Name: Method for the Isolation of Francisella tularensis Outer Membranes (Video) | JoVE Journal
Uploaded: 2010-06-29T17:10:00.000+00:00
Duration: 11 min 6 s
Description: Learn to isolate Francisella tularensis outer membrane vesicles using osmotic lysis and sucrose density gradient ultracentrifugation. This method enables functional studies of outer membrane proteins and bacterial pathogenesis.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

The project described was supported by Grant Numbers P01 AI055637 and U54 AI057156 from NIAID/NIH. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the RCE Programs Office, NIAID, or NIH.

Day 1: F. tularensis plate inoculation <ol> <li>Plate frozen stock cultures of F. tularensis onto Mueller-Hinton agar supplemented with 2.5% (vol/vol) donor calf serum, 2% (vol/vol) IsoVitaleX, 0.1% (wt/vol) glucose, and 0.025% (wt/vol) iron pyrophosphate. Grow F. tularensis at 37°C with 5% CO2 for approximately 24 h.</li> </ol> Day 2: F. tularensis liquid media inoculation <ol> <li>Prepare and autoclave 1 liter of cation-adjusted Mueller-Hinton medium (containing 1.23 mM calcium chloride dihydrate and 1.03 mM magnesium chloride hexahydrate). When cooled to 37°C, further supplement medium with 0.1% (wt/vol) glucose, 0.025% (wt/vol) iron pyrophosphate, and 2% (vol/vol) IsoVitaleX. Filter sterilize (0.22 μ) medium into two 500-ml aliquots.</li> <li>Remove 12 ml of supplemented Mueller-Hinton medium and transfer into a sterile 50-ml Falcon tube.</li> <li>Using a sterile 10-μl inoculation loop, scrape a large loopful of F. tularensis growth from agar plates (from Day 1) and transfer bacteria into 12 ml of Mueller-Hinton medium (see Step 2). Pipette solution multiple times to break-up clumps and prepare homogenous bacterial suspension. Do not vortex.</li> <li>Using a sterile pipette, inoculate each 500-ml vessel with 5 ml of bacterial suspension from Step 3. Grow broth cultures at 37°C for 14 to 18 h with gentle shaking (190-200 rpm on a New Brunswick Innova 2300 series shaker).</li> </ol> Day 3: Spheroplasting, osmotic lysis, and sucrose density gradient centrifugation <ol> <li>Obtain F. tularensis cultures from the shaking incubator and remove a 1-ml aliquot from each 500-ml culture to check the respective optical densities. We have found optimum membrane extraction and isolation results when using F. tularensis cells in early logarithmic phase of growth, which correlates with an OD600 between 0.2 to 0.4 (~107 to 108 CFU/ml). Cultures with an OD600 less than 0.2 will not yield sufficient quantity of total membrane material for subsequent sucrose gradients. Conversely, cultures with an OD600 greater than 0.4 (nearing stationary phase) have been found to yield mixed-membrane fusions.</li> <li>Centrifuge cultures at 7,500 x g for 30 min at 15°C to collect the cells. We recommend centrifugation of cultures in four 250-ml centrifuge bottles, because it facilitates subsequent pellet suspension.</li> <li>Carefully remove the media supernatant from each centrifuge bottle and firmly tap the bottles on absorbent material to remove excess growth medium.</li> <li>Within 10 min following completion of centrifugation, suspend each bacterial pellet in 8.75 ml of 0.75 M sucrose (in 5 mM Tris, pH 7.5). All four bacterial pellets should be suspended and transferred (35 ml total of bacterial suspension) to a sterile 250-ml flask (with a small stirbar) within 10 min.</li> <li>While gently mixing the cell suspension on a stirplate, slowly add 70 ml (2 volumes) of 10 mM disodium EDTA (in 5 mM Tris, pH 7.8) over the course of 10 min. Add the EDTA solution with the tip of pipette below the cell suspension level to avoid elevated local concentrations of EDTA. The stirbar should be rotating at a rate sufficient to thoroughly mix the cell suspension but not fast enough to cause frothing or bubble formation. After the EDTA solution has been added, incubate the solution for 30 min at room temperature.</li> <li>After the 30 min incubation, slowly add 11 ml (1/10th volume) of a 2 mg/ml lysozyme solution to a final concentration of 200 μg/ml. Continue to mix the cell suspension during the lysozyme solution addition. As noted above, add the lysozyme solution with the pipette tip below the cell suspension fluid level to avoid elevated local concentrations of lysozyme. Stock lysozyme solutions (2 mg/ml) can be prepared in single-use aliquots and stored at -20°C for three to four months. Incubate the resulting solution for 30 min at room temperature.</li> <li>During the 30 min incubation, prepare a sterile 1L flask with a small stirbar and 530 ml of room temperature Cellgro Molecular Grade dH2O (4.5 volumes).</li> <li>Osmotically lyse the cell suspension (from Step 6) by slow dilution (flow rate of approximately 11 ml/min) into 530 ml of Molecular Grade dH2O (from Step 7) over the course of 10 to 15 min with gentle mixing. Because of the larger solution volume in this step, increase the stirbar speed to ensure proper mixing. The stirbar should be rotating at a rate sufficient to thoroughly disperse the cell suspension once added to the dH2O but not fast enough to lead to frothing or bubble formation. Add the cell suspension with the tip of pipette resting on the bottom of the flask, adjacent to the stirbar, to facilitate uniform dilution of the cell suspension. We have found that 25-ml pipettes work best during this step. Carefully monitor the dilution process and interrupt the flow rate as necessary to disperse local accumulations of cells, cell clumps, and cell wisps. Incubate the resulting solution for 30 min at room temperature. Note that this step appears to be one of the most challenging and critical for overall success of the experiment.</li> <li>After the 30 min incubation, transfer 40 ml aliquots of the osmotic lysis solution into 50-ml conical tubes and centrifuge at 7500 x g for 30 min at 10°C to remove intact cells and debris.</li> <li>Following centrifugation, carefully remove 27 to 30 ml of supernatant from each conical tube and pool supernatants in a sterile 1L flask</li><li>Transfer 25 ml of pooled supernatant, each, into 16 ultracentrifuge tubes and centrifuge at 200,000 x g (44,400 rpm in F50L-8x39 FiberLite ultracentrifuge rotor) for 2 h at 4°C. Note that each rotor holds 8 tubes, so this step requires two rotors and two ultracentrifuges. Alternatively, the second set of 8 tubes should be held at 4°C until centrifuged.</li> <li>Within 10 min of the completion of the ultracentrifuge run, prepare modified membrane resuspension buffer. Transfer 8 ml of membrane suspension buffer (25% [wt/wt] sucrose, 5 mM Tris, 30 mM MgCl2) to a sterile tube and add 1 tablet of EDTA-free protease inhibitor cocktail and 375 U of Benzonase. Gently mix solution to dissolve protease inhibitor tablet.</li> <li>Following ultracentrifugation, carefully pour off supernatants, invert ultracentrifuge tubes on absorbent material, and firmly tap tubes to remove excess supernatant. Mark the location of each membrane pellet with a marker to aid in visualization. Gently resuspend eight membrane pellets with 600 μl each of modified membrane resuspension buffer. Transfer each membrane resuspension to the next set of eight tubes, gently resuspend these eight pellets, and pool the resulting membrane resuspensions in a sterile tube. The pelleted membranes are difficult to resuspend, so continue passing the modified membrane resuspension buffer over the pellet until it peels away from the wall of the tube. It is often necessary to use the micropipette tip to scrape the membrane pellet off the tube wall and aid in the resuspension process. When pipetting, avoid frothing or bubble formation. Note that this step appears to be one of the most challenging and critical for overall success of the experiment.</li> <li>When the membrane pellets have been resuspended and removed from all 16 tubes, wash every four tubes with 600 μl of modified membrane resuspension buffer. The wash should be focused around the marked area (membrane pellet) of each tube. Transfer the wash into the membrane resuspension tube. Repeat the wash step for the remaining three sets of four tubes.</li> <li>Incubate the membrane resuspension with gentle rocking for 30 min at room temperature to degrade DNA and aid in reduction of flocculent material.</li> <li>Remove a small aliquot of the membrane suspension and perform protein quantification to determine total membrane yield. We typically prepare undiluted, 1:1 diluted (in 2.5% SDS), and 1:3 diluted (in 2.5% SDS) membrane samples, heat the samples at 90°C for 10 min, and use the BioRad DC Protein Assay to quantify the membrane resuspension protein concentration. Total protein yield is generally between 1.0 mg/ml and 1.6 mg/ml. The use of heat and SDS are required to release OMPs from the extracted membranes so that a more accurate protein quantification value can be obtained.</li> <li>Prepare linear sucrose gradients by layering 1.8 ml each of sucrose solutions (wt/wt, prepared in 5 mM EDTA, pH 7.5) into 14 x 95 mm Ultra-Clear ultracentrifuge tubes in the following order: 55%, 50%, 45%, 40%, 35%, 30%. Layering of sucrose gradients is easiest to perform when using a bulb pipettor, not an automated pipettor.</li> <li>Based on the membrane resuspension protein quantification (Step 16), layer 1.5 mg of membrane resuspension on top of each gradient. Each 14 x 95 mm ultracentrifuge tube has a maximum capacity of 12.5 ml and, given that 10.8 ml is accounted for by the sucrose solutions from Step 17, you should not load more than 1.7 ml of membrane resuspension per gradient. Individually weigh and carefully adjust the final weight of each sucrose gradient tube (by adding or removing membrane resuspension) so that all tubes weigh the same (± 0.01 g).</li> <li>Transfer sucrose gradients into a SW-40Ti swinging bucket rotor and centrifuge at 256,000 x g (38,000 rpm) for a minimum of 17 h at 4°C.</li> </ol> Day 4: Collection of sucrose gradient fractions <ol> <li>After 17 h of centrifugation, stop the centrifuge run, and carefully remove sucrose gradients from the SW-40Ti rotor.</li> <li>Carefully clean the bottom of the sucrose gradient tube with 70% ethanol, puncture the bottom of the tube with a 21g needle, and collect sequential 500 μl fractions from the gradient into sterile microfuge tubes. Repeat for subsequent gradients. The gradient should be allowed to drip by gravity flow, so as not to disturb the sucrose gradient. However, if the dripping ceases from the bottom of the gradient tube, a small amount of pressure may be applied to the top of the tube using one's finger.</li> <li>Determine the refractive index of each sucrose gradient fraction using a refractometer and correlate the refractive index with a specific density in g/ml 1. Assess protein localization by preparing representative sucrose gradient fractions (density increments of 0.01; from 1.11 g/ml to 1.26 g/ml) for SDS-PAGE separation, transfer to nitrocellulose, and immunoblotting.</li> </ol> Representative Results When performed correctly, this procedure results in the complete separation of IM and OM vesicles from both Type A (e.g. SchuS4) and Type B (e.g. LVS) strains of F. tularensis. As shown in representative immunoblots in Figure 1, F. tularensis OMPs localize between densities of 1.17 and 1.20 g/ml. By comparison, IMPs localize between densities of 1.13 and 1.14 g/ml. <img src="/files/ftp_upload/2044/2044fig1.jpg" alt="Protein electrophoresis results; OMP FopA, OMP Mip, IMP SecY bands; molecular weight markers." data-altupdatedat="1788235205000" data-alt="Figure 1"> Figure 1. Immunoblotting of F. tularensis sucrose density gradients. Sequential fractions were collected from gradients and densities (g/ml) were calculated based upon refractive indices. Proteins were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted to detect OMP and IMP fractionation. mw, prestained molecular weight standards with sizes (in kDa) noted on the left side of each blot. LVS, whole cell lysates of F. tularensis LVS. The corresponding sucrose gradient fraction densities are noted above their respective lanes. OMPs and IMPs, as noted in the left margin, were detected with polyclonal, monospecific antisera. Similar results were obtained for F. tularensis SchuS4.

<ol><li>Price, C. A. Centrifugation in density gradients. , Academic Press. 335-335 (1982).</li>
<li>Osborn, M. J., Gander, J. E., Parisi, E., Carson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanism+of+assembly+of+the+outer+membrane+of+Salmonella+typhimurium.+Isolation+and+characterization+of+cytoplasmic+and+outer+membrane%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Mechanism of assembly of the outer membrane of Salmonella typhimurium. Isolation and characterization of cytoplasmic and outer membrane.</a> J Biol Chem. 247 (12), 3962-3962 (1972).</li>
<li>Nikaido, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+outer+membranes%5BTitle%5D)+AND+%22Methods+Enzymol%22%5BJournal%5D)">Isolation of outer membranes.</a> Methods Enzymol. 235, 225-225 (1994).</li>
<li>Mizuno, T., Kageyama, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Separation+and+characterization+of+the+outer+membrane+of+Pseudomonas+aeruginosa%5BTitle%5D)+AND+%22J+Biochem%22%5BJournal%5D)">Separation and characterization of the outer membrane of Pseudomonas aeruginosa.</a> J Biochem. 84 (1), 179-179 (1978).</li>
<li>Huntley, J. F., Conley, P. G., Hagman, K. E., Norgard, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+Francisella+tularensis+outer+membrane+proteins%5BTitle%5D)+AND+%22J+Bacteriol%22%5BJournal%5D)">Characterization of Francisella tularensis outer membrane proteins.</a> J Bacteriol. 189, 561-561 (2007).</li>
<li>Huntley, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Native+outer+membrane+proteins+protect+mice+against+pulmonary+challenge+with+virulent+type+A+Francisella+tularensis%5BTitle%5D)+AND+%22Infect+Immun%22%5BJournal%5D)">Native outer membrane proteins protect mice against pulmonary challenge with virulent type A Francisella tularensis.</a> Infect Immun. 76 (8), 3664-3664 (2008).</li>
<li>Bevanger, L., Maeland, J. A., Naess, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Agglutinins+and+antibodies+to+Francisella+tularensis+outer+membrane+antigens+in+the+early+diagnosis+of+disease+during+an+outbreak+of+tularemia%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Agglutinins and antibodies to Francisella tularensis outer membrane antigens in the early diagnosis of disease during an outbreak of tularemia.</a> J Clin Microbiol. 26 (3), 433-433 (1988).</li>
<li>Hubalek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Towards+proteome+database+of+Francisella+tularensis%5BTitle%5D)+AND+%22J+Chromatogr+B+Analyt+Technol+Biomed+Life+Sci%22%5BJournal%5D)">Towards proteome database of Francisella tularensis.</a> J Chromatogr B Analyt Technol Biomed Life Sci. 787 (1), 149-149 (2003).</li>
<li>Pavkova, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Francisella+tularensis+live+vaccine+strain%3A+proteomic+analysis+of+membrane+proteins+enriched+fraction%5BTitle%5D)+AND+%22Proteomics%22%5BJournal%5D)">Francisella tularensis live vaccine strain: proteomic analysis of membrane proteins enriched fraction.</a> Proteomics. 5 (9), 2460-2460 (2005).</li>
<li>Sandstrom, G., Tarnvik, A., Wolf-Watz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunospecific+T-lymphocyte+stimulation+by+membrane+proteins+from+Francisella+tularensis%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Immunospecific T-lymphocyte stimulation by membrane proteins from Francisella tularensis.</a> J Clin Microbiol. 25 (4), 641-641 (1987).</li>
<li>Sjostedt, A., Tarnvik, A., Sandstrom, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+T-cell-stimulating+17-kilodalton+protein+of+Francisella+tularensis+LVS+is+a+lipoprotein%5BTitle%5D)+AND+%22Infect+Immun%22%5BJournal%5D)">The T-cell-stimulating 17-kilodalton protein of Francisella tularensis LVS is a lipoprotein.</a> Infect Immun. 59 (9), 3163-3163 (1991).</li>
<li>Twine, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Francisella+tularensis+proteome%3A+low+levels+of+ASB-14+facilitate+the+visualization+of+membrane+proteins+in+total+protein+extracts%5BTitle%5D)+AND+%22J+Proteome+Res%22%5BJournal%5D)">Francisella tularensis proteome: low levels of ASB-14 facilitate the visualization of membrane proteins in total protein extracts.</a> J Proteome Res. 4 (5), 1848-1848 (2005).</li>
</ol>

No conflicts of interest declared.

This protocol describes a variation of the spheroplasting, osmotic lysis, and sucrose density gradient centrifugation "gold standard" for the physical separation and enrichment of Gram-negative bacterial OMs from other cellular components 2, 3, 4. Whereas we previously described the application of this method for isolating OMs from both Type A and Type B strains of F. tularensis 5, this presentation offers a more detailed and optimized procedure for OM isolation. Indeed, this is an important advance for identifying potentially surface-exposed and presumptive virulence factors from this deadly pathogen. The significance of this procedure was demonstrated in studies by our lab showing that immunization of mice with purified OMPs afforded substantial protection against virulent Type A F. tularensis pulmonary challenge 6. As noted above, growth phase of the bacterial cells, careful monitoring during osmotic lysis, and gentle resuspension of membrane pellets are critical steps that appear to correlate with success/failure of this membrane separation procedure. While this optimized method is rigorous and subject to some experimental variability, the resulting OMs appear to be of remarkably enhanced purity as compared to those garnered from the use of detergents, lithium chloride, or sodium carbonate 7, 8, 9, 10, 11, 12.

<table><tbody><tr><td>Donor calf serum</td><td>Mediatech, Inc.</td><td>35-022-CV</td><td></td></tr><tr><td>IsoVitaleX</td><td>BD Biosciences</td><td>211876</td><td></td></tr><tr><td>Cellgro molecular grade dH&lt;sub&gt;2&lt;/sub&gt;O</td><td>Mediatech, Inc.</td><td>46-000-CM</td><td></td></tr><tr><td>38.5 ml polycarbonate bottle</td><td>FiberLite</td><td>010-0514</td><td></td></tr><tr><td>F50L-8x39 ultracentrifuge rotor</td><td>FiberLite</td><td>096-087051</td><td></td></tr><tr><td>Complete mini EDTA-free protease inhibitor cocktail</td><td>Roche Group</td><td>04693132001</td><td></td></tr><tr><td>Benzonase</td><td>Novagen, EMD Millipore</td><td>70664-3</td><td></td></tr><tr><td>DC protein assay</td><td>Bio-Rad</td><td>500-0116</td><td></td></tr><tr><td>Ultra-Clear 14x95mm centrifuge tubes</td><td>Beckman Coulter Inc.</td><td>344060</td><td></td></tr><tr><td>SW-40Ti swinging bucket rotor</td><td>Beckman Coulter Inc.</td><td>331301</td><td></td></tr><tr><td>Abbe benchtop refractometer</td><td>Fisher Scientific</td><td>13-964-10</td><td></td></tr></tbody></table>

method for the isolation of francisella tularensis outer membranes

Method for the Isolation of Francisella tularensis Outer Membranes

Francisella tularensis is a Gram-negative intracellular coccobacillus and the causative agent of the zoonotic disease tularemia. When compared with other bacterial pathogens, the extremely low infectious dose (&lt;10 CFU), rapid disease progression, and high morbidity and mortality rates suggest that the virulent strains of Francisella encode for novel virulence factors. Surface-exposed molecules, namely outer membrane proteins (OMPs), have been shown to promote bacterial host cell binding, entry, intracellular survival, virulence and immune evasion. The relevance for studying OMPs is further underscored by the fact that they can serve as protective vaccines against a number of bacterial diseases. Whereas OMPs can be extracted from gram-negative bacteria through bulk membrane extraction techniques, including sonication of cells followed by centrifugation and/or detergent extraction, these preparations are often contaminated with periplasmic and/or cytoplasmic (inner) membrane (IM) contaminants. For years, the "gold standard" method for the biochemical and biophysical separation of gram-negative IM and outer membranes (OM) has been to subject bacteria to spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation. Once layered on a sucrose gradient, OMs can be separated from IMs based on the differences in buoyant densities, believed to be predicated largely on the presence of lipopolysaccharide (LPS) in the OM. Here, we describe a rigorous and optimized method to extract, enrich, and isolate F. tularensis outer membranes and their associated OMPs.

A protocol for separating inner and outer membranes from Francisella tularensis by spheroplasting, osmotic lysis, and sucrose density gradient ultracentrifugation.

Francisella tularensis is a Gram-negative intracellular coccobacillus and the causative agent of the zoonotic disease tularemia. When compared with other ...

Isolation of Francis Cellis outer membrane vesicles and their associated outer membrane proteins starts with osmotic lysis of bacterial cells. Logarithmic phase bacteria are sequentially treated with hypertonic sucrose, EDTA and lysozyme solutions before being slowly diluted into deionized water. The resulting bacteria lysates are subjected to low speed centrifugation, which removes cellular debris and unbroken cells, followed by high speed centrifugation to pellet membrane vesicles.Next, total membrane vesicles are layered onto sucrose density gradients in order to separate inner membrane or IM and outer membrane or om vesicles. Om vesicles can be separated from IM vesicles based on differences in buoyant densities believed to be predicated largely on the presence of lipopolysaccharide or LPS in the om immuno blotting of sequential fractions from sucrose density gradients show spatial separation of inner membrane proteins from outer membrane proteins. Hi, I'm Dr.Michael Norgaard, and this is Dr.Jason Huntley from the Department of Microbiology at UT Southwestern Medical Center at Dallas.Today, Dr.Huntley will show you a procedure for extracting, enriching and isolating France cellis outer membranes and their associated membrane proteins. We use this procedure in our laboratory to study the outer membrane localized and potentially surface exposed molecules for this intracellular bacterial pathogen. We have a busy day, so let's get started.To begin this procedure, prepare and grow two 500 milliliter cultures of FCIs bacteria as described in the accompanying written protocol. Retrieve the bacterial cultures from the shaking incubator and remove a one milliliter Eloqua from each culture for OD measurement F to lorenza cells in early logarithmic phase of growth, correlating with an OD 600 between 0.2 and 0.4. Give optimal membrane extraction and isolation results when the cultures reach the desired OD 600.Use four 250 milliliter centrifuge bottles to centrifuge the cultures at 7, 500 Gs for 30 minutes at 15 degrees Celsius in order to collect the cells. After centrifugation, carefully remove the media supernatant from each centrifuge bottle and firmly tap the bottles on absorbent material to remove excess growth medium. Within 10 minutes of completing the centrifugation, suspend each bacterial pellet in 8.75 milliliters of 0.75 molar sucrose in five millimolar triss pH 7.5.After suspension of each pellet, transfer the solution to a sterile 250 milliliter flask. Holding a small stir bar. Repeat for all four bacterial pellets with a final bacterial pellet suspension totaling 35 milliliters Thoroughly mix the cell suspension on a star plate, but not too fast.In order to avoid frothing or bubbles while mixing slowly, add 70 milliliters of 10 millimolar EDTA interests to the solution over the course of 10 minutes. Hold the tip of the pipette below the cell suspension level to avoid elevated local concentrations of EDTA. After adding the EDTA, incubate the solution for 30 minutes at room temperature.Then while still mixing slowly, add 11 milliliters of a two milligram per milliliter lysozyme solution to a final concentration of 200 micrograms per milliliter. Continue to mix the cell suspension during the lysozyme solution addition. As before, hold the tip below the cell suspension level.Incubate the cells with the lysozyme for 30 minutes at room temperature. During this incubation, prepare a sterile one liter flask with a small stir bar and 530 milliliters of room temperature. Cell grow molecular grade deionized water.When the cells have finished incubating osmotically lysed the cell suspension using a 25 milliliter pipette, resting on the bottom of the flask adjacent to the stir bar, slowly dilute the cells into the water in the one liter flask over the course of 10 to 15 minutes with gentle mixing. This step is challenging and is critical for overall success of the experiment. Stirring should be fast enough to thoroughly disperse the cell suspension, but not too fast leading to frothing or bubble formation as needed.Slowly increase the stir bar speed to ensure proper mixing. Carefully monitor the dilution process and adjust the flow rate or rotate the flask as necessary to disperse local accumulations of cells, cell clumps, and cell wisps. Once all the cells have been added, incubate the mix for 30 minutes at room temperature at the end of the incubation transfer 40 milliliter aliquots of the osmotic lysis solution into 50 milliliter conical tubes and centrifuge at 7, 500 Gs for 30 minutes at 10 degrees Celsius.To remove intact cells and debris following centrifugation carefully remove 27 to 30 milliliters of supernatant from each conical tube and pool the supernat in a sterile one liter flask. Transfer 25 milliliters of pooled supernat into each of 16 ultracentrifuge tubes and centrifuge at 200, 000 Gs for two hours at four degrees Celsius. 10 minutes before centrifugation is complete.Prepare modified membrane resus suspension buffer. Combine eight milliliters of membrane suspension buffer with one tablet of EDTA free protease inhibitor cocktail and 375 units of benzo nase in a sterile tube and mixed gently following centrifugation of the lysed cells. Proceed to recover the membrane pellet.When ultracentrifugation of the lysed cells is complete, carefully pour off the supernat, then invert the tubes absorbent material and firmly tap them. To remove excess supernatant, mark the location of each membrane pellet with a marker to aid in visualization. Gently resuspend eight membrane pellets with 600 microliters each of modified membrane resus suspension buffer.It is difficult to resuspend the membrane pellets. So continue gently passing the modified membrane Resus suspension Buffer over the pellet until it peels away from the wall of the tube. This is another step which is challenging and critical for overall success of the experiment.If necessary, use the micro pipette tip to scrape the membrane pellet off the tube wall. When pipetting avoid frothing or bubble formation, excess shear forces will lead to mixed my cell formation, which precludes membrane separation. Transfer each membrane resus suspension to the next set of eight tubes.Gently resus suspend the remaining pellets and pool all suspensions in a sterile tube. After removing the membrane pellets from all 16 tubes, wash every four tubes with 600 microliters of modified membrane Resus suspension buffer. Focus the wash around the marked area of each tube and combine the wash with the resuspended membranes.Repeat the wash for the remaining tubes. Incubate the membrane resus suspension with gentle rocking for 30 minutes at room temperature to degrade DNA and aid in reduction of flocculent material. Next, remove a small Eloqua of the membrane suspension and perform protein quantitation.To determine total membrane yield, prepare one to one and one to three dilution of membrane suspension in 2.5%SDS heat, the undiluted one-to-one and one to three membrane suspensions at 90 degrees Celsius for 10 minutes. SDS and heat are required to release O MPS from the extracted membranes. Use the BioRad DC protein assay to quantitate the membrane resuspension protein concentration.Total protein yield is generally between one and 1.6 milligrams per milliliter. Once the concentration of protein in the membrane preparation is determined, the samples can be separated on a sucrose gradient. Start by preparing a series of sucrose solutions in EDTA.To prepare the linear sucrose gradients, use a bulb pipetter to layer 1.8 milliliters of each sucrose solution into 14 by 95 millimeter UltraClear ultracentrifuge tubes in the following order, 55%50%45%40%35%and 30%Next layer 1.5 milligrams of membrane resus suspension on top of each gradient. Do not load more than 1.7 milliliters of membrane resus suspension per gradient individually weigh each tube and carefully adjust the final weight by adding or removing membrane resuspension so that all tubes weigh the same. Transfer the sucrose gradients into an SW 40 TI swinging bucket rotor and centrifuge at 256, 000 GS for a minimum of 17 hours at four degrees Celsius.After 17 hours of centrifugation, stop the centrifuge run and carefully remove the sucrose gradients from the rotor. Next, collect the fractions. Carefully clean the bottom of the sucrose gradient tube with 70%ethanol.Then puncture the bottom of the tube with the 21 gauge needle and collect sequential 500 microliter fractions from the gradient into sterile micro fuge tubes. The gradient should be allowed to drip by gravity flow so as not to disturb the sucrose gradient. However, if the dripping ceases from the bottom of the gradient tube, a small amount of pressure may be applied to the top of the tube using one's finger.Finally, determine the refractive index of each sucrose gradient fraction using a refractometer correlate the refractive index with a specific density in grams per milliliter. Assess protein localization of representative sucrose gradient fractions by SDS page separation and immuno blotting. Here is an immuno blot of fractions from the sucrose gradient used to separate the membranes extracted from lysed Freis live vaccine strain freis outer membrane proteins or O mps, localized between densities of 1.17 and 1.2 grams per milliliter.By comparison, inner membrane proteins or ips localized between densities of 1.13 and 1.14 grams per milliliter. Similar results were obtained for the FHU four strain. I've just demonstrated how to extract and isolate Francis Cellis out membrane vesicles through osmotic lysis and sucrose density gradient ultracentrifugation.When performing this procedure, it's important to remember to use bacterial cells in the early logarithmic phases of growth to slowly but consistently dilute those bacterial cells into deionized water during the osmotic glyco step, and to take care when Resus suspending the ultracentrifuge membrane pellets. This concludes our membrane separation procedure. Thanks for watching and good luck with your future experiments.

method-for-the-isolation-of-francisella-tularensis-outer-membranes

Research

JoVE Journal

Biology

Source: Cian, M. B. et. al., Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii. J. Vis. Exp. (2020)This video demonstrates the separation of the inner and outer membrane fractions of Gram-negative bacteria using sucrose gradient ultracentrifugation. The membrane fractions are purified and stored at low temperatures for targeted analysis of membrane-specific components.

Separation of Inner and Outer Membrane Fractions of Gram-Negative Bacteria

Source: Watson, D. C., et.al. Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria. J. Vis. Exp. (2021)This video demonstrates the isolation and concentration of extracellular vesicles (EVs) derived from the outer membrane of genetically engineered bacteria. The process involves culturing the bacteria to release EVs, followed by sequential centrifugation and filtration steps to remove cells and debris. Finally, ultrafiltration is used to...

Isolation and Enrichment of Outer Membrane-Derived Extracellular Vesicles from Bacteria

Source: Jung, A. L., et al. Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages. J. Vis. Exp. (2017)This video demonstrates the isolation of outer membrane vesicles (OMVs) from Legionella pneumophila using sequential centrifugation, sterile filtration, and ultracentrifugation. The purified OMVs, enriched in membrane proteins and lipopolysaccharides, are suitable for host-pathogen interaction studies.

Ultracentrifugation-Based Isolation of Legionella pneumophila Outer Membrane Vesicles

This protocol describes intratracheal inoculations of Fischer 344 rats with Francisella tularensis. This procedure mimics pulmonary exposure of humans to this potential biothreat agent and can be used to test vaccine and therapeutic efficacy against pulmonary tularemia.

Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis

This method provides a framework for studying incorporation of exogenous fatty acids from complex host sources into bacterial membranes, particularly Staphylococcus aureus. To achieve this, protocols for the enrichment of lipoprotein particles from chicken egg yolk and subsequent fatty acid profiling of bacterial phospholipids utilizing mass spectrometry are described.

Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids

Mycobacterium tuberculosis shows increased production and release of extracellular vesicles in response to low iron conditions. This work details a protocol for generating low iron conditions and methods for the purification and characterization of mycobacterial extracellular vesicles released in response to iron deficiency.

Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria

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

Bacterial vesicles play important roles in pathogenesis and have promising biotechnological applications. The heterogeneity of vesicles complicates analysis and use; therefore, a simple, reproducible method to separate varying sizes of vesicles is necessary. Here, we demonstrate the use of size exclusion chromatography to separate heterogeneous vesicles produced by Aggregatibacter actinomycetemcomitans.

Size Exclusion Chromatography to Analyze Bacterial Outer Membrane Vesicle Heterogeneity

Bacteria secrete nanometer-sized extracellular vesicles (EVs) carrying bioactive biological molecules. EV research focuses on understanding their biogenesis, role in microbe-microbe and host-microbe interactions and disease, as well as their potential therapeutic applications. A workflow for scalable isolation of EVs from various bacteria is presented to facilitate standardization of EV research.

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

This protocol describes how to use TIRF microscopy to track individual ion channels and determine their activity in supported lipid membranes, thereby defining the interplay between lateral membrane movement and channel function. It describes how to prepare the membranes, record the data, and analyze the results.

Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using Total Internal Reflection Fluorescence (TIRF) Microscopy

Though prokaryotes lack an organellar system, there are subcellular regions with localized proteins. Quantitative and qualitative analysis of the enzymes and proteins in the subcellular regions is important for developing drug and vaccine targets. Here, we illustrate the subcellular fractionation of Leptospiral proteins using Triton X-114 and its analysis.

Enriching Subcellular Proteins in Leptospira Using a Triton X-114-Based Fractionation Approach

Learn to isolate Francisella tularensis outer membrane vesicles using osmotic lysis and sucrose density gradient ultracentrifugation. This method enables functional studies of outer membrane proteins and bacterial pathogenesis.

Method for the Isolation of Francisella tularensis Outer Membranes

Explore More Videos