C ell division in Escherichia coli and other rod-shaped bacteria depends on the precise placement of a division septum at the cell center, a process initiated by the assembly of an equatorial ring (Z-ring) of the tubulin-like FtsZ GTPase on the cytoplasmic membrane (1, 2). The Z-ring assembly is spatially restricted to midcell by nucleoid occlusion (3, 4) and by the MinCDE system (5-7). The first mechanism ensures that Z-rings form only in cellular space devoid of nucleoid mass, while the Min system prevents Z-ring assembly at the cell poles.Although the basis of nucleoid-mediated inhibition of Z-ring assembly remains unclear, MinC is a division inhibitor that has been shown to block Z-ring assembly in vivo (8, 9) and FtsZ polymerization in vitro (10). MinC is recruited to the membrane by MinD ATPase and, in the absence of MinE, the MinCD complex is dispersed throughout the membrane, blocking cell division at all sites in the cell (11)(12)(13)(14). In the presence of MinE, however, the midcell site is relieved of MinCD inhibition, allowing normal equatorial septation. The biochemical mechanism underlying such topological regulation remains poorly understood.Cytological studies have shed light on subcellular localization and dynamics of the Min proteins in E. coli. MinD fused to the GFP localizes into a horseshoe structure on the polar membrane and undergoes a rapid (periodicity Ϸ50 s) pole-to-pole oscillation in live E. coli cells in a MinE-dependent manner (15, 16). MinC also colocalizes and cooscillates with the MinD horseshoe when MinE is present (12, 13), so that the time-averaged concentration of MinC is lowest at midcell to allow medial division.Interestingly, MinE localizes both as a ring structure (E-ring) at the edge of the MinCD horseshoe as well as a polar zone that extends along the horseshoe arms (17, 18). The E-ring, together with the MinE polar zone, moves to the nearest cell pole, causing rapid shrinkage of the MinCD horseshoe and forcing coupled oscillation of MinCD and MinE to the opposite cell pole (17, 18). Recently, MinE was shown to stimulate MinD ATPase activity in the presence of phospholipids, and the level of stimulation correlated with the period of MinD oscillation (19).In this study, we demonstrate that purified MinD polymerizes into protein fibers upon incubation with ATP and phospholipids. Adding purified MinE to the assembly reaction promotes both bundling of MinD filaments as well as their depolymerization. The resultant diminution in the MinD polymer mass ensues from MinE-mediated stimulation of MinD ATPase in the presence of lipids. These results suggest that dynamic MinD assembly regulated by MinE is the biochemical basis for MinD oscillation in E. coli and underscore that protein assembly and protein motion, powered by ATP binding and hydrolysis, evolved in primitive cells as a mechanism for searching and measuring cellular space. Materials and MethodsProtein Expression and Purification. His 6 -tagged Pyrococcus furiosus MinD1 was expressed from pET-28a containing minD1 in BL...

Escherichia coli breaks down over 60% of the murein of its side wall and reuses the component amino acids to synthesize about 25% of the cell wall for the next generation. The amino sugars of the murein are also efficiently recycled. Here we show that the 1,6-anhydro-N-acetylmuramic acid (anhMurNAc) is returned to the biosynthetic pathway by conversion to N-acetylglucosamine-phosphate (GlcNAc-P). The sugar is first phosphorylated by anhydro-N-acetylmuramic acid kinase (AnmK), yielding MurNAc-P, and this is followed by action of an etherase which cleaves the bond between D-lactic acid and the N-acetylglucosamine moiety of MurNAc-P, yielding GlcNAc-P. The kinase gene has been identified by a reverse genetics method. The enzyme was overexpressed, purified, and characterized. The cell extract of an anmK deletion mutant totally lacked activity on anhMurNAc. Surprisingly, in the anmK mutant, anhMurNAc did not accumulate in the cytoplasm but instead was found in the medium, indicating that there was rapid efflux of free anhMurNAc.

MurQ is an N-acetylmuramic acid-phosphate (MurNAc-P) etherase that converts MurNAc-P to N-acetylglucosamine-phosphate and is essential for growth on MurNAc as the sole source of carbon (T. Jaegar, M. Arsic, and C. Mayer, J. Biol. Chem. 280:30100-30106, 2005). Here we show that MurQ is the only MurNAc-P etherase in Escherichia coli and that MurQ and AnmK kinase are required for utilization of anhydro-MurNAc derived either from cell wall murein or imported from the medium.

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