Construction of a series of ompC-ompF chimeric genes by in vivo homologous recombination in Escherichia coli and characterization of their translational products

Abstract

OmpC and OmpF are major outer membrane proteins and although they are homologous proteins, they function differently in several respects. As an approach to elucidate the submolecular structures that determine their differences, we have constructed a series of ompC-ompF chimeric genes by in vivo homologous recombination between these two genes, which are adjacent on a plasmid. The recombination sites in the chimeric genes were localized by means of restriction endonuclease analysis and nucleotide sequence determination. Most of the chimeric gene products were accumulated in the outer membrane. One of the chimeric gene products, with a fusion site in a central region between the OmpC and OmpF proteins, was normally expressed but not accumulated in the outer membrane. The trimeric structures of some of the chimeric gene products appeared to be extremely unstable in a SDS solution. From these results, domains contributing to the formation of specific structures in which the OmpC and OmpF proteins differ were identified. Bacterial cells possessing the chimeric gene products were also investigated as to their sensitivity to phages that require either OmpC or OmpF as a receptor component. With the aid of the chimeric gene products, the immunogenic determinants for three anti-OmpC monoclonal antibodies were found to be localized at different portions of the OmpC polypeptide: the N-terminal, central and C-terminal portions, respectively.