School of Engineering and Science - Jacobs University

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.
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