Name: separation of the cell envelope for gram-negative bacteria into inner and outer membrane fractions with technical adjustments for acinetobacter baumannii
Uploaded: 2020-04-10T13:00:00
Description: Watch this Scientific Journal Video about Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii at J

This work was funded by P20GM10344 and R01AI139248 awarded to Z. D. Dalebroux.

1. General Reagents and Media Preparation for Membrane Extraction
<ol>
	<li>Bacterial growth media: Prepare and sterilize 1 L of broth media in a thoroughly cleaned and autoclaved 2 L flask.</li>
	<li>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.</li>
	<li>Master stock of divalent cation chelation solution (0.5 M EDTA pH 8; 100 mL): Add 18.6 g of disodium eth...

<ol>
	<li>Silhavy, T. J., Kahne, D., Walker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bacterial+cell+envelope.">The bacterial cell envelope.</a> Cold Spring Harbor Perspectives in Biology. 2, 000414 (2010).</li><li>Egan, A. J., Vollmer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physiology+of+bacterial+cell+division.">The physiology of bacterial cell division.</a> Annals of the New York Academy of Sciences. 1277, 8-28 (2013).</li><li>Pazos, M., Peters, K., Vollmer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+peptidoglycan+growth+by+dynamic+and+variable+multi-protein+complexes.">Robust peptidoglycan growth by dynamic and variable multi-protein complexes.</a> Current Opinion in Microbiology. 36, 55-61 (2017).</li><li>Raetz, C. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fortifying+the+barrier:+the+impact+of+lipid+A+remodelling+on+bacterial+pathogenesis.">Fortifying the barrier: the impact of lipid A remodelling on bacterial pathogenesis.</a> Nature Reviews Microbiology. 11, 467-481 (2013).</li><li>Simpson, B. W., Trent, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pushing+the+envelope:+LPS+modifications+and+their+consequences.">Pushing the envelope: LPS modifications and their consequences.</a> Nature Reviews Microbiology. 17, 403-416 (2019).</li><li>Ebbensgaard, A., Mordhorst, H., Aarestrup, F. M., Hansen, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Outer+Membrane+Proteins+and+Lipopolysaccharides+for+the+Sensitivity+of+Escherichia+coli+to+Antimicrobial+Peptides.">The Role of Outer Membrane Proteins and Lipopolysaccharides for the Sensitivity of Escherichia coli to Antimicrobial Peptides.</a> Frontiers in Microbiology. 9, 2153 (2018).</li><li>Liu, D., Reeves, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli+K12+regains+its+O+antigen.">Escherichia coli K12 regains its O antigen.</a> Microbiology. 140 (1), 49-57 (1994).</li><li>Kalynych, S., Morona, R., Cygler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+understanding+the+assembly+process+of+bacterial+O-antigen.">Progress in understanding the assembly process of bacterial O-antigen.</a> FEMS Clinical Microbiology Reviews. 38, 1048-1065 (2014).</li><li>Osborn, M. J., Gander, J. E., Parisi, E., Carson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+assembly+of+the+outer+membrane+of+Salmonella+typhimurium.+Isolation+and+characterization+of+cytoplasmic+and+outer+membrane.">Mechanism of assembly of the outer membrane of Salmonella typhimurium. Isolation and characterization of cytoplasmic and outer membrane.</a> Journal of Biological Chemistry. 247, 3962-3972 (1972).</li><li>Osborn, M. J., Munson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+the+inner+(cytoplasmic)+and+outer+membranes+of+Gram-negative+bacteria.">Separation of the inner (cytoplasmic) and outer membranes of Gram-negative bacteria.</a> Methods in Enzymology. 31, 642-653 (1974).</li><li>Cian, M. B., Giordano, N. P., Masilamani, R., Minor, K. E., Dalebroux, Z. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+enterica+serovar+Typhimurium+use+PbgA/YejM+to+regulate+lipopolysaccharide+assembly+during+bacteremia.">Salmonella enterica serovar Typhimurium use PbgA/YejM to regulate lipopolysaccharide assembly during bacteremia.</a> Infection and Immunity. , (2019).</li><li>Masilamani, R., Cian, M. B., Dalebroux, Z. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+Tol-Pal+Reduces+Outer+Membrane+Glycerophospholipid+Levels+for+Envelope+Homeostasis+and+Survival+during+Bacteremia.">Salmonella Tol-Pal Reduces Outer Membrane Glycerophospholipid Levels for Envelope Homeostasis and Survival during Bacteremia.</a> Infection and Immunity. 86, (2018).</li><li>Nikaido, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outer+Membrane+of+Salmonella-Typhimurium+Transmembrane+Diffusion+of+Some+Hydrophobic+Substances.">Outer Membrane of Salmonella-Typhimurium Transmembrane Diffusion of Some Hydrophobic Substances.</a> Biochimica Et Biophysica Acta. 433, 118-132 (1976).</li><li>Dalebroux, Z. D., Matamouros, S., Whittington, D., Bishop, R. E., Miller, S. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+analysis+of+the+outer+membrane+and+other+layers+of+the+cell+envelope+of+Acinetobacter+sp.">Chemical analysis of the outer membrane and other layers of the cell envelope of Acinetobacter sp.</a> Journal of Bacteriology. 116, 410-417 (1973).</li><li>Geisinger, E., Huo, W., Hernandez-Bird, J., Isberg, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acinetobacter+baumannii:+Envelope+Determinants+That+Control+Drug+Resistance,+Virulence,+and+Surface+Variability.">Acinetobacter baumannii: Envelope Determinants That Control Drug Resistance, Virulence, and Surface Variability.</a> Annual Review of Microbiology. 73, 481-506 (2019).</li><li>Kamischke, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Acinetobacter+baumannii+Mla+system+and+glycerophospholipid+transport+to+the+outer+membrane.">The Acinetobacter baumannii Mla system and glycerophospholipid transport to the outer membrane.</a> Elife. 8, (2019).</li></ol>

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...

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 lipoproteins2,3. 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)4,5. The lipid asymmetry and the biochemistry of the LOS/LPS molecules in the outer leaflet confer barrier properties to the cell surface that protect the bacterium against hazards in its environment6,7.
LPS molecules are comprised of three constituents: the lipid A disaccharolipid, the core oligosaccharide, and the O-polysaccharide or O-antigen. Lipid A is a multiply acylated disaccharolipid. Core-oligosaccharides consist of 10&#8211;15 sugars known as rough LPS or R-LPS. The core is subdivided into the inner region, composed of 2-keto-3-deoxy-D-manno-octulosonic acid (kdo) and one or more heptose residues, and an outer region that consists generally of hexoses (glucose or galactose) and heptoses, or acetamido sugars5. The outer core region is more variable in its components and structure than the inner core. In Salmonella spp., only one core structure has been described; however, in Escherichia coli there are five different core structures (designated K-12, R1, R2, R3, and R4)8. E. coli K-12 DH5&#945;, which we use in this procedure carries a mutation that results in production R-LPS9. The R-LPS molecules lack the O-antigen moiety and have a similar molecular weight to LOS molecules.
The addition of O-antigen to R-LPS turns this molecule into smooth LPS, or S-LPS. The O-antigens are built from short 3-4 carbohydrate subunits and consist of multiple modalities with varying chain lengths10. Some LPS-producing bacteria, like Salmonella enterica serovar Typhimurium (S. Typhimurium), display a trimodal distribution of LPS molecules on their surface10,11. Very-long chain O-antigens can contain over one hundred subunits and weigh over one hundred kilodaltons. The O-antigens provide surface properties to the bacterium that are necessary to resist antibiotics, evade predation by bacteriophages, and cause disease.
Species of Campylobacter, Bordetella, Acinetobacter, Haemophilus, Neisseria and others generate LOS molecules instead of LPS molecules on their surface12. LOS molecules consist of lipid A and core oligosaccharides but lack the O-antigen. These types of Gram-negative bacteria modify their core oligosaccharides with additional sugars and combinations of sugars to alter surface properties12. Both LOS and LPS-producing microbes derivatize the phosphates on lipid A and core molecules with cationic moieties7. These additions include phosphoethanolamine, galactosamine and aminoarabinose substitutions, which function by neutralizing anionic surface charge and thereby protecting against cationic antimicrobial peptides. Gram-negative bacteria also modify the core oligosaccharide structure with variable non-stoichiometric substitutions of sugars, or extra kdo molecules, and alter the number of acyl chains on lipid-A disaccharolipids7.
The ability to isolate the IM from the OM of Gram-negative bacteria has been instrumental for understanding the role of the cell envelope in antimicrobial resistance and disease pathogenesis11,12. Derivations of this approach have been used to deduce mechanisms of assembly, maintenance, and remodeling of the protein, phospholipid, and glycolipid constituents for the OM.
Our lab routinely performs bacterial lipidomic analyses to study protein-mediated lipid regulation and lipid function in a variety of Gram-negative species. The volumes used in the protocol reflect the routine use of this procedure to analyze non-radiolabeled phospholipids by thin layer chromatography and liquid chromatography tandem mass spectrometry13,14.
The protocol begins by exposing a chilled suspension of Gram-negative bacteria to a high osmolar solution of sucrose and adding lysozyme to dissociate the OM from the underlying peptidoglycan layer (Figure 1)12. EDTA is then added to facilitate penetration of the lysozyme, since divalent cation sequestration disrupts the lateral electrostatic bridging interactions between adjacent LOS/LPS molecules15. The original protocol from which ours has been adapted required the formation of spheroplasts, a Gram-negative bacterial cell form that consist of a plasma membrane and cytosol, but lacks the peptidoglycan layer and an OM. It is possible that spheroplasts are produced by the adapted method; however, the technique does not rely or intend on their formation for success. Instead, the lysozyme-EDTA treated bacteria are rapidly harvested by centrifugation and re-suspended in a sucrose solution of lesser concentration before pressurized lysis. The OMs that might have been released by forming spheroplasts should in theory be harvestable from the supernatants of the treated cells, but this approach is not detailed herein. Ultimately, the treated cells are subjected to conventional homogenization and lysis, which enhances the efficiency and reproducibility of the membrane separation procedure16.
After lysis, the total membranes are collected by ultracentrifugation and applied to a discontinuous sucrose density gradient to fractionate the IMs and OMs. The classical approach uses a more continuous gradient that consists of at least five different sucrose solutions11,12. The discontinuous gradient in our protocol consists of three sucrose solutions and partitions the bilayers into two distinct fractions17. The LOS and LPS molecules within the OMs of Gram-negative bacteria drive the envelope to partition into an upper brown low-density IM fraction and a lower white high-density OM fraction (Figure 1 and Figure 2).
Acinetobacter baumannii are important multidrug resistant human pathogens that produce LOS molecules in their OM and erect a cell envelope that is difficult to separate18,19. Recent work suggests that a derivation of the protocol we present here can be used to partition the bilayers of these organisms20. Therefore, we tested our protocol on A. baumannii 17978. Initially, the procedure was inadequate. However, we modified the sucrose concentration of the middle density solution and greatly improved separation (Figure 2). An NADH dehydrogenase assay and a LOS/LPS extraction and detection procedure was used to confirm separation for A. baumannii, wild-type S. Typhimurium and two O-antigen deficient enterobacterial genotypes; namely, galE-mutant S. Typhimurium and a laboratory strain, E. coli DH5&#945; (Figure 3 and Figure 4).
The intent of this work is to supply a streamlined approach for reproducibly isolating the membranes of Gram-negative bacteria. The protocol can be used to study many types of membrane-associated molecules for these microbes.

<table>
	<tbody>
		<tr>
			<td>1 L Centrifuge bottles, PC/PPCO, super speed, with sealing cap, Nalgene</td>
			<td>VWR</td>
			<td>525-0466</td>
			<td></td>
		</tr>
		<tr>
			<td>1 L Pyrex Media Storage Bottle with High Temperature Cap</td>
			<td>VWR</td>
			<td>10416-312</td>
			<td></td>
		</tr>
		<tr>
			<td>2000 mL Erlenmeyer Flask, Narrow Mouth</td>
			<td>VWR</td>
			<td>10545-844</td>
			<td></td>
		</tr>
		<tr>
			<td>4-20% mini PROTEAN Precast Protein Gels, 12 well</td>
			<td>BIORAD</td>
			<td>4561095</td>
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		<tr>
			<td>4x Laemmli Sample Buffer 10 mL</td>
			<td>BIORAD</td>
			<td>1610747</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL sterile polypropylene centrifuge tubes</td>
			<td>VWR</td>
			<td>89049-174</td>
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			<td>7 mL Dounce Tissue Homogenizer with Two Glass Pestles</td>
			<td>VWR</td>
			<td>71000-518</td>
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			<td>70 mL polycarbonate bottle assembly</td>
			<td>Beckman Coulter Life Sciences</td>
			<td>355622</td>
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			<td>VWR</td>
			<td>A10752</td>
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			<td>B10P Benchtop pH Meter with pH Probe</td>
			<td>VWR</td>
			<td>89231-664</td>
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			<td>Barnstead GenPure xCAD Plus UV/UF - TOC (bench version)</td>
			<td>ThermoFisher Scientific</td>
			<td>50136146</td>
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			<td>Benzonase Nuclease</td>
			<td>MilliporeSigma</td>
			<td>70746-3</td>
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		<tr>
			<td>EDTA disodium salt dihydrate 99.0-101.0%, crystals, ultrapure Bioreagent Molecular biology grade, J.T. Baker</td>
			<td>VWR</td>
			<td>4040-01</td>
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			<td>EmulsiFlex-C3</td>
			<td>Avestin, Inc.</td>
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			<td>Fiberlite F13-14 x 50cy Fixed Angle Rotor</td>
			<td>ThermoFisher Scientific</td>
			<td>75006526</td>
			<td></td>
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			<td>Hydrochloric acid 6.0 N</td>
			<td>VWR</td>
			<td>BDH7204</td>
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			<td>IBI Scientific Orbital Platform Shaker</td>
			<td>Fischer Scientific</td>
			<td>15-453-211</td>
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			<td>LB Broth Miller</td>
			<td>VWR</td>
			<td>214906</td>
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			<td>Lysozyme, Egg White, Ultra Pure Grade</td>
			<td>VWR</td>
			<td>VWRV0063</td>
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			<td>Magnesium chloride hexahydrate</td>
			<td>Sigma Aldrich</td>
			<td>M2670</td>
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			<td>NADH</td>
			<td>Sigma Aldrich</td>
			<td>10107735001</td>
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			<td>Optima XPN-80 - IVD</td>
			<td>Beckman Coulter Life Sciences</td>
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			<td>Phenol Solution, Equilibrated with 10 mM Tris HCl, pH 8.0, 1 mM EDTA, BioReagent, for molecular biology</td>
			<td>Sigma - Millipore</td>
			<td>P4557</td>
			<td></td>
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		<tr>
			<td>Pierce Coomassie Plus (Bradford) Assay Kit</td>
			<td>Thermofisher</td>
			<td>23238</td>
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			<td>Pro-Q emerald 300 Lipopolysaccharide Gel Stain Kit</td>
			<td>Thermofisher</td>
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			<td>Proteacease -50, EDTA free</td>
			<td>G Biosciences</td>
			<td>786-334</td>
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			<td>Proteinase K, Molecular Biology Grade</td>
			<td>New England Biolabs</td>
			<td>P8107S</td>
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			<td>Sodium hydroxide &#8805;99.99%</td>
			<td>VWR</td>
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			<td>Sorval RC 6 Plus Centrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>36-101-0816</td>
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		<tr>
			<td>Sucrose</td>
			<td>MilliporeSigma</td>
			<td>SX1075-3</td>
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			<td>SW 41 Ti Swinging-Bucket Rotor</td>
			<td>Beckman Coulter Life Sciences</td>
			<td>331362</td>
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			<td>Tris(hydroxymethyl)aminomethane (TRIS, Trometamol) &#8805;99.9% (dried basis), ultrapure Bioreagent Molecular biology grade, J.T. Baker</td>
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			<td>JT4109-6</td>
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			<td>Type 45 Ti Fixed-Angle Titanium Rotor</td>
			<td>Beckman Coulter Life Sciences</td>
			<td>339160</td>
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			<td>Ultra-Clear Tube ,14 x 89mm</td>
			<td>Beckman Coulter Life Sciences</td>
			<td>344059</td>
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			<td>Vortex-Genie 2</td>
			<td>VWR</td>
			<td>102091-234</td>
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	</tbody>
</table>

separation of the cell envelope for gram-negative bacteria into inner and outer membrane fractions with technical adjustments for acinetobacter baumannii

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&#8217;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.

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...

Watch this Scientific Journal Video about Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii at J

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

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.

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

This method works by partitioning the envelope of Gram-negative bacteria into total, inner, and outer membrane (OM) fractions and concludes with assays to ...

Studying how microbes regulate the chemical composition of the individual bilayers will inform our knowledge of mechanisms of antibiotic killing, antimicrobial resistance and disease pathogenesis. This technique partitions the cell envelope of gram-negative bacteria into two defined fractions without using detergent. Therefore, the lipids, proteins and sugars can be assessed in an environment that is semi-native.Some of the steps are time dependent and require focus and coordination. Initially, use one or two samples. When one's comfort level increases, more samples can be added.No more than six samples should be handled at once. This is a multi-day procedure and visual checkpoints are helpful to assess efficacy and error. Prior to performing this sometimes final steps.Streak the bacteria from frozen glycerol stocks onto fresh agar plates. Once colonies develop, store the plates at four degrees Celsius. Use a single colony to inoculate a five milliliter tube filled with broth media and culture the bacteria as desired overnight.Back dilute the overnight bacterial culture into one liter of the preferred broth media and incubate the flasks. When the desired optical density has been achieved, remove the flasks from the incubator and place them on ice. Measure the optical density of the culture at 600 nanometers and calculate the volume of culture that is equivalent to between six and eight times 10 to the eleventh bacterial colony forming units.Add this volume of culture to a centrifuge tube and ensure that the remaining cultures stay on ice until they are to be used. Pellet the bacteria by centrifugation for 10 minutes ina a fixed angle, high speed centrifuge at 7, 000 to 10, 000 times G and four degrees Celsius. Carefully decant the supernatant and discard it.Resuspend each cell pellet in 12.5 milliliters of buffer A.Add a magnetic stir bar to the cell suspension. Add 180 microliters of 10 milligrams per milliliter lysozyme to each cell resuspension. After collecting the lysozyme EDTA treated bacteria by centrifugation, quickly add the protease inhibitor, magnesium chloride and nuclease to the bacteria and rapidly resuspend the cells in buffer B.Stir for two minutes, keeping the suspensions on ice.Next, add 12.5 milliliters of 1.5 millimolar EDTA solution to each cell re-suspension and continue stirring on ice for an additional seven minutes. Decant the suspensions into 15 milliliter conical tubes. Centrifuge the suspensions at 9, 000 to 11, 000 times G for 10 minutes at four degrees Celsius.Discard the supernatants into a biohazard waste container and retain the pellets on ice. Add 25 milliliters of buffer B to each cell pellet. Then, add 55 microliters of one molar magnesium chloride, one microliter of rnase/dnase/nuclease reagent and one microliter of protease inhibitor cocktail.Resuspend the pellets in the mixture. Vigorously pipette and vortex until the solutions appear homogenous. Store the suspensions on ice.Pour the sample into the homogenizer sample cylinder and bring the cell to 20, 000 PSI. To avoid aerosolization of pathogens, condition the pressurized apparatus in buffer B and adjust the pressure levels before adding bacterial suspension. Engage the pressure cell and allude 10 milliliters of buffer B as a precaution.Collect the cell lysate in 50 milliliter conical tubes on ice while also keeping the samples on ice. To achieve complete lysis, repeat this process three to five times. To pellet the remaining, intact, cell material, centrifuge the lysed bacterial samples at 6, 169 times G and four degrees Celsius for 10 minutes.Distribute the supernatants which now contain the homogenized membranes, into polycarbonate bottles for ultracentrifugation. Ultracentrifuge the cell lysates at 184, 500 times G and four degrees Celsius for at least one hour. Discard the supernatants and retain the membrane pellets on ice.Using a glass, Dounce homogenizer, resuspend the membrane pellets in one milliliter of the low-density isopycnic sucrose gradient solution. Then, use a glass, Pasteur pipette to transfer the sample homogenate to a 1.5 milliliter microcentrifuge tube and place the tube on ice. To prepare the sucrose gradient, hold a polypropylene or Ultra-clear Open-Top tube in a slightly tilted position.Slowly add two milliliters of the higher density sucrose solution. Then add four milliliters of the lower density sucrose solution. When preparing the density gradient, add the sucrose solution slowly to avoid mixing the layers.The sucrose layers should be slightly visible and appear generally defined. Next, add the total membrane fraction which was previously resuspended in one milliliter of low density, isopycnic sucrose solution. Finally, fill the remainder of the tube by adding approximately six milliliters of the low density, isopycnic sucrose gradient solution.Ultracentrifuge the samples using a swinging bucket rotor at 288, 000 times G and four degrees Celsius for 16 to 23 hours. Cut the end off a P1000 pipette tip about five milliliters from the point. After centrifugation is complete, use the pipette to remove the upper, brown layer from the tube.Transfer this layer, which contains the inner membrane fraction, to a polycarbonate bottle for ultracentrifugation. Leave about two milliliters of the sucrose solution above the interface between the 53%sucrose and the 73%sucrose to ensure that the lower, white, outer membrane fraction does not cross contaminate the inner membrane fraction. Again using a P1000 pipette tip with the end removed, remove the outer membrane layer and transfer it to a polycarbonate bottle.Fill the remaining void of polycarbonate bottle with isolated membrane storage buffer and mix by inversion or pippetting. Retain the samples on ice. To collect the membranes, ultracentrifuge the polycarbonate bottles at 184, 500 times G and four degrees Celsius for one hour.Finally, discard the supernatants and add 500 to 1, 000 microliters of storage buffer. Resuspend the membranes by Dounce homogenization. Collect the samples in two milliliter microcentrifuge tubes.Total membrane pellets were scraped, Dounce homogenized and ultracentrifuged over sucrose density gradients to separate the inner and outer membranes. A sucrose density gradient of 20%53%73%was sufficient for partitioning some bacterial strains. But not for partitioning A.baumannii which was successfully partitioned using a 20%45%73%gradient.To assess the efficiency of the separation, membrane samples were tested for NADH dehydrogenase, an enzyme located in the bacterial inner membrane. A decrease in optical density at 340 nanometers indicated the presence of the enzyme. Optical density was measured for 50 microgram samples of inner and outer membrane fractions from wild type S.typhimurium, E.coli K12 DH5a and galE mutant S.typhimurium.A higher concentration of protein was added when assaying the purity of A.baumannii membrane fractions because an initial assay using 50 micrograms suggested that the relative levels of NADH dehydrogenase were lower for A.baumannii than for the other bacterial strains. To assess cross contamination of the inner membrane fractions with outer membrane fractions, LPS and LOS were extracted and visualized. The extracts were loaded onto a polyacrylamide gel and stained with Pro-Q Emerald 300.Acinetobacter baumannii is a top priority, multi drug resistant, human pathogen. This protocol should aid researchers in understanding the roles of lipooligosaccharides, phospholipids and lipoproteins in the resistance and virulence mechanisms of this unique and important pathogen. This method is ideal for downstream analysis of phospholipids, glycolipids carbohydrates, proteins and small molecules that typically exist within the dual membranes of gram-negative bacteria.

Research

JoVE Journal

Immunology and Infection

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