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Novel pyoverdine biosynthesis gene(s) of Pseudomonas aeruginosa PAO
Alain Stintzi1, Pierre Cornelis2,1, Dany Hohnadel1
1Laboratoire de Microbiologie, Unité de Recherche Associée no. 1481 du Centre National de la Recherche Scientifique et de l'Université Louis Pasteur, 28 rue Goethe, 67083 Strasbourg, France.
Abstract:
Conjugational mobilization of a Pseudomonas aeruginosa PAO1 cosmid bank (in pMMB33) into a pyoverdine-deficient (pvd) mutant harbouring a mutation 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, contained a 15.1 kb insert which was subcloned into pKT240 as a 10.8 Sacl-CIal fragment conferring the same phenotype. This derivative, pPYP180, like pPYP17, also conferred an apparent wild-type phenotype on pvd mutants previously shown to map genetically in the 23 min region of the P. aeruginosa PAO chromosomes. Physical mapping indicated that the cloned DNA fragment is located at the 66-70 min region of the PAO chromosome, demonstrating that the restored apparent wild-type phenotype observed for the transconjugants was not the result of a true gene complementation. A gene interruption was obtained by replacing a 0.6 kb BgIll-BgIll region of pPYP180 necessary for the expression of the pigmentation/fluorescence phenotype, by a Hgr interposon (omega Hg). After conjugational transfer and introduction of the mutagenized fragment into the PAO1 chromosome by gene replacement, pyoverdine-deficient mutants were recovered, indicating that the fragment indeed contained at least one gene involved in pyoverdine synthesis. The yellow-green fluorescent compound produced by such cells harbouring plasmids pPYP17 or pPYP180 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 mediate iron uptake into PAO1. Pseudoverdine lacked a peptide chain but possessed spectral properties similar to pyoverdine, suggesting that it was structurally related to the chromophore of the pyoverdine molecule. The recent structural determination of pseudoverdine as a coumarin derivative confirmed this view and sheds some light on the biosynthetic pathway of the pyoverdine chromophore.
Insights
Researchers identified a new fluorescent compound, pseudoverdine, in Pseudomonas aeruginosa. This finding sheds light on the pyoverdine biosynthesis pathway and its related chromophore.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Pseudomonas aeruginosa produces pyoverdine, a crucial siderophore for iron uptake.
- Pyoverdine deficiency impacts bacterial growth and virulence.
- Understanding pyoverdine biosynthesis is key to bacterial iron metabolism research.
Purpose of the Study:
- To identify genes involved in pyoverdine synthesis in Pseudomonas aeruginosa.
- To characterize a novel fluorescent compound produced by a complemented mutant.
- To elucidate aspects of the pyoverdine chromophore biosynthesis.
Main Methods:
- Cosmid complementation of pyoverdine-deficient mutants.
- Subcloning and physical mapping of genetic inserts.
- Gene interruption via homologous recombination.
- Characterization of the novel fluorescent compound (pseudoverdine).
Main Results:
- A cosmid clone restored pyoverdine production in a mutant, but physical mapping indicated it was not a true complementation.
- A 10.8 kb fragment containing a gene involved in pyoverdine synthesis was identified.
- A novel fluorescent compound, pseudoverdine, was discovered and characterized.
- Pseudoverdine, a coumarin derivative, is structurally related to the pyoverdine chromophore.
Conclusions:
- The identified fragment contains a gene essential for pyoverdine biosynthesis.
- Pseudoverdine is a novel compound with structural similarities to the pyoverdine chromophore.
- This discovery provides insights into the pyoverdine biosynthetic pathway.