5 The role of quorum-sensing in the virulence of Pseudomonas ...

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done with gentle rocking motion, no pipetting or vortexing. Washed cells twice in 20ml chilled 1M STC (1M Sorbitol, 10mM CaCl2, 10mM Tris-HCl pH 7.5). Re- suspended in 2ml STC and mixed 450µl aliquots of spheroplasts with 1µg MluI- linearised capture vector DNA plus 5µg genomic DNA in no more than a 45µl volume in 30ml sterlin tube; incubated for 10 minutes at room temperature. Added 4.5ml PEG reagent (20% PEG 8000, 10mM CaCl2, 10mM Tris-HCl pH 7.5) mixed by gentle swirling and rocking motion; incubated at room temperature for 10 minutes. The cells were then centrifuged at 200g for 10 minutes at 4ºC. The supernatant was discarded and the cells were re-suspended in 2ml SOS (25% YPD, 7mM CaCl2, 1M sorbitol); incubate at 30°C for 40 minutes. 15ml of TOP agar (1M sorbitol, 2.5% agar, URA broth) which have been kept at 45ºC was added and inverted quickly several times and plated immediately on pre-warmed (37ºC) SORB plates (1M sorbitol, 2% agar, URA broth). Colonies were expected 5-7 days after incubation at 30°C. 2.3.3 Screening of spheroplast colonies: colony PCR At best 100 colonies were patched onto fresh URA plates using sterile wooden toothpicks and left to grow overnight for at least 20hrs. The screening PCR reactions were set up in a total volume of 20µl; 2µl colony PCR buffer (45mM Tris-HCl pH 8.8, 11mM Ammonium sulphate, 45mM Magnesium chloride, 4.4mM EDTA pH 8.0, 1µM dNTP (each), 113µg/ml BSA and 7mM β-mercapoethanol), 2µl 10pmol µl -1 of each positive screening primer, 1µl 10 pmol µl -1 of each tetA primer, 0.4µl GoTaq R DNA Polymerase (Promega, UK) and 13.6µl nanopure water. Each patch was touched gently with a yellow tip (20µl-200µl tip) and re-suspended by gentle pipetting into the PCR mixture. The following cycling conditions were used; 95ºC for 5 minutes for initial denaturation, followed by 35 cycles of 95ºC for 40 seconds, 60ºC for 30 seconds and 72ºC for 2 minutes, proceeded by a final extension at 75ºC for 5 minutes. Any positive patches were than grown overnight in 5ml URA broth and subjected to yeast plasmid preparation as stated in Section 2.1.6. 44
2.3.4 Electroporation: Transfer of suspect plasmids to E. coli An electroporation cuvette (Gene Pulser Cuvette, 0.2cm, BioRad) and an aliquot of electro-competent cells were placed on ice. After the cells had thawed, 1µl of yeast plasmid preparation was added to the cuvette and 20µl E. coli DH10B was added to the DNA drop. The cuvette was incubated on ice for 5 minutes. The Gene Pulser electroporator (BioRad) was set to 1.6kV, 200Ω and 25µF. Excess moisture was removed from the cuvette and an electric pulse was given. The time constant was expected to be between 4.0 and 4.5 msec. 600µl SOC was added immediately and incubated at 37ºC, 200rpm for 1 hour. 100µl of the bacterial suspension was plated onto LA containing tetracycline (10µg/ml) plates. The plates were incubated overnight at 37ºC. 2.3.5 Restriction Fragment Length Polymorphism (RFLP) A colony from each plate was chosen was subsequently used to produce a 5ml overnight for plasmid preparation and for re-streaking onto a fresh LA containing tetracycline (10µg/ml) plate. The following day the 5 ml overnight was harvested and subjected to bacterial plasmid preparation (Section 2.1.5). The restriction digests were performed as in Section 2.1.7, usually with the following enzymes, I-SceI, BamHI and HindIII and if appropriate compared with expected sequence fragment pattern generated using NEBCutter (Vincze, Posfai & Roberts 2003). 2.3.6 Sub-cloning of E106-serW The RFLP of E106-serW plasmid suggested that in order to get the most from sample sequencing, PstI and KpnI were the best enzymes to use for sub-cloning. In a total volume 60µl restriction digestion, 30µl of an E106-serW midi plasmid preparation and 3µl enzyme (either PstI or KpnI) were digested for 5 hours. A restriction digest was prepared of 100ng pWSK29 with either PstI or KpnI. All restriction digests were heat-inactivated at 65ºC for 20 minutes, vacuum-dried, washed in 500µl 70% ethanol. Then centrifuged at 10,000g for 5 minutes, vacuum- dried and re-suspended in 10µl nanopure water. The ligation was set up a total volume 45
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Characterisation of pathogenicity i
Page 3 and 4: Statement of Originality This accom
Page 5 and 6: % Percent Abbreviations ºC Degrees
Page 7 and 8: Table of contents 1 INTRODUCTION 1
Page 9 and 10: 4 THE CONTRIBUTION OF PAPI-1 AND PA
Page 11 and 12: 1 Introduction 1 INTRODUCTION 1 1.1
Page 13 and 14: Cell-surface Proteases Haemolysins
Page 15 and 16: 1.2.1 Pathogenicity islands: a subg
Page 17 and 18: appear to be related and there have
Page 19 and 20: assessed in number of assays and mo
Page 21 and 22: TAR cloning is a method designed by
Page 23 and 24: Figure 1-3 Top plate shows colony g
Page 25 and 26: Figure 1-5 depicts the principles o
Page 27 and 28: Balb/c mice. The models described b
Page 29 and 30: urns-sepsis model, but not in the n
Page 31 and 32: ORF ID Gene name Gene function PA14
Page 33 and 34: functional activities it could be s
Page 35 and 36: Figure 1-6 Schematic showing the OR
Page 37 and 38: RhlI Autoinducer RhlR Rhl Regulon G
Page 39 and 40: 1.5.2 Pseudomonas aeruginosa LES Th
Page 41 and 42: 2 Material and Methods 2 MATERIAL A
Page 43 and 44: 2.1.2 Strains used Pseudomonas aeru
Page 45 and 46: Plasmids Plasmid Description Refere
Page 47 and 48: In the case of capture vector const
Page 49 and 50: Primer name Sequence Capture vector
Page 51 and 52: The relevant homology sequence was
Page 53: Important to the use of the capture
Page 57 and 58: 2.4.2 Infection dose preparation Th
Page 59 and 60: mould was then added to the metal c
Page 61 and 62: 3 Analysis of genomic islands captu
Page 63 and 64: 3.1 Capture experiments involving g
Page 65 and 66: DNA No of colonies Control No DNA 0
Page 67 and 68: were designed based on the assumpti
Page 69 and 70: 3.2.1 KR115-lys10 and KR159-lys10 A
Page 71 and 72: estriction digestion with I-SceI to
Page 73 and 74: generated. The most significant nuc
Page 75 and 76: they both produced three fragments
Page 77 and 78: elated genomic islands and this res
Page 79 and 80: All the Yersinia species available
Page 81 and 82: Leicester). E105-leuX was the first
Page 83 and 84: The strains highlighted with alignm
Page 85 and 86: predict this protein to be a dnaJ-c
Page 87 and 88: Figure 3-15 depicts the codon usage
Page 89 and 90: ORF Gene range Blastx Description y
Page 91 and 92: ecognition sequences are found with
Page 93 and 94: (S: 150, E: 1e-37). This predicts t
Page 95 and 96: having multiple copies of itself. A
Page 97 and 98: The capture vectors used during thi
Page 99 and 100: cloning and their findings was inco
Page 101 and 102: The genomic island capture method c
Page 103 and 104: unpublished). This was achieved by
Page 105 and 106:
Pseudomonas aeruginosa PA14 (UCBPP-
Page 107 and 108:
stressful’ to the bacteria and th
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log CFU/mg 4 3 2 1 0 0 2 4 6 8 Days
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mice exhibited visible symptoms at
Page 113 and 114:
4.4.1 Survival times post-infection
Page 115 and 116:
(P
Page 117 and 118:
4.4.4 Intravenous infection A quest
Page 119 and 120:
Symptom score Symptom score Symptom
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Fold increase Fold increase 15 10 5
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Figure 4-15 Histopathology lung sec
Page 125 and 126:
4.4.7 Progression of disease post-i
Page 127 and 128:
Figure 4-19 Leukocytes numbers moni
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interesting result is that the ∆P
Page 131 and 132:
Unfortunately, 65% of the ORFs with
Page 133 and 134:
morphology. ∆PAPI-1∆PAPI-2 leav
Page 135 and 136:
The intravenous sepsis model data s
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in PAO1(Lee et al. 2006). They show
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shown that deletion of the exoU gen
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5 The role of quorum-sensing in the
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Figure 5-1 shows overnight growth o
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Quorum-sensing is bacterial cell de
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Interestingly, the quorum-sensing d
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Symptom score Symptom score Symptom
Page 151 and 152:
LES431 LESB58 LESB65 LES400 � �
Page 153 and 154:
Additional comparisons of the CFU r
Page 155 and 156:
log CFU/mg log CFU/mg log CFU/ml 5
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For LESB58-infected mice the histop
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Strain Mouse Timepoint HB CI GC Sco
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Score: 4 HB Score: 4 Score: 4 B HCI
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post-infection to that of healthy m
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Tang et al. (1996) performed additi
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Figure 5-14 PFGE comparison of the
Page 169 and 170:
isolates have a similar virulence i
Page 171 and 172:
The data presented in this thesis s
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than PAO1. Despite the bacterial lu
Page 175 and 176:
6 Thesis conclusion The underlying
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Hopefully the methodologies describ
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1 2 3 4 5 6 7 8 9 10 11 12 13 λHin
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AQ: 1 AQ: 2 AQ: 3 AQ: 4 AQ: 5 AQ: 6
Page 183 and 184:
AQ: 18 AQ: 19 AQ: 20 F1 AQ: 21 tapr
Page 185 and 186:
AQ: 32 AQ: 33 AQ: 34 tapraid5/z9d-j
Page 187 and 188:
AQ: 40 AQ: 41 DISCUSSION tapraid5/z
Page 189 and 190:
AQ: 48 tapraid5/z9d-jid/z9d-jid/z9d
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tapraid5/z9d-jid/z9d-jid/z9d01009/z
Page 193 and 194:
8 Bibliography AL-ALOUL, M., CRAWLE
Page 195 and 196:
D'ARGENIO, D.A., WU, M., HOFFMAN, L
Page 197 and 198:
genomic islands in Pseudomonas aeru
Page 199 and 200:
MURRAY, A.W. and SZOSTAK, J.W., 198
Page 201 and 202:
specificity of chaperone-usher mach
Page 203 and 204:
VAN HEECKEREN, A.M. and SCHLUCHTER,
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