SummaryThe 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 transglycosylasetranspeptidase 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.

During the cell cycle of rod-shaped bacteria, two morphogenetic processes can be discriminated: length growth of the cylindrical part of the cell and cell division by formation of two new cell poles. The morphogenetic protein complex responsible for the septation during cell division (the divisome) includes class A and class B penicillin-binding proteins (PBPs). In Escherichia coli, the class B PBP3 is specific for septal peptidoglycan synthesis. It requires the putative lipid II flippase FtsW for its localization at the division site and is necessary for the midcell localization of the class A PBP1B. In this work we show direct interactions between FtsW and PBP3 in vivo and in vitro by FRET (Fö rster resonance energy transfer) and co-immunoprecipitation experiments. These proteins are able to form a discrete complex independently of the other cell-division proteins. The K2-V42 peptide of PBP3 containing the membrane-spanning sequence is a structural determinant sufficient for interaction with FtsW and for PBP3 dimerization. By using a two-hybrid assay, the class A PBP1B was shown to interact with FtsW. However, it could not be detected in the immunoprecipitated FtsW-PBP3 complex. The periplasmic loop 9/10 of FtsW appeared to be involved in the interaction with both PBP1B and PBP3. It might play an important role in the positioning of these proteins within the divisome.

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 penicillinbinding 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-F178 amphiphilic peptide) between TMSs 4 and 5 and Gly 311 in TMS 8 are important components of the amino acid sequence-folding information.The wall peptidoglycan is a bacterium-specific polymer that preserves cell integrity and plays an important role in bacterial morphogenesis. To allow bacterial cell growth and division, two morphogenetic networks channel peptidoglycan assembly into wall expansion and septum formation in a cell cycle-dependent fashion. The morphogenetic protein machineries responsible for maintenance of the cell's cylindrical shape during cell elongation and for septation during cell division involve a set of network-specific proteins (2,16,38,42).In Escherichia coli, the first morphogenetic network includes class B penicillin-binding protein 2 (PBP2) and the RodA protein, which are responsible for wall elongation and maintenance of the rod shape. They are encoded by the pbpA and rodA genes, respectively, which form an operon with the dacA gene, encoding the monofunctional PBP5 in the 14-min region of the chromosome (49, 50). Inactivation of RodA or PBP2 leads to the formation of spherical cells. MreB, MreC, and MreD also belong to this morphogenetic network (53). If MreB is not produced, the cells become spherical (54). MreB is an actinlike protein (52) that polymerizes into fibrous spirals at the inner face of the membrane in E. coli and Bacillus subtilis (25,48). This protein is required for proper chromosome segregation (32). In Caulobacter crescentus, MreB seems to spatially coordinate the activities of the cell wall assembly proteins (17).Formation of the septum in E. coli requires class B PBP3 (also called FtsI) and FtsZ, FtsA, ZipA, FtsK, FtsQ, FtsL, FtsB, FtsW, and FtsN, which constitute the second morphogenetic network. The majority of the genes encoding these proteins lie in the 2-min region of the chromosome and form the dcw (division cell wall) operon. Inactivation of one gene inhibits septation and leads to filamentous growth (4, 36). To initiate cell division, the GTP-binding tubulin-like FtsZ protein forms an intracellular ring at the division site (34, 35...

The monofunctional peptidoglycan glycosyltransferase (MtgA) catalyzes glycan chain elongation of the bacterial cell wall. Here we show that MtgA localizes at the division site of Escherichia coli cells that are deficient in PBP1b and produce a thermosensitive PBP1a and is able to interact with three constituents of the divisome, PBP3, FtsW, and FtsN, suggesting that MtgA may play a role in peptidoglycan assembly during the cell cycle in collaboration with other proteins.