Insertion mutagenesis on a cell-surface-exposed region of outer membrane protein PhoE of Escherichia coli K-12

Abstract

Amino acid residue arginine-158 of the outer membrane protein PhoE of Escherichia coli K-12 has been shown to be cell-surface-exposed [Korteland et al. (1985) Eur. J. Biochem. 152, 691-697]. To study the effects of small insertions in this region of the protein on its biogenesis and characteristics, a unique restriction site was created by site-directed mutagenesis in a plasmid carrying the phoE gene and oligonucleotides of 12-74 bp were inserted. The insertions did not interfere with incorporation into the outer membrane since (a) several monoclonal antibodies, directed against the cell-surface-exposed part of PhoE protein, bound to whole cells producing the altered proteins and (b) the proteins formed functional pores for the uptake of beta-lactam antibiotics. The binding of one monoclonal antibody and of the PhoE-specific phages TC45 and TC45hrN3 was disturbed by the insertions, showing that this region of the protein is immunogenic and is involved in the binding of both of these phages. The functioning of the mutant pores was characterized both in vivo by studying the uptake of beta-lactam antibiotics and in vitro after the reconstitution of the proteins in black lipid films. The pore characteristics changed depending on the nature of the inserted amino acids. Addition of a negatively charged amino acid resulted in decreased anion-selectivity, whereas insertion of a positive charge and deletion of a negative charge had only a small influence.