Identification of a DNA sequence motif required for expression of iron-regulated genes in pseudomonads

Rombel, Irene; McMorran, Brendan J.; Lamont, Iain L.

doi:10.1007/bf00290456

Cited by 58 publications

(85 citation statements)

References 48 publications

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“…Promoters that are recognized by RNA polymerase containing PvdS contain a sequence motif, the IS box, at about 33 bp from the transcription start sites and this forms part of the promoter sequence (Rombel et al, 1995;Wilson et al, 2001). A second sequence CGT at about 210 bp is also required for promoter recognition by PvdS (S. Tsao, M. J.…”

Section: Introductionmentioning

confidence: 99%

Identification and characterization of novel pyoverdine synthesis genes in Pseudomonas aeruginosa

2003

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Fluorescent pseudomonads secrete yellow-green siderophores named pyoverdines or pseudobactins. These comprise a dihydroxyquinoline derivative joined to a type-specific peptide and, usually, a carboxylic acid or amide. In Pseudomonas aeruginosa strain PAO1, six genes that encode proteins required for pyoverdine synthesis (pvd genes) have been identified previously. Expression of all of these genes requires an alternative sigma factor PvdS. The purpose of this research was to identify other genes that are required for pyoverdine synthesis in P. aeruginosa PAO1. Fourteen candidate genes were identified from the PAO1 genome sequence on the basis of their location in the genome, the functions of homologues in other bacteria, and whether their expression was likely to be PvdS-dependent. The candidate genes were mutated and the effects of the mutations on pyoverdine production were determined. Eight new pvd genes were identified. The presence of homologues of pvd genes in other strains of P. aeruginosa was determined by Southern blotting and in other fluorescent pseudomonads by interrogation of genome sequences. Five pvd genes were restricted to strains of P. aeruginosa that make the same pyoverdine as strain PAO1, suggesting that they direct synthesis of the type-specific peptide. The remaining genes were present in all strains of P. aeruginosa that were examined and homologues were present in other Pseudomonas species. These genes are likely to direct synthesis of the dihydroxyquinoline moiety and the attached carboxylic acid/amide group. It is likely that most if not all of the genes required for pyoverdine synthesis in P. aeruginosa PAO1 have now been identified and this will form the basis for a biochemical description of the pathway of pyoverdine synthesis.

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Section: Introductionmentioning

confidence: 99%

Identification and characterization of novel pyoverdine synthesis genes in Pseudomonas aeruginosa

2003

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“…Transcription of pvdD could be terminated at either of these sites, giving a transcript of about 7.5 kb. The size of the transcript corresponding to pvdD was previously estimated to be 5 kb following Northern (RNA) analysis (36), but the sequence of the gene indicates that this is an underestimate, presumably due to the high degree of instability of this transcript (36).…”

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confidence: 99%

“…This protein is predicted to contain 803 amino (35) cloned into pUC9 (54) and contains DNA required for pyoverdine synthesis (34,35). Restriction fragments are labelled by using the same nomenclature as previously (36). The locations and orientations of two genes (pvdD and fpvA) sequenced in this study are shown.…”

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confidence: 99%

Nucleotide sequence of pvdD, a pyoverdine biosynthetic gene from Pseudomonas aeruginosa: PvdD has similarity to peptide synthetases

1995

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Pseudomonas aeruginosa secretes a fluorescent siderophore, pyoverdine, when grown under iron-deficient conditions. Pyoverdine consists of a chromophoric group bound to a partly cyclic octapeptide. As a step toward understanding the molecular events involved in pyoverdine synthesis, we have sequenced a gene, pvdD, required for this process. The gene encodes a 2,448-residue protein, PvdD, which has a predicted molecular mass of 273,061 Da and contains two highly similar domains of about 1,000 amino acids each. The protein is similar to peptide synthetases from a range of bacterial and fungal species, indicating that synthesis of the peptide moiety of pyoverdine proceeds by a nonribosomal mechanism. The pvdD gene is adjacent to a gene, fpvA, which encodes an outer membrane receptor protein required for uptake of ferripyoverdine.Pseudomonas aeruginosa is an opportunistic pathogen which infects injured, immunodeficient, or otherwise compromised patients. The bacteria secrete a siderophore, pyoverdine, which is likely to play an important role in infection by competing with transferrin for iron in order to overcome the iron-withholding defense mechanism present in mammals (3,11,42). Almost all isolates of P. aeruginosa from infected patients secrete pyoverdine (9,22,29), and it has been shown that pyoverdine is present in the sputa of cystic fibrosis patients infected with P. aeruginosa (21). A mutant of P. aeruginosa which is unable to synthesize pyoverdine showed reduced virulence in an animal model of infection (24).Pyoverdine from P. aeruginosa PAO is a water-soluble, yellow-green fluorescent compound. It consists of a dihydroxyquinoline chromophoric group linked to an eight-residuevia the N-terminal serine, with a small dicarboxylic acid attached to the chromophore. Iron complexation is thought to occur through oxygen atoms present on the dihydroxyquinoline and two hydroxamate units supplied by the L-N 5 -OH-Orn residues. Several Pseudomonas species produce similar compounds, variously called pyoverdines or pseudobactins, all of which have the same dihydroxyquinoline group but differ in the nature of the attached peptide (reviewed in references 1 and 7), and it is likely that all of these are synthesized by similar mechanisms. Synthesis of the chromophoric group begins with condensation of D-tyrosine and L-2,4-diaminobutyric acid (7), with glutamic acid being the precursor of the amide-linked dicarboxylic acid (50). It has been suggested that biosynthesis of the peptide moiety of pyoverdine occurs by a nonribosomal mechanism (30).Little is known about the molecular nature of enzymes involved in the biosynthesis of pyoverdine in P. aeruginosa or any other pseudomonad. Recently, pvdA, which encodes an enzyme (L-ornithine N 5 -oxygenase) responsible for catalyzing the hydroxylation of ornithine, an early step in pyoverdine biosynthesis in P. aeruginosa, has been characterized at the molecular level (55). The pbsC gene, which is involved in synthesis of pseudobactin by Pseudomonas sp. strain M114, has also been seque...

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“…The sequences were analysed by standard methods with version 8.1-UNIX of the Computer Genetics Group package (Devereux et al, 1984) in conjunction with other programs as described previously ; sequence alignments were performed using  (Devereux et al, 1984) and residues were highlighted using BoxShade (http :\\ www.ch.embnet.org\software\BOXIform.html). Southern analysis was done by standard methods (Sambrook et al, 1989) with chromosomal DNA prepared by the method of Chen & Kuo (1993), and Northern (RNA) analysis was done as described previously (Rombel et al, 1995). Creating a mutation in pvdF.…”

Section: Methodsmentioning

confidence: 99%

“…2), that contains DNA required for synthesis of pyoverdine (Rombel & Lamont, 1992). Subsequent studies showed that this clone hybridized with five distinct ironregulated transcripts from P. aeruginosa (Rombel et al, 1995), with three of these corresponding to the pvdD, fpvA and pvdE genes (McMorran et al, 1996 ;Merriman et al, 1995 ;Poole et al, 1993). The first aim of this study was to characterize the gene corresponding to another of the iron-regulated transcripts.…”

Section: Sequence Analysismentioning

confidence: 99%

Involvement of a transformylase enzyme in siderophore synthesis in Pseudomonas aeruginosa The GenBank accession number for the sequence reported in this paper is U07359.

et al. 2001

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Fluorescent pseudomonads produce yellow-green siderophores when grown under conditions of iron starvation. Here, the characterization of the pvdF gene, which is required for synthesis of the siderophore pyoverdine by Pseudomonas aeruginosa strain PAO1, is described. A P. aeruginosa pvdF mutant was constructed and found to be defective for production of pyoverdine, demonstrating the involvement of PvdF in pyoverdine synthesis. Transcription analysis showed that expression of pvdF was regulated by the amount of iron in the growth medium, consistent with its role in siderophore production. DNA sequencing showed that pvdF gives rise to a protein of 31 kDa that has similarity with glycinamide ribonucleotide transformylase (GART) enzymes involved in purine synthesis from a wide range of eukaryotic and prokaryotic species. Chemical analyses of extracts from wild-type and pvdF mutant bacteria indicated that the PvdF enzyme catalyses the formylation of N 5 -hydroxyornithine to give rise to N 5 -formyl-N 5 -hydroxyornithine, a component of pyoverdine. These studies enhance understanding of the enzymology of pyoverdine synthesis, and to the best of the authors ' knowledge provide the first example of involvement of a GART-type enzyme in synthesis of a secondary metabolite.

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Identification of a DNA sequence motif required for expression of iron-regulated genes in pseudomonads

Cited by 58 publications

References 48 publications

Identification and characterization of novel pyoverdine synthesis genes in Pseudomonas aeruginosa

Identification and characterization of novel pyoverdine synthesis genes in Pseudomonas aeruginosa

Nucleotide sequence of pvdD, a pyoverdine biosynthetic gene from Pseudomonas aeruginosa: PvdD has similarity to peptide synthetases

Involvement of a transformylase enzyme in siderophore synthesis in Pseudomonas aeruginosa The GenBank accession number for the sequence reported in this paper is U07359.

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