HACKER, J. and KAPER, J.B., 2000. Pathogenicity islands and <strong>the</strong> evolution <strong>of</strong> microbes. Annual Review <strong>of</strong> Microbiology, 54, pp. 641-679. HAFT, D.H., SELENGUT, J., MONGODIN, E.F. and NELSON, K.E., 2005. A guild <strong>of</strong> 45 CRISPR-associated (Cas) prote<strong>in</strong> families and multiple CRISPR/Cas subtypes exist <strong>in</strong> prokaryotic genomes. PLoS computational biology, 1(6), pp. e60. HAUSER, A.R., COBB, E., BODI, M., MARISCAL, D., VALLES, J., ENGEL, J.N. and RELLO, J., 2002. Type III prote<strong>in</strong> secretion is associated with poor cl<strong>in</strong>ical outcomes <strong>in</strong> patients with ventilator-associated pneumonia caused by <strong>Pseudomonas</strong> aerug<strong>in</strong>osa. Critical care medic<strong>in</strong>e, 30(3), pp. 521-528. HAUSER, A.R., KANG, P.J. and ENGEL, J.N., 1998. PepA, a secreted prote<strong>in</strong> <strong>of</strong> <strong>Pseudomonas</strong> aerug<strong>in</strong>osa, is necessary for cytotoxicity and <strong>virulence</strong>. Molecular microbiology, 27(4), pp. 807-818. HAUSSLER, S., 2004. Bi<strong>of</strong>ilm formation by <strong>the</strong> small colony variant phenotype <strong>of</strong> <strong>Pseudomonas</strong> aerug<strong>in</strong>osa. Environmental microbiology, 6(6), pp. 546-551. HE, J., BALDINI, R.L., DEZIEL, E., SAUCIER, M., ZHANG, Q., LIBERATI, N.T., LEE, D., URBACH, J., GOODMAN, H.M. and RAHME, L.G., 2004. <strong>The</strong> broad host range pathogen <strong>Pseudomonas</strong> aerug<strong>in</strong>osa stra<strong>in</strong> PA14 carries two pathogenicity islands harbor<strong>in</strong>g plant and animal <strong>virulence</strong> genes. Proceed<strong>in</strong>gs <strong>of</strong> <strong>the</strong> National Academy <strong>of</strong> Sciences <strong>of</strong> <strong>the</strong> United States <strong>of</strong> America, 101(8), pp. 2530-5. HUANG, Y.H., FERRIERES, L. and CLARKE, D.J., 2006. <strong>The</strong> <strong>role</strong> <strong>of</strong> <strong>the</strong> Rcs phosphorelay <strong>in</strong> Enterobacteriaceae. Research <strong>in</strong> microbiology, 157(3), pp. 206- 212. IMAMURA, Y., YANAGIHARA, K., TOMONO, K., OHNO, H., HIGASHIYAMA, Y., MIYAZAKI, Y., HIRAKATA, Y., MIZUTA, Y., KADOTA, J., TSUKAMOTO, K. and KOHNO, S., 2005. Role <strong>of</strong> <strong>Pseudomonas</strong> aerug<strong>in</strong>osa <strong>quorum</strong>-<strong>sens<strong>in</strong>g</strong> systems <strong>in</strong> a mouse model <strong>of</strong> chronic respiratory <strong>in</strong>fection. Journal <strong>of</strong> medical microbiology, 54(Pt 6), pp. 515-518. JANDER, G., RAHME, L.G. and AUSUBEL, F.M., 2000. Positive correlation between <strong>virulence</strong> <strong>of</strong> <strong>Pseudomonas</strong> aerug<strong>in</strong>osa mutants <strong>in</strong> mice and <strong>in</strong>sects. Journal <strong>of</strong> Bacteriology, 182(13), pp. 3843-3845. JANSEN, R., EMBDEN, J.D., GAASTRA, W. and SCHOULS, L.M., 2002. Identification <strong>of</strong> genes that are associated with DNA repeats <strong>in</strong> prokaryotes. Molecular microbiology, 43(6), pp. 1565-1575. KLOCKGETHER, J., REVA, O., LARBIG, K. and TUMMLER, B., 2004. Sequence analysis <strong>of</strong> <strong>the</strong> mobile genome island pKLC102 <strong>of</strong> <strong>Pseudomonas</strong> aerug<strong>in</strong>osa C. Journal <strong>of</strong> Bacteriology, 186(2), pp. 518-534. KLOCKGETHER, J., WURDEMANN, D., REVA, O., WIEHLMANN, L. and TUMMLER, B., 2007. Diversity <strong>of</strong> <strong>the</strong> abundant pKLC102/PAGI-2 family <strong>of</strong> 174
genomic islands <strong>in</strong> <strong>Pseudomonas</strong> aerug<strong>in</strong>osa. Journal <strong>of</strong> Bacteriology, 189(6), pp. 2443-2459. KOUPRINA, N. and LARIONOV, V., 2006. Selective isolation <strong>of</strong> mammalian genes by TAR clon<strong>in</strong>g. Current protocols <strong>in</strong> human genetics / editorial board, Jonathan L.Ha<strong>in</strong>es ...[et al.], Chapter 5, pp. Unit 5.17. KUKAVICA-IBRULJ, I., BRAGONZI, A., PARONI, M., WINSTANLEY, C., SANSCHAGRIN, F., O'TOOLE, G.A. and LEVESQUE, R.C., 2008. In vivo growth <strong>of</strong> <strong>Pseudomonas</strong> aerug<strong>in</strong>osa stra<strong>in</strong>s PAO1 and PA14 and <strong>the</strong> hypervirulent stra<strong>in</strong> LESB58 <strong>in</strong> a rat model <strong>of</strong> chronic lung <strong>in</strong>fection. Journal <strong>of</strong> Bacteriology, 190(8), pp. 2804-2813. KULASEKARA, B.R., KULASEKARA, H.D., WOLFGANG, M.C., STEVENS, L., FRANK, D.W. and LORY, S., 2006. Acquisition and evolution <strong>of</strong> <strong>the</strong> exoU locus <strong>in</strong> <strong>Pseudomonas</strong> aerug<strong>in</strong>osa. Journal <strong>of</strong> Bacteriology, 188(11), pp. 4037-4050. LARBIG, K.D., CHRISTMANN, A., JOHANN, A., KLOCKGETHER, J., HARTSCH, T., MERKL, R., WIEHLMANN, L., FRITZ, H.J. and TUMMLER, B., 2002. Gene islands <strong>in</strong>tegrated <strong>in</strong>to tRNA(Gly) genes confer genome diversity on a <strong>Pseudomonas</strong> aerug<strong>in</strong>osa clone. Journal <strong>of</strong> Bacteriology, 184(23), pp. 6665-6680. LARIONOV, V., KOUPRINA, N., GRAVES, J. and RESNICK, M.A., 1996. Highly selective isolation <strong>of</strong> human DNAs from rodent-human hybrid cells as circular yeast artificial chromosomes by transformation-associated recomb<strong>in</strong>ation clon<strong>in</strong>g. Proceed<strong>in</strong>gs <strong>of</strong> <strong>the</strong> National Academy <strong>of</strong> Sciences <strong>of</strong> <strong>the</strong> United States <strong>of</strong> America, 93(24), pp. 13925-30. LASKOWSKI, M.A. and KAZMIERCZAK, B.I., 2006. Mutational analysis <strong>of</strong> RetS, an unusual sensor k<strong>in</strong>ase-response regulator hybrid required for <strong>Pseudomonas</strong> aerug<strong>in</strong>osa <strong>virulence</strong>. Infection and immunity, 74(8), pp. 4462-4473. LAU, G.W., RAN, H., KONG, F., HASSETT, D.J. and MAVRODI, D., 2004. <strong>Pseudomonas</strong> aerug<strong>in</strong>osa pyocyan<strong>in</strong> is critical for lung <strong>in</strong>fection <strong>in</strong> mice. Infection and immunity, 72(7), pp. 4275-4278. LEE, D.G., URBACH, J.M., WU, G., LIBERATI, N.T., FEINBAUM, R.L., MIYATA, S., DIGGINS, L.T., HE, J., SAUCIER, M., DEZIEL, E., FRIEDMAN, L., LI, L., GRILLS, G., MONTGOMERY, K., KUCHERLAPATI, R., RAHME, L.G. and AUSUBEL, F.M., 2006. Genomic analysis reveals that <strong>Pseudomonas</strong> aerug<strong>in</strong>osa <strong>virulence</strong> is comb<strong>in</strong>atorial. Genome biology, 7(10), pp. R90. LEE, V.T., SMITH, R.S., TUMMLER, B. and LORY, S., 2005. Activities <strong>of</strong> <strong>Pseudomonas</strong> aerug<strong>in</strong>osa effectors secreted by <strong>the</strong> Type III secretion system <strong>in</strong> vitro and dur<strong>in</strong>g <strong>in</strong>fection. Infection and immunity, 73(3), pp. 1695-1705. LEEM, S.H., NOSKOV, V.N., PARK, J.E., KIM, S.I., LARIONOV, V. and KOUPRINA, N., 2003. Optimum conditions for selective isolation <strong>of</strong> genes from complex genomes by transformation-associated recomb<strong>in</strong>ation clon<strong>in</strong>g. Nucleic acids research, 31(6), pp. e29. 175
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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
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RhlI Autoinducer RhlR Rhl Regulon G
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1.5.2 Pseudomonas aeruginosa LES Th
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2 Material and Methods 2 MATERIAL A
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2.1.2 Strains used Pseudomonas aeru
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Plasmids Plasmid Description Refere
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In the case of capture vector const
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Primer name Sequence Capture vector
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The relevant homology sequence was
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Important to the use of the capture
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2.3.4 Electroporation: Transfer of
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2.4.2 Infection dose preparation Th
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mould was then added to the metal c
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3 Analysis of genomic islands captu
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3.1 Capture experiments involving g
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DNA No of colonies Control No DNA 0
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were designed based on the assumpti
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3.2.1 KR115-lys10 and KR159-lys10 A
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estriction digestion with I-SceI to
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generated. The most significant nuc
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they both produced three fragments
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elated genomic islands and this res
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All the Yersinia species available
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Leicester). E105-leuX was the first
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The strains highlighted with alignm
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predict this protein to be a dnaJ-c
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Figure 3-15 depicts the codon usage
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ORF Gene range Blastx Description y
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ecognition sequences are found with
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(S: 150, E: 1e-37). This predicts t
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having multiple copies of itself. A
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The capture vectors used during thi
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cloning and their findings was inco
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The genomic island capture method c
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unpublished). This was achieved by
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Pseudomonas aeruginosa PA14 (UCBPP-
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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
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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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