ABSTRACT
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.
Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Glycoside Hydrolases/metabolism , Muramic Acids/metabolism , Cell Wall/metabolism , Culture Media/metabolism , Escherichia coli/growth & developmentABSTRACT
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.
Subject(s)
Acetylglucosamine/analogs & derivatives , Acetylglucosamine/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Muramic Acids/metabolism , Peptidoglycan/metabolism , Cell Wall/metabolism , Escherichia coli Proteins/genetics , Ethers/metabolism , Gene Deletion , Phosphotransferases/genetics , Phosphotransferases/metabolismABSTRACT
Accurate positioning of the division septum at the equator of Escherichia coli cells requires a rapid oscillation of MinD ATPase between the polar halves of the cell membrane, together with the division inhibitor MinC, under MinE control. The mechanism underlying MinD oscillation remains poorly understood. Here, we demonstrate that purified MinD assembles into protein filaments in the presence of ATP. Incubation with phospholipid vesicles further stimulates MinD polymerization. Addition of purified MinE in the presence of lipids promotes bundling of MinD filaments as well as their disassembly through activation of MinD ATPase. MinE thus provokes a net decay in the steady-state MinD polymer mass. Taken together, our results suggest that reversible MinD assembly modulated by MinE underlies the dynamic processing of positional information in E. coli to identify precisely the nascent site for cell division.
Subject(s)
Adenosine Triphosphatases/chemistry , Adenosine Triphosphate/physiology , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/physiology , Phospholipids/physiology , Cell Cycle Proteins , Microscopy, Electron , Polymers/chemistryABSTRACT
Aqualysin I has at least two Ca2+-binding sites that have different affinities for Ca2+. The binding of various metal ions to aqualysin I was studied using 23Na- and 139La-NMR spectrometry. Evidence is presented that Ca2+, La3+, and Na+ bind to the low-affinity Ca2+-binding site of aqualysin I, but Mg2+ does not. Our results confirm that binding of metals at the low-affinity Ca2+-binding site is essential for thermostabilization, since the addition of Mg2+ did not result in thermostabilization. La3+ was found to bind to both the low-affinity Ca2+-binding site and an additional metal ion-binding site that can also be involved in the thermostabilization of aqualysin I.
