Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii

Summary

Gram-negative bacteria produce two spatially segregated membranes. The outer membrane is partitioned from the inner membrane by a periplasm and a peptidoglycan layer. The ability to isolate the dual bilayers of these microbes has been critical for understanding their physiology and pathogenesis.

Abstract

This method works by partitioning the envelope of Gram-negative bacteria into total, inner, and outer membrane (OM) fractions and concludes with assays to assess the purity of the bilayers. The OM has an increased overall density compared to the inner membrane, largely due to the presence of lipooligosaccharides (LOS) and lipopolysaccharides (LPS) within the outer leaflet. LOS and LPS molecules are amphipathic glycolipids that have a similar structure, which consists of a lipid-A disaccharolipid and core-oligosaccharide substituent. However, only LPS molecules are decorated with a third subunit known as the O-polysaccharide, or O-antigen. The type and amount of glycolipids present will impact an organism’s OM density. Therefore, we tested whether the membranes of bacteria with varied glycolipid content could be similarly isolated using our technique. For the LPS-producing organisms, Salmonella enterica serovar Typhimurium and Escherichia coli, the membranes were easily isolated and the LPS O-antigen moiety did not impact bilayer partitioning. Acinetobacter baumannii produces LOS molecules, which have a similar mass to O-antigen deficient LPS molecules; however, the membranes of these microbes could not initially be separated. We reasoned that the OM of A. baumannii was less dense than that of Enterobacteriaceae, so the sucrose gradient was adjusted and the membranes were isolated. The technique can therefore be adapted and modified for use with other organisms.

Introduction

Gram-negative bacteria produce two membranes that are separated by a periplasmic space and a peptidoglycan cell wall¹. The inner membrane (IM) encases the cytosol and is a symmetric bilayer of phospholipids. Peptidoglycan protects against turgor pressure and provides the bacterium with a cell shape, and is attached to the outer membrane (OM) by lipoproteins^2,3. The OM surrounds the periplasm and is predominantly asymmetric. The inner leaflet consists of phospholipids and the outer leaflet consists of glycolipids known as lipooligosaccharides (LOS) or lipopolysaccharides (LPS)

Protocol

1. General Reagents and Media Preparation for Membrane Extraction

1. Bacterial growth media: Prepare and sterilize 1 L of broth media in a thoroughly cleaned and autoclaved 2 L flask.
2. General resuspension buffer (1 M Tris Buffer pH 7.5; 50 mL): Dissolve 6.05 g of Tris base in 30 mL of H₂O. Adjust pH to 7.5 with 5 M HCl. Adjust final volume to 50 mL with ultrapure H₂O.
3. Master stock of divalent cation chelation solution (0.5 M EDTA pH 8; 100 mL): Add 18.6 g of disodium ethylene tetraacetate·2H₂O to 80 mL of H₂O. Stir vigorously and adjust pH to 8.0 with NaOH. Adjust final volume to 100 mL with....

Results

This technique provides an effective means to isolate the IMs and OMs for Gram-negative bacteria. An outline of the entire procedure is illustrated (Figure 1). In general, the normalization of cultures to an OD₆₀₀ of 0.6-0.8 in 1 L of media, or harvesting between 6.0 and 8.0 x 10¹¹ bacterial cells will ensure that the appropriate amount of membrane material is collected for subsequent separation.

Upon lysing the bacteria and ultracentrifuging.......

Discussion

This method will continue to aid researchers in understanding the role of the cell envelope in bacterial physiology and pathogenesis. Following the sequential ultracentrifugation steps a purified total, inner, and OM fraction can be obtained. These membranes can be assayed in isolation to test hypotheses related to membrane protein localization and function, transport and trafficking across the periplasm, and the composition of the individual bilayers under various environmental conditions. Biological studies exploring t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 L Centrifuge bottles, PC/PPCO, super speed, with sealing cap, Nalgene	VWR	525-0466
1 L Pyrex Media Storage Bottle with High Temperature Cap	VWR	10416-312
2,000 mL Erlenmeyer Flask, Narrow Mouth	VWR	10545-844
4-20% mini PROTEAN Precast Protein Gels, 12 well	BIORAD	4561095
4x Laemmli Sample Buffer 10 mL	BIORAD	1610747
50 mL sterile polypropylene centrifuge tubes	VWR	89049-174
7 mL Dounce Tissue Homogenizer with Two Glass Pestles	VWR	71000-518
70 mL polycarbonate bottle assembly	Beckman Coulter Life Sciences	355622
Agar Powder	VWR	A10752
B10P Benchtop pH Meter with pH Probe	VWR	89231-664
Barnstead GenPure xCAD Plus UV/UF - TOC (bench version)	ThermoFisher Scientific	50136146
Benzonase Nuclease	MilliporeSigma	70746-3
EDTA disodium salt dihydrate 99.0-101.0%, crystals, ultrapure Bioreagent Molecular biology grade, J.T. Baker	VWR	4040-01
EmulsiFlex-C3	Avestin, Inc.
Fiberlite F13-14 x 50cy Fixed Angle Rotor	ThermoFisher Scientific	75006526
Hydrochloric acid 6.0 N	VWR	BDH7204
IBI Scientific Orbital Platform Shaker	Fischer Scientific	15-453-211
LB Broth Miller	VWR	214906
Lysozyme, Egg White, Ultra Pure Grade	VWR	VWRV0063
Magnesium chloride hexahydrate	Sigma Aldrich	M2670
NADH	Sigma Aldrich	10107735001
Optima XPN-80 - IVD	Beckman Coulter Life Sciences	A99839
Pharmco Products PURE ALCOHOL 200 PROOF GL 4/CS	Fischer Scientific	NC1624582
Phenol Solution, Equilibrated with 10 mM Tris HCl, pH 8.0, 1 mM EDTA, BioReagent, for molecular biology	Sigma - Millipore	P4557
Pierce Coomassie Plus (Bradford) Assay Kit	Thermofisher	23238
Pro-Q emerald 300 Lipopolysaccharide Gel Stain Kit	Thermofisher	P20495
Proteacease -50, EDTA free	G Biosciences	786-334
Proteinase K, Molecular Biology Grade	New England Biolabs	P8107S
Sodium hydroxide ≥99.99%	VWR	AA45780-22
Sorval RC 6 Plus Centrifuge	ThermoFisher Scientific	36-101-0816
Sucrose	MilliporeSigma	SX1075-3
SW 41 Ti Swinging-Bucket Rotor	Beckman Coulter Life Sciences	331362
Tris(hydroxymethyl)aminomethane (TRIS, Trometamol) ≥99.9% (dried basis), ultrapure Bioreagent Molecular biology grade, J.T. Baker	VWR	JT4109-6
Type 45 Ti Fixed-Angle Titanium Rotor	Beckman Coulter Life Sciences	339160
Ultra-Clear Tube ,14 x 89mm	Beckman Coulter Life Sciences	344059
Vortex-Genie 2	VWR	102091-234