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MATERIALS AND METHODS
Bacterial strain and medium
P. aeruginosa PAO1 (DSM 1707) was used in all studies. Preculture was performed in a 100- ml Erlenmeyer flask containing 50 ml of a modified mineral salts medium with glucose and yeast extract (MSGY) of the following composition (in distilled water): 1.74 g/L NaNH4HPO4.4H2O; 0.54 g/L NaH2PO4.H2O; 0.2 g/L MgSO4.7H2O; 0.2 g/L yeast extract; 0.04 g/L KCl; 0.005 g/L FeSO4.7H2O; 5.0 g/L glucose and 1% (v/v) trace mineral solution (2.86 g/L H3BO3; 1.81 g/L MnCl2.4H2O; 0.22 g/L ZnSO4.7H2O; 0.08 g/L CuSO4.5H2O; 0.06 g/L CoCl2.6H2O; 0.025 g/L Na2MoO4.2H2O) (Atlas, 1993). The flask was incubated at 37°C on a rotary shaker (200 rpm) for 4 h until mid-exponential phase was reached (OD540 = 0.1).
The culture was subsequently used to inoculate 100 ml MSGY broth in 500-ml Erlenmeyer flasks, with and without 2.5 g glass wool (mean diameter 15 μm, total surface area 3 250 cm2) (Merck, Darmstadt, Germany), to a final inoculum of 4 × 106 cfu/ml. 3.2.2 Microscopy and analytical procedures To evaluate whether glass wool served as a surface for the establishment of biofilms, brightfield microscopy (Zeiss Axioskop, Zeiss, Oberkochen, Germany) was performed on various samples of glass wool obtained at times 0 h, 4 h, 8 h, 18 h, 24 h and 48 h after inoculation.
The glass wool was stained with 0.01% (w/v) crystal violet and immediately viewed by bright-field microscopy. Images were captured using a COHU monochrome CCD camera (RS-170, Cohn Inc., San Diego, CA, USA).
For analytical procedures, samples of planktonic cells cultured in the absence and presence of glass wool and biofilm cells were obtained after 18 h of incubation, as described in Sections 3.2.3.1 – 3.2.3.3. The planktonic cells taken from flasks containing glass wool were referred to as surface influenced planktonic (SIP) cells. The respective samples were diluted to a final volume of 100 ml prior to analysis. The culturable count was determined by plating 0.1-ml aliquots of serial dilutions onto triplicate plates of Luria Bertani (LB) agar and incubating for 24 h at 37°C. The optical density of cell suspensions was determined at 540 nm. Total protein concentrations were determined according to the method of Bradford (1976). Briefly, cell suspensions were disrupted by ultrasonication by applying 3 × 20 s pulses using a 4710 Series Ultrasonic Homogenizer (Cole-Palmer Instrument Co., Chicago, IL, USA) at an output of 40%. Lysates were boiled for 10 min, 50-μl aliquots mixed with 1.5 ml Coomassie Plus Protein Assay Reagent (Pierce, Rockford, IL, USA) and the absorbance measured at 595 nm. The protein concentration was calculated using bovine serum albumin (BSA) as standard.
Extraction of whole-cell proteins
Planktonic biomass
After incubation for 18 h at 37°C, planktonic P. aeruginosa cells, cultured without glass wool, were collected by centrifugation at 13 000 × g for 10 min. The pellet was washed twice in 0.2 M sodium phosphate buffer (pH 6.8) and then resuspended in 10 mM Tris-HCl (pH 7.4). The suspension was heated to 95°C for 30 min and sonicated by six pulses of 15 s each using a 4710 Series Ultrasonic Homogenizer at an output of 40%. Lysis buffer B, composed of 9 M urea; 65 mM DTE; 65 mM CHAPS and 5% (v/v) ampholytes (pH 3.0 – 10.0) (Amersham- Pharmacia Biotech, Uppsala, Sweden)(Gravel and Golaz, 1996) was added. The protein sample was then stored at -70°C until required.
Biofilm biomass
The glass wool, cultured for 18 h with P. aeruginosa, was removed from the MSGY broth, rinsed twice in 0.2 M sodium phosphate buffer (pH 6.8) and then placed in a sterile flask containing 45 g of glass beads (mean diameter 6 mm). Ten ml of 10 mM Tris-HCl (pH 7.4) was added to the flask and it was shaken vigorously for 10 min to detach the bacterial cells from the glass wool surface. The bacteria were then collected by centrifugation (13 000 × g, 10 min) and samples were processed as described above for the planktonic bacterial cells.
Surface influenced planktonic (SIP) biomass
The P. aeruginosa cells remaining in the medium after removal of the glass wool were also collected by centrifugation at 13 000 × g for 10 min, and proteins were extracted as described in Section 3.2.3.1 for the planktonic bacterial cells. These bacterial cells are referred to as surface influenced planktonic (SIP) cells to indicate their origin.
Concentration of protein samples
All protein samples were concentrated using the method of Wessel and Flugge (1984). Since the biofilm biomass was less than both the planktonic and SIP biomasses, a larger volume of the biofilm sample was concentrated. One hundred and fifty μl of the planktonic and SIP samples were concentrated to a final volume of 100 μl each. Three hundred μl of the biofilm sample was concentrated to a final volume of 40 μl. The protein content of each extract was determined by a Coomassie Plus Protein Assay Reagent (Pierce) and standardised to ca. 200 μg for each gel.
CHAPTER ONE: LITERATURE REVIEW
1.1 GENERAL INTRODUCTION
1.2 BIOFILM FORMATION BY P. aeruginosa
1.2.1 Steps in biofilm development
1.2.1.1 Reversible attachment
1.2.1.2 Irreversible attachment
1.2.1.3 Biofilm maturatio
1.2.1.4 Detachment
1.3 STRUCTURAL COMPONENTS AND CELL-TO-CELL SIGNALLING MOLECULES REQUIRED FOR BIOFILM FORMATION
1.3.1 Importance of flagella, pili and adhesins
1.3.2 Importance of membrane proteins
1.3.3 Importance of extracellular polysaccharides
1.3.4 Importance of quorum sensing
1.4 REGULATION OF BIOFILM FORMATION
1.4.1 Two-component signal transduction pathways 1
1.4.2 Factors regulating carbon metabolism
1.4.3 Phase-dependent regulators
1.4.4 Quorum sensing
1.5 THE BIOFILM PHENOTYPE
1.5.1 Phenotypic differentiation during biofilm development
1.5.2 Antimicrobial resistance of P. aeruginosa biofilms
1.5.2.1 Mechanisms of biofilm resistance
1.5.2.2 Persister cells, phenotypic variants and mutant cells
1.6 THE STUDY OF BACTERIAL BIOFILMS
1.6.1 Culturing systems
1.6.2 Approaches to studying biofilm-specific gene expression
1.6.2.1 Reporter gene-based approaches
1.6.2.2 Proteomic approaches
1.6.2.3 Transcriptomic approaches
1.7 AIMS OF THIS INVESTIGATION
1.8 REFERENCES
CHAPTER TWO: ESTABLISHMENT OF TWO-DIMENSIONAL GEL ELECTROPHORESIS FOR DETERMINATION OF THE P. aeruginosa PROTEOMES
2.1 INTRODUCTION
2.2 MATERIALS AND METHODS
2.2.1 Bacterial strain and culture conditions
2.2.2 Whole-cell protein extractions
2.2.2.1 Sample preparation method 1 (SP 1)
2.2.2.2 Sample preparation method 2 (SP 2)
2.2.2.3 Sample preparation method 3 (SP 3)
2.2.3 Two-dimensional polyacrylamide gel electrophoresis (2-DE)
2.2.3.1 Preparation of the ampholyte-containing tube gels for
iso-electric focusing
2.2.3.2 First-dimension iso-electric focusing (IEF)
2.2.3.3 Second-dimension protein separation (SDS-PAGE)
2.2.4 Visualisation of proteins on 2-DE gels
2.2.4.1 Coomassie R250 staining (S 1)
2.2.4.2 Silver diamine staining (S 2)
2.2.4.2.1 Preparation of silver diamine staining and
spot development solutions
2.2.4.2.2 Staining method
2.3 RESULTS AND DISCUSSION
2.3.1 Sample preparation
2.3.1.1 Cell lysis
2.3.1.2 Protein solubilisation
2.3.2 First-dimension IEF
2.3.3 Second-dimension SDS-PAGE
2.3.4 Staining of 2-D PAGE gels
2.4 CONCLUDING REMARKS
2.5 REFERENCES
CHAPTER THREE: THE USE OF GLASS WOOL AS AN ATTACHMENT SURFACE FOR STUDYING PHENOTYPIC CHANGES IN Pseudomonas aeruginosa BIOFILMS BY TWODIMENSIONAL GEL ELECTROPHORESIS
3.1 INTRODUCTIO
3.2 MATERIALS AND METHODS
3.2.1 Bacterial strain and medium
3.2.2 Microscopy and analytical procedures
3.2.3 Extraction of whole-cell proteins
3.2.3.1 Planktonic biomass
3.2.3.2 Biofilm biomass
3.2.3.3 Surface influenced planktonic (SIP) biomass
3.2.4 Concentration of protein samples
3.2.5 Two-dimensional gel electrophoresis
3.2.6 Image analysis
3.3 RESULTS
3.3.1 Biofilm development on glass wool
3.3.2 2-DE maps
3.3.3 Comparison of the proteome profiles
3.4 DISCUSSION
3.5 REFERENCES
CHAPTER FOUR: PROTEOME COMPARISON OF Pseudomonas aeruginosa PLANKTONIC, SURAFCE INFLUENCED PLANKTONIC AND BIOFILM POPULATIONS BASED UPON COMPOSITE TWO-DIMENSIONAL GEL ELECTROPHORESIS
4.1 INTRODUCTION
4.2 MATERIALS AND METHODS
4.2.1 Bacterial strain and medium
4.2.2 Collection of biomass
4.2.3 Extraction of whole-cell proteins through differential solubilisation
4.2.4 Concentration of protein samples
4.2.5 Two-dimensional gel electrophoresis
4.2.6 Image analy
4.2.7 Protein sequencing and identification
4.2.7.1 N-terminal amino acid sequencing and protein identification
4.2.7.2 Peptide mass fingerprinting and protein identification
4.3 RESULTS
4.3.1 “Composite map” creation of P. aeruginosa planktonic, SIP and biofilm population
4.3.2 Proteome profile analysis
4.3.3 Identity of differentially expressed proteins
4.3.3.1 Outer membrane proteins
4.3.3.2 Probable outer membrane proteins
4.3.3.3 Cytoplasmic proteins
4.4 DISCUSSION
4.5 ACKNOWLEDGEMENTS
4.6 REFERENCES
CHAPTER FIVE: CONSTRUCTION AND CHARACTERISATION OF AN OprG-DEFICIENT MUTANT STRAIN OF Pseudomonas aeruginosa PAO1 (DSM 1707)
CHAPTER SIX: CONCLUDING REMARKS
