Laurent Coquet, Sébastien Vilain, Pascal Cosette, Thierry Jouenne, and Guy-Alain Junter
Proteomic analyses are increasingly implemented to investigate the particular physiology of (naturally or artificially) immobilized microorganisms. The protein maps of immobilized cells are compared with those of suspended counterparts to reveal alterations in protein expression induced by the microbial mode of growth. Proteins whose amount significantly varies between free and immobilized cells are identified by mass spectrometry and referring the peptide fingerprints to published databases. This proteomic approach is illustrated here using Pseudomonas aeruginosa cells grown as biofilms on clay beads for 24 or 48 h. Both the growth mode (suspended or attached) and the incubation time were shown to control the expression level of a large number of proteins by P. aeruginosa. Proteins whose amount significantly varied in biofilm organisms compared to suspended bacteria could be divided into three main classes, namely proteins linked to metabolic processes, proteins involved in adaptation and protection, and membrane proteins.
Key Words: Biofilm; 2D-electrophoresis; immobilized cells; P. aeruginosa; proteome. 1. Introduction
Microbiologists have shown a great interest for the sessile microbial growth mode over the past two decades. Indeed, it is now admitted that in most ecosystems, microorganisms predominate as surface-attached and matrix-enclosed communities called biofilms (1). Despite this definite importance of the sessile state in microbial way of life and its consequences for human beings, our present knowledge of the physiology of sessile bacteria remains still fragmentary. Thus, the mechanisms involved in the resistance of biofilm cells to antimicrobials (one of the main characteristic of biofilm cells) are complex and still not fully understood (2).
The publication of the first complete genome sequence of a living organism in 1995 opened a new era in biology (3). The exponential increase in genome sequence information that followed has prompted researchers to design new "global" experiments dealing with functional genomics. Transcriptomics, relying on DNA
From: Methods in Biotechnology: Immobilization of Enzymes and Cells, Second Edition Edited by: J. M. Guisan © Humana Press Inc., Totowa, NJ
arrays, provides information on the global gene expression pattern of a cell. Proteome—defined by Wilkins as all proteins expressed by a genome, cell or tissue (4)—provides additional information on the amount, modification and subcellular localization of molecules that act as cellular effectors (i.e., proteins). Geneticists, molecular biologists, and biochemists have logically applied these new tools to biofilms (5). However, all these data need an excellent experimental design. The present chapter gives appropriate methods for assessing alterations induced by cell adhesion on bacterial proteome. As an example, sessile cells are obtained by colonizing clay beads (6) but the procedure applies as well to microbial cells recovered from other model biofilms (7).
2.1. Cultivation of Planktonic and Sessile Bacteria (see Note 1)
2. Minimal salt medium, pH 7.5: 0.6 g/L Tris-base; 14.97 g/L Tris-HCl, 0.5 g/L NH4Cl, 0.05 g/L CaCl2, 0.05 g/L MgSO4, 7H2O, 0.005 g/L FeSO4, 7H2O, 0.005 g/L MnSO4, H2O, 15 g/L glucose, 2 g/L yeast extract.
3. 250-mL Erlenmeyer flasks.
4. Glass column with a recycle loop (see Note 2).
2.2. Recovery of Bacteria
1. 0.1 MPotassium phosphate buffer, pH 7.0.
2. Sterile flasks.
3. Glass beads (3-mm diameter).
4. Glass-fiber membrane (GF/C Whatman).
2.3. Preparation of Crude Protein Extracts
2. IEF buffer: 5 Murea, 2 Mthiourea, 2% (w/v) 3-[3-cholamidopropyl) dimethyl-ammonio]-1-propanesulphonate (CHAPS), 2 mMtributyl phosphine (see Note 3), 10 mM dithiothreitol (DTT), and 2% (v/v) carrier ampholytes, pH 3.5-10 (Amersham).
3. Bio-Rad protein assay kit.
2.4. Rehydratation of IPG Strips
1. IEF buffer.
2. Coomassie Brilliant Blue R-250 (Sigma).
3. Reswelling cassette (Amersham Biosciences).
2.5. First Dimension: Isoelectric Focalization
1. Immobilized pH gradient (IPG) Strips (Immobiline Dry Strip 18 cm, pH 3.0 to 10.0, nonlinear).
2. Multiphor II horizontal electrophoresis apparatus (Amersham Biosciences), power supply (3500 V), thermostatation by tap water circulation, IEF sample applicator strip (Amersham Biosciences).
3. IEF electrode strips.
4. Mineral oil.
5. Deionized water.
9. Plastic film.
2.6. IPG Strip Equilibration
1. Equilibration buffer No. 1: 6 M urea, 30% (v/v) glycerol, 2% (w/v) sodium dodecylsulfate (SDS) and 2% (w/v) DTT in 50 mM Tris-HCl, pH 6.8.
2. Equilibration buffer No. 2: 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 2.5% (w/v) iodoacetamide and 0.03% (w/v) Commassie Brilliant Blue R-250 (Sigma) in 50 mM Tris-HCl, pH 6.8.
2.7. Second Dimension: Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
1. Protean II xi cell electrophoresis apparatus (BioRad).
2. Polyacrylamide solution: 40 g polyacrylamide + 100 mL water
5. Tetramethyleneethylenediamine (TEMED) solution (Sigma).
6. 10 % (w/v) Ammonium persulfate solution.
7. Stacking gel buffer:1 M Tris-HCl, pH 6.8.
9. Electrode buffer solution: 3 g/L Trizma base, 14 g/L glycine, 1 g/L SDS in deion-ized water.
10. Vacuum flask.
2.8. Gel Silver Staining
1. Fixation solution: 10% (v/v) acetic acid and 30% (v/v) ethanol in deionized water.
3. 0.02 % (w/v) Sodium thiosulfate solution in deionized water.
4. 0.1 g/L Silver nitrate solution in deionized water.
5. Development solution: 24 g sodium carbonate, 200 ||L of 37% formaldehyde solution and 80 |L of 2% sodium thiosulfate solution in 2 L deionized water.
2.9. Gel Analysis
1. Gel scanning densitometer (e.g., Bio-Rad GS 800).
2. Gel analysis software (PDQuest software [Bio-Rad] or Melanie 4 (Amersham Biosciences).
2.10. Enzymatic Digestion of Proteins
1. Sequencing grade modified trypsin (Promega, Madison, WI).
2. Sterile scalpel.
3. Acetonitrile (CH3CN).
4. 10 mM Ammonium bicarbonate solution.
5. Extraction solution: water/CH3CN/trifluoroacetic acid (TFA) (79/20/1).
2.11. MALDI-TOF Analyses
1. The matrix is constituted of 3 mg/mL a-cyano-4-hydroxycinnamic acid diluted in a 60 % (v/v) CH3CN and 0.1 % (v/v) TFA solution.
3. Mass spectrophotometer (proTOF™ 2000, Perkin Elmer Sciex, Boston, MA). 3. Methods
3.1. Cultivation of Biofilm Cells
1. Put sterile clay beads into the flow cell (see Note 4).
2. Fill the glass tank with 800 mL of minimal salt medium and connect it with the recycle loop.
3. Use a peristaltic pump to apply a recycle flow rate of 0.7 mL/min.
4. Put the tank in a water bath maintained at 37°C.
5. Run the reactor for 18 or 48 h.
3.2. Cultivation of Planktonic Cells
Apply the same protocol than for biofilm cells but in the absence of clay beads.
3.3. Recovery of Bacteria
1. Remove aseptically (see Note 4) the clay beads.
2. Wash the beads twice with 80 mL of 0.1 Mpotassium phosphate buffer, pH 7.0 in a 250-mL Erlenmeyer flask.
3. Place the beads into a sterile flask containing 30 g of glass beads and 50 mL of potassium phosphate buffer, pH 7.0.
4. Shake vigorously the beads for 20 min.
5. Filter twice the bacteria suspension under vacuum.
6. Centrifuge the suspension at 1500^for 15 min.
7. Resuspend the bacterial pellet in 5 mL 0.1 Mphosphate buffer, pH 7.0.
3.4. Preparation of Crude Protein Extracts
1. Centrifuge the bacterial suspension at 1500^ for 15 min.
2. Wash the pellet in 0.1 Mphosphate buffer, pH 7.0, and pellet cells at 1500^for 15 min.
3. Resuspend the pellet in IEF buffer (about 0.2 g wet weight pellet per 2 mL buffer).
5. Disrupt cells by thermal shock (from -24°C to 20°C) and then by ultrasonication (30 W; 15 pulses of 2 s separated by 2-s breaks). This last procedure must be performed at 4°C.
6. Centrifuge the protein extract at 10,000^ for 20 min to eliminate cell debris.
7. Protein amounts in the supernatant are evaluated using the Bio-Rad protein assay by measuring the absorbance at 595 nm (see Note 5).
8. Store the supernatant at -80°C.
3.5. Rehydration of IPG Strips
1. Calculate the sample volume necessary to obtained 100 |g of proteins.
2. Transfer this sample to a 1.5-mL-Eppendorf tube and complete to 400 |L with IEF buffer containing 1% (m/v) Coomassie Brilliant Blue R-250 (see Note 6).
4. Fill each immobiline drystrip reswelling tray with a protein sample (see Note 7).
5. Spread the protein sample with a spatula.
6. Pull off the protective covers of the IPG strips.
7. Put the IPG strips carefully into the rehydration cassette, the gel slide directed towards the protein sample (see Note 8).
8. Cover the IPG strips with mineral oil.
9. Rehydrate the strips overnight at room temperature.
3.6. Isoelectric Focalization
1. Moisten the cooling plate of the Multiphor II apparatus with 20 mL of mineral oil.
2. Place the tray and electrode holder onto the cooling plate.
3. Pour 15 mL of mineral oil into the tray.
4. Place the Drystrip aligners (see Note 9).
5. Remove the IPG strips from the rehydratation cassette by using a clip.
6. Place the IPG strips on the DryStrip aligners, their acidic end towards the anode.
7. Cut two IEF electrode strips to a length corresponding to the width of the IPG strips.
8. Soak the electrode strips with deionized water.
9. Place the IEF electrode strips on top of the aligned IPG gel strips at the cathodic and anodic ends.
10. Position the electrodes and press them gently down on top of the IEF electrode strips.
11. Cover the IPG strips with mineral oil.
12. Place the lid, connect the cables to the power supply.
13. Maintain the temperature of the tray by a circulation of tap water.
14. Start the IEF. Running conditions depend on the pH gradient and the length of the IPG strips used. For a pH of 3.0 to 10.0 and a strip of 18 cm, the time schedule is the following: 150 V for 1 h, 350 V for 15 min, 750 V for 45 min, 1.5 kV for 1 h and 3.5 kV for 17 h (1 mA, constant) for a total of 61.8 kVh.
15. After IEF, store the IPG strips between two sheets of plastic film at -24°C.
3.7. Equilibration of IPG Gel Strips
1. Defrost the IPG strips.
2. Equilibrate successively the IPG strips in 15 mL of each equilibration buffer for 10 min each time under slight agitation.
3. Blot the IPG strips to remove excess equilibration buffer.
3.8. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (see Note 10)
1. Transfer in vacuum flask (for 1 gel) 15.70 mL of 40% polyacrylamide solution, 8.39 mL of 2% bis-acrylamide solution, 19.35 mL of Tris-buffer, pH 8.9, and 7.70 mL of deionized water (see Note 11).
2. Remove air under vacuum.
3. Add 258 ||L of 20% SDS solution, 40.50 mL of TEMED and 154.8 ||L of 10 % ammonium persulfate solution.
4. Cast immediately the running gel by using a casting stand of the Protean II xi Cell apparatus (gel size 200 x 250 x 1 mm3). Avoid introduction of any air bubbles.
5. Carefully pipette 2 mL of overlay propan-2-ol 50% in water onto the top of the gel.
6. Allow the gel to polymerize for 45-60 min at room temperature.
7. Remove propan-2-ol and add water from the top of the gel for the night.
9. Transfer in vacuum flask 923 |L of 40% polyacrylamide solution, 491 |L of 2% bis-acrylamide solution, 1.01 mL of 1 M Tris-buffer, pH 6.8, and 5.59 mL of water (see Note 11).
10. Remove air under vacuum.
11. Add 40.6 |L of 20% sodium dodecyl sulfate (SDS) solution, 26.5 |L of TEMED and 40.6 |L of 10% ammonium persulfate solution.
12. Cast immediately the stacking gel onto the top of the running gel.
14. Allow the gel to polymerize for 45 to 60 min at room temperature.
15. Remove propan-2-ol and rinse with distilled water.
16. Place each IPG strip on top of the stacking gel. Carefully press the IPG strip with a spatula onto the surface of the gel.
17. Fill the buffer tank of the electrophoresis apparatus with the electrode buffer.
18. Insert the gel cassette in the electrophoresis apparatus. Thermostate at 8°C with polyethyleneglycol.
19. Put the lid on the electrophoresis unit and connect cables.
20. Start SDS PAGE at 20 mA/gel for about 1 h with a limit of 150 V.
22. When Coomassie Brilliant Blue tracking dye has migrated off the lower end of the gel, terminate the run.
23. Remove the gel from the buffer tank.
24. Peel carefully the gel from the glass plate and remove the IPG strip and the stacking gel with a spatula.
3.9. Gel Staining
1. Fix the gel from 3 h to overnight in the fixation solution.
2. Rinse twice the gel in the 10% ethanol solution for 10 min.
3. Rinse 3 x 10 min in deionized water.
4. Soak gel for 1 min in 0.02 % sodium thiosulfate solution.
5. Rinse for 1 min in deionized water.
6. Impregnate for 30 min in silver nitrate solution.
7. Rinse in water for 30 s.
8. Develop image for 20 to 30 min in the development solution.
9. Stop development in 1% acetic acid solution during 10 min.
10. Soak in a 3% glycerol solution.
3.10. Gel Analysis
1. Scan the gel using a densitometer.
2. Analyze the gel using a software (PDQuest or Melanie 4).
3.11. Protein Identification
1. Excise the spots from the gel with a sterile scalpel and slice into small pieces.
2. Wash gel plugs twice for 15 min with 100 ||L of deionized water.
3. Wash plugs twice with 100 |L of H2O/CH3CN (1/1 v/v) for 15 min.
5. Dry plugs using a SpeedVac centrifuge for a few minutes.
7. After rehydratation (30 min), cover the plugs with 20 |L of 10 mM ammonium bicarbonate solution and after 3 h, add 35 |L of water.
8. Allow digestion overnight at 37°C.
9. Collect the liquid phase containing peptides.
10. Add 20 |L of H2O/CH3CN/TFA mixture (79/20/1) to remove peptides remaining in the gel.
11. Pull the different fractions.
12. Dry in a vacuum centrifuge.
13. Redissolve in 10 |L of 5% formic acid.
14. 1 |L Peptide solution is mixed with 1 |L of the matrix solution.
15. 1 |L Mixed peptide-matrix is deposed on the target-plate.
The peptide fingerprints can be matched against in silico digests using the MS-
FIT software with the GenePept database restricted to Pseudomonas aeruginosa, accessible at http:Wprospector.ucsf.edu.
3.12. A Practical Example: Standard Proteomic Maps for Planktonic and Clay-Bead-Attached P. aeruginosa Cells
1. Figure 1 shows standard proteomic maps for planktonic (F) and clay-bead-attached (CB) P. aeruginosa cells after incubation for 18 or 48 h.
2. A total of 886 protein spots were discriminated on 2-DE electropherograms and quantified by computing scanning densitometry.
3. The corresponding proteins were expressed in at least one of the 4 tested incubation conditions (i.e., they were present in at least one of the four synthetic gels created by the PDQuest software).
4. The proteins produced by attached bacteria could be divided into three families by comparing their spot intensities to those of spots from suspended-cell electro-pherograms (i.e., underproduced, overproduced, and unaffected peptides). The levels of a large number of proteins differed between biofilm organisms and suspended bacteria (Table 1). These differences depended both on the growth mode (suspended or attached) and the incubation time.
5. A number of proteins whose amount significantly varied in biofilm organisms compared to suspended bacteria were identified (Table 2).
6. These proteins can be divided into three main classes. The first class includes proteins linked to metabolic processes, in particular enzymes, showing (not surprisingly) that central metabolism is altered by the sessile mode of growth. The second class includes proteins involved in adaptation and protection. This general stress response initiated by growth within a biofilm might explain the resis-
Number of Proteins Whose Amount Significantly Varied in P. aeruginosa Cells According to the Incubation Conditions.
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