Insertion mutagenesis and membrane topology model of the Pseudomonas aeruginosa outer membrane protein OprM

Kendy K.Y. Wong, Robert Hancock

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

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 KCI. 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 β strands was proposed.

LanguageEnglish
Pages2402-2410
Number of pages9
JournalJournal of bacteriology
Volume182
Issue number9
DOIs
Publication statusPublished - 1 May 2000
Externally publishedYes

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

Cite this

@article{d85ed5a9725f4183ad4ba67d7218671b,
title = "Insertion mutagenesis and membrane topology model of the Pseudomonas aeruginosa outer membrane protein OprM",
abstract = "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 KCI. 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 β strands was proposed.",
author = "Wong, {Kendy K.Y.} and Robert Hancock",
year = "2000",
month = "5",
day = "1",
doi = "10.1128/JB.182.9.2402-2410.2000",
language = "English",
volume = "182",
pages = "2402--2410",
journal = "Journal of Bacteriology",
issn = "0021-9193",
publisher = "American Society for Microbiology",
number = "9",

}

Insertion mutagenesis and membrane topology model of the Pseudomonas aeruginosa outer membrane protein OprM. / Wong, Kendy K.Y.; Hancock, Robert.

In: Journal of bacteriology, Vol. 182, No. 9, 01.05.2000, p. 2402-2410.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Insertion mutagenesis and membrane topology model of the Pseudomonas aeruginosa outer membrane protein OprM

AU - Wong, Kendy K.Y.

AU - Hancock, Robert

PY - 2000/5/1

Y1 - 2000/5/1

N2 - 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 KCI. 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 β strands was proposed.

AB - 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 KCI. 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 β strands was proposed.

UR - http://www.scopus.com/inward/record.url?scp=0033996953&partnerID=8YFLogxK

U2 - 10.1128/JB.182.9.2402-2410.2000

DO - 10.1128/JB.182.9.2402-2410.2000

M3 - Article

VL - 182

SP - 2402

EP - 2410

JO - Journal of Bacteriology

T2 - Journal of Bacteriology

JF - Journal of Bacteriology

SN - 0021-9193

IS - 9

ER -