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Published on: March 8, 2020
Quantitative lipid composition of cell envelopes of Corynebacterium glutamicum elucidated through reverse micelle
Ritu Bansal-Mutalik1, Hiroshi Nikaido
1Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
Abstract:
Cells of the Corynebacterium-Nocardia-Mycobacterium group of bacteria are surrounded by an outer membrane (OM) containing mycolic acids that are covalently linked to the underlying arabinogalactan-peptidoglycan complex. This OM presumably acts as a permeability barrier that imparts high levels of intrinsic drug resistance to some members of this group, such as Mycobacterium tuberculosis, and its component lipids have been studied intensively in a qualitative manner over the years. However, the quantitative lipid composition of this membrane has remained obscure, mainly because of difficulties in isolating it without contamination from the inner cytoplasmic membrane. Here we use the extraction, with reverse surfactant micelles, of intact cells of Corynebacterium glutamicum and show that this method extracts the free OM lipids quantitatively with no contamination from lipids of the cytoplasmic membrane, such as phosphatidylglycerol. Although only small amounts of corynomycolate were esterified to arabinogalactan, a large amount of cardiolipin was present in a nonextractable form, tightly associated, possibly covalently, with the peptidoglycan-arabinogalactan complex. Furthermore, we show that the OM contains just enough lipid hydrocarbons to produce a bilayer covering the cell surface, with its inner leaflet composed mainly of the aforementioned nonextractable cardiolipin and its outer leaflet composed of trehalose dimycolates, phosphatidylinositol mannosides, and highly apolar lipids, similar to the Minnikin model of 1982. The reverse micelle extraction method is also useful for extracting proteins associated with the OM, such as porins.
Insights
This study quantitatively analyzes the lipid composition of the bacterial outer membrane (OM) in Corynebacterium glutamicum. A novel extraction method reveals the OM
Area of Science:
- Microbiology
- Bacterial Cell Envelope
- Lipid Biochemistry
Background:
- The outer membrane (OM) of Corynebacterium-Nocardia-Mycobacterium bacteria contains mycolic acids, contributing to drug resistance.
- Previous studies qualitatively assessed OM lipids but lacked quantitative data due to isolation challenges.
- Understanding the OM's quantitative lipid composition is crucial for deciphering its barrier function and drug resistance mechanisms.
Purpose of the Study:
- To quantitatively determine the lipid composition of the bacterial outer membrane (OM) in Corynebacterium glutamicum.
- To develop and validate a method for isolating OM lipids without contamination from the cytoplasmic membrane.
- To elucidate the structural organization and lipid distribution within the OM bilayer.
Main Methods:
- Extraction of intact Corynebacterium glutamicum cells using reverse surfactant micelles.
- Quantitative analysis of extracted free OM lipids.
- Characterization of non-extractable lipids associated with the peptidoglycan-arabinogalactan complex.
Main Results:
- The reverse micelle method quantitatively extracted free OM lipids without cytoplasmic membrane lipid contamination.
- A significant amount of cardiolipin was found tightly associated, possibly covalently, with the peptidoglycan-arabinogalactan complex.
- The OM bilayer consists of cardiolipin in the inner leaflet and trehalose dimycolates, phosphatidylinositol mannosides, and apolar lipids in the outer leaflet.
Conclusions:
- Reverse micelle extraction is an effective method for quantitative OM lipid analysis and protein extraction.
- The study provides the first quantitative lipid composition of the bacterial OM, supporting a bilayer model.
- Findings offer insights into the structural basis of the OM's permeability barrier and drug resistance in this bacterial group.
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