School of Engineering and Science - Jacobs University

More documents

Recommendations

Info

Results The signal for PCR fragment 1 is shifted as compared to Pss22d wt. This shift is due to the insertion of the resistance cassette and the loss of a chromosomal ApaLI site within the deleted sequence. Transformant no. 5 was designated Pss22d∆Pvd and further analyzed for its siderophore phenotype. As expected, Pss22d∆pvsA was no longer fluorescent when grown on King’s B medium indicating the deficiency of the strain to produce pyoverdin (Fig.9A). But surprisingly, it still produced a halo on CAS-agar almost as large as the wild type strain (Fig.9B). This halo clearly indicated that mutant Pss22d∆pvsA produces a functional siderophore although the pyoverdin biosynthesis of this strain disrupted successfully. 5.1.2 Southern blot analysis of Psg1a and Pss22d for the presence of yersiniabactin biosynthetic genes At the time of construction of Pss22d∆Pvd, the first genome sequence of a P. syringae was completed, namely the sequence of PsmDC3000. With the finished annotation of the genome project, the presence of a yersiniabactin biosynthesis cluster in PsmDC3000 was reported (Buell et al., 2003). Pss22d∆Pvd Pss22d A B Fig. 9. Siderophore production of Pss22d and Pss22d∆Pvd. A: Fluorescence of pyoverdin on Kings B medium; B: Siderophore production on CAS agar. Bacterial suspension was applied to the respective medium and incubated at 28°C for 48h. 51
Results Yersiniabactin is a siderophore first identified in Yersinia pestis but also reported for a number of other bacteria like for example uropathogenic E. coli (Schubert et al., 1998). BLAST analysis of the available sequence for PssB728a did not show any possible candidates of yersiniabactin biosynthetic genes. Since it was unclear whether Pss22d produces yersiniabactin, a Southern blot analysis was performed. The NRPS encoded by the irp1 gene is necessary for yersiniabactin synthesis, thus a probe was derived against the irp1 homologue of DC3000 via PCR and used for hybridization of KpnI-digested genomic DNA of Psg1a, Pss22d, PssB728a and PsmDC3000. Hybridization temperature was 55°C. The only signal detected was that of the control, PsmDC3000 (Fig.10). Although we can not completely exclude the presence of a yersiniabactin biosynthetic gene cluster in Psg and Pss, for the tested strains the presence of yersiniabactin biosynthetic genes is unlikely. 1 2 3 4 10 kb Fig. 10. Screening for the yersiniabactin biosynthesis gene irp1 by southern blot analysis. KpnI digested genomic DNA of Psg1a (1), Pss22d (2), PssB728a (3) and PsmDC3000 (4) was hybridised with a probe against the yersiniabactin NRPS irp1. 52
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 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: Results If integration is due to a
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,
show all

School of Engineering and Science - Jacobs University

Create successful ePaper yourself

Delete template?

Save as template?