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Results siderophore receptor gene acr in D. chrysanthemi, it is downstream of this ORF in the pseudomonads. 3) Certain additional genes could potentially be part of the siderophore cluster (Fig.13 indicated in green). Following cbrD, there is a putative methyltransferase gene, conserved in all three strains. In both pseudomonads a putative regulator containing a sensory domain with week similarity to chemotaxis/aerotaxis and a regulator containing a GGDEF domain involved in cyclic-diGMP signaling are present directly after the siderophore cluster. Additionally, in both P. syringae strains two putative regulatory genes are located in front of the siderophore receptor. The first is a homologue of the alternative sigma factor FecI, involved in ferric citrate uptake. The second shows similarities to the regulator FecR that interacts with FecI (Visca et al., 2002). As Pss22d∆Sid is the best characterized of the three transposon mutants, this derivation was used for further analysis. 5.1.4 Construction of a marker exchange mutant of the achromobactinlike siderophore in Pss22d In order to address the impact of the achromobactin-like siderophore on the phenotype of Pss22d, a non-polar marker exchange mutant, deficient for the biosynthesis of this siderophore was constructed. The achromobactin biosynthesis cluster encodes for three copies of a siderophore synthetase, namely AcsA, AcsC, and AscD. These synthetases are supposed to be responsible for the amide bond formation connecting the achromobactin precursor molecules (Fig.14). The first of the three corresponding genes is acsD. In mutant Pss22d∆Ach, this ORF has been exchanged against a kanamycin cassette (Fig.15). For the construction of this mutant, a 1.3-kb PCR fragment was amplified by PCR using the primer combination Achr1_fwd and Achr2_KpnI_rev. The PCR fragment was purified and ligated into the vector pGemT-easy. The resulting plasmid pACS1 was selected for orientation of the 57
Results Achr2_KpnI_rev binding site next to the SpeI recognition site of the vector. A second PCR fragment of 2 kb was amplified by the primer pair Achr5_EcoRV_fwd and Achr6_rev. The PCR product was purified and brought into pGemT-easy, too. This plasmid was designated pACS2. The 1.7-kb PstI fragment of pMKm was purified and blunt-ended by application of Klenow fragment. Plasmid pACS1 was linearized by SpeI, blunt-ended and fused with the kanamycin resistance cassette by ligation. Fig. 14. Structure of the siderophore achromobactin. The oxo-glutaric acid side chains of achromobactin can cyclize. The structure shown on the right prevails under neutral conditions. The siderophore synthetases AcsA, AcsC and AcsD are supposed to form the amide bonds present in achromobactin. Modified after Franza, et al., (2005). The resulting plasmid carrying a 3 kb insert was designated pACS3. This plasmid was linearized by the restrictase ScaI and afterwards treated with EcoRI, resulting in a 3-kb fragment of the insert and parts of the vector backbone. The 3-kb fragment was purified, blunt ended and ligated into the EcoRV-opened plasmid pACS2. Correct orientation of the fragments to each other, resulting in the kanamycin cassette being flanked by both PCR fragmentes was verified by restriction digest with the restrictase PstI. As PCR fragment 1 carries an internal recognition site for this enzyme in addition to a single recognition site in the vector sequence, the fragment pattern of a plasmid with insert in correct orientation could be unambiguously identified. 58
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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
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 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: Results Nevertheless the site of tr
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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School of Engineering and Science - Jacobs University

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