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Isolation, composition, and structure of membrane of Listeria monocytogenes
Abstract:
The plasma membrane of Listeria monocytogenes strain 42 was prepared by osmotic lysis of protoplasts with tris(hydroxymethyl)aminomethane (Tris) buffer, pH 8.2, containing MgCl(2) and glucose, followed by washing with NaCl and MgCl(2) in Tris buffer. Electron microscopy showed that the preparation was not contaminated with cytoplasmic material. The membrane preparation was composed of 55 to 60% protein, 1.5% ribonucleic acid, 0.1% deoxyribonucleic acid, 1.3 to 2.3% carbohydrate, 0.17 to 0.38% amino sugar, 0.2 to 0.4% rhamnose, 3.5 to 4.0% phosphorus, 10.5 to 12.0% nitrogen, and 30 to 35% lipid. Amino acid composition of the washed membrane showed some variation from that of the whole cells. Sulfur-containing amino acids were not present in the membrane hydrolysate. The membrane carbohydrate contained glucose, galactose, ribose, and arabinose. The membrane lipid was 80 to 85% phospholipid and 15 to 20% neutral lipid. The lipid contained 2.3 to 3.0% phosphorus, 2.5 to 3.0% carbohydrate, and a very small amount of nitrogen (0.2 to 0.3%). The phospholipid was of the phosphatidyl glycerol type. Electron micrographs of the washed membrane showed three layers. The outer and inner layers varied in thickness from 25 to 37 A and the middle layer from 20 to 25 A. The total thickness varied between 85 and 100 A. These preparations contained many vesicles which stained heavily with lead citrate. Some vesicles were also attached to the protoplast ghosts in the form of extrusions or intrusions, or both. Membrane preparations obtained by lysis of protoplasts in the absence of MgCl(2) were fragmented and contained less lipid (20 to 22%) and ribonucleic acid (0.3 to 0.5%) than preparations prepared with MgCl(2).
Insights
This study details the isolation and biochemical characterization of Listeria monocytogenes plasma membranes. The MgCl(2)-dependent preparation yielded pure membranes rich in protein and lipids, suitable for further research.
Area of Science:
- Microbiology
- Cell Biology
- Biochemistry
Background:
- Listeria monocytogenes is a significant foodborne pathogen.
- Understanding its cell envelope structure is crucial for developing control strategies.
- Previous studies on L. monocytogenes plasma membrane composition are limited.
Purpose of the Study:
- To isolate and purify the plasma membrane of Listeria monocytogenes strain 42.
- To biochemically characterize the isolated plasma membrane.
- To investigate the role of MgCl(2) in membrane preparation.
Main Methods:
- Protoplast formation via osmotic lysis in Tris buffer (pH 8.2) with MgCl(2) and glucose.
- Washing of membranes with NaCl and MgCl(2) in Tris buffer.
- Biochemical analysis of membrane composition (protein, RNA, DNA, carbohydrate, lipid, amino acids, phosphorus, nitrogen).
- Electron microscopy for structural analysis and purity assessment.
Main Results:
- A pure plasma membrane preparation was obtained, free from cytoplasmic contamination.
- The membrane comprised 55-60% protein, 30-35% lipid, and significant amounts of phosphorus and nitrogen.
- Lipids were predominantly phospholipids (phosphatidyl glycerol type), and carbohydrates included glucose, galactose, ribose, and arabinose.
- Electron microscopy revealed a trilaminar structure (85-100 A total thickness) with vesicles.
- MgCl(2) was essential for obtaining intact membrane preparations with higher lipid and RNA content.
Conclusions:
- The described method effectively isolates pure Listeria monocytogenes plasma membranes.
- The biochemical composition provides a detailed profile of the membrane.
- The presence of MgCl(2) is critical for the integrity and yield of the plasma membrane preparation.