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Material and Methods 4.5.7 Thin-layer chromatography Thin layer chromatography was performed on silica-based TLC plates (Macherey-Nagel, Düren). Samples were applied as methanol solutions and brought to dryness completely prior to separation. Separation was performed in glass tanks with equilibrated gas phase. The respective mobile phase was prepared freshly prior to TLC separation. After separation, substances were detected by autofluorescence at an excitation wavelength of λ=365nm or quenching of a fluorescence indicator within the silica matrix (excitation λ=254nm). Alternatively, the TLC plate was scanned on the phosphoimager (Fla-3000, Raytest) as described under 4.2.6.3. Detection of siderophore activity was achieved by CAS overlay as described under 4.5.5. 45
Results 5 Results 5.1 Analysis of siderophores produced by Pss22d Under iron-limiting conditions many pseudomonads produce a yellow-greenish, fluorescent pigment, thus they are classified as “fluorescent pseudomonads”. These pigments are peptide-type siderophores called pyoverdins. All pyoverdins share the same dihydroxyquinoline chromophore responsible for their fluorescence, with an amidically connected small dicarboxylic acid (or its monoamide). The peptide chain bound to the chromophore carboxyl group varies. It consists of 6-12 amino acids often including modified amino acids or D-forms (Fuchs et al., 2001). Approximately 50 different pyoverdins have been identified so far and the type of pyoverdin produced can vary even among strains of the same species, for example 3 different pyoverdins have been characterized for P. aeruginosa (de Chial et al., 2003). In contrast to their otherwise prominent diversity, all pathovars of P. syringae tested so far produce the same pyoverdin (Fig.6) (Bultreys et al., 2001; Bultreys et al., 2003; Bultreys et al., 2004; Jülich et al., 2001). In order to validate that pathogen Psg1a and antagonist Pss22d do produce this pyoverdin (Pvd Ps ), a 59 Fe-uptake experiment was performed with the help of our collaboration partner HO L-Thr 1 HO H NH CO N CO OC HN HO COOH D-OHAsp 1 OC L-Thr 2 OH HN COOH OH L-Ser 2 L-Ser 1 D-OHAsp 2 A OC HN HO CO NH COOH HO HO NH 2 CH CH2 CH 2 L-Lys CH 2 CH2 NH H N + O NH NHCOCH 2 CH 2 CONH 2 1, L-Ser 1 2, Gly B HO OC HN OC HN HO OC HN O HO L-Thr 1 L-Thr 2 CO CO NH NH COOH CO HO D-OHAsp OH H N COOH D-OHAsp 1 L-Ser 2 CO HO HO NH 2 CH CH2 CH 2 L-Lys CH2 CH 2 NH H N O + NH NHCOCH2CH2CONH2 Fig. 6. The siderophore pyoverdin of P. syringae. The pyoverdin typical for P. syringae has a seven amino-acid side chain that can be linear (A) or can form a ring between the second threonine and the terminal serine (B). Modified after Bultreys et al., (2004) 46
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Siderophore production of Pseudomon
Page 3 and 4: Acknowledgements This project was s
Page 5 and 6: Abbreviations Ach achromobactin-lik
Page 7 and 8: Contents 4.1.2 Pseudomonas syringae
Page 9 and 10: Contents 5.3 Influence of sideropho
Page 11 and 12: Introduction 2 Introduction Like hu
Page 13 and 14: Introduction In contrast to the rhi
Page 15 and 16: Introduction 2.1.2 Host, pathogen a
Page 17 and 18: Introduction The relationship betwe
Page 19 and 20: Introduction developement of resist
Page 21 and 22: Introduction Fe 2+ + H 2 O 2 → Fe
Page 23 and 24: Introduction 1997a; May et al., 199
Page 25 and 26: Introduction Pss22d demonstrated a
Page 27 and 28: Introduction 2.4 Aim of this study
Page 29 and 30: Material and Methods Tab. 4. Antibi
Page 31 and 32: Material and Methods Iron-free 5b m
Page 33 and 34: Material and Methods Solution3, car
Page 35 and 36: Material and Methods 3.6 Plasmids T
Page 37 and 38: Material and Methods PCR-Screening
Page 39 and 40: Material and Methods on ice for 10
Page 41 and 42: Material and Methods stained in eth
Page 43 and 44: Material and Methods 4.2.5.3 Conver
Page 45 and 46: Material and Methods 4.2.5.9 Ligati
Page 47 and 48: Material and Methods strains were s
Page 49 and 50: Material and Methods 9.5). For sign
Page 51 and 52: Material and Methods gentle shaking
Page 53: Material and Methods columns were w
Page 57 and 58: Results The 13-kb open reading fram
Page 59 and 60: Results If integration is due to a
Page 61 and 62: Results Yersiniabactin is a siderop
Page 63 and 64: Results Next, a large number of tra
Page 65 and 66: Results Nevertheless the site of tr
Page 67 and 68: Results Achr2_KpnI_rev binding site
Page 69 and 70: Results Interestingly the mutant Ps
Page 71 and 72: Results XAD4-purification yielded i
Page 73 and 74: Results After changing the mobile p
Page 75 and 76: Results P P A Fig. 21. Isoelectic f
Page 77 and 78: Results reduced in comparison to th
Page 79 and 80: Results A-E represents single inocu
Page 81 and 82: Results Tab. 10. Development of pop
Page 83 and 84: Results Tab. 11. Distribution of th
Page 85 and 86: Discussion bacteria with redundande
Page 87 and 88: Discussion 6.2 Possible regulation
Page 89 and 90: Discussion stationary phase transit
Page 91 and 92: Discussion and bacterium (Loper et
Page 93 and 94: Literature Bultreys, A., Gheysen, I
Page 95 and 96: Literature Galperin, M. Y. (2006).
Page 97 and 98: Literature Mazzola, M. (2002). Mech
Page 99 and 100: Literature Schubert, S., Rakin, A.,
Page 101: Literature Zou, J., Rodriguez-Zas,
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