School of Engineering and Science - Jacobs University

Contents
4.2.6.2 Alkaline transfer of DNA............................................................. 39
4.2.6.3 Hybridisation and detection....................................................... 39
4.2.7 DNA-sequencing............................................................................ 40
4.3 Analysis of bacterial growth in vitro................................................... 40
4.4 Analysis of bacterial growth in planta................................................ 40
4.4.1 Wound inoculation......................................................................... 40
4.4.2 Spray inoculation........................................................................... 41
4.5 Analysis of siderophore-production................................................... 41
4.5.1 Uptake of 59 Fe................................................................................. 41
4.5.2 Determination of siderophore concentration by quantitative
CAS-assay ...................................................................................... 42
4.5.3 Quantification of pyoverdin by fluorescence measurement...... 43
4.5.4 Purification of siderophores by XAD4 ......................................... 43
4.5.5 Isoelectric focussing of siderophores ......................................... 44
4.5.6 Gel-filtration ................................................................................... 44
4.5.7 Thin-layer chromatography .......................................................... 45
5 Results......................................................................................................... 46
5.1 Analysis of siderophores produced by Pss22d................................. 46
5.1.1 Construction of a pyoverdin-deficient mutant of Pss22d .......... 47
5.1.2 Southern blot analysis of Psg1a and Pss22d for the presence of
yersiniabactin biosynthetic genes ............................................... 51
5.1.3 Screening for an additional siderophore of Pss22d by random
mutagenesis................................................................................... 53
5.1.4 Construction of a marker exchange mutant of the
achromobactin-like siderophore in Pss22d................................. 57
5.1.5 In vitro comparison of Pss22d and its siderophore mutants..... 59
5.1.6 Purification of the achromobactin-like siderophore for structure
determination ................................................................................. 61
5.2. Comparison of siderophore production in Psg1a and Pss22d ....... 65