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Results Fig. 18. Seperation of the achromobactin-containing XAD4 purificationS by TLC. XAD4 purified supernatant of Pss22d∆Pvd separated by TLC on silica plate (mobile phase: Diethylether : n-butanole: acetic acid: water; ratio 1:1:1:1; v/v). Fluorescence of the sample was visualizes by UV 365nm . Drawn line at the bottom indicates the start and the uppermost fluorescent line represents the running front. Separations of the Pss22d∆Pvd XAD4-purification were compared to that of the Pss22d∆Sid and a control achromobactin sample purified from D. chrysanthemi cbsE-1 tonB60 (Fig.19). In all three samples the majority of fluorescent components migrated to more than half of the running front. The CAS-overlay of the TLC plates showed a positive reaction for authentic achromobactin and the Pss22d∆Pvd XAD4-purification (Fig19A). In both samples the CAS-active substance had moved only slightly (retention factor of 0.25). As expected this band was absent in the siderophore-deficient strain. No fluorescent band could be associated to the CAS-active region on the plate, indicating that neither the achromobactin-like siderophore nor authentic achromobactin were visible at the wavelength tested. A B ← 1 2 3 1 2 3 Fig. 19. CAS overlay of TLC separated XAD4 purification. XAD4 purified supernatant separated by TLC on silica plate with different mobile phases: A) Diethylether : n-butanole: acetic acid: water; ratio 1:1:1:1; (v/v). B) MTBE : Methanol: acetic acid : water; ratio 3:1:1:1 (v:v). Samples: 1) Pss22d∆Pvd 2) Pss22d∆Sid 3) Achromobactin purified from D. chrysanthemi cbsE-1 tonB60. The arrow indicates CAS-active band of Pss22d∆Pvd, the less intensive signal below this band is due to long incubation time of the overlay. 63
Results After changing the mobile phase to methyl t-buthyl ether (MTBE), methanol, acetic acid and water (3:1:1:1, v/v) the retention factor of the CAS-active substance was increased to 0.45 (Fig.19B). Interestingly, the retention factor for authentic achromobactin differs from that (0.35). The smearing of the CASactive band of Pss22d∆Pvd (Fig.19B) was likely due to a relatively long incubation of the overlay in order to visualize a second signal above the major CAS-active band. This additional signal could be observed in both siderophorepositive samples and may correspond to a second conformation of the siderophore. The original CAS-active band of Pss22d∆Pvd corresponds to the more intensive spot indicated by the arrow (Fig.19B). Approximately the same amount of XAD4-purification was applied for both strains and the intensity of the different fluorescent bands did not differ significantly between both lanes. In contrast, the siderophore activity detected for D. chrysanthemi cbsE-1 tonB60 was much lower as compared to Pss22d∆Pvd. The modified separation conditions are currently applied on the next large-scale purification. After this step, another TLC-separation using a different stationary phase will be used for further purification. Macherey-Nagel offers a range of differently modified silica plates with varying hydrophobicities of the matrices. The affinity of the CAS-active substance to silica modified by addition of a diolor an amino group was tested (Fig.20). The affinity of the CAS-active substance towards the stationary phase was lower for the diol modification and higher for the amino modified plate. Thus the diol modified plate could be optimized for better focusing of the CAS-active band, while the amino-modified phase could be used to separate other disturbing substances. 64
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Siderophore production of Pseudomon
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Acknowledgements This project was s
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Abbreviations Ach achromobactin-lik
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Contents 4.1.2 Pseudomonas syringae
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Contents 5.3 Influence of sideropho
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Introduction 2 Introduction Like hu
Page 13 and 14:
Introduction In contrast to the rhi
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Introduction 2.1.2 Host, pathogen a
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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 and 54: Material and Methods columns were w
Page 55 and 56: Results 5 Results 5.1 Analysis of s
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: Results XAD4-purification yielded i
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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