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

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In conclusion, E105-leuX is a genomic islet as contains a majority of the markers identified in the literature as essential. The next subsection will explore the genomic structure of the genomic islet. 3.4.2 The genomic structure of the E105-leuX genomic islet Full length DNA sequence analysis led to the discovery of 6 genes (ORFs) within E105-leuX, they are depicted in Figure 3-16 and described in Table 3-8. A number of these genes were mobility genes, insertion elements and an integrase. The genomic islet is predicted to encode three restriction endonucleases. This subsection will gives an overview of the genes present within E105-leuX, determining whether they are functional and their role within the genomic islet. Figure 3-16 shows the genomic structure of the E105-leuX genomic islet; 11, 618bp sequence 78
ORF Gene range Blastx Description yjgB Complement 79..1101 yjgB, E. coli DH10B 79 Putative zinc alcohol dehydrogenase oxidoreductase leuX 1255..1336 - leuX tRNA int 1515..2709 insA 2979..3746 insB 3228..3731 E105leuX _mod E105leuX _res 3965..5982 5985..8894 IS3 9354..9424 E105leuX _1 E105leuX _2 9452..9865 Complement 9982..10086* CKO_03944, C. koseri (S: 350, E: 6e-110, I: 68%- 57%-60%) IS1 InsA, E. coli W3110 (S: 1507, E: 0, I:99%) IS1 InsB, E. coli W3110 (S: 1507, E: 0, I:99%) EcoP15I, mod (S: 446, E: 3e-123, I:55%- 73%) EcoP15I, res (S: 1816, E: 0, I: 90%) IS3, E. coli MG1655 K12 (S: 937, E: 2e-18, I: 91%) EcE24377A_4906, E. coli E24377A, (S: 249, E: 3e-65, I: 85%) EcE24377A_4907, E. coli E24377A (S: 74, E: 1e-12 and I: 94%) Partial phage integrase, P4 type, pseudogene, two frameshifts Insertion element protein, IS1 Insertion element protein, IS1 Type III restriction modification system, methylase, pseudogene Type III restriction modification system, restriction endonuclease IS3, partial Putative restriction endonuclease, partial Putative restriction endonuclease, pseudogene yjhS 10097..11104 yjhS, E. coli DH10B Hypothetical protein yjhT Complement 11142..11534 Table 3-8 The ORFs found within E105-leuX genomic islet. yjhT, E. coli DH10B Putative inner membrane protein Gene range defines where the genes are situated within the available E105-leuX sequence. The blastx scores were generated against the E105-leuX DNA sequence and include the most significant alignment S=Bit-score, E=Expected value and I=Identity percentage * Based on start of alignment and not a start codon. The ORF marked as int was a P4-type integrase. The P4 family of bacteriophages integrate downstream of tRNA sites and this action is facilitated by the integrase protein. The integrase is rendered non-functional by two frameshifts. A frameshift is caused by insertion or deletion of nucleotides from a sequence that is not divisible by 3 (Figure 3-17). The earlier the disruption within the ORF occurs, the more
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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
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Cell-surface Proteases Haemolysins
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1.2.1 Pathogenicity islands: a subg
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appear to be related and there have
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assessed in number of assays and mo
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TAR cloning is a method designed by
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Figure 1-3 Top plate shows colony g
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Figure 1-5 depicts the principles o
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Balb/c mice. The models described b
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urns-sepsis model, but not in the n
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ORF ID Gene name Gene function PA14
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functional activities it could be s
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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 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: Figure 3-15 depicts the codon usage
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
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
Page 121 and 122: Fold increase Fold increase 15 10 5
Page 123 and 124: 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
Page 129 and 130: 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
Page 137 and 138: 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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