in Cardiolipin

The phospholipid composition of Escherichia coli minicells has been studied as a model for the cell division site. Minicells appeared to be enriched in cardiolipin at the expense of phosphatidylglycerol. Mass spectrometry showed no differences between the gross acyl chain compositions of minicells and wild-type cells.

SecA is an intrinsic subunit of the Escherichia coli preprotein translocase and exposes its carboxyl terminus to the periplasm

SecA is the dissociable ATPase subunit of the Escherichia coli preprotein translocase, and cycles in a nucleotide‐modulated manner between the cytosol and the membrane. Overproduction of the integral subunits of the translocase,the SecY, SecE and SecG polypeptides, results in an increased level of membrane‐bound SecA. This fraction of SecA is firmly associated with the membrane as it is resistant to extraction with the chaotropic agent urea, and appears to be anchored by SecYEG rather than by lipids. Topology analysis of this membrane‐associated form of SecA indicates that it exposes a carboxy‐terminal domain to the periplasmic face of the membrane.

Sec-dependent preprotein translocation in bacteria

Abstract Translocation of precursor proteins across the cytoplasmic membrane in bacteria is mediated by a multi-subunit protein complex termed translocase, which consists of the integral membrane heterotrimer SecYEG and the peripheral homodimeric ATPase SecA. Preproteins are bound by the cytosolic molecular chaperone SecB and targeted in a complex with SecA to the translocation site at the cytoplasmic membrane. This interaction with SecYEG allows the SecA/preprotein complex to insert into the membrane by binding of ATP to the high affinity nucleotide binding site of SecA. At that stage, presumably recognition and proofreading of the signal sequence occurs. Hydrolysis of ATP causes the release of the preprotein in the translocation channel and drives the withdrawal of SecA from the membrane-integrated state. Hydrolysis of ATP at the low-affinity nucleotide binding site of SecA converts the protein into a compact conformational state and releases it from the membrane. In the absence of the proton motive force, SecA is able to complete the translocation stepwise by multiple nucleotide modulated cycles.

Non-hydrolysable GTP-gamma-S stabilizes the FtsZ polymer in a GDP-bound state

FtsZ, a tubulin homologue, forms a cytokinetic ring at the site of cell division in prokaryotes. The ring is thought to consist of polymers that assemble in a strictly GTP‐dependent way. GTP, but not guanosine‐5′‐O‐(3‐thiotriphosphate) (GTP‐γ‐S), has been shown to induce polymerization of FtsZ, whereas in vitro Ca2+ is known to inhibit the GTP hydrolysis activity of FtsZ. We have studied FtsZ dynamics at limiting GTP concentrations in the presence of 10 mM Ca2+. GTP and its non‐hydrolysable analogue GTP‐γ‐S bind FtsZ with similar affinity, whereas the non‐hydrolysable analogue guanylyl‐imidodiphosphate (GMP‐PNP) is a poor substrate. Preformed FtsZ polymers can be stabilized by GTP‐γ‐S and are destabilized by GDP. As more than 95% of the nucleotide associated with the FtsZ polymer is in the GDP form, it is concluded that GTP hydrolysis by itself does not trigger FtsZ polymer disassembly. Strikingly, GTP‐γ‐S exchanges only a small portion of the FtsZ polymer‐bound GDP. These data suggest that FtsZ polymers are stabilized by a small fraction of GTP‐containing FtsZ subunits. These subunits may be located either throughout the polymer or at the polymer ends, forming a GTP cap similar to tubulin.

Insights into nucleotide recognition by cell division protein FtsZ from a mant-GTP competition assay and molecular dynamics

Essential cell division protein FtsZ forms the bacterial cytokinetic ring and is a target for new antibiotics. FtsZ monomers bind GTP and assemble into filaments. Hydrolysis to GDP at the association interface between monomers leads to filament disassembly. We have developed a homogeneous competition assay, employing the fluorescence anisotropy change of mant-GTP upon binding to nucleotide-free FtsZ, which detects compounds binding to the nucleotide site in FtsZ monomers and measures their affinities within the millimolar to 10 nM range. We have employed this method to determine the apparent contributions of the guanine, ribose, and the α-, β-, and γ-phosphates to the free energy change of nucleotide binding. Similar relative contributions have also been estimated through molecular dynamics and binding free energy calculations, employing the crystal structures of FtsZ-nucleotide complexes. We find an energetically dominant contribution of the β-phosphate, comparable to the whole guanosine moiety. GTP and GDP bind with similar observed affinity to FtsZ monomers. Loss of the regulatory γ-phosphate results in a predicted accommodation of GDP which has not been observed in the crystal structures. The binding affinities of a series of C8-substituted GTP analogues, known to inhibit FtsZ but not eukaryotic tubulin assembly, correlate with their inhibitory capacity on FtsZ polymerization. Our methods permit testing of FtsZ inhibitors targeting its nucleotide site, as well as compounds from virtual screening of large synthetic libraries. Our results give insight into the FtsZ-nucleotide interactions, which could be useful in the rational design of new inhibitors, especially GTP phosphate mimetics.

Stimulation and inhibition of cell division in synchronized Escherichia coli.

Escherichia coli was synchronized by centrifugal elutriation. When grown in a Tris-based medium, addition of EDTA resulted in division about 20 min earlier (division of control at t = 75 min). EDTA addition caused a change in cell shape, with cells becoming narrower and longer, whereas the surface area to volume ratio increased. Irradiation with UV inhibited not only division and constriction, but also the increase in DAP incorporation found in dividing control cells. Possibly, division requires the construction of new polar caps, whereas premature division might involve remodeling of existing murein. In both cases, cell shape is presumed to be a relevant factor for division.