Genomic, genetic and structural analysis of pyoverdine-mediated iron acquisition in the plant growth-promoting bacterium Pseudomonas fluorescens SBW25
© Moon et al; licensee BioMed Central Ltd. 2008
Received: 26 June 2007
Accepted: 14 January 2008
Published: 14 January 2008
Pyoverdines (PVDs) are high affinity siderophores, for which the molecular mechanisms of biosynthesis, uptake and regulation have been extensively studied in Pseudomonas aeruginosa PAO1. However, the extent to which this regulatory model applies to other pseudomonads is unknown. Here, we describe the results of a genomic, genetic and structural analysis of pyoverdine-mediated iron uptake by the plant growth-promoting bacterium P. fluorescens SBW25.
In silico analysis of the complete, but un-annotated, SBW25 genome sequence identified 31 genes putatively involved in PVD biosynthesis, transport or regulation, which are distributed across seven different regions of the genome. PVD gene iron-responsiveness was tested using 'lacZ fusions to five PVD loci, representative of structural and regulatory genes. Transcription of all fusions increased in response to iron starvation. In silico analyses suggested that regulation of fpvR (which is predicted to encode a cytoplasmic membrane-spanning anti-sigma factor) may be unique. Transcriptional assays using gene expression constructs showed that fpvR is positively regulated by FpvI (an extracytoplasmic family (ECF) sigma factor), and not directly by the ferric uptake regulator (Fur) as for PAO1. Deletion of pvdL, encoding a predicted non-ribosomal peptide synthetase (NRPS) involved in PVD chromophore biosynthesis confirmed the necessity of PvdL for PVD production and for normal growth in iron-limited media. Structural analysis of the SBW25 PVD shows a partly cyclic seven residue peptide backbone, identical to that of P. fluorescens ATCC13525. At least 24 putative siderophore receptor genes are present in the SBW25 genome enabling the bacterium to utilize 19 structurally distinct PVDs from 25 different Pseudomonas isolates.
The genome of P. fluorescens SBW25 contains an extensively dispersed set of PVD genes in comparison to other sequenced Pseudomonas strains. The PAO1 PVD regulatory model, which involves a branched Fpv signaling pathway, is generally conserved in SBW25, however there is a significant difference in fpvR regulation. SBW25 produces PVD with a partly cyclic seven amino acid residue backbone, and is able to utilize a wide variety of exogenous PVDs.
Iron is a common co-factor for redox-dependent enzymes and an essential element for almost all living organisms. In nature iron is abundant; however, in aerobic environments and under general physiological conditions, iron typically exists in the insoluble ferric (Fe3+) form, thus rendering acquisition by organisms difficult. Under conditions of iron limitation, many bacteria produce and secrete low molecular weight ferric-specific ligands known as siderophores (for review, see ). The ferric-siderophores deliver iron to the cell via specific receptor and transport systems (for review, see ).
Collectively, Pseudomonas spp. produce a wide variety of siderophores , the most complex and common of which are pyoverdines (PVDs) . PVDs contain a peptide moiety, usually between 6–12 amino acids in length, and a dihydroxyquinoline chromophore moiety , which gives PVD its characteristic yellow-green fluorescent appearance. PVD-mediated iron uptake processes have been extensively characterized in Pseudomonas aeruginosa PAO1, where key regulatory and structural proteins have been identified, and regulatory mechanisms have been elucidated [5–9].
In P. aeruginosa PAO1, the primary level of PVD regulation involves the ferric uptake regulator (Fur), which, upon interaction with ferrous iron (Fe2+), binds to a specific DNA sequence (the Fur-box) in the promoter region of certain iron-regulated genes and blocks transcription. Under iron-deplete conditions (no Fe2+ available to interact with Fur) Fur-dependent repression is relieved [6, 10]. PAO1 PVD genes that possess Fur boxes, which have been demonstrated to bind Fur in vitro [5, 11], are pvdS and fpvI, which encode extracytoplasmic family (ECF) sigma factors, and fpvR, an inner membrane-spanning anti-sigma factor. The alternative sigma factor, PvdS, when associated with core RNA polymerase, binds iron-starvation (IS) box motifs  and directs the expression of a suite of genes involved in the synthesis of PVD [7, 13]. The activity of PvdS is regulated by a transmembrane signaling system that comprises of FpvR and FpvA, an outer membrane PVD receptor, and is primarily mediated by PVD . More recently, a second ECF sigma factor (FpvI) was found to direct transcription of fpvA [5, 14]. Like PvdS, FpvI is also under the direct regulation of FpvR and forms a second divergent branch of the signaling pathway.
Many plant-associated pseudomonads are known to produce PVDs, including the plant pathogen, P. syringae and the saprophytes, P. putida and P. fluorescens. The importance of siderophores for rhizosphere colonization by P. putida has been previously reported [15, 16]. However, little is known about the molecular mechanisms of PVD production and regulation in these organisms.
Here we report a genetic characterization of genes for PVD production in a plant growth-promoting bacterium, Pseudomonas fluorescens SBW25. SBW25 was originally isolated from field-grown sugar beet  and its biocontrol activity against the soil-borne pathogen, Pythium ultimum, is related to its considerable plant colonization ability . The present investigation was prompted by initial findings that three genes implicated in iron-acquisition processes were up-regulated on plant surfaces, as revealed by in vivo expression technology (IVET) analysis [19, 20]. Two genes had significant homology to putative siderophore receptor genes, and one gene appeared to be homologous to PAO1 pvdL, which encodes a non-ribosomal peptide synthetase (NRPS) involved in PVD biosynthesis . These findings suggested a significant role for iron uptake during seedling colonization.
Our work began with the un-annotated whole genome sequence of SBW25 . Based initially on an analysis of this sequence, 31 genes were identified with predicted roles in PVD biosynthesis, transport and regulation, for which the regulation of a subset of these was investigated by genetic analysis using chromosomally-integrated 'lacZ fusions and gene expression constructs. The results indicate a not previously realized mechanism of fpvR regulation. Moreover, we determine the chemical structure of SBW25 PVD and examine the biological role of PVD via a siderophore-deficient mutant. The ability of SBW25 to utilize a panel of structurally distinct exogenous PVDs was also assessed.
Results and Discussion
Genomic analysis of PVD genes in P. fluorescens SBW25
SBW25 homologues were identified for all PAO1 PVD genes except PA2411, which encodes a thioesterase, and pvdJ and pvdD, which encode NRPSs involved in biosynthesis of the PVD peptide backbone. In PAO1, PA2411 is not essential for PVD production, and is possibly redundant since pvdG also encodes a thioesterase . Although homologues of pvdJ and pvdD were not identified in SBW25, three additional NRPS genes were identified: two downstream of pvdP (Pflu2552 and Pflu2553) and one downstream of pvdI (Pflu2544; Figure 1). Differences in the sequences and organization of these NRPS genes likely reflect differences between the structures of the PVDs produced by SBW25 and PAO1 (discussed below).
The PVD genes of SBW25 are distributed across seven different loci within the genome (Figure 1), which is the most widespread distribution of PVD genes in a genomic comparison among four various sequenced Pseudomonas spp. . This distribution is in stark contrast to that of P. syringae where PVD genes form one large cluster . In P. aeruginosa PAO1 the PVD genes are confined to two loci separated by just 11.5 kb (Figure 1), which appears to be typical of P. aeruginosa isolates in general, where almost all PVD genes are located within the same region of the genome . PVD gene homologues in P. putida KT2440 and P. fluorescens strains Pf0–1  and Pf-5  are distributed across three loci.
Despite the overall differences in the genomic organization of PVD genes between SBW25 and PAO1, genes within each of the seven SBW25 PVD clusters are largely syntenous with those in PAO1. There are, however, two exceptions: pvdQ and fpvR, which in SBW25 are found in two non-adjacent regions of the genome and separated from all other PVD genes. The isolated position of fpvR (especially in relation to fpvI – see Figure 1), is particularly unusual: the genes for ECF sigma factors and their cognate anti-sigma factors are typically adjacent, and usually co-regulated by Fur [2, 26, 27]. The gene arrangement in SBW25 suggests that fpvR and fpvI might be under separate regulatory control – a notion given credibility by the fact that the promoter sequence of fpvR in SBW25 does not have a recognizable Fur-box motif. Interestingly, in place of the expected Fur-box motif is an IS-box motif, albeit one in which the last three bases of the consensus sequence are in reverse order (TAAAT-N16-TGC, rather than TAAAT-N16-CGT). The possibility that in SBW25 fpvR might be regulated by PvdS led to further investigation (described below).
Expression of putative PVD genes in response to iron starvation
Bacterial strains and plasmids used in this study.
Relevant genotype and/or other characteristics
SBW25ΔpvdL or ΔPflu4387
PBR840 with mini-Tn phoA3 disruption of secondary siderophore biosynthesis genes, GmR
S. Matthijs, unpublished
SBW25 carrying the pvdS-'lacZ fusion, SmR
SBW25 carrying the pvdL-'lacZ fusion, SmR
SBW25 carrying the fpvI::'lacZ fusion, SmR
SBW25 carrying the fpvR::'lacZ fusion, SmR
SBW25 carrying the fpvA::'lacZ fusion, SmR
TR107.2.1 with pTr130.1, GmR
TR107.2.1 with pTr130.2, GmR
TR107.2.1 with pTr130.3, GmR
TR107.2.1 with pBroadgate-D, GmR
TR135.1.1 with pTr130.1, GmR
TR135.1.1 with pTr130.2, GmR
TR135.1.1 with pTr130.3, GmR
TR135.1.1 with pBroadgate-D
TR107.5.1 with pTr130.1, GmR
TR107.5.1 with pTr130.2, GmR
TR107.5.1 with pTr130.3, GmR
TR107.5.1 with pBroadgate-D
Integration vector with 'lacZ, TcR
Source of the omega cassette, SmR
pUIC3 with tet gene replaced by Smr/Spr Ω fragment from pHP45Ω, SmR
pUIC3 containing pvdL deletion fragment
PCR product cloning vector, KmR
helper plasmid, KmR, Tra+
pIVET-Sm carrying the pvdS-'lacZ fusion
pIVET-Sm carrying the pvdL-'lacZ fusion
pIVET-Sm carrying the fpvI-'lacZ fusion
pIVET-Sm carrying the fpvR-'lacZ fusion
pIVET-Sm carrying the fpvA-'lacZ fusion
Gateway expression vector with Plac promoter, GmR
Thwaites and Mansfield
pBroadgate with ccdB gene replaced with the linker fragment, GmR;
Unique regulation of fpvR in SBW25
Next we tested the hypothesis that fpvR is regulated by PvdS rather than Fur, which is the direct regulator of anti-sigma factors in many other bacterial iron-acquisition systems . The regulatory gene constitutive expression constructs (pTR130.1, pTR130.2 and pTR130.3 for pvdS, fpvI and fpvR respectively) were introduced into the fpvR-'lacZ fusion strain (TR107.5.1). β-galactosidase activities of the resulting strains were determined under high (450 μM FeSO4) and low (100 μM 2,2'-dipyridyl) iron regimes. Contrary to expectation, the results presented in Figure 3C showed that expression of FpvI, and not PvdS, resulted in the up-regulation of fpvR, even in the presence of iron. It is unknown whether FpvI directly regulates fpvR, or if an intermediate regulatory component is involved. However, a conserved sequence motif, 5'-TAATGAGAA-3', was identified 56 nt upstream of fpvA and 128 nt upstream of fpvR, which may potentially serve as an FpvI binding site.
Genomic comparisons suggest that the non-adjacent arrangement of fpvI and fpvR in SBW25 may not be unique. Homologues of fpvR were not reported in P. putida KT2440 and P. fluorescens Pf0–1, and fpvR and fpvI were not identified in P. syringae pv. tomato DC3000 , however, this is likely to reflect a lack of strong sequence homology to the characterized counterparts in PAO1. Indeed, in SBW25, the amino acid sequence identity between FpvR and its PAO1 homologue was the lowest among all genes examined (48.7%; Additional file 1). A search for fpvR genes in other sequenced P. fluorescens strains revealed putative homologues in Pf0–1 and Pf-5 (48.1% and 48.4% amino acid sequence identity to PAO1 FpvR, respectively), however neither gene was adjacent to its cognate fpvI homologue, nor possessed a recognizable Fur-box motif in the upstream sequence. A putative IS-box was observed 44 bp upstream of the Pf0–1 homologue, with 7 out of 8 bases matching the IS-box consensus. However, an IS-box was not observed for the Pf-5 homologue, nor was the potential FpvI-binding site sequence identified upstream of SBW25 fpvA and fpvR. The presence of key genes encoding components of the Fpv branched signaling pathway (fpvA, fpvR, pvdS and fpvI) in the Pf0–1 and Pf-5 genomes suggests that this pathway may be conserved in these P. fluorescens strains – a hypothesis that can be tested in future work.
Deletion analysis of pvdL
The growth kinetics of PBR840 were determined in iron-limited CAA broth and the same medium supplemented with 450 μM FeCl3 in microtitre plate cultures. Results are shown in Figure 4B. A repeated measures MANOVA (see figure caption for details) showed that the optical density of PBR840 cells was significantly reduced compared to wild-type in iron deplete medium from 14 h [F1,8 = 28.02, P < 0.0001]. No significant reduction was detected during exponential growth, for example, at 4, 6 and 8 h, P values were 0.849, 0.333 and 0.398, respectively. No significant difference in growth was observed between wild-type and PBR840 in iron replete medium [F1,8 = 0.03, P = 0.862].
Growth analysis of PBR840 took place initially in 200 μl microtitre plate cultures, which although incubated with periodic shaking, could have become oxygen limited thus masking the effect of the pvdL mutation during exponential growth. To test this hypothesis, growth of PBR840 and SBW25 was further investigated using strongly aerated cultures grown in CAA broth, where iron is more likely to be present in the ferric form. The growth dynamics (data not shown) were comparable to those observed in the static cultures, with no statistical difference in cell density being detected during exponential growth, however differences in final yield were evident at 24 hours (the mean A450 reading and standard error for PBR840 was 0.9239 ± 0.014 (n = 4), and for SBW25 was 1.3510 ± 0.016 (n = 4)). Furthermore, PBR840 was incapable of growing in CAA medium supplemented with the strong iron-chelator EDDHA (ethylenediaminedihydroxyphenylacetic acid, 0.5 mg/ml), whereas growth was restored upon provision of purified SBW25 PVD (Figure 4C). Taken together, the data confirmed the predicted role of pvdL (Pflu4387) in PVD production.
Structural analysis of SBW25 PVD
Utilization of exogenous PVDs by P. fluorescens SBW25
The identification of two SBW25 plant-induced genes with homology to siderophore receptor genes [19, 20] prompted us to examine the ability of SBW25 to uptake and utilize exogenous siderophores. Initially, a genome-wide search for putative TonB-dependent siderophore receptor genes was undertaken, which revealed 24 putative siderophore receptor genes dispersed throughout the genome (listed in Additional file 3). Nine of these were immediately adjacent to predicted sigma factor/anti-sigma factor gene pairs (Additional file 3), that are presumably involved in their regulation. The number of putative receptor genes is similar to that (26) for P. fluorescens Pf0–1 , and include fpvA (Pflu2545), and Pflu5798 and Pflu6132 which were both previously shown to be induced in planta . These results strongly support the notion that SBW25 has the capability to utilize a considerable number of exogenous iron-chelating siderophores produced by other microbes.
Utilization of exogenous Pseudomonas PVDs
Strain PVD isolated from
15F3 growth stimulation
P. aeruginosa PAO1a
P. aeruginosa 7NSK2a
P. aeruginosa 59.20a
P. agarici LMG 2112
P. asplenii LMG 5147
P. fluorescens LMG 14562
P. fluorescens 17400::qbsFc
P. fluorescens 5
A. Sarniguet, INRA, France
P. fluorescens C7R12
P. Lemanceau, INRA/Université de Bourgogne, France
P. fluorescens SBW25
P. libanensis LMG 21606
P. salomonii LMG 22120
P. syringae LMG 1247
P. vancouverensis LMG 20222
Pseudomonas sp. W2Ap9
S. Matthijs, VUB, Brussels
Pseudomonas sp. W2Aug1
S. Matthijs, VUB, Brussels
Pseudomonas sp. W2Aug7
S. Matthijs, VUB, Brussels
Pseudomonas sp. W2Aug36
S. Matthijs, VUB, Brussels
Pseudomonas sp. W2Dec18
S. Matthijs, VUB, Brussels
Pseudomonas sp. W2Dec29
S. Matthijs, VUB, Brussels
Pseudomonas sp. W2Dec33
S. Matthijs, VUB, Brussels
Pseudomonas sp. W15Feb31B
S. Matthijs, VUB, Brussels
Pseudomonas sp. W2Jun14
S. Matthijs, VUB, Brussels
Pseudomonas sp. W15Ap2
S. Matthijs, VUB, Brussels
Pseudomonas sp. W15Aug 1
S. Matthijs, VUB, Brussels
Pseudomonas sp. W15Oct32
S. Matthijs, VUB, Brussels
The genomic era has revolutionized the scientific method of comparative biology, where the general applicability of biological traits and systems that have often been evaluated in few model organisms may now be readily assessed across taxa. Although PVDs are common siderophores of Pseudomonas spp., the regulation of the genes involved in the biosynthesis and uptake of PVD has been elucidated almost exclusively in P. aeruginosa PAO1. The availability of the P. fluorescens SBW25 genome sequence has revealed that PVD genes are considerably more widespread throughout the SBW25 genome than in other sequenced pseudomonads, and has enabled a comparative analysis of PVD regulation in SBW25 and PAO1 to be undertaken. The PVD regulatory model established for PAO1 involves a unique branched signaling pathway . Prior to this study it was not known whether this model applies to other Pseudomonas isolates. Using chromosomally-integrated gene reporter fusions and gene expression constructs, we found that the PAO1 branched signaling model is present in SBW25. In addition, we reveal a significant difference between PAO1 and SBW25 in terms of the regulation of fpvR. In SBW25, over-expression of fpvI results in constitutive expression of fpvR, suggesting that fpvR is positively regulated by FpvI, rather than directly by Fur, as is the case of fpvR of PAO1 . The structure of SBW25 PVD was elucidated and it was found that the peptide backbone is identical to that of P. fluorescens ATCC 13525, comprising a partly cyclic peptide of seven residues. The PVD structure implicated the involvement of two NRPSs in the synthesis of the PVD peptide backbone. The SBW25 genome harbors 24 putative TonB-dependent siderophore receptors, two of which are previously shown to be plant-inducible . Exogenous PVD uptake assays demonstrate that SBW25 is able to utilize a variety of structurally distinct siderophores produced by other Pseudomonas isolates. These data lay the foundation for further detailed functional analysis of these putative siderophore receptors in term of regulation and transporter specificity.
Bacterial strains, plasmids, and growth conditions
Escherichia coli DH5αλpir  was used as a recipient strain for gene cloning and then a donor for conjugative transfer into Pseudomonas cells. Pseudomonas strains and plasmids used in this study are listed in Table 1. E. coli cultures were grown at 37°C in Luria-Bertani (LB) medium  and P. fluorescens cultures were grown at 28°C in LB or casamino acids medium (CAA) . Antibiotics were used at the following final concentrations (μg/ml): ampicillin (Ap) 100, chloramphenicol (Cm) 25, gentamycin (Gm) 20, kanamycin (Km) 50, streptomycin (Sm) 100 and tetracycline (Tc) 10. 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal; Melford Laboratories, Chelsworth, UK) was used at a final concentration of 40 μg/ml.
To monitor PVD gene activities, reporter strains were grown in the presence of 450 nM – 450 μM FeSO4 for iron supplemented media. To chelate iron, 100 μM 2, 2'-dipyridyl (Sigma, St Louis MO, USA), or 0.5 mg/ml ethylenediaminedihydroxyphenylacetic acid (EDDHA) was used. Purified PVD was supplied at 50 μM.
Growth curves of bacterial strains were obtained using a VersaMax microtiter plate reader with SOFTmax PRO software (Molecular Devices, Sunnyvale CA, USA). Inoculum was prepared by culturing strains stored at -80°C in LB broth overnight, then sub-culturing into CAA broth with growth overnight. For growth experiments, cells from the CAA culture were washed once with sterile water and 2 μl was used inoculate 200 μl CAA or CAA supplemented with 450 μM FeCl3 in wells of the 96-well microtiter plate. Plates were incubated at 28°C and absorbance readings at 450 nm were taken every five minutes after brief shaking. Growth curves were also obtained from highly aerated cultures grown in 250 ml flasks containing 20 ml CAA broth, and inoculated to an initial OD450 of ~0.15. Cultures were grown at 28°C with shaking at 200 rpm and absorbance readings at 450 nm were taken over a 24 hour period.
In silico analyses
Putative homologues of PVD-related genes in P. fluorescens SBW25 were identified by interrogating the un-annotated SBW25 genome  with P. aeruginosa PAO1 PVD gene sequences [23, 40] using nucleotide-nucleotide Basic Local Alignment Search Tool (BLASTN). Complete SBW25 open reading frames (ORFs) were identified from matching sequence hits, and these were used to re-query the PAO1 genome via translated query vs. protein sequence BLAST (tBLASTX) algorithms. Sequences upstream of putative homologues were examined by eye for the PAO1 Fur-box consensus sequence, 5'-GATAATGATAATCATTATC-3' , and iron starvation (IS) boxes, 5'-TAAAT-N16-CGT-3' [12, 41]. The best matches to Fur-box motifs were confirmed using MotifScanner , in conjunction with a Fur-box position weight matrix (accession MX000013) from the Prodoric website [43, 44]. Genes were mapped to the genome using Artemis software , and the percentage identity and similarity at the amino acid sequence level between PAO1 and SBW25 homologues was determined by the Needleman-Wunsch global alignment algorithm implemented in EMBOSS needle . Common DNA sequence motifs were identified by MEME version 3.0 . In silico PVD peptide backbone structural predictions were conducted using the methods of Rausch et al. and Stachelhaus et al.[36, 37] implemented in NRPSpredictor . Putative siderophore receptor genes were identified by hits to protein family (Pfam) accessions PF00593 (TonB-dependent receptor) and PF07715 (TonB-dependent receptor plug domain) , in combination with BLAST homology to putative siderophore receptor genes. Putative sigma factor and anti-sigma factor genes adjacent to receptor genes were identified by hits to Pfam accessions PF04542 (Sigma-70 region) and PF04773 (FecR protein), respectively.
Gene reporter and expression constructs
Oligonucleotide primers used in this study.
Gene expression constructs for pvdS, fpvI and fpvR were created using the Gateway cloning system (Invitrogen). Fragments containing the ribosome-binding site and entire ORF of pvdS, fpvI and fpvR were PCR-amplified using primers in Table 3. Fragments were directionally cloned into pENTR/D-TOPO (Invitrogen) following the manufacturers instructions. The sequences of the cloned fragments were verified, and LR recombination reactions using Gateway LR Clonase enzyme mix (Invitrogen) were performed to transfer the cloned fragment to the broad host range destination expression vector, pBroadgate (gift from R. Thwaites and J. Mansfield). pBroadgate is a derivative of pBBR1MCS-5  that has been modified as a Gateway destination vector by incorporation of an att R cassette, containing a Cm resistance determinant and a lethal ccd B gene (Invitrogen). P. fluorescens reporter strains were transformed with expression and pBroadgate-D control constructs by electroporation.
β-galactosidase activities of 'lacZ reporter fusion strains were determined from cultures grown to mid- to late log phase in CAA media, supplemented with FeSO4 or 2,2'-dipyridyl as required. Assays were based on the hydrolysis of 4-methylumbelliferyl-β-D-galactoside to yield the fluorescent product, 7-hydroxy-4-methylcoumarin (4MU) as described . 4MU was detected at 460 nm after excitation at 365 nm using a Hoefer DyNA Quant 200 fluorometer (Pharmacia Biotech, San Francisco CA, USA), or FLUOstar (BMG Labtech, Offenburg, Germany).
Construction of PBR840 (ΔpvdL)
An ORF Pflu4387 (pvdL) knockout mutant was constructed by deleting a 10.9 kb internal portion of the 12.9 kb pvdL gene. This was achieved by a combination of gene splicing by overlap extension (SOE) PCR  and a two-step allelic exchange strategy . Briefly, two 1 kb fragments from each of the 5'- and 3'-end of pvdL were PCR-amplified from SBW25 genomic DNA using the primer pairs pvdL1 and pvdL2, and pvdL3 and pvdL4, respectively (Table 3). The two fragments were ligated in a third PCR using primers pvdL1 and pvdL4 via the complementary residues incorporated at the 5'-ends of primers pvdL2 and pvdL3 (Table 3). The resulting 2 kb pvdL deletion fragment was cloned into pCR8/GW/TOPO (Invitrogen). After the sequence of the cloned fragment was verified, the fragment was retrieved by Bgl II digestion and cloned into the integration vector pUIC3 to create pUIC3–40.
To generate a chromosomal pvdL deletion, pUIC3–40 was mobilized into P. fluorescens SBW25 by conjugation with the help of pRK2013 (tra+). Chromosomal integration of pUIC3–40 by a single homologous recombination event was selected by plating transconjugants on LB agar containing Tc and X-gal. Blue, Tc-resistant colonies contained both intact and deleted versions of pvdL, and were grown in LB broth without selection to allow the second homologous recombination event to take place. After two consecutive overnight subcultures, cells were plated on LB agar containing X-gal. White colonies were screened for Tc sensitivity, and the pvdL deletion was confirmed by PCR analysis.
PVD structure determination
To determine the structure of SBW25 PVD, mass spectral data were obtained with a MAT 900 ST instrument providing an EB-QIT (quadrupole ion trap) geometry and equipped with an ESI II ion source (Finnigan MAT, Bremen, Germany); spray voltage 3.4–3.6 kV, capillary temperature 230°C. Source conditions were set to minimize fragmentation, resolution ca. 5000 (10% valley). Trifluoroacetic acid was added to the sample dissolved in water (100:0.2, v/v). Fragmentation induced by low energy collision activation was effected in the octapole unit, located in front of the QIT and in the QIT itself (~2·10-3 Pa He as bath gas diffusing in the collision octapole).
PVD purification and exogenous siderophore uptake by SBW25
PVD was purified from bacterial cultures grown for 48 h in 1 l of CAA at 28°C. For P. aeruginosa, the strains were grown at 37°C. The cell-free supernatant was passed on a C-18 column (5 × 2.5 cm) conditioned with methanol and rinsed with sterile H2O. PVD was eluted with 80% methanol. Subsequently the methanol was evaporated and the PVD lyophilized. Purified PVD was quantified spectrophotometrically .
To estimate the diversity of exogenous PVDs that may be utilized by SBW25, PVDs were purified from a panel of 25 Pseudomonas isolates and strains, representing a high diversity of species that had previously been shown to each produce a unique PVD (each having a unique siderotype) as shown by isoelectric focusing (S. Matthijs, unpublished). These isolates included Pseudomonas type strains, and environmental isolates collected from the Woluwe river, Brussels, Belgium (Table 2). CAA agar plates containing 0.5 mg/ml EDDHA were overlaid with 5 × 106 cells of strain 15F3, a siderophore-deficient derivative of PBR840 that contains transposon mini-Tn phoA3 disruption of biosynthetic genes for a secondary siderophore (S. Matthijs, unpublished). Filter-paper disks impregnated with 5 μl of 8 mM purified siderophore were placed on the agar . Plates were incubated at 28°C and scored for the presence of detectable growth after one day.
List of abbreviations used
casamino acids medium
in vivo expression technology
non-ribosomal peptide synthetase
polymerase chain reaction
quadrupole ion trap
splicing by overlap extension
We are grateful to I.L. Lamont and P. Cornelis for helpful discussions. Many thanks also to R.W. Jackson for statistical analyses and helpful discussion, Z.A. Park-Ng for statistical analyses, S.R. Giddens and C.G. Knight for bioinformatics support, R. Thwaites and J. Mansfield for the kind gift of pBroadgate, and J. Bull for technical support. This research was supported by a grant from the Biotechnology and Biological Sciences Research Council, U.K. to P.B.R.
- Neilands JB: Siderophores: structure and function of microbial iron transport compounds. J Biol Chem. 1995, 270 (45): 26723-26726.View ArticlePubMed
- Crosa JH: Signal transduction and transcriptional and posttranscriptional control of iron-regulated genes in bacteria. Microbiol Mol Biol Rev. 1997, 61 (3): 319-336.PubMed CentralPubMed
- Cornelis P, Matthijs S: Diversity of siderophore-mediated iron uptake systems in fluorescent pseudomonads: not only pyoverdines. Environ Microbiol. 2002, 4 (12): 787-798. 10.1046/j.1462-2920.2002.00369.x.View ArticlePubMed
- Meyer JM: Pyoverdines: pigments, siderophores and potential taxonomic markers of fluorescent Pseudomonas species. Arch Microbiol. 2000, 174 (3): 135-142. 10.1007/s002030000188.View ArticlePubMed
- Beare PA, For RJ, Martin LW, Lamont IL: Siderophore-mediated cell signalling in Pseudomonas aeruginosa: divergent pathways regulate virulence factor production and siderophore receptor synthesis. Mol Microbiol. 2003, 47 (1): 195-207. 10.1046/j.1365-2958.2003.03288.x.View ArticlePubMed
- Vasil ML, Ochsner UA: The response of Pseudomonas aeruginosa to iron: genetics, biochemistry and virulence. Mol Microbiol. 1999, 34 (3): 399-413. 10.1046/j.1365-2958.1999.01586.x.View ArticlePubMed
- Lamont IL, Beare PA, Ochsner U, Vasil AI, Vasil ML: Siderophore-mediated signaling regulates virulence factor production in Pseudomonas aeruginosa. Proc Natl Acad Sci USA. 2002, 99 (10): 7072-7077. 10.1073/pnas.092016999.PubMed CentralView ArticlePubMed
- Redly GA, Poole K: FpvIR control of fpvA ferric pyoverdine receptor gene expression in Pseudomonas aeruginosa: demonstration of an interaction between FpvI and FpvR and identification of mutations in each compromising this interaction. J Bacteriol. 2005, 187 (16): 5648-5657. 10.1128/JB.187.16.5648-5657.2005.PubMed CentralView ArticlePubMed
- Lamont IL, Martin LW, Sims T, Scott A, Wallace M: Characterization of a gene encoding an acetylase required for pyoverdine synthesis in Pseudomonas aeruginosa. J Bacteriol. 2006, 188 (8): 3149-3152. 10.1128/JB.188.8.3149-3152.2006.PubMed CentralView ArticlePubMed
- Prince RW, Cox CD, Vasil ML: Coordinate regulation of siderophore and exotoxin A production: molecular cloning and sequencing of the Pseudomonas aeruginosa fur gene. J Bacteriol. 1993, 175 (9): 2589-2598.PubMed CentralPubMed
- Ochsner UA, Vasil ML: Gene repression by the ferric uptake regulator in Pseudomonas aeruginosa: cycle selection of iron-regulated genes. Proc Natl Acad Sci USA. 1996, 93 (9): 4409-4414. 10.1073/pnas.93.9.4409.PubMed CentralView ArticlePubMed
- Wilson MJ, McMorran BJ, Lamont IL: Analysis of promoters recognized by PvdS, an extracytoplasmic-function sigma factor protein from Pseudomonas aeruginosa. J Bacteriol. 2001, 183 (6): 2151-2155. 10.1128/JB.183.6.2151-2155.2001.PubMed CentralView ArticlePubMed
- Cunliffe HE, Merriman TR, Lamont IL: Cloning and characterization of pvdS, a gene required for pyoverdine synthesis in Pseudomonas aeruginosa: PvdS is probably an alternative sigma factor. J Bacteriol. 1995, 177 (10): 2744-2750.PubMed CentralPubMed
- Redly GA, Poole K: Pyoverdine-mediated regulation of FpvA synthesis in Pseudomonas aeruginosa: involvement of a probable extracytoplasmic-function sigma factor, FpvI. J Bacteriol. 2003, 185 (4): 1261-1265. 10.1128/JB.185.4.1261-1265.2003.PubMed CentralView ArticlePubMed
- Loper JE, Henkels MD: Utilization of heterologous siderophores enhances levels of iron available to Pseudomonas putida in the rhizosphere. Appl Environ Microbiol. 1999, 65 (12): 5357-5363.PubMed CentralPubMed
- Molina MA, Godoy P, Ramos-Gonzalez MI, Munoz N, Ramos JL, Espinosa-Urgel M: Role of iron and the TonB system in colonization of corn seeds and roots by Pseudomonas putida KT2440. Environ Microbiol. 2005, 7 (3): 443-449. 10.1111/j.1462-2920.2005.00720.x.View ArticlePubMed
- Bailey MJ, Lilley AK, Thompson IP, Rainey PB, Ellis RJ: Site directed chromosomal marking of a fluorescent pseudomonad isolated from the phytosphere of sugar beet; stability and potential for marker gene transfer. Mol Ecol. 1995, 4 (6): 755-763.View ArticlePubMed
- Ellis RJ, Timms-Wilson TM, Bailey MJ: Identification of conserved traits in fluorescent pseudomonads with antifungal activity. Environ Microbiol. 2000, 2 (3): 274-284. 10.1046/j.1462-2920.2000.00102.x.View ArticlePubMed
- Gal M, Preston GM, Massey RC, Spiers AJ, Rainey PB: Genes encoding a cellulosic polymer contribute toward the ecological success of Pseudomonas fluorescens SBW25 on plant surfaces. Mol Ecol. 2003, 12 (11): 3109-3121. 10.1046/j.1365-294X.2003.01953.x.View ArticlePubMed
- Rainey PB: Adaptation of Pseudomonas fluorescens to the plant rhizosphere. Environ Microbiol. 1999, 1 (3): 243-257. 10.1046/j.1462-2920.1999.00040.x.View ArticlePubMed
- Mossialos D, Ochsner U, Baysse C, Chablain P, Pirnay JP, Koedam N, Budzikiewicz H, Fernández DU, Schäfer M, Ravel J, Cornelis P: Identification of new, conserved, non-ribosomal peptide synthetases from fluorescent pseudomonads involved in the biosynthesis of the siderophore pyoverdine. Mol Microbiol. 2002, 45 (6): 1673-1685. 10.1046/j.1365-2958.2002.03120.x.View ArticlePubMed
- http://www.sanger.ac.uk/Projects/P_fluorescens/. [http://www.sanger.ac.uk/Projects/P_fluorescens/]
- Ravel J, Cornelis P: Genomics of pyoverdine-mediated iron uptake in pseudomonads. Trends Microbiol. 2003, 11 (5): 195-200.View ArticlePubMed
- Smith EE, Sims EH, Spencer DH, Kaul R, Olson MV: Evidence for diversifying selection at the pyoverdine locus of Pseudomonas aeruginosa. J Bacteriol. 2005, 187 (6): 2138-2147. 10.1128/JB.187.6.2138-2147.2005.PubMed CentralView ArticlePubMed
- Paulsen IT, Press CM, Ravel J, Kobayashi DY, Myers GSA, Mavrodi DV, DeBoy RT, Seshadri R, Ren Q, Madupu R, Dodson RJ, Durkin AS, Brinkac LM, Daugherty SC, Sullivan SA, Rosovitz MJ, Gwinn ML, Zhou L, Schneider DJ, Cartinhour SW, Nelson WC, Weidman J, Watkins K, Tran K, Khouri H, Pierson EA, Pierson LS, Thomashow LS, Loper JE: Complete genome sequence of the plant commensal Pseudomonas fluorescens Pf-5. Nature Biotechnol. 2005, 23 (7): 873-878. 10.1038/nbt1110.View Article
- Koster M, van Klompenburg W, Bitter W, Leong J, Weisbeek P: Role for the outer membrane ferric siderophore receptor PupB in signal transduction across the bacterial cell envelope. EMBO J. 1994, 13 (12): 2805-2813.PubMed CentralPubMed
- Visca P, Leoni L, Wilson MJ, Lamont IL: Iron transport and regulation, cell signalling and genomics: lessons from Escherichia coli and Pseudomonas. Mol Microbiol. 2002, 45 (5): 1177-1190. 10.1046/j.1365-2958.2002.03088.x.View ArticlePubMed
- Fuchs R, Budzikiewicz H: Structural studies of pyoverdins by mass spectroscopy. Curr Org Chem. 2001, 5: 265-288. 10.2174/1385272013375562.View Article
- Budzikiewicz H, Schäfer M, Fernández DU, Matthijs S, Cornelis P: Characterization of the chromophores of pyoverdins and related siderophores by electrospray tandem mass spectrometry. BioMetals. 2007, 20 (2): 135-144. 10.1007/s10534-006-9021-3.View ArticlePubMed
- Budzikiewicz H, Schäfer M, Meyer JM: Siderotyping of fluorescent pseudomonads - problems in the determination of molecular masses by mass spectrometry. Mini- Rev Org Chem. 2007, 4: 246-253. 10.2174/157019307781369968.View Article
- Fuchs R, Budzikiewicz H: Structural studies of pyoverdins with cyclopeptidic substructures by electrospray ionization and collision induced fragmentation. Spectroscopy. 2000, 14: 229-246.View Article
- Hohlneicher U, Hartmann R, Taraz K, Budzikiewicz H: Pyoverdin, ferribactin, azotobactin - a new triade of siderophores from Pseudomonas chlororaphis ATCC 9446 and its relation to Pseudomonas fluorescens ATCC 13525. Z Naturforsch. 1995, 50: 337-344.
- Budzikiewicz H: Siderophores of the Pseudomonadaceae sensu stricto (fluorescent and non-fluorescent Pseudomonas spp.). Prog Ch Org Nat Prod. 2004, 87: 81-237.
- Linget C, Azadi P, MacLeod JK, Dell A, Abdallah MA: Bacterial siderophores: The structures of the pyoverdins of Pseudomonas fluorescens ATCC 13525. Tetrahedron Lett. 1992, 33 (13): 1737-1740. 10.1016/S0040-4039(00)91719-2.View Article
- Boca Raton, Florida , CRC Press, Abdallah MA: Pyoverdines and pseudobactins. CRC handbook of microbial iron chelates. Edited by: Winkelmann G. 1991, Boca Raton, Florida , CRC Press., 139-152.
- Rausch C, Weber T, Kohlbacher O, Wohlleben W, Huson DH: Specificity prediction of adenylation domains in nonribosomal peptide synthetases (NRPS) using transductive support vector machines (TSVMs). Nucleic Acids Res. 2005, 33 (18): 5799-5808. 10.1093/nar/gki885.PubMed CentralView ArticlePubMed
- Stachelhaus T, Mootz HD, Marahiel MA: The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases. Chem Biol. 1999, 6 (8): 493-505. 10.1016/S1074-5521(99)80082-9.View ArticlePubMed
- Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual. 1989, Plainview , Cold Spring Harbor Laboratory Press, 2
- Meyer JM, Stintzi A, De Vos D, Cornelis P, Tappe R, Taraz K, Budzikiewicz H: Use of siderophores to type pseudomonads: the three Pseudomonas aeruginosa pyoverdine systems. Microbiology. 1997, 143: 35-43.View ArticlePubMed
- Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, Hickey MJ, Brinkman FS, Hufnagle WO, Kowalik DJ, Lagrou M, Garber RL, Goltry L, Tolentino E, Westbrock-Wadman S, Yuan Y, Brody LL, Coulter SN, Folger KR, Kas A, Larbig K, Lim R, Smith K, Spencer D, Wong GK, Wu Z, Paulsen IT, Reizer J, Saier MH, Hancock RE, Lory S, Olson MV: Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature. 2000, 406 (6799): 959-964. 10.1038/35023079.View ArticlePubMed
- Lamont IL, Martin LW: Identification and characterization of novel pyoverdine synthesis genes in Pseudomonas aeruginosa. Microbiology. 2003, 149: 833-842. 10.1099/mic.0.26085-0.View ArticlePubMed
- Thijs G, Lescot M, Marchal K, Rombauts S, De Moor B, Rouzé P, Moreau Y: A higher order background model improves the detection of regulatory elements by Gibbs Sampling. Bioinformatics. 2001, 17 (12): 1113-1122. 10.1093/bioinformatics/17.12.1113.View ArticlePubMed
- http://prodoric.tu-bs.de. [http://prodoric.tu-bs.de]
- Münch R, Hiller K, Barg H, Heldt D, Linz S, Wingender E, Jahn D: PRODORIC: prokaryotic database of gene regulation. Nucleic Acids Res. 2003, 31 (1): 266-269. 10.1093/nar/gkg037.PubMed CentralView ArticlePubMed
- Rutherford K, Parkhill J, Crook J, Horsnell T, Rice P, Rajandream MA, Barrell B: Artemis: sequence visualisation and annotation. Bioinformatics. 2000, 16 (10): 944-945. 10.1093/bioinformatics/16.10.944.View ArticlePubMed
- Rice P, Longden I, Bleasby A: EMBOSS: The European Molecular Biology Open Software Suite. Trends Genet. 2000, 16 (6): 276-277. 10.1016/S0168-9525(00)02024-2.View ArticlePubMed
- http://www-ab.informatik.uni-tuebingen.do/software. [http://www-ab.informatik.uni-tuebingen.de/toolbox/index.php?view=domainpred]
- Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM, Peterson KM: Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene. 1995, 166 (1): 175-176. 10.1016/0378-1119(95)00584-1.View ArticlePubMed
- Pharmacia B: Hoefer DyNA Quant 200 Fluorometer User Manual.
- Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR: Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene. 1989, 77: 61-68. 10.1016/0378-1119(89)90359-4.View ArticlePubMed
- Prentki P, Krisch HM: In vitro insertional mutagenesis with a selectable DNA fragment. Gene. 1984, 29 (3): 303-313. 10.1016/0378-1119(84)90059-3.View ArticlePubMed
- Figurski DH, Helinski DR: Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci USA. 1979, 76 (4): 1648-1652. 10.1073/pnas.76.4.1648.PubMed CentralView ArticlePubMed
- Giddens SR, Jackson RW, Moon CD, Jacobs MA, Zhang XX, Gehrig SM, Rainey PB: Mutational activation of niche-specific genes provides insight into regulatory networks and bacterial function in a complex environment. Proc Natl Acad Sci USA. 2007, 104 (46): 18247-18252. 10.1073/pnas.0706739104.PubMed CentralView ArticlePubMed
- Holloway BW: Genetic recombination in Pseudomonas aeruginosa. J Gen Microbiol. 1955, 13 (3): 572-581.PubMed
- Höfte M, Mergeay M, Verstraete W: Marking the rhizopseudomonas strain 7NSK2 with a Mu d(lac) element for ecological studies. Appl Environ Microbiol. 1990, 56 (4): 1046-1052.PubMed CentralPubMed
- Matthijs S, Baysse C, Koedam N, Tehrani KA, Verheyden L, Budzikiewicz H, Schafer M, Hoorelbeke B, Meyer JM, De Greve H, Cornelis P: The Pseudomonas siderophore quinolobactin is synthesized from xanthurenic acid, an intermediate of the kynurenine pathway. Mol Microbiol. 2004, 52 (2): 371-384. 10.1111/j.1365-2958.2004.03999.x.View ArticlePubMed
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