Related Experiment Video
Updated: Aug 10, 2026
Method for the Isolation of Francisella tularensis Outer Membranes
Published on: June 29, 2010
Isolation and chemical characterization of outer envelope of Leptospira pomona
Abstract:
Cells of Leptospira interrogans serotype pomona harvested from a chemically defined medium were resuspended in 0.01 M phosphate buffer of pH 7.3. Electron microscopy showed that 90 min of exposure effectively ruptured the outer envelope, freeing it from the cells as small flakes. Both zonal centrifugation in sucrose gradients and centrifugation in isopycnic KBr and CsCl gradients could be used to separate the outer envelope from the axial filaments and protoplasmic cylinders. The latter method resulting in higher yields of purified envelope with the particular protocols used. Thin sections of isolated outer envelope showed the same trilaminar structure seen in sections of intact cells. The outer layers were 1.5 nm thick and appeared as single layers of electron-dense particles. The central electron-transparent layer was 2.0-2.5 nm thick and appeared structureless. The gross chemical composition of the purified outer envelope was 47% protein, 27% carbohydrate, and 23% lipid. Colorimetric carbohydrate determinations revealed hexose, pentose, and 6-deoxyhexose; hexosamine was identified during amino acid analysis. Muramic acid, heptose, and 2-keto-3-deoxyoctonate were not detected. Thin-layer chromatography revealed only polar lipids, about 98% phosphatidylethanolamine and 2% lysophosphatidylethanolamine. Fatty acids identified by gas-liquid chromatography were octadecanoic, octadecenoic, hexadecanoic, and hexadecenoic. Amino acid analysis revealed 17 amino acids, histidine and glutamic acid being most abundant. The outer envelope was interpreted to be comparable with the outer double-track layer found in the cell covering of gram-negative eubacteria.
Insights
This study isolated and characterized the outer envelope of Leptospira interrogans serotype pomona, revealing its structure and chemical composition. The findings provide insights into the cell envelope of this important bacterial pathogen.
Area of Science:
- Microbiology
- Cell Biology
- Biochemistry
Background:
- Leptospira interrogans is a pathogenic bacterium responsible for leptospirosis.
- Understanding the structure and composition of its cell envelope is crucial for developing effective treatments and vaccines.
- Previous studies have not fully elucidated the detailed characteristics of the Leptospira outer envelope.
Purpose of the Study:
- To isolate and purify the outer envelope of Leptospira interrogans serotype pomona.
- To characterize the ultrastructure and gross chemical composition of the isolated outer envelope.
- To compare the Leptospira outer envelope with that of Gram-negative bacteria.
Main Methods:
- Cells were cultured in a chemically defined medium and harvested.
- Outer envelopes were ruptured and separated using zonal and isopycnic gradient centrifugation.
- Ultrastructure was examined by electron microscopy, and chemical composition was determined through various biochemical analyses (colorimetric, chromatography, amino acid analysis).
Main Results:
- Electron microscopy confirmed the trilaminar structure of the isolated outer envelope, similar to intact cells.
- The outer envelope consists of 47% protein, 27% carbohydrate, and 23% lipid.
- Key lipids identified were phosphatidylethanolamine and lysophosphatidylethanolamine; 17 amino acids were detected, with histidine and glutamic acid being most abundant.
Conclusions:
- The outer envelope of Leptospira interrogans serotype pomona can be successfully isolated and purified.
- Its trilaminar structure and chemical composition are comparable to the outer membrane of Gram-negative eubacteria.
- These findings contribute to a better understanding of Leptospira cell envelope biology and potential targets for antimicrobial strategies.
