SummaryCell 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 immunoand 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.

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.

The complete understanding of the morphological differentiation of streptomycetes is an ambitious challenge as diverse sensors and pathways sensitive to various environmental stimuli control the process. Germination occupies a particular position in the life cycle as the good achievement of the process depends on events occurring both during the preceding sporulation and during germination per se. The cyclic AMP receptor protein (crp) null mutant of Streptomyces coelicolor, affected in both sporulation and germination, was therefore presented as a privileged candidate to highlight new proteins involved in the shift from dormant to germinating spores. Our multidisciplinary approach-combining in vivo data, the analysis of spores morphological properties, and a proteome study-has shown that Crp is a central regulatory protein of the life cycle in S. coelicolor; and has identified spores proteins with statistically significant increased or decreased expression that should be listed as priority targets for further investigations on proteins that trigger both ends of the life cycle.

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...