ABSTRACT
The construction of hybrid proteins of PBP1B and PBP3 has been described. One hybrid protein (PBP1B/3) contained the transglycosylase domain of PBP1B and the transpeptidase domain of PBP3. In the other hybrid protein, the putative transglycosylase domain of PBP3 was coupled to the transpeptidase domain of PBP1B (PBP3/1B). The hybrid proteins were localized in the cell envelope in a similar way as the wild-type PBP1B. In vitro isolates of the strains containing the hybrid proteins had a transglycosylase activity intermediate between that of wild-type PBP1B-producing strain and that of a PBP1B overproducer. Analysis with specific antibiotics against PBP1A/1B and PBP3 and mutant analysis in strains containing PBP3/1B revealed no detectable effects in vivo compared with wild-type strains. The same was shown for PBP1B/3 when the experiments were performed in a recA background. The data indicate that the hybrid proteins cannot replace native penicillin-binding proteins. This finding suggests that functional high-molecular-weight penicillin-binding protein specificity is at least in part determined by the unique combination of the two functional domains.
Subject(s)
Bacterial Proteins , Carrier Proteins , Escherichia coli Proteins , Escherichia coli/enzymology , Glycosyltransferases/metabolism , Hexosyltransferases/metabolism , Multienzyme Complexes/metabolism , Muramoylpentapeptide Carboxypeptidase , Peptidoglycan Glycosyltransferase , Peptidyl Transferases/metabolism , Serine-Type D-Ala-D-Ala Carboxypeptidase , Blotting, Western , Cefsulodin/metabolism , Cell Compartmentation , Cell Membrane/enzymology , Cephalexin/metabolism , Cephalosporins/metabolism , Escherichia coli/genetics , Glycosyltransferases/genetics , Hexosyltransferases/genetics , Multienzyme Complexes/genetics , Penicillin-Binding Proteins , Peptidyl Transferases/genetics , Recombinant Fusion Proteins/metabolism , Structure-Activity RelationshipABSTRACT
Earlier studies revealed that PBP1B of Escherichia coli occurred as a monomeric as well as a dimeric form (C.A.L. Zijderveld, M.E.G. Aarsman, T. den Blaauwen, and N. Nanninga, J. Bacteriol. 173:5740-5746, 1991). In this study, the dimer of PBP1B was further analyzed. It appeared that the dimeric form could be divided into two classes. One class, which cofractionated with the cell wall fraction, could be artificially cross-linked to peptidoglycan, indicating a close association with the latter. This class of PBP1B dimers was sensitive to beta-mercaptoethanol. The second class, like the monomeric form of PBP1B, could be isolated with the inner membrane fraction. This dimeric form dissociated in the presence of zinc in combination with beta-mercaptoethanol.
Subject(s)
Bacterial Proteins , Carrier Proteins , Escherichia coli Proteins , Escherichia coli/enzymology , Hexosyltransferases/chemistry , Multienzyme Complexes/chemistry , Muramoylpentapeptide Carboxypeptidase , Peptidoglycan Glycosyltransferase , Peptidoglycan/analysis , Peptidyl Transferases/chemistry , Serine-Type D-Ala-D-Ala Carboxypeptidase , Enzyme Stability , Hexosyltransferases/analysis , Mercaptoethanol/pharmacology , Multienzyme Complexes/analysis , Penicillin-Binding Proteins , Peptidyl Transferases/analysis , Zinc/pharmacologyABSTRACT
A high-molecular-weight band has been detected in Western immunoblots of nonboiled Escherichia coli samples incubated with polyclonal antiserum against penicillin-binding protein 1B (PBP 1B). This band was shown to be a dimer of PBP 1B. The dimer was more strongly associated with the envelope than the monomer, and it was still able to bind penicillin G. Analysis of the binding of fusion proteins of PBP 1B and beta-lactamase showed that the part of PBP 1B necessary for complex formation lies in the amino-terminal half of the protein.
