title: Overproduction of penicillin-binding protein 2 and its inactive variants causes morphological changes and lysis in Escherichia coli.
abstract: Penicillin-binding protein 2 (PBP 2) has long been known to be essential for rod-shaped morphology in gram-negative bacteria, including Escherichia coli and Pseudomonas aeruginosa. In the course of earlier studies with P. aeruginosa PBP 2, we observed that E. coli was sensitive to the overexpression of its gene, pbpA. In this study, we examined E. coli overproducing both P. aeruginosa and E. coli PBP 2. Growth of cells entered a stationary phase soon after induction of gene expression, and cells began to lyse upon prolonged incubation. Concomitant with the growth retardation, cells were observed to have changed morphologically from typical rods into enlarged spheres. Inactive derivatives of the PBP 2s were engineered, involving site-specific replacement of their catalytic Ser residues with Ala in their transpeptidase module. Overproduction of these inactive PBPs resulted in identical effects. Likewise, overproduction of PBP 2 derivatives possessing only their N-terminal non-penicillin-binding module (i.e., lacking their C-terminal transpeptidase module) produced similar effects. However, E. coli overproducing engineered derivatives of PBP 2 lacking their noncleavable, N-terminal signal sequence and membrane anchor were found to grow and divide at the same rate as control cells. The morphological effects and lysis were also eliminated entirely when overproduction of PBP 2 and variants was conducted with E. coli MHD79, a strain lacking six lytic transglycosylases. A possible interaction between the N-terminal domain of PBP 2 and lytic transglycosylases in vivo through the formation of multienzyme complexes is discussed.
authors.affiliation: Guelph-Waterloo Centre for Graduate Work in Chemistry and Biochemistry, Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
authors.forename: Blaine A
authors.initials: BA
authors.lastname: Legaree
authors.forename: Calvin B
authors.initials: CB
authors.lastname: Adams
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Clarke
mesh_terms: D001433:Bacteriolysis / Q000235:genetics* / Q000502:physiology; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000502:physiology / Q000648:ultrastructure; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D008855:Microscopy, Electron, Scanning; D008957:Models, Genetic; D009154:Mutation; D046915:Penicillin-Binding Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D046928:Peptidoglycan Glycosyltransferase / Q000235:genetics / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000235:genetics
chemical_list: D029968:Escherichia coli Proteins; D046915:Penicillin-Binding Proteins; D046928:Peptidoglycan Glycosyltransferase