Recently, we showed that functional Gfp-MinD rapidly oscillates between the two cell halves in a membrane association-dissociation cycle which is dependent on MinE but does not require MinC or FtsZ (33
). Here we showed that biologically active Gfp-MinC undergoes a very similar localization cycle in which the protein oscillates between cell ends at about the same rate as that observed for Gfp-MinD. Like Gfp-MinD (33
), segregation and oscillation of Gfp-MinC required the presence of MinE and occurred independently of FtsZ rings. However, whereas Gfp-MinD localizes independently of MinC, the localization of Gfp-MinC appeared to be directly dictated by MinD. Thus, like MinD itself (33
), Gfp-MinC preferentially associated along the entire periphery of MinE−
filaments, but the fusion failed to accumulate at any specific location in the absence of MinD regardless of the presence of MinE.
Combined with the knowledge that MinC and MinD show a strong interaction in two-hybrid assays (18
), these observations support a model for MinC action which is summarized in Fig. . In the absence of MinD and MinE, MinC has no intrinsic affinity for any special site and is present throughout the cell (Fig. A). In MinE−
cells, MinD associates with the cytoplasmic membrane and recruits MinC. The even distribution of MinC-MinD along the membrane results in a block of FtsZ ring assembly at all PDSs and the formation of nonseptate filaments (Fig. B). In WT cells, MinE accumulates in a ring at midcell and stimulates the dynamic oscillatory behavior of MinD. MinC follows along with MinD and either remains associated with MinD throughout oscillation or disengages during disassembly of MinD from the membrane, only to reengage when MinD assembles at the membrane in the opposite half. In either case, MinC actively interferes with FtsZ ring assembly at only one of the cell poles at a time (Fig. C).
FIG. 4 Model for MinCDE action in preventing aberrant septation events. Symbols: ●, MinC; , MinD; , the MinE ring. PDSs are represented by either a minus (blocked by MinC-MinD) or a plus (not blocked, available for FtsZ ring assembly) sign. (A) In the (more ...)
Given the evidence for a direct interaction between MinC and MinD, the formal possibility that the two proteins segregate to opposite cell halves and oscillate out of phase by half a cycle is far less likely. So far, attempts to exclude this possibility by observing cells in which the two proteins are tagged with different color Gfp derivatives have failed, due to insufficient signal intensities of the nongreen varieties. However, in MinE+ cells in which Gfp-MinC and Gfp-MinD are expressed simultaneously, we have observed that fluorescence still clearly segregates to only one cell end at a time, indicating that the two proteins indeed co-oscillate in the same direction (data not shown).
It is also noteworthy that the average oscillation rate of Gfp-MinC measured in WT cells (~40 s/cycle) was very close to that of Gfp-MinD in cells in which gfp-minD
was coexpressed with minE
to maintain a normal MinD-to-MinE ratio (33
). This finding not only further supports the idea that the behavior of Gfp-MinC directly reflects that of native MinD in the present experiments but also suggests that oscillation parameters measured with the Gfp-tagged proteins fairly accurately reflect those of native MinC-MinD.
How MinC prevents FtsZ ring assembly is not known. Two-hybrid studies failed to show an interaction between the two proteins (18
), and it is well possible that MinC interferes with the activity of another factor required for FtsZ ring assembly, such as a hypothetical membrane-associated molecule that might nucleate FtsZ polymerization. How MinD stimulates MinC activity is also not clear. The present results suggest one straightforward possibility, however; i.e., by recruiting MinC to the membrane, MinD may simply act to increase the local concentration of MinC to effective levels. Compatible with this idea is the fact that an increase in the cellular concentration of MinC to more than ~30-fold its normal level is sufficient to cause division inhibition, even in the absence of MinD (12
). Whether this idea is correct or not, the finding that Gfp-MinC associates with MinD at the cell’s periphery indicates that MinC-MinD-mediated division inhibition is a membrane-associated event, arguing against mechanisms whereby MinC directly modifies cytoplasmic FtsZ pools to a polymerization-incompetent state.
This work extends and supports our observations on the remarkable properties of the Min proteins in living E. coli
). One of the surprising implications is that MinC-MinD prevents aberrant FtsZ ring formation intermittently, at only one of the cell poles at a time. This mode of action appears not to have been conserved in the gram-positive rod Bacillus subtilis
) and begs the question why such an oscillatory mechanism might have developed. MinD and MinE determine each other’s localization pattern (32
) and, as suggested before (33
), one attractive possibility is that oscillation of MinD between the cell segments on either side of a MinE ring is coupled to positioning of the ring. In this view, oscillation of MinD provides cells with a measuring device which allows the positioning of MinE at midcell, in the case of WT cells containing one ring, or at regularly spaced intervals, in the case of filaments containing multiple rings.
The idea that the Min system, in addition to preventing aberrant FtsZ ring assembly, may also function as a measuring device is supported by recent work by Yu and Margolin (41
) in which they analyzed the placement of FtsZ rings in Min−
mutants containing additional mutations that affect nucleoid partitioning (Par−
). Placement of division septa in Min−
mutants is typically not random but is restricted to a narrow area near each cell pole and regular positions between segregated nucleoids (2
). One factor that is likely to contribute to this nonrandom pattern is a phenomenon called nucleoid occlusion which is based on the observation that septum formation appears to be inhibited in the close vicinity of the nucleoid(s) of certain DNA replication and topoisomerase mutants (19
). The mechanism of this inhibitory effect is not understood, although it is conceivable that the formation of septal rings at envelope sites directly surrounding a nucleoid would be sterically hindered by an abundance of membrane-associated transcription and translation complexes (29
). In some models, positioning of the nucleoid is proposed to be the sole determining factor for positioning of the division apparatus (2
), but this idea is refuted by a host of experimental data (references 4
, and 36
and references therein). For instance, mutants affected in nucleoid replication and segregation typically form filamentous cells which release chromosomeless cells from their ends. In several such mutants, septal placement is not random and the size distribution of the DNA-less rods is close to that of normal newly born cells, suggesting that cells can somehow measure a certain distance from the cell pole (4
Compelling evidence that the Min proteins are involved in defining this distance comes from the observation that the positioning of FtsZ rings becomes essentially random in the nucleoid-free segments of ΔminCDE parC
double mutants (41
). One of several interesting predictions of this work is that MinC-MinD in WT cells inhibits FtsZ ring assembly not only at the extreme cell ends but throughout most of the cell envelope except for a relatively narrow zone at the cell’s center defined by the MinE ring (41
). Our observations on the distribution of Gfp-MinC and Gfp-MinD are compatible with this possibility insofar that, during dwell periods, the association of the two proteins with the membrane is clearly not restricted to the polar caps. This was especially obvious in the case of Gfp-MinD, which was frequently seen to cover the membrane from a pole to approximately the middle of the cell (33
). In general, Gfp-MinC did not appear to extend as far toward midcell, although the protein was still observed to cover at least one-quarter of the membrane in most cells. Whether this was due to low signal intensities or whether factors, in addition to the location of MinD, further bias Gfp-MinC localization toward the cell ends is not clear. In any event, additional analyses of the behavior of the Min proteins in relation to nucleoid dynamics should prove valuable in testing the proposal by Yu and Margolin that the combination of MinCDE action and nucleoid occlusion may be sufficient to explain septal placement in E. coli
Clearly, the dynamic behavior of MinC and MinD raises many new questions requiring additional experimentation. Further elucidation of the mechanisms underlying membrane assembly-disassembly of MinC-MinD and of the role(s) of the MinE ring is likely to contribute significantly to our understanding of the spatial organization of bacterial cells.