Linker-insertion mutagenesis of Pseudomonas aeruginosa outer membrane protein OprF

The oprF gene, expressing Pseudomonas aeruginosa major outer membrane protein OprF, was subjected to semi-random linker mutagenesis by insertion of a 1.3 kb Hincll kanamycin-resistance fragment from plasmid pUC4KAPA into multiple blunt-ended restriction sites in the oprF gene. The kanamycin-resistance gene was then removed by Pstl digestion, which left a 12 nucleotide pair linker residue. Nine unique clones were identified that contained such linkers at different locations within the oprF gene and were permissive for the production of full-length OprF variants. In addition, one permissive site-directed insertion, one non-permissive insertion and one carboxy-terminal insertion leading to proteolytic truncation were also identified. These mutants were characterized by DNA sequencing and reactivity of the OprF variants with a bank of 10 OprF-specific monoclonal antibodies. Permissive clones produced OprF variants that were shown to be reactive with the majority of these monoclonal antibodies, except where the insertion was suspected of interrupting the epitope for the specific monoclonal antibody. In addition, these variants were shown to be 2-mercaptoethanol modifiable, to be resistant to trypsin cleavage in intact cells and partly cleaved to a high-molecular-weight core fragment in outer membranes and, where studied, to be accessible to indirect immunofluorescenee labelling in intact cells by monoclonal antibodies specific for surface epitopes. Based on these data, a revised structural model for OprF is proposed.