Novel pyoverdine biosynthesis gene(s) of Pseudomonas aeruginosa PAO

Abstract

~~ ~Conjugational mobilization of a Pseudomonas aeruginosa PAOl cosmid bank (in pMMB33) into a pyoverdine-def icient (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-def icient medium. The relevant pMMB33-derivative cosmid, pPYP17, contained a 151 kb insert which was subcloned into pKT240 as a 10.8 kb Sacl-Clal fragment conferring the same phenotype. This derivative, pPYPl80, 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 PA0 chromosome. Physical mapping indicated that the cloned DNA fragment is located at the 66-70 min region of the PA0 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 BgllI-Bglll region of pPYP180 necessary for the expression of the pigmentation/fluorescence phenotype, by a Hgr interposon (OHg). After conjugational transfer and introduction of the mutagenized fragment into the PAOl chromosome by gene replacement, pyoverdine-def icient 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 t o complex iron, it was unable t o restore growth to pvd mutants in the presence of the iron chelator ethylenediamine di(o-hydroxyphenylacetic acid), or to mediate iron uptake into PAOl .Pseudoverdine lacked a peptide chain but possessed spectral properties similar t o 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.

Identification and characterization of novel pyoverdine synthesis genes in Pseudomonas aeruginosa

“…An early study mapped two mutations that affected pyoverdine synthesis to a locus at 23 min on the recalibrated genetic map of P. aeruginosa PAO1 (Hohnadel et al, 1986) but further study of these mutants has not been reported. The pvc genes lie at about 66-70 min on the genetic map (Stintzi et al, 1996), about 240 kb away from pvdS. All other pvd mutations, including all of the 24 mutations identified by Tsuda et al (1995), have been mapped to the 47 min region of the P. aeruginosa chromosome that corresponds to the part of the genome represented in Fig.…”

“…The genes and enzymes that are required for synthesis of pyoverdine are best characterized in the type I strain PAO1. Most of the pyoverdine synthesis genes that have been identified in this strain are at about 47 min on the genetic map (Ankenbauer et al, 1986;Hohnadel et al, 1986;Rombel & Lamont, 1992;Stintzi et al, 1996;Tsuda et al, 1995) and these genes are listed in Table 1. The pvdA gene encodes an enzyme that catalyses synthesis of N 5 -hydroxyornithine (Visca et al, 1994) and the pvdF gene product catalyses the formylation of N 5 -hydroxyornithine to give N 5 -formyl-N 5 -hydroxyornithine, which is present in the type I pyoverdine (pyoverdine PAO ) made by P. aeruginosa PAO1 .…”

“…The product of the pvdE gene is likely to be an ABC transporter protein and it is essential for pyoverdine synthesis (McMorran et al, 1996) although the transported substrate has not been identified. A separate cluster of four genes (pvcABCD) at 66-70 min on the genetic map has been reported to be required for synthesis of the pyoverdine chromophore (Stintzi et al, 1996(Stintzi et al, , 1999. However, pvc mutants are able to make pyoverdine in some growth media so that these genes are not essential for pyoverdine synthesis (P. Cornelis & U.…”

“…fOHOrn, N 5 -Formyl-N 5 -hydroxyornithine; aThr, allo-threonine; OHOrn, hydroxyornithine; c, cyclic. (Cunliffe et al, 1995;Miyazaki et al, 1995) pvcABCD PA2254-PA2257 Synthesis of the pyoverdine chromophore (Stintzi et al, 1996(Stintzi et al, , 1999 *ORF in the P. aeruginosa genome sequence (http://www.pseudomonas.com; http://pseudomonas.bit.uq.edu.au). (Stover et al, 2000) provides a new approach for identifying pyoverdine synthesis genes.…”

Transcriptional profiling of Legionella pneumophila biofilm cells and the influence of iron on biofilm formation

“…Lpl0237 is 66 % homologous with PvcB from P. aeruginosa and contains the conserved domain of the TauD, TfdA oxygenase family (pfam02668). In the P. aeruginosa genome, pvcA and pvcB are the first two genes of an operon of four genes (pvcABCD), encoding proteins required for the synthesis of the siderophore pyoverdine (Stintzi et al, 1996(Stintzi et al, , 1999. In L. pneumophila, the genes downstream of lpl0237 show no similarity to pvcC and pvcD.…”