Bacterial Pathogenesis

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Methods in Microbiology Volume 27
Series Advisors Gordon Dougan Department of Biochemistry, Wolfson Laboratories, Imperial College of Science, Technology and Medicine, London, UK Graham J Boulnois Zeneca Pharmaceuticals, Mereside, Alderley Park, Macclesfield, Cheshire, UK Jim Prosser Department of Molecular and Cell Biology, Marischal College, University of Aberdeen, Aberdeen, UK Ian R Booth Department of Molecular and Cell Biology, Marischal College, University of Aberdeen, Aberdeen, UK David A Hodgson Department of Biological Sciences, University of Warwick, Coventry, UK David H Boxer Department of Biochemistry, Medical Sciences Institute, The University, Dundee, UK
Page 4 and 5: Methods in M i cro bi ol ogy Volume
Page 6 and 7: Contents Contributors .............
Page 8 and 9: 6.10 Bacterial Exotoxins ..........
Page 10 and 11: Contributors Carlos F. Ambbile-Cuev
Page 12 and 13: Michele Farris Department of Bioche
Page 14 and 15: Mark Pallen Microbial Pathogenicity
Page 16 and 17: Preface 'I1 n'existe pas de science
Page 18 and 19: unknown (or unpredictable) function
Page 20 and 21: eflects a growing realization in re
Page 22 and 23: SECTION I Detection of Virulence Ge
Page 24 and 25: 1.1 Detection of Virulence Genes Ex
Page 26 and 27: permitting the simultaneous screeni
Page 28 and 29: tion have been used successfully to
Page 30 and 31: are impaired in their ability to su
Page 32 and 33: References most numerous facultativ
Page 34 and 35: SECTION 2 Laboratory Safety - Worki
Page 36 and 37: 2.1 Introduction Paul V. Tearle Nat
Page 38 and 39: Reference Thus, in both Chapters 2.
Page 40 and 41: 2.2 Safe Working Practices in the L
Page 42 and 43: it is the physical, noninfectious i
Page 44 and 45: difficulty of doing anything about
Page 46 and 47: elimination (1, the most effective)
Page 48 and 49: Sub-cause Age of staff Poor supervi
Page 50 and 51: 2.3 Safe Working Practices in the L
Page 52 and 53:
analysis and audit may also be used
Page 54 and 55:
W w Second stage controls. . contro
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By-products Information References
Page 58 and 59:
SECTION 3 Detection, Speciation and
Page 60 and 61:
3.1 Introduction Peter Vandamme Pos
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3.2 Detection Margareta leven Assoc
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visualization of bacteria, but also
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days to weeks; many definitive test
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(NASBA) and the transcription media
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legionellae in respiratory specimen
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3.3 Speciation Peter Vandamme Postd
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classdymg bacteria was analyzed. Am
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sequences indeed never show signifi
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3.4 Identification Peter Vandamme P
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tional growth and metabolization of
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DNA sequence. Furthermore, 16s rRNA
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method suffice for numerous isolate
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List of Suppliers Polyphasic taxono
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SECTION 4 Host Interactions - Anima
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4.1 Introduction T. J. Mitchell Div
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e examined histologically. Organ cu
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4.2 Interaction of Bacteria and The
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The complexity of pathogenic mechan
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++++++ COMPARISON OF DIFFERENT ORGA
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microscopy can assess cell damage (
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cient and deficient isogenic varian
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4.3 Transgenic and Knockout Animals
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(Hogan et al., 1994). The DNAmay be
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Knoc k-i n Animals In some experime
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etween two copies of the tamoxifen-
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g I Table 4.4. Examples of the use
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* Table 4.5. Examples of the use of
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g m Table 4.5 continued IFN-y IFN-Y
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Numerous knockout mouse strains tha
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Gossen, M. and Bujard, H. (1992). T
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Zinkernagel, R., Seimentz, M. and B
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4.4 Interactions of Bacteria and Th
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Route of Infection of cloned animal
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Measure required length of intestin
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sampling, and be well tolerated by
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Guerry, P., Pope, P. M., Burr, D. H
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4.5 Interaction of Bacteria and The
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washed before resuspending in mediu
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the bacterial culture broth with th
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cells in suspension for several rea
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that bacterial cytotoxic factors or
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SECTION 5 Host I n te ract io ns -
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5.1 Introduction Michael J. Daniels
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References bacteria often carry sin
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5.2 Testing Pathogenicity Michael J
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hosts than leaf spotting or wilt pa
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different plants can be infiltrated
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Table 5. I. The behavior of X. c pv
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Rudolph, K., Roy, M. A., Sasser, M.
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5.3 hup Genes and Their Function Al
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mental treatments. The timing and a
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essential positive regulators, or t
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of the Hrp system is presently obsc
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Dong, X., Mindrinos, M., Davis, K.
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5.4 Avirulence Genes Ulla Bonas lns
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of tissue due to disease developmen
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genes are tested for their plant in
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Bacteria expressing avirulence gene
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5.5 Extracellular Enzymes Their Rol
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- m Table 5.5. Extracellular enzyme
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+++a++ GENETIC DETERMINANTS OF EXTR
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Consequently, the precise link betw
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processing and so GspG, -H, -I, and
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++++++ ACKNOWLEDGEMENTS References
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5.6 Bacterial Phytotoxins Carol L.
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ment. Since most bacterial phytotox
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ecently used in transgenic plants a
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Jones, J. D. G. and Gutterson, N. (
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5.7 Epiphytic Growth and Survival S
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++++++ EFFECTS OF SCALE ON SAMPLING
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References should be avoided so tha
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SECTION 6 Biochemical Approaches 6.
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6.1 Introduction: Fractionation of
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Gram-negative Bacteria Washed bacte
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References trated lipoproteins, whi
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6.2 The Proteorne Approach C. David
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determining how its removal (e.g. b
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Spot Detection to one week before f
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MS acid residues in a polypeptide s
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An extension of the mass matching p
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approaches such as the construction
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infected with C. truchomutis, over
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6.3 Microscopic Approaches to the S
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The antigen of interest can be loca
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Figure 6.5. Whole-cell preparation
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Table 6.3. Pre-embedding immunolabe
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References Bacterial cell-associate
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6.4 Characterization of Bacterial S
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against a range of concentrations o
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single class of binding site on the
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Gel Filtration Whole cells are remo
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References This chapter has set out
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List of Suppliers Wall, J., Ayoub,
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6.5 Characterizing Flagella and Mot
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Phase-contrast microscopic observat
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of flagellar insertion as the ghost
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A last word of advice - do always c
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Tethered Cells Uniflagellate bacter
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Aizawa, $I., Dean, G. E., Jones, C.
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6.6 Fimbriae: Detection, Purificati
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Type 1 fimbriae, found on the major
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Detection of Fimbriae by Adhesive C
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~~~ Figure 6.16. Immunoelectron mic
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References The authors wish to than
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6.7 Isolation and Purification of C
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exopolysaccharides, EPSs) are relea
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cise chemistry of the LPS, and for
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Hitchcock-Brown whole-cell lysates
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Gotschlich, E. C., Fraser, 8. A., N
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6.8 Structural Analysis of Cell Sur
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26 I 3 Figure 6.18.1-D 'H NMR spect
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Composition Analysis Colorimetric a
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sugars with optically active alcoho
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Periodate oxidation Partial hydroly
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A 0 8H-lb.20 8 ti- IC. 40 Q ti- tc.
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the 6-deoxysugars, residues b and d
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References cal and NMR methods. The
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List of Suppliers The following is
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6.9 Techniques for Analysis of Pept
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peptidoglycan isolation from these
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control and constant evaluation of
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Labeling Reaction and Sample Requir
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Billot-Klein, D., Gutmann, L., Sabl
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6.10 Bacterial Exotoxins Mahtab Moa
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ourselves to a summary of character
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(repeats in toxin) exotoxin family,
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Aeromonas hydrophila aerolysin, Ser
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1997). P. aeruginosa exotoxin A and
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Aktories, K. and Mohr, C. (1992). C
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Menestrina, G. F. (1991). Electroph
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6.11 A Systematic Approach to the S
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6 7 B 1 2 3 4 5 C 1 2 3 N-terminal
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Analysis of Bacterial Cell Supernat
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tion as the components of the secre
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to attribute a role in secretion fo
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uninterpretable. A number of differ
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than one secretion machinery with v
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References overexpression of a wild
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Merck, K. B., de Cock, H., Verheij,
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6.12 Detection, Purification, and S
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an exogenous AHL (preferably OHHL)
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+++we SEPARATION, IDENTIFICATION AN
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lock the HPLC columns. Attention sh
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Figure 6.27. EI-MS fragmentation pa
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Synthesis of N-(3-oxoacyl)-~-homose
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6.13 Iron Starvation and Siderophor
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eceptor proteins form part of a hig
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phosphate-buffered systems can inte
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advantages both of speed and chemic
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produced by other bacterial and fun
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Ecker, D. J., Loomis, L. D., Cass,
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SECTION 7 Molecular Genetic Approac
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7.1 Introduction Charles J. Dorman
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spectrum of difficulty lies Chlamyd
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7.2 Genetic Approaches to the Study
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transposon mutagenesis, which combi
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Once a virulence gene has been iden
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References Alpuche-Aranda, C. M., S
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and virulence determinants in Vibri
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7.3 Signature Tagged MutagenesW * S
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sequence twice in a pool of 96 diff
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Table 7.4. Generation of the mutant
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Table 7.5. Colony blots 1. Grow the
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we found that approximately 25% of
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References List of Suppliers primer
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7.4 Physical Analysis of the Salmon
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Table 7.8. Isolation of intact geno
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Cycle 2 Cycles 3 and 4 Frequently,
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0 2 4 6 8 10kb t 1 1 1 1 1 1 1 1 1
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1 2 3 4 5 I II Figure 7.3. (a) Auto
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List of Suppliers Liu, S.-L., Hesse
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7.5 Molecular Analvsis of Pseudomon
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of P. aeruginosa virulence genes ha
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Table 7.13. Murine’ models in use
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Table 7.14. Virulence of P. oerugin
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Cash, H. A., Woods, D. E., McCullou
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Stieritz, D. D. and Holander, I. A.
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7.6 Molecular Genetics of Bordetell
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are under the control of a single g
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which may play a fundamental role i
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et al., 1988). On the contrary, the
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Figure 7.6. Schematic representatio
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Beier, D., Schwarz, B., Fuchs, T. M
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7.7 Genetic Manipulation of Enteric
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confers a catalase-positive phenoty
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esistance (Tc) (Sougakoff et al., 1
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Figure 7.7. (b) Schematic outline o
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Natural transformation strains test
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References There has been rapid pro
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List of Suppliers of ferritin to ir
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7.8 Molecular Approaches for the St
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Table 7.22. Isolation of chromosoma
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Table 7.25. Protoplast transformati
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Colonies obtained after transformat
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References Table 7.29. RNA isolatio
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List of Suppliers Oxoid Monograph N
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7.9 Molecular Genetic Analvsis of S
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Table 7.30. Transposon mutagenesis
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(2) Linkage between the transposon
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introduce point mutations generated
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marker. The majority of bacteria th
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Resolution of Cointegrants by Trans
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performed using a fragment encompas
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transformation frequency is too low
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quency confines this procedure to c
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Bramley, A. J., Patel, A. H., O'Rei
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Novick, R. P. (1990). The staphyloc
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7.10 Molecular A P proaches to Stud
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DNA (Tan et al., 1995). Chlamydial
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was cloned from a pUC 8 expression
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factors have, so far, been unsucces
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tion of RNA polymerase core subunit
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SECTION 8 Gene txpression and Analy
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8.1 Gene Expression and Analysis Ia
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in multi-copy can titrate out possi
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permits direct targeting of putativ
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Northern blot analysis RT-PCR label
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accurately and in conjunction with
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DNA Footprinting This is a widely u
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(Frackman et al., 1990). It has bee
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Berg, C. M and Berg, D. E. (1996).
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List of Suppliers Simpson, D. A. C.
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SECTION 9 Host Reactions - Animals
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9.1 Introduction Mark Roberts Depar
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in the case of septic shock. It is
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9.2 Strategies for the Studv of Int
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and saturable inhibition of bacteri
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adhesins. To facilitate the purific
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References We thank all the workers
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9.3 Cytoskeletal Rearrangements Ind
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(3) The interaction of intimin of e
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easy. For example, actin rearrangem
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Using mutated cell lines Inability
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Rosenshine, I., Donnenberg, M. S.,
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9.4 Activation and Inactivation of
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Actin Biochemistry guished. Intrace
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Figure 9.2. Schematic depiction of
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++++++ Rho-MODULATING BACTERIALTOXI
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Since the toxins are catalytically
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TcdB-I 0463 Tcd B-I 470 TcsL-82 48
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Effect on Bacterial Use of Actin Li
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small GTP-binding protein Rho regul
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tion of phorbol ester-stimulated ph
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9.5 Strategies for the Use of Gene
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+++a++ USE OF CYTOKINE AND CYTOKINE
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T Cell Proliferation Cytokine Studi
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Antibody Subclasses FACS Analysis S
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OCallaghan, D., Maskell, D., Liew,
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SECTION 10 Host Reactions - Plants
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10.1 Host Reactions - Plants Charle
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Xanthomonas campestris pv. manihoti
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immunocytochemical studies within t
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Figure 10.2. Development of paramur
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etention and antibody recognition w
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Figure 10.4. Detection of phenolics
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Table 10.5. Histochemical detection
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Figure 10.6. Peroxidase activity in
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Figure 10.7. Localization of H,02 a
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A comparative summary of reactions
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Table 10.9. Preparation of gold pro
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symptom characteristic of infection
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Figure 10.10, Detection of flavanoi
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for conjugation via the hydroxy moi
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Figure 10.11 (A) Transmitted white
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References and protein/DNA complexe
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Buja, M., Eigenbrodt, M. L. and Eig
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Mansfield, J., Jenner, C., Hockenhu
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SECTION II Strategies and Problems
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11.1 Introduction Alejandro Craviot
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11.2 Strategies for Control of ComG
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endothelial cells to reach the subm
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The epidemiology of cholera is an i
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Table I I. I. Available animal mode
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acelular combinada con 10s toxoides
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11.3 Antibiotic Resistance and Pres
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(1) Enzymatic inactivation of the d
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ampicillin in these same strains in
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Single-event antibiotic usage may s
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SECTION I2 Internet Resources for R
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12.1 Internet Resources for Researc
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and DNA sequences (http://www.ncbi.
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++++++ SEQUENCE DATABASES The US Na
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searched as one of the options on t
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Web sites (e.g. httpJ/www.biochern.
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Index Accidents, 19,31 analysis, 27
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preliminary considerations, 232-233
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Exoproteins see Secreted proteins,
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Immunological detection of fimbriae
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tissue choice, 78 toxin measurement
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identification, 20-21 pathogen cate
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Transgenic animals, 83 in bacterial
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