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Pseudomonas aeruginosa outer membrane permeability: isolation of a porin protein F-deficient mutant
Abstract:
A mutant of Pseudomonas aeruginosa severely deficient in outer membrane protein F levels was isolated by screening heavily mutagenized strains for membrane protein alterations on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. To provide a basis for phenotypic comparison, three independent spontaneous revertants with normal protein F levels were isolated. Neither the protein F-deficient mutant nor its revertants had gross surface alterations as judged by their sensitivities to 31 phages with diverse receptors and their low degrees of leakage of periplasmic beta-lactamase into the supernatant. Outer membrane permeability was measured in whole cells by examining the rates of hydrolysis of a chromogenic beta-lactam, nitrocefin, by periplasmic RP1-encoded beta-lactamase. It was found that the outer membrane permeabilities of wild-type and protein F revertant strains were similar, but low when compared with those of Escherichia coli and an antibiotic-supersusceptible mutant Z61 of P. aeruginosa. The loss of protein F caused a further significant decrease in outer membrane permeability. The results suggest that protein F is a pore-forming protein in vivo and that only a small proportion, as few as 1 in 400, of the protein F molecules form active functional channels in vivo.
Insights
Pseudomonas aeruginosa lacking outer membrane protein F showed reduced outer membrane permeability. This suggests protein F forms functional channels, crucial for nutrient uptake and antibiotic resistance.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Outer membrane proteins (OMPs) are crucial for bacterial cell envelope structure and function.
- Protein F in Pseudomonas aeruginosa is a major outer membrane protein with potential roles in permeability.
- Understanding OMP function is vital for developing new antimicrobial strategies.
Purpose of the Study:
- To investigate the role of outer membrane protein F in Pseudomonas aeruginosa outer membrane permeability.
- To characterize a mutant deficient in protein F and its revertants.
- To determine if protein F forms functional channels in the outer membrane.
Main Methods:
- Isolation and characterization of a protein F-deficient mutant and spontaneous revertants.
- Analysis of outer membrane protein profiles using SDS-PAGE.
- Assessment of phage sensitivity and beta-lactamase leakage to evaluate surface integrity.
- Measurement of outer membrane permeability using nitrocefin hydrolysis by periplasmic beta-lactamase.
Main Results:
- A Pseudomonas aeruginosa mutant severely deficient in protein F was successfully isolated.
- The protein F-deficient mutant and revertants showed no gross surface alterations in phage sensitivity or protein leakage.
- Outer membrane permeability was significantly reduced in the protein F-deficient mutant compared to wild-type and revertant strains.
- Protein F appears to form functional channels, with a low proportion of molecules actively contributing.
Conclusions:
- Outer membrane protein F plays a significant role in regulating Pseudomonas aeruginosa outer membrane permeability.
- Protein F functions as a pore-forming protein in vivo.
- The findings provide insights into the structural and functional importance of OMPs in bacterial physiology.