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Results 5.1.3 Screening for an additional siderophore of Pss22d by random mutagenesis In order to identify biosynthetic genes responsible of the potential second siderophore of Pss22d, a random mutagenesis was conducted. For this we used a modified mini-Tn5 construct derived from pCAM140 (Wilson et al., 1995). The plasmid pCAM-Not was constructed by Dr. Helge Weingart via deletion of the gusA gene by NotI digest of pCAM140. Random mutagenesis was performed by conjugating the plasmid pCAM-Not in both, Pss22d∆pvsA and Pss22d wt. The random character of transposon insertion-loci was verified by analysis of size distribution among the transposon-tagged fragments in Pss22d∆pvsA tansconjugants, as follows. Genomic DNA of 17 Tn5 mutants of Pss22d∆Pvd was digested with PstI (Fig.11A) and ClaI (Fig.11B) and hybridized with a probe against Tn5 at 60°C. The resulting distribution pattern did not show any redundancy of fragment sizes, thus it could be assumed that the mutagenesis had resulted in random Tn5 insertion. Size (kb) A B Fig. 11. Distribution of mini-Tn5 insertions in Pss22d∆Pvd. 17 Tn5 mutants of Pss22d∆Pvd were analysed for their loci of transposon insertion. Genomic DNA was digested with PstI (A) and ClaI (B) and examined by Southern blot analysis for the size of the Tn5 insertion fragment. The probe was derived against the BamHI fragment of the transposon. 53
Results Next, a large number of transconjugants was screened on CAS agar for siderophore-negative (Sid - ) phenotype (Fig.12). The colony size of the resulting mutants on the indicator plates had a remarkable influence on the size of the siderophore-derived halos. Therefore, all clones that did not produce a halo in the first screening where re-streaked on a CAS agar plate. Out of 500 Tn5- mutants of Pss22d∆Pvd, three siderophore-negative clones were obtained. In contrast and as expected, screening of 1500 Tn5-mutants derived from Pss22d wild type did not result in any siderophore-negative mutants. The loci of transposon insertions in the siderophore-negative mutants were subsequently identified by cloning. Genomic DNA of mutants Pss22d∆Sid, Pss22d∆Sid-23 and Pss22d∆Sid-25 was treated with the following restrictases: PstI, ClaI, XbaI, KpnI, SacI, SacII and SpeI. These digests were ligated into plasmid pBBR-Mcs1 cleaved with the respective restrictase. In order to select for ligation products containing the transposon, the ligation was transformed into E. coli DH5α and transformants were screened for spectinomycin resistance mediated by the mini-Tn5. Two plasmides were derived from this experiment: pBBR-11-Sid and pBBR-23-Sid, containing a PstI insert of Pss22d∆Sid and Pss22d∆Sid-23, respectively. Insert sizes of both plasmids were approx. 6 kb. The plasmids were sent to MWG biotech for sequencing. Fig. 12. Screening of Tn5-mutants for siderophore-negative phenotype on CAS-agar. Transposon mutants of Pss22d∆Pvd were picked on CAS-agar plate and incubated for 48h.The white arrow indicates a sid - clone. 54
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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
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 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: Results Yersiniabactin is a siderop
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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