The bacterial cell division protein FtsZ from Escherichia coli has been purified with a new calcium precipitation method. The protein contains one GDP and one Mg 2؉ bound, it shows GTPase activity, and requires GTP and Mg 2؉ to polymerize into long thin filaments at pH 6.5. FtsZ, with moderate ionic strength and low Mg 2؉ concentrations, at pH 7.5, is a compact and globular monomer. Mg 2؉ induces FtsZ self-association into oligomers, which has been studied by sedimentation equilibrium over a wide range of Mg 2؉ and FtsZ concentrations. The oligomer formation mechanism is best described as an indefinite self-association, with binding of an additional Mg 2؉ for each FtsZ monomer added to the growing oligomer, and a slight gradual decrease of the affinity of addition of a protomer with increasing oligomer size. The sedimentation velocity of FtsZ oligomer populations is compatible with a linear singlestranded arrangement of FtsZ monomers and a spacing of 4 nm. It is proposed that these FtsZ oligomers and the polymers formed under assembly conditions share a similar axial interaction between monomers (like in the case of tubulin, the eukaryotic homolog of FtsZ). Similar mechanisms may apply to FtsZ assembly in vivo, but additional factors, such as macromolecular crowding, nucleoid occlusion, or specific interactions with other cellular components active in septation have to be invoked to explain FtsZ assembly into a division ring.The FtsZ protein, which is found in all known eubacteria and archaea as well as in chloroplasts with the exception of the Chlamydiae (1, 2), plays a central role in bacterial cell division (3-5). During the life of a cell, FtsZ molecules remain in the cytoplasm until the time previous to division when they localize at the midcell point, forming a ring at the inner face of the cytoplasmic membrane where the septum will form (6, 7). The interactions of FtsZ with other septator proteins that are functionally relevant, such as PBP3 and FtsA, are being discovered (reviewed in Refs. 4,5,8,and 9). Several regulators of FtsZ ring formation have been found that either prevent/inhibit polymer formation (like the SOS response protein SulA (10), the MinCDE complex (11), and more recently, the ErzA protein from Bacillus subtilis (12)) or stabilize the FtsZ polymer (like the membrane protein ZipA (13, 14)). Despite this information, FtsZ ring structure and assembly are still poorly understood.FtsZ binds guanine nucleotides and has GTPase activity (15-17), which is dependent on protein concentration and probably is linked to self-association equilibria (15,16,18). Multiple sequence alignment and secondary structure predictions indicate that bacterial FtsZ is a homolog of eukaryotic tubulins (19,20). The x-ray crystal structure of the FtsZ from the archaeon Methanococcus jannaschii (21) and the electron crystallographic model structure of the ␣␤-tubulin dimer (22) have similar folds.The nature of the polymers that FtsZ forms in vivo is unknown. FtsZ self-assembles in vitro to form filaments and rings (23-28...

Experimental conditions that simulate the crowded bacterial cytoplasmic environment have been used to study the assembly of the essential cell division protein FtsZ from Escherichia coli. In solutions containing a suitable concentration of physiological osmolytes, macromolecular crowding promotes the GTP-dependent assembly of FtsZ into dynamic two-dimensional polymers that disassemble upon GTP depletion. Atomic force microscopy reveals that these FtsZ polymers adopt the shape of ribbons that are one subunit thick. When compared with the FtsZ filaments observed in vitro in the absence of crowding, the ribbons show a lag in the GTPase activity and a decrease in the GTPase rate and in the rate of GTP exchange within the polymer. We propose that, in the crowded bacterial cytoplasm under assembly-promoting conditions, the FtsZ filaments tend to align forming dynamic ribbon polymers. In vivo these ribbons would fit into the Z-ring even in the absence of other interactions. Therefore, the presence of mechanisms to prevent the spontaneous assembly of the Z-ring in non-dividing cells must be invoked.

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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.