Analysis of MinC reveals two independent domains involved in interaction with MinD and FtsZ - PubMed (original) (raw)

Comparative Study

Analysis of MinC reveals two independent domains involved in interaction with MinD and FtsZ

Z Hu et al. J Bacteriol. 2000 Jul.

Abstract

In Escherichia coli FtsZ assembles into a Z ring at midcell while assembly at polar sites is prevented by the min system. MinC, a component of this system, is an inhibitor of FtsZ assembly that is positioned within the cell by interaction with MinDE. In this study we found that MinC consists of two functional domains connected by a short linker. When fused to MalE the N-terminal domain is able to inhibit cell division and prevent FtsZ assembly in vitro. The C-terminal domain interacts with MinD, and expression in wild-type cells as a MalE fusion disrupts min function, resulting in a minicell phenotype. We also find that MinC is an oligomer, probably a dimer. Although the C-terminal domain is clearly sufficient for oligomerization, the N-terminal domain also promotes oligomerization. These results demonstrate that MinC consists of two independently functioning domains: an N-terminal domain capable of inhibiting FtsZ assembly and a C-terminal domain responsible for localization of MinC through interaction with MinD. The fusion of these two independent domains is required to achieve topological regulation of Z ring assembly.

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Figures

FIG. 1

FIG. 1

Alignment of MinC sequences. The sequences of MinC from various bacteria were aligned using MegAlign (DNA Star) and the Clustal Method. Identical amino acids in three or more sequences are boxed. The MinC sequences (with GenBank accession numbers in parentheses) are, from the top, E. coli (AAB59061.1), S. enterica serovar Typhimurium, V. cholerae, Bacillus subtilis (AAA22400.1), and T. maritima (AAD36124.1). The asterisk indicates that the B. subtilis MinC is truncated and the last 15 residues are not shown.

FIG. 2

FIG. 2

Gel filtration chromatography of MalE-MinC and MalE-MinC19. Affinity-purified MalE-MinC was analyzed on a Superose-6 gel filtration column equilibrated with polymerization buffer. Fractions obtained from the elution were analyzed by SDS-PAGE (fraction number indicated at the top). (A and B) Lane S contains molecular weight markers (from the top, phosphorylase b, 97.4K; serum albumin, 66K; ovalbumin, 45K; and carbonic anhydrase, 29K). (A) A 1-ml sample of MalE-MinC (12.5 μM) was applied to the column; (B) a 1-ml sample of MalE-MinC19 (12 .5 μM) was applied to the column. (C) A standard curve for estimating the size of MalE-MinC was obtained by running the following molecular weight standards: apoferritin (400K), β-amylase (200K), alcohol dehydrogenase (150K), bovine serum albumin (66K), and carbonic anhydrase (29K).

FIG. 3

FIG. 3

Expression of the C-terminal domain of MinC induces minicell formation in wild-type cells. JS219 containing pJC90 (malE) (A) or pZH111 (_malE-minC_116-231) (B) was diluted from an overnight culture and grown for several hours. Arabinose (0.005%) was added, and samples were taken for photography 2 h later.

FIG. 4

FIG. 4

Expression of the N-terminal domain of MinC induces filamentation. JS964 (Δ_min_) containing various MalE fusions was photographed 90 to 120 min after adding arabinose (0.005%) to exponentially growing cultures. The plasmids and fusions used were as follows: (A) pJC90 (malE), (B) pZH111 (_malE-minC_1–115), (C) pZH112 (_malE-minC_116–231), and (D) pZH101 (malE-minC).

FIG. 5

FIG. 5

The N-terminal domain of MinC is sufficient to prevent FtsZ polymerization. Affinity-purified MalE-MinC1–115 and MalE-MinC116–231 were tested for their effect on FtsZ polymerization utilizing a sedimentation assay. FtsZ at 200 μg/ml was incubated in polymerization buffer (50 mM morpholineethanesulfonic acid [pH 6.5], 50 mM KCl, 1 mM MgCl2) with increasing concentrations of the MalE fusions. The reactions were initiated by the addition of GTP at 1 mM. After a 5-min incubation at room temperature the samples were centrifuged at 80K rpm for 15 min in a Beckman TLA 100.2 rotor. The pellets were resuspended in SDS sample buffer and analyzed by SDS-PAGE. (A and B) Lanes GDP contain a control with GDP added, and lanes GTP contain GTP but no fusion protein. The final concentration of fusion protein added (in micrograms per milliliter) was 50, 100, 200, 400, 800, and 1,200 in lanes 3 to 8, respectively. (C) The amount of FtsZ in the pellet was plotted as a percentage of the control lacking the fusion protein. The data for MalE-MinC was taken from reference .

FIG. 6

FIG. 6

The C-terminal and N-terminal domains of MinC promote oligomerization. MalE-MinC1–115 and MalE-MinC116–231 were analyzed by gel filtration chromatography as described in the legend to Fig. 2, except that smaller fractions were collected. (A and B) Fractions from the elution were analyzed by SDS-PAGE. (C) The same standard curve shown in Fig. 2 was used to estimate the size of the fusions.

FIG. 7

FIG. 7

Model of MinC. In this model MinC is depicted as a dimer although it is possible that it forms larger oligomers. The N-terminal domain (Z domain) interacts with FtsZ to prevent polymerization. The C-terminal domain (D domain) is responsible for interaction with MinD resulting in placement of MinC at the membrane. It is not clear if dimerization plays a role in these interactions. The C-terminal domain is clearly sufficient for dimerization, although in vitro results show that the N-terminal domain may also contribute to dimerization. The N-terminal domain also promotes the formation of oligomers larger than dimers. This activity is partially suppressed in the full-length MinC.

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