Membrane topology of the outer membrane protein OprH from Pseudomonas aeruginosa: PCR-mediated site-directed insertion and deletion mutagenesis

The 21-kDa outer membrane protein OprH from Pseudomonas aeruginosa is overexpressed under Mg2+. starvation conditions and when overproduced causes resistance to polymyxin B, gentamicin, and EDTA. By circular dichroism analysis, OprH revealed a calculated beta-sheet structure content of 37.3%. PCR-based site-directed deletion and epitope insertion mutagenesis was used to test a topological model of OprH as an eight-stranded beta-barrel. Three permissive and seven nonpermissive malarial epitope insertion mutants and four permissive and four nonpermissive deletion mutants confirmed the general accuracy of this model. Thus, OprH is the smallest outer membrane protein to date to be confirmed as a beta-stranded protein.