Determination of the Relative OmpA Expression and Membrane Integration in an OmpA-Deficient Escherichia coli Strain Complemented with a Plasmid Containing an OmpA Gene.

Abstract

The outer membrane protein A (OmpA) of E. coli, is thought to be necessary for Ffactor conjugation. A previous study demonstrated that a wild type E. coli strain and an OmpA-deficient strain, each complemented by the pCR2.1-TOPO plasmid containing a cloned ompA gene, had no significant differences in conjugation efficiency upon induction of the ompA gene on the plasmid. This investigation assessed whether the poor conjugation efficiency was due to lack of integration of the OmpA into the outer membrane in the complemented strain. The wild-type and complemented mutant strains were lysed and outer membranes extracted to separate cytosolic and membrane samples. SDS-PAGE was used to separate proteins in these samples to identify the presence or absence of OmpA. Results from this investigation suggest OmpA was not integrated into the uninduced complemented mutant. The presence or absence of the 40 kDa OmpA protein was difficult to discern in cytosolic samples because of poor gel replicates and the lack of a distinctive OmpA identifier such as an antibody.