title: Contribution of membrane-binding and enzymatic domains of penicillin binding protein 5 to maintenance of uniform cellular morphology of Escherichia coli.
abstract: Four low-molecular-weight penicillin binding proteins (LMW PBPs) of Escherichia coli are closely related and have similar DD-carboxypeptidase activities (PBPs 4, 5, and 6 and DacD). However, only one, PBP 5, has a demonstrated physiological function. In its absence, certain mutants of E. coli have altered diameters and lose their uniform outer contour, resulting in morphologically aberrant cells. To determine what differentiates the activities of these LMW PBPs, we constructed fusion proteins combining portions of PBP 5 with fragments of other DD-carboxypeptidases to see which hybrids restored normal morphology to a strain lacking PBP 5. Functional complementation occurred when truncated PBP 5 was combined with the terminal membrane anchor sequences of PBP 6 or DacD. However, complementation was not restored by the putative carboxy-terminal anchor of PBP 4 or by a transmembrane region of the osmosensor protein ProW, even though these hybrids were membrane bound. Site-directed mutagenesis of the carboxy terminus of PBP 5 indicated that complementation required a generalized amphipathic membrane anchor but that no specific residues in this region seemed to be required. A functional fusion protein was produced by combining the N-terminal enzymatic domain of PBP 5 with the C-terminal beta-sheet domain of PBP 6. In contrast, the opposite hybrid of PBP 6 to PBP 5 was not functional. The results suggest that the mode of PBP 5 membrane anchoring is important, that the mechanism entails more than a simple mechanical tethering of the enzyme to the outer face of the inner membrane, and that the physiological differences among the LMW PBPs arise from structural differences in the DD-carboxypeptidase enzymatic core.
authors.affiliation: Department of Microbiology and Immunology, University of North Dakota School of Medicine, Grand Forks, North Dakota 58202-9037, USA.
authors.forename: David E
authors.initials: DE
authors.lastname: Nelson
authors.forename: Anindya S
authors.initials: AS
authors.lastname: Ghosh
authors.forename: Avery L
authors.initials: AL
authors.lastname: Paulson
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Young
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D004150:Dipeptidases; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000502:physiology*; D006602:Hexosyltransferases; D008969:Molecular Sequence Data; D002267:Muramoylpentapeptide Carboxypeptidase / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D016133:Polymerase Chain Reaction / Q000379:methods; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D046915:Penicillin-Binding Proteins; D011993:Recombinant Fusion Proteins; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D010450:Endopeptidases; D004150:Dipeptidases; C019989:DD-carboxypeptidase-endopeptidase; D002267:Muramoylpentapeptide Carboxypeptidase