Use of a beta-lactamase fusion vector to investigate the organization of penicillin-binding protein 1B in the cytoplasmic membrane of Escherichia coli.
The coding region for the mature form of TEM beta-lactamase was fused to random positions within the coding region of the penicillin-binding protein 1B (PBP 1B) gene and the nucleotide sequences across the fusion junctions of 100 in-frame fusions were determined. All fusion proteins that contained at least the NH2-terminal 94 residues of PBP 1B provided individual cells of E. coli with substantial levels of ampicillin resistance, suggesting that the beta-lactamase moiety had been translocated to the periplasm. Fusion proteins that contained less than or equal to 63 residues of PBP 1B possessed beta-lactamase activity, but could not protect single cells of E. coli from ampicillin, indicating that the beta-lactamase moiety of these fusion proteins remained in the cytoplasm. The beta-lactamase fusion approach suggested a model for the organization of PBP 1B in which the protein is embedded in the cytoplasmic membrane by a single hydrophobic transmembrane segment (residues 64-87), with a short NH2-terminal domain (residues 1-63), and the remainder of the polypeptide (residues 88-844) exposed on the periplasmic side of the cytoplasmic membrane. The proposed model for the organization of PBP 1B was supported by experiments which showed that the protein was completely digested by proteinase K added from the periplasmic side of the cytoplasmic membrane but was only slightly reduced in size by protease attack from the cytoplasmic side of the membrane.
Edelman
A
A
Microbial Genetics Group, School of Biological Sciences, University of Sussex, Falmer, Brighton, UK.
Bowler
L
L
Broome-Smith
J K
JK
Spratt
B G
BG
D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase / Q000235:genetics* / Q000378:metabolism; D046915:Penicillin-Binding Proteins; D010406:Penicillins / Q000378:metabolism; D010458:Peptidyl Transferases; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D001618:beta-Lactamases / Q000235:genetics*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D011993:Recombinant Fusion Proteins; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase; D001618:beta-Lactamases