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Material and Methods 4.2.6.2 Alkaline transfer of DNA The DNA for Southern blot analysis was separated by gelelectrophoresis (4.2.3) on a 0.8% agarose gel. After documentation of the gel, the DNA was transferred to a positively charged nylon membrane (Hybond N + ) by capillary transfer using 5x SSC/10 mM NaOH buffer. The transfer was performed over night. After transfer the DNA was fixed to the membrane using a UVcrosslinker. The dried membrane could be stored at room temperature until hybridisation. Composition of 20x SSC is 3 M NaCl and 0.3 M tri-sodium citrate. 4.2.6.3 Hybridisation and detection Prior to hybridisation with the probe, the membrane was incubated with hybridisation buffer (0.1% n-laurylsarcosin, 0.02% SDS and 1% blocking reagent in 5x SSC) for 1 hour. In the meantime, the DNA-probe was denaturated for 10 min. at 95°C and then shock-cooled in an ethanol/ice bath. Then the probe was resuspended in 10 ml of pre-warmed hybridisation buffer. Then the probe/buffer mixture was added to the membrane and hybridised for 12-16 hours at the respective temperature. After hybridisation the membrane was washed two times under non-stringent conditions (5 min. room temperature in 2x SSC, 0.1% SDS) and two times under stringent conditions (15 min at hybridisation temperature in 0.1x SSC, 0.1% SDS). Thereafter the membrane was equilibrated at room temperature for 1 min. in buffer 1 (100 mM Tris/HCl at pH 7.5 with 150 mM NaCl). Then the membrane was incubated at room temperature for 30 min. in the blocking buffer 2 (0.5% blocking reagent in buffer 1). After a washing the membrane in buffer 1, the antibody solution (2 µl of anti-digoxygenin-ap, fab-fragment by Roche, Mannheim, in 20 ml of buffer 1) was added to the membrane and incubated at room temperature for 30 min.. Any excess of antibody was removed by two washing steps in buffer 1 (15 min., room temperature). Finally, the membrane was equilibrated for 1 min. in the detection buffer (0.1 M Tris/HCL, 100 mM NaCl, and 50 mM MgCl 2 at an pH of 39
Material and Methods 9.5). For signal generation, the membrane was incubated with the ECFsubstrate (Amersham Pharmacia) on a glass tray. The signal was detected on a phosphoimager (FLA-3000, Raytest). The membrane was scanned at excitation wavelength 475 nm and emission signals above 520 nm were detected. 4.2.7 DNA-sequencing DNA sequencing was performed at MWG Biotech (Ebersberg). 4.3 Analysis of bacterial growth in vitro For growth curve analysis of P. syringae bacterial cultures were inoculated to an OD 600nm of 0.05 to 0.1 with an exponentially growing pre-culture. The cultures were incubated at 28°C by shaking at 280 rpm. For each strain 3 separate cultures were analysed in parallel. 4.4 Analysis of bacterial growth in planta For in planta analysis, soybean plants of the cultivar “Maple Arrow” were grown in light shelves (Conviron) at approx. 23°C with a light period of 16 hours. Relative humidity was approx. 40%. 4.4.1 Wound inoculation Bacterial inoculum was prepared by resuspending cells from a fresh KB-agar plate in sterile water. OD 600nm of the suspension was adjusted to 0.01. For inoculation of the antagonist Pss22d, this suspension was mixed 2:1 with sterile water. For inoculation of the pathogen Psg1a, the suspension was diluted 1:2 with sterile water. For co-inoculation, 1 volume of pathogen suspension was mixed with 2 volumes of antagonist suspension. Subsequently, leafs of 4-weekold soybean plants were punched with a sterile needle (10 wounds per leaflet). Each wound was inoculated with 5 µl of bacterial suspension. For 40
Page 1 and 2: 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: Material and Methods strains were s
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 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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