NOVEL PYOVERDINE BIOSYNTHESIS GENE(S) OF PSEUDOMONAS-AERUGINOSA PAO

Conjugational mobilization of a Pseudomonas aeruginosa PAO1 cosmid ban k (in pMMB33) into a pyoverdine-deficient (pvd) mutant harbouring a mu tation in the 47 min region of the chromosome yielded one clone which restored yellow-green pigmentation and fluorescence when grown on iron -deficient medium. The relevant pMMB33-derivative cosmid, pPYP17, cont ained a 15.1 kb insert which was subcloned into pKT240 as a 10.8 kb Sa cI-ClaI fragment conferring the same phenotype, This derivative, pPYP1 80, like pPYP17, also conferred an apparent wild-type phenotype on pvd mutants previously shown to map genetically in the 23 min region of t he P. aeruginosa PAO chromosome. Physical mapping indicated that the c loned DNA fragment is located at the 66-70 min region of the PAO chrom osome, demonstrating that the restored apparent wild-type phenotype ob served for the transconjugants was not the result of a true gene compl ementation. A gene interruption was obtained by replacing a 0.6 kb Bgl II-BglII region of pPYP180 necessary for the expression of the pigment ation/fluorescence phenotype, by a Hg-r interposon (Omega Hg). After c onjugational transfer and introduction of the mutagenized fragment int o the PAO1 chromosome by gene replacement, pyoverdine-deficient mutant s were recovered, indicating that the fragment indeed contained at lea st one gene involved in pyoverdine synthesis. The yellow-green fluores cent compound produced by such cells harbouring plasmids pPYP17 or pPY P180 differed from pyoverdine in several aspects and was consequently named pseudoverdine. Although pseudoverdine was able to complex iron, it was unable to restore growth to pvd mutants in the presence of the iron chelator ethylenediamine di(o-hydroxyphenylacetic acid), or to me diate iron uptake into PAO1. Pseudoverdine lacked a peptide chain but possessed spectral properties similar to pyoverdine, suggesting that i t was structurally related to the chromophore of the pyoverdine molecu le. The recent structural determination of pseudoverdine as a coumarin derivative confirmed this view and sheds some light on the biosynthet ic pathway of the pyoverdine chromophore.