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Electron microscopy during release and purification of mesosomal vesicles and protoplast membranes from
Abstract:
The mesosomes of log-phase Staphylococcus aureus ATCC 6538P and Staphylococcus aureus phage-type 80/81, as seen in situ in ultrathin sections, were of the vesicular type. The constituent vesicles ranged from 35 to 50 nm in diameter when the glutaraldehyde-osmium-uranium-lead sequence of fixation and staining was used. During protoplasting in hypertonic buffer containing a muralytic enzyme, vesicles of the same size were extruded and required magnesium ion to maintain structural integrity. The vesicles, purified from the protoplasting supernatant medium by density gradient centrifugation, maintained size and configuration in a homogeneous preparation. Cytoplasmic membranes, produced by osmotic shock and nuclease treatment of protoplasts, were similarly concentrated in gradients. However, they were not free of membrane-associated ribosomes nor of mesosomal vesicles except when prepared in the absence of magnesium.
Insights
Mesosomes in Staphylococcus aureus are vesicular structures. Magnesium ions are crucial for maintaining the structural integrity of these extruded vesicles during protoplasting.
Area of Science:
- Microbiology
- Cell Biology
- Bacterial Ultrastructure
Background:
- Mesosomes are intracellular membrane structures found in Gram-positive bacteria.
- Their precise function and composition remain areas of active research.
- Previous studies have yielded varied descriptions of mesosome morphology.
Purpose of the Study:
- To characterize the morphology and behavior of mesosomes in Staphylococcus aureus.
- To investigate the role of magnesium ions in mesosome structural integrity.
- To differentiate mesosomes from cytoplasmic membranes.
Main Methods:
- Ultrathin sectioning and electron microscopy of Staphylococcus aureus.
- Protoplasting using muralytic enzymes in hypertonic buffer.
- Density gradient centrifugation for vesicle purification.
- Osmotic shock and nuclease treatment for cytoplasmic membrane isolation.
Main Results:
- Mesosomes in log-phase Staphylococcus aureus were identified as vesicular structures (35-50 nm in diameter).
- These vesicles were extruded during protoplasting and required magnesium ions for structural stability.
- Purified mesosomal vesicles maintained their size and configuration.
- Cytoplasmic membranes, when prepared without magnesium, were free of mesosomal vesicles.
Conclusions:
- Staphylococcus aureus mesosomes are extruded as discrete vesicular structures.
- Magnesium ions play a critical role in maintaining the structural integrity of these mesosomal vesicles.
- Distinguishing mesosomes from cytoplasmic membranes is facilitated by the presence or absence of magnesium during preparation.