Cytokinesis in bacteria involves the coordinated constriction of cell envelope layers by the septal ring, a cytoskeleton-like organelle that assembles at the division site. This equatorial ring structure remains associated with the invaginating cell membrane until the two daughter cells separate (reviewed in Rothfield et al., 1999; Margolin 2000). Also known as the divisome, the septal ring consists of at least nine essential cell division gene products in Escherichia coli: FtsA, FtsI, FtsK, FtsL, FtsN, FtsQ, FtsW, FtsZ and ZipA (Rothfield et al., 1999; Margolin 2000). All nine proteins localize to the septum, a site in the mid-cell region apparently selected by the minCDE system (Hu and Lutkenhaus, 1999; Raskin and de Boer, 1999a; 1999b; Rowland et al., 2000; Fu et al., 2001; Hale et al., 2001; reviewed in RayChaudhuri et al., 2001). When any one of the cell division proteins is non-functional or absent, cells grow without dividing, leading to the formation of filaments.
Studies of cell division proteins have provided some insight to their individual activities, but how they co-operatively implement cytokinesis remains ambiguous. FtsZ, the best characterized of the nine, is a homologue of the eukaryotic cytoskeletal component tubulin (de Pereda et al., 1996; Lowe and Amos, 1998; Nogales et al., 1998). Like tubulin, it has GTPase and polymerization activities and may provide the force necessary to constrict the cell (de Boer et al., 1992; RayChaudhuri and Park, 1992; Mukherjee et al., 1993; Mukherjee and Lutkenhaus, 1994;1998; Erickson et al., 1996; Yu and Margolin, 1997; Lu et al., 2000). FtsA and ZipA both interact directly with FtsZ (Hale and de Boer, 1997; Wang et al., 1997; Din et al., 1998; Liu et al., 1999; Ma and Margolin, 1999; Hale et al., 2000; Mosyak et al., 2000; Haney et al., 2001), and ZipA stabilizes polymerized FtsZ (RayChaudhuri, 1999; Hale et al., 2000). Interestingly, FtsA is homologous to actin, another eukaryotic cytoskeletal component (Bork et al., 1992; Sanchez et al., 1994; van den Ent and Lowe, 2000). FtsI, also known as penicillin-binding protein 3, appears to be a transpeptidase required specifically for peptidoglycan synthesis at the septum (Spratt and Cromie, 1988; reviewed in Nguyen-Disteche et al., 1998). The largest of the group, the 1330-amino-acid FtsK, appears to be a bifunctional protein: its C-terminal domain facilitates resolution of chromosome dimers during DNA segregation, whereas the N-terminal domain carries out a necessary, but undefined, function in the developing septum; (Draper et al., 1998; Liu et al., 1998; Wang and Lutkenhaus, 1998; Yu et al., 1998a; 1998b; Recchia et al., 1999; Steiner et al., 1999; Barre et al., 2000). The functions of the remaining proteins (FtsL, -N, -Q and -W) are unknown. Furthermore, there are probably other factors involved in cell division that have not yet been identified.
To understand how these cell division proteins work together to form the septal ring, we can examine their dependence on one another for localization. The ability of a particular cell division protein to assemble into a ring structure, at putative division sites in a filament, indicates its independence from the missing or nonfunctional protein for recruitment into the septal ring. Compilation of various localization studies using this approach have yielded a mostly linear dependency pathway, suggestive of a sequential order of assembly (Rothfield et al., 1999; Margolin 2000; see below). The localization of FtsZ at the division site to form the Z ring appears to be the first step in divisome assembly: FtsZ does not depend on any of the other eight proteins for localization, whereas all others depend on its activity. FtsZ is followed closely by FtsA and ZipA; they do not require each other, or any other cell division protein, except for FtsZ. Subsequently, FtsK, -Q, -L, -I, and -N appear to be recruited in that order. The position of FtsW in the pathway is not clear from the published literature.
We had determined previously that there is a strictly linear dependency among FtsQ, FtsL and FtsI for septal recruitment, and that the recruitment of all three depends on both FtsZ and FtsA (Chen et al., 1999; Ghigo et al., 1999; Weiss et al., 1999). However, a number of gaps remained in the dependency analysis of protein assembly at the septum. Furthermore, it seemed possible that some pairs of proteins might depend on each other for assembly, thus not exhibiting the linear sequence that characterized most of the pathway. For instance, FtsK has been assigned its position upstream of FtsQ because it can localize to potential division sites in filaments without functional FtsQ or FtsI, but not in ftsZ or ftsA filaments (Wang and Lutkenhaus, 1998; Wang et al., 1998; Yu et al., 1998a). Yet, the septal localization of FtsQ and other downstream proteins in FtsK-deprived filaments has not been examined. Also, while FtsN appears to be a late recruit to the septal ring, as it needs functional FtsZ, -A, -Q, and -I for localization, and Z rings can form in filaments depleted of FtsN (Addinall et al., 1997), there still might exist a co-dependency with one of the other proteins.
Here, we report that GFP fusions to FtsQ, FtsL and FtsI fail to localize in filaments depleted of FtsK and confirm that FtsZ, FtsA and ZipA do localize to potential division sites in such filaments. Localization studies using GFP fusion proteins also showed that FtsZ, -A, -I, -L and -Q and ZipA can all be recruited into septal rings in the absence of FtsN. These localization results strengthen considerably the sequential model of protein assembly into the septal ring. To give further support to the idea that the divisome is a complex of multiple proteins, we used ECFP and EYFP fusions to demonstrate that FtsQ, FtsL and FtsI co-localize with FtsZ in filaments depleted of FtsN. A summary of all these findings provides a framework for understanding how the septal ring is formed.