Division in bacterial cells occurs through the concerted action of a number of division proteins localized at the division site (
22,
29). These division proteins are recruited by the Z ring, which is formed through the self assembly of FtsZ, the ancestral homologue of eukaryotic tubulins (
21,
25). The Z ring, along with these additional division proteins, is designated the septal ring (
16), an organelle that is capable of carrying out division. The recruitment of these additional division proteins to the Z ring occurs in at least two steps. Proteins FtsA and ZipA are recruited by direct interaction with FtsZ. Many of the remaining proteins do not interact directly with FtsZ, but rather depend on FtsA (
2,
14,
23,
32).
Deletion of the
min locus results in the production of minicells, small anucleate cells produced by division occurring near the poles of the cell (
3). These minicell divisions appear to occur at the expense of medial divisions because the nucleated mother cells have greater average cell length than wild-type cells (
30). Interestingly, increased expression of essential cell division protein FtsZ suppresses this increased average cell length, suggesting that FtsZ is limiting in this mutant (
5). Examination of Z rings in the
min deletion mutant indicates that Z rings form at the cell poles and at interior positions (
8,
34). This observation suggests that the assembly of Z rings at interior positions is not necessarily delayed by deletion of the
min locus but that maturation of these Z rings into a fully functional septal ring might be hampered. It's possible that multiple Z rings within the same cell compete with each other to become fully functional.
Although the
min mutant contains Z rings near the poles of the cell, polar Z rings are not observed in wild-type cells (
8,
34). This fascinating ability of the
min system to prevent Z rings from forming at the poles but to allow them to form at midcell has led to an intense investigation of the
minsystem. The
min system consists of three genes,
minCDE, all of which are necessary to achieve topological regulation of cell division (
11). Genetic evidence indicates that MinC and MinD cooperate to form an inhibitor of cell division, which is topologically regulated by MinE. Analysis of functional green fluorescent protein fusions indicates that this topological regulation by MinE is achieved by inducing MinCD to oscillate from pole to pole without allowing occupation of the midcell (
17,
27,
28).
Although MinC and MinD cooperate to form an efficient inhibitor of division, several lines of evidence suggest that MinC is the inhibitor and that it is activated by MinD. First, overproduction of MinC, but not MinD, inhibits division in a Δ
min strain (
12). Second, MinC can combine with DicB, encoded by a cryptic phage, to efficiently inhibit division, consistent with MinC being the component of the inhibitor that contacts the division machinery (
12). In all cases, the inhibition, like that caused by MinC and MinD, can be suppressed by overproduction of FtsZ, suggesting a common mechanism of inhibition. Furthermore, this suppressibility by overexpression of FtsZ suggests that FtsZ might be the target of MinC (
6,
12). Immunoelectron microscopy studies revealed that Z rings were not present in filaments produced by overexpression of MinCD, suggesting that this inhibitor blocked division by preventing Z-ring formation (
8). Additional support for FtsZ as the target of MinCD comes from the increased resistance of several
ftsZ mutants to MinCD (
6). These mutants were isolated on the basis of resistance to SulA, an inhibitor of cell division that is induced by DNA damage. Finally, a MalE-MinC fusion that is capable of blocking division and Z-ring formation in vivo binds to FtsZ and prevents accumulation of FtsZ polymers in vitro, consistent with MinC inhibiting division by preventing Z-ring assembly (
18,
19).
More recently this mode of action of MinC was questioned based on several observations (
20). First, fluorescence microscopy was used to observe Z rings in cells overexpressing MinCD. These rings contained ZipA but not FtsA. Second, a strain carrying
ftsZ103, a
sulA-resistant mutant and reported to be MinCD resistant, filamented in the presence of overexpressed MinCD. Third, increasing FtsA suppressed MinCD-induced filamentation. Last, overexpression of SulA prevented oligomerization of the endogenous FtsZ in cell extracts whereas MinCD did not. As a result it was suggested that MinCD did not prevent formation of Z rings but rather acted at a later step to prevent FtsA from localizing to the Z ring. This mode of action for MinCD seemed unlikely since it would not explain how the
min system prevents Z rings from forming at the poles of cells. However, we have reanalyzed the effect of MinCD on cell division since our previous report was done using immunoelectron microscopy (
8), which lacks the sensitivity of fluorescence microscopy (
1). Our results are consistent with FtsZ being the target of MinC and with MinCD inhibiting division by blocking Z-ring formation.
DISCUSSION
Our results indicate that FtsZ is the target of the MinCD inhibitor and that MinCD blocks division by preventing assembly of Z rings. This result is consistent with our previous findings (
6) and disagrees with the conclusions of Justice et al. (
20). They concluded that MinCD blocked division by preventing the addition of FtsA to the Z ring. However, by utilizing GFP fusions to FtsZ and ZipA, we avoided fixing cells and still observed that overexpression of MinCD blocked Z-ring formation. We also confirmed that overproduction of FtsZ suppressed MinCD-induced lethality, whereas FtsA had little effect. Finally, we show that several
ftsZ (Rsa) alleles, in the absence of the wild-type
ftsZ, confer various levels of resistance to MinCD.
Previously we reported that overexpression of MinCD prevented Z-ring formation (
8); however, Justice et al. (
20) reported that this result depended on the fixation conditions. Using our fixation conditions they did not observe Z rings; however, using a variety of other fixation conditions they observed somewhat atypical Z rings that contained ZipA but lacked FtsA. Furthermore, these rings had an abnormal appearance, so it was suggested that MinCD might interfere with the architecture of the Z ring such that it had a lower affinity for FtsA. Using GFP fusions to FtsZ and ZipA, and thereby avoiding fixation, we observed that MinCD prevented Z-ring formation. At low levels of expression of FtsZ-GFP, expression of MinCD completely prevented Z-ring formation. At higher levels of FtsZ-GFP expression, inhibition was not complete and some FtsZ structures were formed, often spirals, although a few typical Z rings were present. This ability of higher levels of FtsZ-GFP to at least partially overcome MinCD and form structures is consistent with the ability of an increased level of FtsZ to suppress MinCD. The failure of FtsZ-GFP to completely suppress MinCD is most likely due to its inability to functionally by substitute for FtsZ in division, although it can localize in vivo and polymerize in vitro (
33).
The mechanism of MinCD action was confirmed using ZipA-GFP as a marker for Z rings. ZipA is known to bind tightly to the C-terminal region of FtsZ and to be a good marker for the presence of Z rings (
15,
16). Expression of MinCD completely blocked the localization of ZipA-GFP, again indicating that MinCD completely blocked Z-ring assembly.
Although Justice et al. (
20) reported that increased expression of
ftsA showed some suppression of MinCD-induced filamentation, we observed no effect of increased FtsA on MinCD-induced lethality. We found that multicopy plasmids expressing
ftsQAZ suppressed MinCD lethality. However, removing
ftsZ from this plasmid eliminated suppression, whereas eliminating
ftsA had no effect. Justice et al. (
20) used slightly higher levels of FtsA in their studies (10-fold increase versus 5- to 7-fold in this study), which may provide some protection.
In addition to higher levels of FtsZ suppressing MinCD-induced filamentation, some mutations in
ftsZ also suppress MinCD action (
8). The associated mutants, designated
ftsZ (Rsa), were isolated as ones that confer resistance to DNA damage-inducible inhibitor SulA (
7). These mutants suppressed the lethality of overexpression of MinCD when carried on a plasmid in a strain with a wild-type copy of
ftsZ on the chromosome. However, these mutants were divided into two classes based on the degree of filamentation after induction of MinCD. One class, consisting of
ftsZ2 and
ftsZ3, was designated very resistant, whereas another class was designated partially resistant and included
ftsZ1,
ftsZ9, ftsZ100, and
ftsZ103 (
ftsZ114). Justice et al. (
20) found that
ftsZ114 failed to block filamentation following induction of MinCD. This raised questions about the earlier results and interpretations. We therefore, examined those
ftsZ mutants that were able to support viability for their ability to confer resistance to MinCD in the absence of wild-type
ftsZ. In support of our previous report we observed that
ftsZ2, ftsZ9, and
ftsZ100 conferred resistance to MinCD-induced lethality, whereas
ftsZ114 (
ftsZ103is identical) did not. However,
ftsZ114 did support more growth in the presence of overexpressed MinCD than
ftsZ, indicating that it confers some resistance. Thus, these alleles of
ftsZ confer resistance to MinCD but the degree of resistance varies and depends on the test used. The most resistant strain is one carrying allele
ftsZ2; this strain even appeared to grow slightly better in the presence of MinCD (Fig.
5).
Although MalE fusions to SulA and MinC block Z-ring formation and block FtsZ polymerization their modes of action are surely different. MalE-SulA blocks FtsZ GTPase, whereas MalE-MinC does not (
19,
24). Thus, it was suggested that SulA prevented the interaction of FtsZ monomers that would lead to GTPase activity whereas MinC might destabilize FtsZ polymers. How might the same
ftsZ mutations confer resistance to MinCD and SulA? One possibility is that they alter FtsZ such that MinC and SulA no longer interact with FtsZ. This is unlikely to be the explanation for all the mutations because they are not clustered. Another possibility is that these mutations lead to the formation of more-stable polymers, which are thus more resistant to MinC. Consistent with this we have found that FtsZ2, which is very resistant to MinC, produces stable polymers (Mukherjee et al., submitted). In contrast, FtsZ114, which is only weakly resistant, expresses nearly normal GTPase activity, implying that FtsZ114 polymers turn over rapidly. More study will be required to verify this possibility.
Justice et al. (
20) induced SulA and MinCD to block cell division and then examined the ability of these inhibitors to block FtsZ oligomerization in the extracts upon raising the temperature. They found that SulA prevented oligomerization, as expected from previous studies (
19,
31), but found that MinCD did not. Although this is consistent with MinC acting after assembly, possibly destabilizing FtsZ polymers as reported earlier (
19), it may not actually be supportive due to limitations of this approach. In vivo the activity of MinC is concentrated at the membrane by MinD (
17,
27). In the absence of MinD, MinC has to be overexpressed 25- to 50-fold to block division. The concentrating effect of MinD is lost once the cells are broken. Thus, Justice et al. (
20) overexpressed MinCD sufficiently to inhibit division in vivo but were unlikely to have overexpressed MinC sufficiently to affect FtsZ polymerization in vitro (a 1:1 stoichiometry [
19], which is unlikely to be achieved by the single-copy vector used to express
minC, is required). In contrast to MinC, SulA does not appear to be localized to the membrane and presumably binds FtsZ in the cytoplasm, preventing its assembly into polymers.
Finally, it is unlikely that MinCD acts by preventing recruitment of FtsA to the Z ring, as it would not explain the ability of themin system to prevent Z-ring formation at the cell poles. The present results using GFP fusions, whose use avoids any possible artifacts due to fixation, confirm that MinCD inhibits cell division by preventing formation of Z rings.