References of "den Blaauwen, Tanneke"
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See detailBacterial cell wall growth, shape and division.
Derouaux, Adeline ULg; Terrak, Mohammed ULg; Den Blaauwen, Tanneke et al

in Bacterial Membranes: Structural and Molecular Biology (2013)

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See detailCooperativity of peptidoglycan synthases active in bacterial cell elongation.
Banzhaf, Manuel; van den Berg van Saparoea, Bart; Terrak, Mohammed ULg et al

in Molecular Microbiology (2012), 85(1), 179-94

Growth of the bacterial cell wall peptidoglycan sacculus requires the co-ordinated activities of peptidoglycan synthases, hydrolases and cell morphogenesis proteins, but the details of these interactions ... [more ▼]

Growth of the bacterial cell wall peptidoglycan sacculus requires the co-ordinated activities of peptidoglycan synthases, hydrolases and cell morphogenesis proteins, but the details of these interactions are largely unknown. We now show that the Escherichia coli peptidoglycan glycosyltrasferase-transpeptidase PBP1A interacts with the cell elongation-specific transpeptidase PBP2 in vitro and in the cell. Cells lacking PBP1A are thinner and initiate cell division later in the cell cycle. PBP1A localizes mainly to the cylindrical wall of the cell, supporting its role in cell elongation. Our in vitro peptidoglycan synthesis assays provide novel insights into the cooperativity of peptidoglycan synthases with different activities. PBP2 stimulates the glycosyltransferase activity of PBP1A, and PBP1A and PBP2 cooperate to attach newly synthesized peptidoglycan to sacculi. PBP2 has peptidoglycan transpeptidase activity in the presence of active PBP1A. Our data also provide a possible explanation for the depletion of lipid II precursors in penicillin-treated cells. [less ▲]

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See detailThe integral membrane FtsW protein and peptidoglycan synthase PBP3 form a subcomplex in Escherichia coli
Fraipont, Claudine ULg; Alexeeva, Svetlana; Wolf, Benoît et al

in Microbiology (2011), 157

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See detailSeptal and lateral wall localization of PBP5, the major D,D-carboxypeptidase of Escherichia coli, requires substrate recognition and membrane attachment
Potluri, Lakshmiprasad; Karczmarek, Aneta; Verheul, Jolanda et al

in Molecular Microbiology (2010), 77(2), 300323

The distribution of PBP5, the major D,D-carboxypeptidase in Escherichia coli, was mapped by immunolabelling and by visualization of GFP fusion proteins in wild-type cells and in mutants lacking one or ... [more ▼]

The distribution of PBP5, the major D,D-carboxypeptidase in Escherichia coli, was mapped by immunolabelling and by visualization of GFP fusion proteins in wild-type cells and in mutants lacking one or more D,D-carboxypeptidases. In addition to being scattered around the lateral envelope, PBP5 was also concentrated at nascent division sites prior to visible constriction. Inhibiting PBP2 activity (which eliminates wall elongation) shifted PBP5 to midcell, whereas inhibiting PBP3 (which aborts divisome invagination) led to the creation of PBP5 rings at positions of preseptal wall formation, implying that PBP5 localizes to areas of ongoing peptidoglycan synthesis. A PBP5(S44G) active site mutant was more evenly dispersed, indicating that localization required enzyme activity and the availability of pentapeptide substrates. Both the membrane bound and soluble forms of PBP5 converted pentapeptides to tetrapeptides in vitro and in vivo, and the enzymes accepted the same range of substrates, including sacculi, Lipid II, muropeptides and artificial substrates. However, only the membrane-bound form localized to the developing septum and restored wild-type rod morphology to shape defective mutants, suggesting that the two events are related. The results indicate that PBP5 localization to sites of ongoing peptidoglycan synthesis is substrate dependent and requires membrane attachment. [less ▲]

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See detailInteraction between two murein (peptidoglycan) synthases, PBP3 and PBP1B, in Escherichia coli
Bertsche, Ute; Kast, Thomas; Wolf, Benoît ULg et al

in Molecular Microbiology (2006), 61(3), 675-690

The murein (peptidoglycan) sacculus is an essential polymer embedded in the bacterial envelope. The Escherichia coli class B penicillin-binding protein (PBP) 3 is a murein transpeptidase and essential for ... [more ▼]

The murein (peptidoglycan) sacculus is an essential polymer embedded in the bacterial envelope. The Escherichia coli class B penicillin-binding protein (PBP) 3 is a murein transpeptidase and essential for cell division. In an affinity chromatography experiment, the bifunctional transglycosylase-transpeptidase murein synthase PBP1B was retained by PBP3-sepharose when a membrane fraction of E. coli was applied. The direct protein-protein interaction between purified PBP3 and PBP1B was characterized in vitro by surface plasmon resonance. The interaction was confirmed in vivo employing two different methods: by a bacterial two-hybrid system, and by cross-linking/co-immunoprecipitation. In the bacterial two-hybrid system, a truncated PBP3 comprising the N-terminal 56 amino acids interacted with PBP1B. Both synthases could be cross-linked in vivo in wild-type cells and in cells lacking FtsW or FtsN. PBP1B localized diffusely and in foci at the septation site and also at the side wall. Statistical analysis of the immunofluorescence signals revealed that the localization of PBP1B at the septation site depended on the physical presence of PBP3, but not on the activity of PBP3. These studies have demonstrated, for the first time, a direct interaction between a class B PBP (PBP3) and a class A PBP (PBP1B) in vitro and in vivo, indicating that different murein synthases might act in concert to enlarge the murein sacculus during cell division. [less ▲]

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See detailMaturation of the Escherichia coli divisome occurs in two steps
Aarsman, Mirjam E. G.; Piette, André ULg; Fraipont, Claudine ULg et al

in Molecular Microbiology (2005), 55(6), 1631-1645

Cell division proteins FtsZ ( FtsA, ZipA, ZapA), FtsE/X, FtsK, FtsQ, FtsL/B, FtsW, PBP3, FtsN and AmiC localize at mid cell in Escherichia coli in an interdependent order as listed. To investigate whether ... [more ▼]

Cell division proteins FtsZ ( FtsA, ZipA, ZapA), FtsE/X, FtsK, FtsQ, FtsL/B, FtsW, PBP3, FtsN and AmiC localize at mid cell in Escherichia coli in an interdependent order as listed. To investigate whether this reflects a time dependent maturation of the divisome, the average cell age at which FtsZ, FtsQ, FtsW, PBP3 and FtsN arrive at their destination was determined by immuno- and GFP- fluorescence microscopy of steady state grown cells at a variety of growth rates. Consistently, a time delay of 14 - 21 min, depending on the growth rate, between Z-ring formation and the mid cell recruitment of proteins down stream of FtsK was found. We suggest a two-step model for bacterial division in which the Z-ring is involved in the switch from cylindrical to polar peptidoglycan synthesis, whereas the much later localizing cell division proteins are responsible for the modification of the envelope shape into that of two new poles. [less ▲]

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See detailFunctional analysis of the cell division protein FtsW of Escherichia coli
Pastoret, Soumya; Fraipont, Claudine ULg; den Blaauwen, Tanneke et al

in Journal of Bacteriology (2004), 186(24), 8370-8379

Site-directed mutagenesis experiments combined with fluorescence microscopy shed light on the role of Escherichia coli FtsW, a membrane protein belonging to the SEDS family that is involved in ... [more ▼]

Site-directed mutagenesis experiments combined with fluorescence microscopy shed light on the role of Escherichia coli FtsW, a membrane protein belonging to the SEDS family that is involved in peptidoglycan assembly during cell elongation, division, and sporulation. This essential cell division protein has 10 transmembrane segments (TMSs). It is a late recruit to the division site and is required for subsequent recruitment of penicillin-binding protein 3 (PBP3) catalyzing peptide cross-linking. The results allow identification of several domains of the protein with distinct functions. The localization of PBP3 to the septum was found to be dependent on the periplasmic loop located between TMSs 9 and 10. The E240-A249 amphiphilic peptide in the periplasmic loop between TMSs 7 and 8 appears to be a key element in the functioning of FtsW in the septal peptidoglycan assembly machineries. The intracellular loop (containing the R166-FI78 amphiphilic peptide) between TMSs 4 and 5 and Gly 311 in TMS 8 are important components of the amino acid sequence-folding information. [less ▲]

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See detailStructural determinants required to target penicillin-binding protein 3 to the septum of Escherichia coli
Piette, André ULg; Fraipont, Claudine ULg; den Blaauwen, Tanneke et al

in Journal of Bacteriology (2004), 186(18), 6110-6117

In Escherichia coli, cell division is mediated by the concerted action of about 12 proteins that assemble at the division site to presumably form a complex called the divisome. Among these essential ... [more ▼]

In Escherichia coli, cell division is mediated by the concerted action of about 12 proteins that assemble at the division site to presumably form a complex called the divisome. Among these essential division proteins, the multimodular class B penicillin-binding protein 3 (PBP3), which is specifically involved in septall peptidoglycan synthesis, consists of a short intraceflular M1-R23 peptide fused to a F24-L39 membrane anchor that is linked via a G40-S70 peptide to an R71-1236 noncatalytic module itself linked to a D237-V577 catalytic penicillin -binding module. On the basis of localization analyses of PBP3 mutants fused to green fluorescent protein by fluorescence microscopy, it appears that the first 56 amino acid residues of PBP3 containing the membrane anchor and the G40-E56 peptide contain the structural determinants required to target the protein to the cell division site and that none of the putative protein interaction sites present in the noncatalytic module are essential for the positioning of the protein to the division site. Based on the effects of increasing production of FtsQ or FtsW on the division of cells expressing PBP3 mutants, it is suggested that these proteins could interact. We postulate that FtsQ could play a role in regulating the assembly of these division proteins at the division site and the activity of the peptidoglycan assembly machineries within the divisome. [less ▲]

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