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Journal of Bacteriology, May 2000, p. 2402-2410, Vol. 182, No. 9
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Insertion Mutagenesis and Membrane Topology Model of the Pseudomonas aeruginosa Outer Membrane Protein OprM

Kendy K. Y. Wong and Robert E. W. Hancock*

Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3

Received 17 November 1999/Accepted 10 February 2000

Pseudomonas aeruginosa OprM is a protein involved in multiple-antibiotic resistance as the outer membrane component for the MexA-MexB-OprM efflux system. Planar lipid bilayer experiments showed that OprM had channel-forming activity with an average single-channel conductance of only about 80 pS in 1 M KCl. The gene encoding OprM was subjected to insertion mutagenesis by cloning of a foreign epitope from the circumsporozoite form of the malarial parasite Plasmodium falciparum into 11 sites. In Escherichia coli, 8 of the 11 insertion mutant genes expressed proteins at levels comparable to those obtained with the wild-type gene and the inserted malarial epitopes were surface accessible as assessed by indirect immunofluorescence. When moved to a P. aeruginosa OprM-deficient strain, seven of the insertion mutant genes expressed proteins at variable levels comparable to that of wild-type OprM and three of these reconstituted MIC profiles resembling those of the wild-type protein, while the other mutant forms showed variable MIC results. Utilizing the data from these experiments, in conjunction with multiple sequence alignments and structure predictions, an OprM topology model with 16 beta  strands was proposed.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3. Phone: 604-822-2682. Fax: 604-822-6041. E-mail: bob{at}cmdr.ubc.ca.

Journal of Bacteriology, May 2000, p. 2402-2410, Vol. 182, No. 9
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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