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

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Number Organism Capture Vector Size of insert % positive clone 1 E. coli K12 MG1655 serW 4kb 16% (3/19) 2 E. coli K12 MG1655 leuX 43kb 6% (6/95) 3 P. aeruginosa PA01 lys10 11kb 8% (1/12) 4 P. aeruginosa PA14 lys10 13kb 8% (2/24) 5 P. aeruginosa PA14 lys47 110kb Unsuccessful Table 3-1 shows the targets of the initial capture experiments. The size of the captured insert was generated from the publicly available GenBank genome sequences. The following subsections will describe the initial captured inserts from the reference genomes. The genomic island capture technique was designed to enable rapid estimation of the size of the genomic insert by having two I-SceI restriction sites flanking the captured insert, which are located immediately upstream of TS1 and immediately downstream TS2. 3.1.1 K12 MG1655–serW The serW locus within E. coli K12 MG1655 is an empty site, no insertion events have occurred, the size of the insert is the distance between the two targeting sequences (TS1 and TS2). The first successful capture experiment resulted from homologous recombination between the serW capture vector and K12 MG1655- serW amplicon. The K12 MG1655-serW amplicon was generated from E. coli K12 MG1655 genomic DNA using the serW TS1 U and serW TS2 D primers. The use of the K12 MG1655-serW amplicon provided a resolution of the initial hurdles to successful genomic island capture. The initial capture experiments involving genomic DNA resulted in no or very low recovery of colonies (Table 3-2). A number of experiments using the K12 MG1655-serW amplicon as a control demonstrated that the genomic DNA preparation was inhibiting the capture process (Table 3-3). 54
DNA No of colonies Control No DNA 0 Test 10µg K12 MG1655 - serW 0 Test 20ng E101 amplicon –serW 1744 Table 3-2 shows number of colonies retrieved during a capture experiment using 1µg serW capture vector as a control and testing E. coli K12 MG1655 genomic DNA as well as the E101 amplicon and serW capture vector DNA No of colonies Control No DNA 0 Control 20ng pLLX13 173 Test 20ng pLLX13 + 10µg gDNA 0 Test 180ng K12 MG1655 –serW 178 Test 180ng K12 MG1655–serW + 10µg gDNA 0 Table 3-3 shows number of colonies retrieved during a capture experiment using 20ng pLLX13 as a control and testing K12 MG1655-serW amplicon and serW capture vector with replicas of 10µg E. coli K12 MG1655 genomic DNA (gDNA) After the resolution of the genomic DNA problems, K12 MG1655-serW insert was also successfully captured from E. coli K12 MG1655 genomic DNA. 3.1.2 K12 MG1655–leuX The leuX-associated genomic island within E. coli K12 MG1655 is 43kb in size and was the largest insert captured during this project. It is a genomic island described in the literature by Bishop et al. (2005). Figure 3-3 shows K12 MG1655-leuX digested with HindIII. Figure 3-2 shows a 0.6% agarose gel with the restriction profile of K12 MG1655-leuX with HindIII Lane 1 λHindIII 500ng; Lane 2-7 K12 MG1655-leuX; Lane 8 Generuler DNA ladder mix 500ng 23kb 9.4kb 6.8kb 4.4kb 2.3kb 2.0kb 500b p 1 2 3 4 5 6 7 8 55 10kb 8kb 6kb 5kb 4kb 3.5kb 3kb 2.5kb 2kb 1.5kb 1.2kb 1kb 900bp 800bp 700bp 600bp 500bp 400bp 300bp 200bp 100bp
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Characterisation of pathogenicity i
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Statement of Originality This accom
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% Percent Abbreviations ºC Degrees
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Table of contents 1 INTRODUCTION 1
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4 THE CONTRIBUTION OF PAPI-1 AND PA
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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 and 54: Important to the use of the capture
Page 55 and 56: 2.3.4 Electroporation: Transfer of
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: 3.1 Capture experiments involving g
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
Page 109 and 110: log CFU/mg 4 3 2 1 0 0 2 4 6 8 Days
Page 111 and 112: mice exhibited visible symptoms at
Page 113 and 114: 4.4.1 Survival times post-infection
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(P
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4.4.4 Intravenous infection A quest
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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
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4.4.7 Progression of disease post-i
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Figure 4-19 Leukocytes numbers moni
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interesting result is that the ∆P
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Unfortunately, 65% of the ORFs with
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morphology. ∆PAPI-1∆PAPI-2 leav
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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
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LES431 LESB58 LESB65 LES400 � �
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Additional comparisons of the CFU r
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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
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isolates have a similar virulence i
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The data presented in this thesis s
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than PAO1. Despite the bacterial lu
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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
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AQ: 18 AQ: 19 AQ: 20 F1 AQ: 21 tapr
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AQ: 32 AQ: 33 AQ: 34 tapraid5/z9d-j
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AQ: 40 AQ: 41 DISCUSSION tapraid5/z
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AQ: 48 tapraid5/z9d-jid/z9d-jid/z9d
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tapraid5/z9d-jid/z9d-jid/z9d01009/z
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8 Bibliography AL-ALOUL, M., CRAWLE
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D'ARGENIO, D.A., WU, M., HOFFMAN, L
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genomic islands in Pseudomonas aeru
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MURRAY, A.W. and SZOSTAK, J.W., 198
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specificity of chaperone-usher mach
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VAN HEECKEREN, A.M. and SCHLUCHTER,
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