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

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.