Gram-negative diderm bacteria are characterized by a tripartite cell envelope, composed of an inner membrane (IM) and a lipopolysaccharide (LPS)-containing outer membrane (OM), separated by an aqueous space where the peptidoglycan is embedded. LPS is a peculiar glycolipid endowed with several biological activities. The biosynthesis and transport of LPS to its final location take place in every compartment of the cell envelope. Proteins and protein machineries with different subcellular localization are involved in this process to facilitate the trafficking of LPS across subcellular compartments that differ in their physicochemical proprieties. The fractionation of bacterial cell envelopes can give information on the status of the LPS biogenesis by allowing the analysis of LPS profiles and of the localization of proteins involved in the transport. Here, we describe a standardized protocol for membrane fractionation in Escherichia coli using sucrose density gradient centrifugation that separates the IM from the OM cellular fractions. Bacterial cells are first converted into spheroplasts and lysed; then the membrane fractions are collected by ultracentrifugation and separated at high speed by exploiting the differences in membrane density. The fractions obtained are analyzed for LPS total amount and electrophoretic profile.

Membrane Fractionation by Isopycnic Sucrose Density Gradient Centrifugation for Qualitative Analysis of LPS in Escherichia coli / E.C. Cardoso Mendes Moura, A. Polissi, P. Sperandeo (METHODS IN MOLECULAR BIOLOGY). - In: Lipopolysaccharide Transport / [a cura di] P. Sperandeo. - [s.l] : Springer, 2022. - ISBN 978-1-0716-2580-4. - pp. 53-69 [10.1007/978-1-0716-2581-1_4]

Membrane Fractionation by Isopycnic Sucrose Density Gradient Centrifugation for Qualitative Analysis of LPS in Escherichia coli

E.C. Cardoso Mendes Moura
Primo
;
A. Polissi
Secondo
;
P. Sperandeo
Ultimo
2022

Abstract

Gram-negative diderm bacteria are characterized by a tripartite cell envelope, composed of an inner membrane (IM) and a lipopolysaccharide (LPS)-containing outer membrane (OM), separated by an aqueous space where the peptidoglycan is embedded. LPS is a peculiar glycolipid endowed with several biological activities. The biosynthesis and transport of LPS to its final location take place in every compartment of the cell envelope. Proteins and protein machineries with different subcellular localization are involved in this process to facilitate the trafficking of LPS across subcellular compartments that differ in their physicochemical proprieties. The fractionation of bacterial cell envelopes can give information on the status of the LPS biogenesis by allowing the analysis of LPS profiles and of the localization of proteins involved in the transport. Here, we describe a standardized protocol for membrane fractionation in Escherichia coli using sucrose density gradient centrifugation that separates the IM from the OM cellular fractions. Bacterial cells are first converted into spheroplasts and lysed; then the membrane fractions are collected by ultracentrifugation and separated at high speed by exploiting the differences in membrane density. The fractions obtained are analyzed for LPS total amount and electrophoretic profile.
LPS biogenesis; Membrane fractionation; Sucrose gradient; Tricine-SDS-PAGE; Ultracentrifugation; Bacterial Outer Membrane Proteins; Biological Transport; Cell Membrane; Centrifugation, Density Gradient; Glycolipids; Lipopolysaccharides; Peptidoglycan; Sucrose; Escherichia coli; Escherichia coli Proteins
Settore BIO/19 - Microbiologia Generale
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/939990
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