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Results For both plasmids a 500 bp sequence starting from the pBBR T7 primer binding site was obtained. The sequences were then used for BLAST analysis. Figure 13 summarizes the result obtained for pBBR-11-Sid. For pBBR-23-Sid, the same sequence was obtained, indicating that the same PstI Fragment had been subcloned. i- mini-Tn5 -o 2,8 kb 1,3 kb A acr acsF acsD acsC acsA acsE yhcA acsB cbrA-D B acr acsF acsD acsC acsA acsE yhcA acsB cbrA-D C acsF acr acsD acsC acsA acsE yhcA acsB cbrA-D 2 kb Fig. 13. Location of mini-Tn5 insertion 11-Sid - in Pss22d∆Pvd. BLAST analysis of the insert sequence obtained for pBBR-11-Sid - showed significant similarities to the sequence of the achromobactin biosynthesis cluster in Dikeya chrysanthemi (C) and the two genome sequences of PssB728a (A) and Psp1448A (B). The part of the insert fragment that could be sequenced was located approx. 2.8 kb upstream of the transposon i- end and it aligned with the iucAC-like siderophore synthetase acsD. This indicated, that the transposon itself is inserted in the yhcA-permease. Comparison of the three clusters shows a high similarity in sequence and organisation of the genes, yet the siderophore receptor acr is arranged differently (indicated in red). ORF’s indicated in green could be part of the achromobactin cluster as well. 55
Results Nevertheless the site of transposon insertion varies a little, as restriction analysis could demonstrate (data not shown). Blast analysis of the pBBR-11- Sid 500bp sequence showed significant sequence similarity to genes of the Dickeya chrysanthemi (formerly Erwinia chrysanthemi) achromobactin biosynthesis cluster (GenBank accession number: F416740 and AF416739; Franza, et. al. 2005). Best similarity was obtained for the acsD siderophore synthetase from within this gene cluster. Calculation of the respective fragment sizes indicated, that the localization of transposon insertion should be within the gene yhcA encoding for a permease. The insertion into yhcA was next confirmed by a PCR subcloning technique (Daria Zhurina, personal communication). By the time this sequence information was obtained, draft annotation of the genome sequence of PssB728a became available at GenBank (www.ncbi.nlm.nih.gov/Taxonomy/ taxonomyhome.html, TxID: 205918). In line with our results for Pss22d, the draft annotation indicated the presence of a second siderophore biosynthetic gene cluster in PssB728a. This cluster showed similar organization and high sequence identities to the achromobactin biosynthetic gene cluster from D. chrysanthemi. As the genome sequence of P. syringae pv. phaseolicola 1448A became available, we could identify a similar siderophore biosynthesis cluster in this pathovar, too (Fig.13). There is no homologue of this cluster in strain PsmDC3000. Comparison of the biosynthesis cluster in the three positive strains allowed the following observations: 1) All genes that have been assigned to be involved in achromobactin biosynthesis are present in every strain (Fig.13; grey arrows, genes labeled according to Franza, et. al. 2005). 2) The aminotransferase acsF is located differently in D. chrysanthemi as compared to P. syringae (Fig.13). While it is located upstream of the 56
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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: Results Next, a large number of tra
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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School of Engineering and Science - Jacobs University

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