MinDE-dependent pole-to-pole oscillation of division inhibitor MinC in Escherichia coli

Abstract

By inhibiting FtsZ ring formation near the cell ends, the MinC protein plays a critical role in proper positioning of the division apparatus in Escherichia coli. MinC activity requires that of MinD, and the MinE peptide provides topological specificity by suppressing MinC-MinD-mediated division inhibition specifically at the middle of the cell. We recently presented evidence that MinE not only accumulates in an FtsZ-independent ring structure at the cell's middle but also imposes a unique dynamic localization pattern upon MinD in which the latter accumulates alternately in either one of the cell halves in what appears to be a rapidly oscillating membrane association-dissociation cycle. Here we show that functional green fluorescent protein-MinC displays a very similar oscillatory behavior which is dependent on both MinD and MinE and independent of FtsZ. The results support a model in which MinD recruits MinC to its site of action and in which FtsZ ring assembly at each of the cell ends is blocked in an intermittent and alternate fashion.

FIG. 1

Identification of Gfp-MinC with MinC-specific antiserum. Immunoblot showing Gfp-MinC (52.5 kDa; upper arrowhead) and native MinC (24.8 kDa; lower arrowhead) as detected with MinC-specific antiserum. Cells were grown to an optical density (600 nm) of 0.5 at 30°C (lane 6) or 37°C (lanes 1 to 5 and 7) either in the absence (lane 7) or presence (lanes 1 to 6) of 50 μM IPTG, and whole-cell extracts were prepared. Lanes 1 and 2 contain 44 μg of total protein from strain PB114 (ΔminCDE) and PB103 (WT), respectively. Lanes 3 to 7 contain 44 μg (lanes 3, 6, and 7), 8.8 μg (lane 4), or 4.4 μg (lane 5) of protein from strain PB114(λDB175)/pDR175 [ΔminCDE(P_lac::minDE)/λp_R::gfp-minC]. Samples in lanes 4 and 5 were mixed with appropriate amounts of PB114 extract such that each lane contained 44 μg of total protein.

FIG. 2

Dynamic properties of functional Gfp-MinC in live cells. Phase-contrast (F and G), fluorescence (A to E), and differential interference contrast (A′ to E′) micrographs showing properties of Gfp-MinC. Cells were grown at 30°C (F) or 37°C (A to E and G) either in the absence (E) or presence (A to D and G) of 50 μM IPTG. (A to D) Time-lapse images showing segregation and oscillation of Gfp-MinC in the presence of MinD and MinE in strains PB114(λDB175)/pDR175 [ΔminCDE(P_lac::minDE)/λp_R::gfp-minC] (A, B, and D) and PB103(λDR122) (WT/λp_R::gfp-minC) (C). Times are indicated in seconds. (E) Random distribution of Gfp-MinC in the absence of MinD and MinE in strain PB114(λDB175)/pDR175. (F and G) Correction of the minicell phenotype (Min⁻) of strain PB114(λDB175)/pDR175 by Gfp-MinC. Bar, 2 (A to E) or 5 (F and G) μm.

FIG. 3

Gfp-MinC localization in filaments. Fluorescence (A to C) and differential interference contrast (A′ to C′) images showing the distribution of Gfp-MinC in cells in which FtsZ ring assembly is blocked. Cells were grown at 37°C with 50 μM IPTG. (A) Gfp-MinC localization in MinE⁻ filaments of strain PB114(λDR155)/pDR175 [ΔminCDE(P_lac::minD)/λp_R::gfp-minC]. (B) Time-lapse images of Gfp-MinC localization in a SfiA-induced filament of strain PB103(λDR144)/pDR175 [WT(P_lac::sfiA)/λp_R::gfp-minC]. Times are indicated in seconds. (C) Random distribution of Gfp-MinC in a SfiA-induced filament of strain PB114(λDR144)/pDR175 [ΔminCDE(P_lac::sfiA)/λp_R::gfp-minC]. Bar, 5 μm.

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 absence of MinD and MinE, MinC localizes nonspecifically to the cytoplasm and has no effect on septal or FtsZ ring formation. (B) In the presence of MinD, and absence of MinE, MinC associates with MinD along the entire membrane, preventing FtsZ ring formation at all PDSs. (C) In WT cells, MinC co-oscillates with MinD from one side of the MinE ring to the other, actively interfering with FtsZ ring assembly at each cell end in a sequential and rapidly repeating fashion. (D) In cells lacking FtsZ rings, multiple MinE rings define three or more cell segments. As in WT cells, MinC co-oscillates with MinD between the segments flanking each MinE ring. Note that although the figure suggests the presence of a limited number of regularly spaced PDSs in each cell, the proposed mechanism of MinCDE action does not depend on the exact number or nature of the PDSs and is equally tenable whether potential sites for FtsZ ring assembly are (co)determined by positioning of the nucleoids (41) or any other mechanism.