The concentration of the cell division proteins FtsZ, FtsA, and ZipA and their assembly into a division ring during the Escherichia coli B/r K cell cycle have been measured in synchronous cultures obtained by the membrane elution technique. Immunostaining of the three proteins revealed no organized structure in newly born cells. In a culture with a doubling time of 49 min, assembly of the Z ring started around minute 25 and was detected first as a two-dot structure that became a sharp band before cell constriction. FtsA and ZipA localized into a division ring following the same pattern and time course as FtsZ. The concentration (amount relative to total mass) of the three proteins remained constant during one complete cell cycle, showing that assembly of a division ring is not driven by changes in the concentration of these proteins. Maintenance of the Z ring during the process of septation is a dynamic energy-dependent event, as evidenced by its disappearance in cells treated with sodium azide.

The role of the carboxy terminus of the Escherichia coli cell division protein FtsA in bacterial division has been studied by making a series of short sequential deletions spanning from residue 394 to 420. Deletions as short as 5 residues destroy the biological function of the protein. Residue W415 is essential for the localization of the protein into septal rings. Overexpression of the ftsA alleles harboring these deletions caused a coiled cell phenotype previously described for another carboxy-terminal mutation (Gayda et al., J. Bacteriol. 174:5362-5370, 1992), suggesting that an interaction of FtsA with itself might play a role in its function. The existence of such an interaction was demonstrated using the yeast two-hybrid system and a protein overlay assay. Even these short deletions are sufficient for impairing the interaction of the truncated FtsA forms with the wild-type protein in the yeast two-hybrid system. The existence of additional interactions between FtsA molecules, involving other domains, can be postulated from the interaction properties shown by the FtsA deletion mutant forms, because although unable to interact with the wild-type and with FtsA⌬1, they can interact with themselves and cross-interact with each other. The secondary structures of an extensive deletion, FtsA⌬27, and the wild-type protein are indistinguishable when analyzed by Fourier transform infrared spectroscopy, and moreover, FtsA⌬27 retains the ability to bind ATP. These results indicate that deletion of the carboxy-terminal 27 residues does not alter substantially the structure of the protein and suggest that the loss of biological function of the carboxy-terminal deletion mutants might be related to the modification of their interacting properties.FtsA is an essential cell division protein of Escherichia coli that is widely conserved in bacteria. Together with ftsZ, which codes for a GTPase analog of the eukaryotic tubulin, ftsA forms one of the most frequently conserved gene pairs among the cell division genes in the eubacteria. Based on sequence homology it has been proposed that FtsA belongs to the sugar kinase/hsp70/actin superfamily (4). This superfamily comprises several proteins with a common two-domain topology and the ability to bind and hydrolyze ATP. FtsA binds to columns of ATP-agarose and can be isolated from cells either as a phosphorylated or a nonphosphorylated form (29), but so far no other biochemical function has been described for this protein.FtsA is present both in the cytoplasm and in the cytoplasmic membrane (29), where it forms a structural part of the septum (32). It has been proposed that FtsA is a component of a membrane-associated complex (septator or divisome), which would include periplasmic, transmembrane, and cytoplasmic proteins acting coordinately to perform septation (27,35). Genetic analysis suggests that FtsA may interact, directly or indirectly, with other cell division proteins, such as FtsZ, PBP3, FtsQ, and FtsN (9,10,24,33,34). The FtsZ/FtsA ratio is important for cell division, an...

SummaryThe cell division protein FtsZ is a GTPase structurally related to tubulin and, like tubulin, it assembles in vitro into filaments, sheets and other structures. To study the roles that GTP binding and hydrolysis play in the dynamics of FtsZ polymerization, the nucleotide contents of FtsZ were measured under different polymerizing conditions using a nitrocellulose filterbinding assay, whereas polymerization of the protein was followed in parallel by light scattering. Unpolymerized FtsZ bound 1 mol of GTP mol 21 protein monomer. At pH 7.5 and in the presence of Mg 21 and K 1 , there was a strong GTPase activity; most of the bound nucleotide was GTP during the first few minutes but, later, the amount of GTP decreased in parallel with depolymerization, whereas the total nucleotide contents remained invariant. These results show that the long FtsZ polymers formed in solution contain mostly GTP. Incorporation of nucleotides into the protein was very fast either when the label was introduced at the onset of the reaction or subsequently during polymerization. Molecular modelling of an FtsZ dimer showed the presence of a cleft between the two subunits maintaining the nucleotide binding site open to the medium. These results show that the FtsZ polymers are highly dynamic structures that quickly exchange the bound nucleotide, and this exchange can occur in all the subunits.