Escherichia coli Division Inhibitor MinCD Blocks Septation by Preventing Z-Ring Formation

Bacteria were grown in Luria-Bertani (LB) medium at 37°C. Spectinomycin (SPC) at 50 μg/ml, kanamycin at 25 μg/ml, and chloramphenicol at 20 μg/ml were added as needed. Glucose (always 0.2%), IPTG (isopropyl-β-d-thiogalactopyranoside), or arabinose was added at the concentrations indicated.

The strains and plasmids used in this study are listed in Table1. All the JKD7.2 strains containing a pBEF plasmid were obtained by the transformation of JKD7.2/pKD3c with the desired plasmid and selecting on plates containing SPC at 37°C. Transformants were checked for sensitivity to chloramphenicol to ensure loss of pKD3c.

Plasmid pSEB14 differs from pSEB12 (minCDE) in that it expresses only the minCD genes. This plasmid was recovered in a strain that overexpressed MinE from pJPB216 (26). To test for the maintenance of pSEB14 in various strains, coextracted plasmids pSEB14 and pJPB216 were digested with EcoRI (which linearized only pJPB216) and precipitated with ethanol in order to obtain pSEB14 at about the same concentration as the pSEB12 preparation. To obtain the best transformation efficiencies, equal volumes of the pSEB12 and pSEB14 preparations were used in electroporation experiments with a Gene Pulser apparatus from Bio-Rad (0.1-cm-diameter cuvette; 1.5 kV, 25 μF, and 200 Ω). pSEB104 was generated by cloning the NgoMIV fragment carryingaraC and P_BAD-ftsZ-GFP from pJC104 (A. Mukherjee, C. Saez, and J. Lutkenhaus, submitted for publication) into pGB2 cut with XmaI. pSEB103 was obtained by replacing the SstI-XbaI fragment that carriesftsZ on pSEB104 by a PCR fragment containing zipAbetween SstI and XbaI sites. zipA was amplified using oligonucleotides 5′ZipAGFP (5′-ATGAGCTCGTTAGAACAACAGAGAAT) and 3′ZipAGFP (5′-TATCTAGAGGCGTTGGCGTCTTTGA).

An overnight culture was diluted by a factor of 100 in LB medium plus SPC and grown to an optical density at 540 nm (OD₅₄₀) of 0.05. Arabinose was added to the desired concentration, and the culture was split between two flasks. In one of these flasks 0.1 mM IPTG was added. At the time samples were taken to be observed by fluorescence microscopy, a sample was taken for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed as indicated below. Samples were directly (not fixed) observed and photographed with a Nikon Optiphot fluorescence microscope equipped with an E Plan oil immersion lens (Nikon) with a 100× objective and a DAGE-MTIDC-330 charge-coupled device camera using Flashpoint software (Integral Technologies). Fluorescence pictures were taken using a Nikon B-2A filter block with a 450- to 490-nm exitation filter and a 520-nm barrier filter. Images were imported to Adobe Photoshop software to be assembled.

Samples (1 ml) of cultures were centrifuged, and the cells were lysed by resuspension in 100 μl of SDS sample buffer (62.5 mM Tris-HCl) [pH 6.8], 1% SDS, 10% glycerol, 5% β-mercaptoethanol) and heating at 100°C for 5 min. Equivalent amounts of OD₅₄₀ material from all of the samples were then subjected to SDS-PAGE (12.5% gel). The proteins in the gel were transferred to nitrocellulose, and FtsZ was detected by an indirect immunostaining procedure with a rabbit polyclonal antiserum against FtsZ (1:3,000) and goat anti-rabbit immunoglobulin G antibodies coupled to alkaline phosphatase (1:3,000).

Previously, using immunoelectron microscopy we reported that overexpression of MinCD prevented Z-ring formation (8); however, this conclusion was questioned by Justice et al. (20), who used the more sensitive fluorescence microscopy. In their study various fixation conditions were utilized before immunostaining, with various results. Using our fixation conditions no Z rings were observed following overexpression of MinCD. Consistent with this, we had found that a MalE-MinC fusion blocked Z-ring assembly when assessed under these fixation conditions (19). However, using a variety of other fixation conditions they observed Z rings. Interestingly, ZipA but not FtsA was also found to localize under these alternative fixation conditions. Two of these alternate fixation conditions have in common the omission of lysozyme prior to immunostaining. We attempted to reproduce these results using these alternative fixation conditions but were unsuccessful. We observed no immunostaining if lysozyme was omitted, implying that the cells were not permeabilized. Therefore, we decided to try a different approach that avoided fixation.

To avoid fixation, we chose to examine the localization of GFP fusions to division proteins as these fusions generally retain some function and are able to localize to the division site. We clonedftsZ-gfp on a plasmid downstream of the arabinose promoter. This plasmid was introduced into PB114 Δmin (λDB173). The phage contains minCD downstream of the lacpromoter. This combination of strain and plasmid allowed us to manipulate the levels of MinCD independently of FtsZ-GFP. In these experiments ftsZ-gfp was induced with or without the simultaneous induction of minCD. Even at low levels of induction of ftsZ-gfp the cells filamented (Fig.1A; 0.01% arabinose) due to the increased level of FtsZ-GFP. Despite the filamentation the cells contained numerous Z rings that were well spaced. In contrast induction of minCD along with ftsZ-gfp completely prevented the formation of Z rings (Fig. 1B). No Z rings were observed in over 50 cells examined although occasional spots of fluorescence or spirals were observed. This experiment was repeated at a higher level of induction of ftsZ-gfp (0.05% arabinose). This higher level also resulted in filamentation, and the cells contained numerous Z rings that appeared brighter than at the lower arabinose concentration. The simultaneous induction of minCD at this higher level offtsZ-gfp induction also interfered with Z-ring formation. Although Z-ring formation was not completely prevented, the filaments contained fewer rings and the rings did not have a typical appearance. FtsZ spirals were also observed.

The above results indicate that expression of minCD can prevent FtsZ-GFP from localizing into Z rings. One trivial possibility is that induction of minCD interfered with induction offtsZ-gfp. Therefore, samples were taken from the cultures with 0.05% arabinose and analyzed by immunoblotting (Fig.2). This result shows that the level of FtsZ-GFP was not affected by minCD induction (Fig. 2, lanes 5 and 6). Furthermore, the level of FtsZ-GFP was similar to the level of the endogenous FtsZ. A sample from the culture induced with 0.01% arabinose was also analyzed. The FtsZ-GFP level was about one-third of that with the higher arabinose concentration (Fig. 2, lane 4). SinceminCD induction did not interfere with ftsZ-gfpexpression, we conclude that MinCD prevents FtsZ-GFP from assembling into Z rings.

The results with FtsZ-GFP were fairly clear with respect to the effect of MinCD on Z-ring formation; however, the results are somewhat complicated since inducing FtsZ-GFP blocks cell division. We therefore analyzed the effect of MinCD on the localization of ZipA-GFP. ZipA has been shown to bind directly to FtsZ and to be a good marker for the position of Z rings (15). Also, Justice et al. (20) found that ZipA localized in the presence of MinCD. In addition, we were able to detect ZipA-GFP at a level that did not significantly inhibit division. ZipA-GFP was induced with or without induction of MinCD (Fig.3). The heterogeneous cell length is due to using a min mutant. In the absence of MinCD induction, ZipA-GFP localized to rings, indicating that Z rings were present. With MinCD induction ZipA-GFP was not localized to rings and instead was present along the membrane. Immunoblot analysis showed that induction of ZipA-GFP did not affect the level of FtsZ (Fig. 2), eliminating this as a possible reason for the failure of Z rings to form. From these results we conclude that MinCD blocks Z-ring formation, thereby preventing ZipA from localizing to division sites.

Previous results showed that increased expression of FtsZ can suppress filamentation caused by MinCD or overexpression of MinC (12, 13). However, Justice et al. (20) reported that increasingftsA expression reduced filamentation caused by MinCD. To compare the effects of increased levels of FtsA and FtsZ on MinCD-induced lethality, we utilized a series of multicopy plasmids that contained the fts genes in various combinations. We then looked at the ability of these plasmids to suppress the lethal filamentation caused by induction of MinCD in a Δminbackground. As seen from the spot tests shown in Fig.4, IPTG at 0.125 mM reduced the plating efficiency of PB114 Δmin (λDB173) containing the vector (pJB209) by at least 3 orders of magnitude. In contrast, the presence of a plasmid containing ftsQAZ completely suppressed killing by IPTG. This suppression occurred whether the orientation offtsQAZ was the same as or opposite to that of theaad promoter on the plasmid. The presence of justftsQZ suppressed the sensitivity of PB114 Δmin(λDB173) to IPTG, indicating that it was the presence offtsZ that suppresses minCD-induced killing as observed previously (6, 12). This result is supported by the results with the plasmid containing ftsQA. The presence of this plasmid offered no more protection than the vector alone. These results indicate that increased FtsZ, but not FtsA, provides protection from MinCD.

Previously, it was reported that alleles of ftsZ, designatedftsZ (Rsa) for the resistance of their products to cell division inhibitor SulA, provided resistance to MinCD (6). The test compared the effects of MinCD induction on plating efficiency and filamentation for Δmin strains carrying various alleles of ftsZ on low-copy-number plasmids. The strains tested were merodiploids as each carried a copy of wild-type ftsZ on the chromosome in addition to the allele on the plasmid. In contrast, Justice et al. (20) examined a strain carrying just ftsZ103 and found that this strain filamented following induction of MinCD, suggesting thatftsZ103 provided no protection. This raised the question if any of these ftsZ alleles produced resistance tominCD in the absence of wild-type ftsZ. We used two different tests to examine the minCD resistance due toftsZ alleles that are capable of supporting growth. The first test takes advantage of the previous observation thatminCD on a single-copy vector cannot be efficiently introduced into a wild-type strain (26). The MinE expressed from the chromosome is insufficient to suppressminCD expressed from the plasmid, demonstrating that cells are sensitive to a single extra copy of minCD(26). JKD7-2 (ftsZ::kan) containing the various pBEF plasmids was transformed with either pSEB14 (minCD) or pSEB12 (minCDE). The ratio of transformation efficiencies with these two plasmids is a measure of the resistance tominCD (26). The results presented in Table2 show that the transformation ratio is 0.5% for the control containing pBEF0 (ftsZ). This is slightly higher than that for a strain expressing ftsZ from the chromosome (26), presumably due to the slightly elevated level of FtsZ provided by pBEF0 (twofold higher). This ratio increases to 36.3% for pBEF2 (ftsZ2), the highest obtained with any of these ftsZ alleles. The other three alleles all gave intermediate levels, which were roughly equivalent, with the ratio falling in a range of 2.3 to 5.3%.

In a second test the pBEF plasmids were introduced into JKD7-2 (ftsZ::kan)(λDB173[Plac::minCD]), and the strains were examined for resistance to IPTG. The plating efficiency in the presence of pBEF0 (ftsZ⁺) was reduced by 3 log units at the two IPTG concentrations tested (Fig. 5). TheftsZ alleles varied in their resistance to MinCD in this test. Strains carrying ftsZ2, ftsZ9, andftsZ100 were completely resistant to minCD, as the plating efficiency was unaffected even at the highest IPTG concentration. In contrast, a strain carrying ftsZ114(identical to ftsZ103) was sensitive and only grew poorly at the lower IPTG concentration. However, pBEF114 (ftsZ114) supported more growth than pBEF0 (ftsZ), indicating that the strain carrying it it was more resistant. Thus, not allftsZ alleles producing resistance to SulA produce the same level of resistance to MinCD. On the other hand, selection for alleles of ftsZ producing resistance to SulA appears to usually yield some level of resistance to MinCD.