SummaryThe murein (peptidoglycan) sacculus is an essential polymer embedded in the bacterial envelope. The Escherichia coli class B penicillin-binding protein (PBP) 3 is a murein transpeptidase and essential for cell division. In an affinity chromatography experiment, the bifunctional transglycosylasetranspeptidase murein synthase PBP1B was retained by PBP3-sepharose when a membrane fraction of E. coli was applied. The direct protein-protein interaction between purified PBP3 and PBP1B was characterized in vitro by surface plasmon resonance. The interaction was confirmed in vivo employing two different methods: by a bacterial two-hybrid system, and by cross-linking/co-immunoprecipitation. In the bacterial two-hybrid system, a truncated PBP3 comprising the N-terminal 56 amino acids interacted with PBP1B. Both synthases could be cross-linked in vivo in wild-type cells and in cells lacking FtsW or FtsN. PBP1B localized diffusely and in foci at the septation site and also at the side wall. Statistical analysis of the immunofluorescence signals revealed that the localization of PBP1B at the septation site depended on the physical presence of PBP3, but not on the activity of PBP3. These studies have demonstrated, for the first time, a direct interaction between a class B PBP (PBP3) and a class A PBP (PBP1B) in vitro and in vivo, indicating that different murein synthases might act in concert to enlarge the murein sacculus during cell division.
PBP1B is a major bifunctional murein (peptidoglycan) synthase catalyzing transglycosylation and transpeptidation reactions in Escherichia coli. PBP1B has been shown to form dimers in vivo. The K D value for PBP1B dimerization was determined by surface plasmon resonance. The effect of the dimerization of PBP1B on its activities was studied with a newly developed in vitro murein synthesis assay with radioactively labeled lipid II precursor as substrate. Under conditions at which PBP1B dimerizes, the enzyme synthesized murein with long glycan strands (>25 disaccharide units) and with almost 50% of the peptides being part of cross-links. PBP1B was also capable of synthesizing trimeric muropeptide structures. Tri-, tetra-, and pentapeptide compounds could serve as acceptors in the PBP1B-catalyzed transpeptidation reaction.Most bacteria contain a murein (peptidoglycan) sacculus, an exoskeleton that is essential for the osmotic stability of the cell (1). Murein has a net-like structure and is composed of glycan strands of alternating 1,4-linked N-acetylmuramic acid and N-acetylglucosamine residues that are cross-linked by short peptides (2). Escherichia coli is rod-shaped and has a simple cell cycle; a newborn cell elongates with a constant diameter, and when the length of the cell has doubled, the cell divides at mid-cell, forming two new polar caps. Cell growth requires a well coordinated incorporation of the murein precursor, lipid II, into the existing sacculus (3).Two reactions are required for murein synthesis. First, disaccharide units of lipid II are oligomerized to murein glycan strands by transglycosylation. Second, peptide cross-links are formed by transpeptidation, the latter reaction being the target of -lactam antibiotics. Murein synthases have a modular domain structure (4). E. coli has three bifunctional transglycosylases-transpeptidases, the class A penicillin-binding proteins (PBPs) 3 1A, 1B, and 1C (4 -6), two monofunctional transpeptidases, the class B PBPs 2 and 3, which contain a non-catalytic domain of unknown function (7), and one monofunctional transglycosylase, MtgA (8). Different molecular interactions between certain murein synthases and between murein synthases and hydrolases have been identified, indicating that the enlargement of the sacculus might be achieved by murein synthesis holoenzymes (9).PBP1B exists in three isoforms (␣, , and ␥) that are encoded by the same gene (ponB) and is a major murein synthase in E. coli (10 -12). The protein has a short cytoplasmic part and is anchored in the cytoplasmic membrane via the amino acids 64 -88. Most of the enzyme is located in the periplasm (amino acids 89 -844), and the periplasmic part contains the transglycosylase (amino acids 198 -435) and transpeptidase (amino acids 447-780) domains (13). PBP1B was shown to form dimers in vivo (14, 15), and dimerization does not involve disulfide bridges (16). Moreover, PBP1B interacts with PBP3 and with the MltA-interacting protein A (MipA), that itself interacts with the murein hydrolase MltA (17)...
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