Abstract
Cell division of rod-shaped bacteria requires the Z ring, a ring of FtsZ filaments associated with the inner-membrane wall. The MinCDE proteins help localize the Z ring to the center of the Escherichia coli cell. MinC, which inhibits Z-ring assembly, is a passenger on MinD. Previous studies have shown that MinC-MinD from E. coli and Aquifex aeolicus assemble in vitro into extended filaments with a 1:1 stoichiometry. However, a recent study has raised questions about the function of the MinC-MinD copolymer in vivo, because its assembly appears to require a high concentration of these two proteins and has a long lag time, and its blockade does not affect in vivo activities. Here, we found that MinC and MinD from Pseudomonas aeruginosa coassemble into filaments with a 1:1 stoichiometry. We also found that the minimal concentration of ∼4 μm required for assembly applies only to MinD because above 4 μm MinD, even very low MinC concentrations sustained coassembly. As previously reported, the MinC-MinD coassembly exhibited a long lag of ∼100 s when initiated by ATP. Premixing MinD with ATP eliminated this lag, suggesting that it may be due to slow MinD dimerization following ATP activation. We also discovered that MinC-MinD copolymers quickly bound FtsZ filaments and formed huge bundles. Our results resolve previous questions about the low concentration of MinC and the lag time, insights that may inform future investigations into the exact role of the MinC-MinD copolymer in vivo.
Highlights
Cell division of rod-shaped bacteria requires the Z ring, a ring of FtsZ filaments associated with the inner-membrane wall
In this study we have confirmed that MinC and MinD from P. aeruginosa coassemble into filaments with a 1:1 stoichiometry, very similar to the coassembly of the E. coli and A. aeolicus proteins [24, 25]
We used MinC and MinD proteins from P. aeruginosa because they show a stable activity that may be better than the E. coli proteins
Summary
The C-terminal domain, MinCC, does three things: it forms a tight homodimer, it binds MinD, and it binds the conserved C-terminal peptide of FtsZ [22] One question for this simple scenario is why the MinC concentration in vivo is so low. Because its average concentration is highest at the poles, the inhibitory action of MinC blocks Z-ring formation at the poles and favors Z rings at the center This scenario was complicated by two independent studies, which found that a mixture of MinC and MinD could assemble long filaments [24, 25]. Park et al [26] created mutants of MinC and MinD that would disrupt the interfaces seen in the crystal structure and showed that heterodimers of the mutants and WT protein were still active in vivo These heterodimers should block assembly of filaments of MinC–MinD copolymers, suggesting that the filament assembly was not needed for function in vivo. We have confirmed several results of the previous studies and discovered some new features of the coassembly that may be relevant to the Min system in vivo
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