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Preparation and characterization of everted membrane vesicles from cells of Staphylococcus aureus
K Kodama1, A Hashimoto, Y Morita
1Department of Microbiology, Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Japan.
Abstract:
We developed a method for the preparation of everted membrane vesicles from cells of Staphylococcus aureus. The cells were first treated with ampicillin to weaken the peptidoglycan layer, then the cells were passed through a French press cell. The resulting vesicles were roughly 0.1 microm in diameter, judging from electron microscopic observations. We detected fairly high membrane-bound ATPase activity in the membrane vesicles. We observed respiratory-driven quenching of quinacrine fluorescence, which indicates that inward H+ transport took place. These results indicate that the vesicles are everted. We characterized the membrane-bound ATPase. We also detected Na+/H+ antiport, erythromycin/H+ antiport and chloramphenicol/H+ antiport activities in the membranes of S. aureus.
Insights
We developed a method to create everted membrane vesicles from Staphylococcus aureus cells. These vesicles exhibit ATPase activity and transport protons, sodium, and antibiotics, aiding in understanding bacterial membrane function.
Area of Science:
- Microbiology
- Cell Biology
- Biochemistry
Background:
- Staphylococcus aureus is a significant human pathogen.
- Understanding its membrane transport mechanisms is crucial for developing new antimicrobial strategies.
- Everted membrane vesicles are valuable tools for studying these mechanisms.
Purpose of the Study:
- To develop a reliable method for preparing everted membrane vesicles from Staphylococcus aureus.
- To characterize the functional properties of these vesicles, including ATPase activity and transport capabilities.
Main Methods:
- Staphylococcus aureus cells were treated with ampicillin to weaken the peptidoglycan layer.
- Cells were then subjected to French press lysis to generate membrane vesicles.
- Vesicle eversion was confirmed by respiratory-driven quenching of quinacrine fluorescence.
- ATPase activity and various antiport activities (Na+/H+, erythromycin/H+, chloramphenicol/H+) were measured.
Main Results:
- Successfully prepared everted membrane vesicles of approximately 0.1 microm in diameter.
- Detected significant membrane-bound ATPase activity.
- Confirmed inward proton (H+) transport via respiratory-driven quinacrine fluorescence quenching.
- Identified Na+/H+ antiport, as well as erythromycin/H+ and chloramphenicol/H+ antiport activities.
Conclusions:
- The developed method yields functional everted membrane vesicles from Staphylococcus aureus.
- These vesicles are suitable for investigating membrane-bound enzyme activities and transport systems.
- The identified transport activities provide insights into the mechanisms of antibiotic resistance and nutrient uptake in S. aureus.