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Isolation, structural and functional characterization of Staphylococcus aureus protoplasts obtained using lysoamidase
V V Petrov1, Artzatbanov VYu, E N Ratner
1Institute of Biochemistry and Physiology of Microorganisms, USSR Academy of Sciences, Moscow Region.
Archives of Microbiology
|January 1, 1991
Summary
Lysoamidase efficiently generates Staphylococcus aureus protoplasts by hydrolyzing peptidoglycan cell walls. These protoplasts retain cellular structure and metabolic activity, offering a valuable tool for bacterial research.
Area of Science:
- Microbiology
- Biochemistry
- Cell Biology
Background:
- Staphylococcus aureus is a significant human pathogen.
- Bacterial cell wall hydrolysis is crucial for protoplast formation.
- Understanding protoplast generation is key for studying bacterial physiology.
Purpose of the Study:
- To investigate the efficacy of the lysoamidase bacteriolytic complex in generating protoplasts from Staphylococcus aureus.
- To characterize the conditions and steps involved in protoplast formation.
- To assess the viability and characteristics of the generated protoplasts.
Main Methods:
- Treatment of Staphylococcus aureus VKM B-209P cells with lysoamidase.
- Induction of protoplast formation in the presence of 1 M sucrose.
- Microscopic examination of cell wall destruction and protoplast ultrastructure.
- Assessment of protoplast viability through peptidoglycan synthesis and respiratory activity.
- Analysis of substrate-stimulated respiration in cells and protoplasts.
Main Results:
- Midlogarithmic phase Staphylococcus aureus cells exhibited maximum sensitivity to lysoamidase.
- Lysoamidase induced local cell wall destruction via peptidoglycan hydrolysis.
- Protoplast formation occurred in two steps: spheroplast formation followed by protoplast release, yielding approximately 80% protoplasts.
- Generated protoplasts maintained intact ultrastructure and synthesized peptidoglycan fibrillae.
- Spheroplasts with residual cell walls reverted to bacterial forms, while true protoplasts did not.
- Protoplasts displayed respiratory activity comparable to intact cells, with significant oxygen consumption observed with alpha-glycerophosphate and ethanol.
Conclusions:
- Lysoamidase is an effective agent for producing Staphylococcus aureus protoplasts with high yield.
- The generated protoplasts are viable, retaining ultrastructure and metabolic functions.
- Protoplast formation and characteristics are influenced by cell wall integrity and osmotic conditions.
- The study provides insights into bacterial cell wall structure and protoplast biology.