Escherichia coli division inhibitor MinCD blocks septation by preventing Z-ring formation

Abstract

The min system spatially regulates division through the topological regulation of MinCD, an inhibitor of cell division. MinCD was previously shown to inhibit division by preventing assembly of the Z ring (E. Bi and J. Lutkenhaus, J. Bacteriol. 175:1118-1125, 1993); however, this was questioned in a recent report (S. S. Justice, J. Garcia-Lara, and L. I. Rothfield, Mol. Microbiol. 37:410-423, 2000) which indicated that MinCD acted after Z-ring formation and prevented the recruitment of FtsA to the Z ring. This discrepancy was due in part to alternative fixation conditions. We have therefore reinvestigated the action of MinCD and avoided fixation by using green fluorescent protein (GFP) fusions to division proteins. MinCD prevented the localization of both FtsZ-GFP and ZipA-GFP, consistent with it preventing Z-ring assembly. Consistent with a direct interaction between FtsZ and the MinCD inhibitor, we find that increased FtsZ, but not FtsA, suppresses MinCD-induced lethality. Furthermore, strains carrying various alleles of ftsZ, selected on the basis of resistance to the inhibitor SulA, displayed variable resistance to MinCD. These results are consistent with FtsZ as the target of MinCD and confirm that this inhibitor prevents Z-ring assembly.

FIG. 1

Effect of MinCD expression on Z-ring formation. Fluorescence microscopy of PB114 Δmin (λDB173/pSEB104) (expressing FtsZ-GFP under Pbad promoter control) was performed 3 h after induction with 0.01% arabinose (A and B) or 0.05% arabinose (C and D). At the time of FtsZ-GFP induction, the culture was split in two, and no IPTG (A and C) or 1 mM IPTG (B and D) was added in order to induce MinCD from λDB173.

FIG. 2

Expression of GFP fusions from Pbad and induction of MinCD do not affect the level of FtsZ. Samples from the experiments in Fig. 1 and 3 were analyzed by immunoblotting using an anti-FtsZ polyclonal antibody. Lane 1, purified FtsZ and FtsZ-GFP; lanes 2 to 6, samples taken at the same time as the fluorescence pictures of Fig. 1 and 3; lane 2, sample from Fig. 3A (ZipA-GFP, no MinCD); lane 3, sample from Fig. 3B (ZipA-GFP plus MinCD); lane 4, sample from Fig. 1A (FtsZ-GFP, no MinCD); lane 5, sample from Fig. 1D (FtsZ-GFP plus MinCD); lane 6, sample from Fig. 1C (FtsZ-GFP, no MinCD).

FIG. 3

Effect of MinCD expression on Z-ring formation using ZipA-GFP as a marker for Z rings. Shown is fluorescence microscopy of PB114 Δmin (λDB173/pSEB103) (expressing ZipA-GFP under Pbad promoter control) 3 h after induction by 0.05% arabinose. At the same time ZipA-GFP was induced, the culture was split in two and no IPTG (A) or 1 mM IPTG (B) was added to induce MinCD (under the control of lacZ promoter) from λDB173.

FIG. 4

MinCD-induced division inhibition and the effect of multicopy ftsQAZ. Vector pJPB209 or the indicated derivatives were introduced into PB114 Δmin (λDB173), which expresses minCD under the control of Plac. One colony of each strain was resuspended in 300 μl of LB medium and serially diluted by 10. Samples (4 μl) were spotted on plates containing SPC and glucose with or without IPTG (as indicated) and incubated overnight at 37°C.

FIG. 5

Suppression of MinCD-induced lethality by ftsZ (Rsa) alleles. Plasmid pBEF0 (ftsZ), pBEF2 (ftsZ2), pBEF9 (ftsZ9), pBEF100 (ftsZ100), or pBEF114 (ftsZ114) was introduced into JKD7.2 (ftsZ::kan recA), lysogenic for λDB173 (Plac::minCD). In accordance with the protocol described in the legend to Fig. 4, samples were spotted on SPC and kanamycin plates with or without IPTG and incubated overnight at 37°C.