Ph. Delfosse

Stability, frequency and multiplicity of transposon insertions in the pyoverdine region in the chromosomes of different fluorescent pseudomonads.

Tn5 mutagenesis of different fluorescent pseudomonads was achieved by conjugational transfer of the suicide vector pSUP 10141. Pyoverdine negative (Pvd-) mutants were detected by the absence of fluorescence on Kings B medium and by their inability to grow in the presence of the iron chelator EDDHA [ethylenediamine di(o-hydroxyphenylacetic acid)]. In P. fluorescens ATCC 17400 and three rhizosphere isolates (one P. putida and two P. fluorescens), the percentage of Pvd- mutants ranged between 0 and 0.54%. In a P. chlororaphis rhizosphere isolate, this percentage was higher (4%). In these mutants both of the Tn5 antibiotic resistances (Km and Tc) were stable and the transposon could be detected by hybridization. In Pvd- mutants of P. fluorescens ATCC 17400, the transposon was found to be inserted twice in the chromosome while single insertions were detected in the DNA of other, randomly tested mutants. In P. aeruginosa PAO1, where 13.1% of the mutants were Pvd-, both antibiotic resistances were rapidly lost and accordingly no transposon insertion could be detected by hybridization. However, the Pvd- phenotype was generally stable in these mutants. The plasmid pNK862 containing a mini-Tn10 transposon was introduced by electroporation into P. aeruginosa PAO1 and Kmr mutants were recovered, 89% of which were Pvd- and confirmed to be P. aeruginosa by PCR amplification of the P. aeruginosa lipoprotein gene. The mini-Tn10 insertions were also found to be unstable in PAO1.

Structure and Characterization of Isopyoverdin from Pseudomonas putida BTP1 and Its Relation to the Biogenetic Pathway Leading to Pyoverdins

Abstract Pyoverdin type siderophores produced by six fluorescent Pseudomonas strains isolated from different rhizospheres were purified and characterized. The purified ferri-pyoverdins were tested for their ability to promote the growth of other strains grown under iron deficiency conditions. Only the one obtained from Pseudomonas putida BTP1 did not act as a growth promoter. The structure of the BTP1 siderophore was elucidated by spectroscopic methods and degradation studies. It turned out that it contains a chromophore which differs from the one typical for pyoverdins insofar as it carries the carboxyl group in 3- rather than in 1-position ((3S)-5-amino-1,2-dihydro-8,9-dihydroxy-3 H -pyrimido[1,2 a]quinoline-3-carboxylic acid). The amino group of the chromophore is substituted with the 5-carboxyl group of ʟ-glutamic acid and its carboxyl group with the N -terminus of the peptide ʟ -A sp -ʟ -A la-ʟ -Asp - ᴅ-N5- Ac - N5- OH - Orn - ʟ - Ser - ʟ - ᴄ - N5- OH - Orn . This isopyoverdin fits into the biogenetic scheme which postulates ferribactins as the precursors of pyoverdins