A combinatorial approach to the structure elucidation of a pyoverdine siderophore produced by a Pseudomonas putida isolate and the use of pyoverdine as a taxonomic marker for typing P. putida subspecies
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- Ye, L., Ballet, S., Hildebrand, F. et al. Biometals (2013) 26: 561. doi:10.1007/s10534-013-9653-z
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The structure of a pyoverdine produced by Pseudomonas putida, W15Oct28, was elucidated by combining mass spectrometric methods and bioinformatics by the analysis of non-ribosomal peptide synthetase genes present in the newly sequenced genome. The only form of pyoverdine produced by P. putida W15Oct28 is characterized to contain α-ketoglutaric acid as acyl side chain, a dihydropyoverdine chromophore, and a 12 amino acid peptide chain. The peptide chain is unique among all pyoverdines produced by Pseudomonas subspecies strains. It was characterized as –l-Asp-l-Ala-d-AOHOrn-l-Thr-Gly-c[l-Thr(O-)-l-Hse-d-Hya-l-Ser-l-Orn-l-Hse-l-Ser-O-]. The chemical formula and the detected and calculated molecular weight of this pyoverdine are: C65H93N17O32, detected mass 1624.6404 Da, calculated mass 1624.6245. Additionally, pyoverdine structures from both literature reports and bioinformatics prediction of the genome sequenced P. putida strains are summarized allowing us to propose a scheme based on pyoverdines structures as tool for the phylogeny of P. putida. This study shows the strength of the combination of in silico analysis together with analytical data and literature mining in determining the structure of secondary metabolites such as peptidic siderophores.
KeywordsPseudomonas putidaPyoverdineStructure elucidationBioinformatic predictionPhylogenetic marker
Pseudomonas is a genus of non-fermentative Gram-negative Gammaproteobacteria with wide distribution, such as surfaces of virtually all plant tissues, soil, organic matter and water (Goldberg 2000). The ubiquitous character of these microorganisms explains the diversity of secondary metabolites produced by Pseudomonas species, especially the fluorescent Pseudomonas (rRNA group I). Fluorescent Pseudomonas strains produce pyoverdines or other siderophores to chelate iron(III). So far, more than 70 pyoverdines have been well characterized, and they all consist of three distinct structural parts: a quinoline-1-carboxylic acid containing a chromophore responsible for the green fluorescence, a dicarboxylic acid or its monoamide, bound amidically to the 5-amino group of the chromophore and a peptide chain comprising 6–14 amino acids bound to its 1-carboxyl group (Meyer 2000; Visca et al. 2007; Cornelis 2010). The dihydroxyquinoline structure of the chromophore is identical for all pyoverdines due to the conservation of the biosynthetic gene pvdL among different species of Pseudomonas strains (Mossialos et al. 2002; Ravel and Cornelis 2003). However, slight differences in the chromophore structure have been reported for some strains (Jacques et al. 1995; Budzikiewicz et al. 2007). Different acyl side chains can be observed in isoforms of pyoverdine isolated from one single strain (Meyer 2000). The primary difference among different pyoverdines is to find in their peptide chain organization. Both the chromophore and the peptide chain of pyoverdines are synthesized by a thiotemplate mechanism catalyzed by non-ribosomal peptide synthetases (NRPS) (Ravel and Cornelis 2003; Visca et al. 2007). NRPSs are modularly assembled mega-enzymes, and each module is organized with domains with different catalysis functions. The most essential domains are: a condensation domain (C domain) which catalyzes the formation of the peptide bond, thiolation (T domain) and peptide carrier protein (PCP), and adenylation domain (A domain) which activates an amino acid substrate (Challis et al. 2000; Crosa and Walsh 2002). The resolution of the crystal structure of the phenylalanine-activating adenylation domain PheA illustrated that there are signature residues in the binding pocket of the A domain, controlling substrate recognition (Konz and Marahiel 1999; Stachelhaus et al. 1999). The substrate specificity of different A domains makes the prediction of amino acid sequence of the synthesized peptide possible by in silico studies, using publicly available softwares such as the PKS–NRPS (Bachmann and Ravel 2009) and antiSMASH (Rottig et al. 2011). The final configuration of the amino acid (l- or d-form) can also be predicted by the detection of an epimerization domain (E domain) downstream of the A domain. Thanks to these bioinformatic tools, researchers in the natural product field have already discovered a great number of novel non-ribosomal peptides, especially in the cases of orphan gene clusters and silent gene clusters in sequenced microorganisms (Gross et al. 2007; Rosconi et al. 2013). Different types of pyoverdines can be observed at species and even at sub-species levels as visualized by different band patterns in isoelectric focusing (IEF) gels, a method termed “siderotyping”, thus suggesting that this technique can be used as a taxonomic tool (Koedam et al. 1994; Fuchs et al. 2001; Meyer et al. 2007, 2008). Siderotyping confirmed the existence of 3 types of pyoverdines produced by different strains of Pseudomonas aeruginosa (Cornelis et al. 1989; Meyer et al. 2007), while conversely different Pseudomonas syringae strains corresponding to different pathovars were found to all produce the same pyoverdine (Bultreys et al. 2001). However, in the case of Pseudomonas fluorescens and Pseudomonas putida, which are less clearly defined species and rather form complexes of species, more information is still needed to be collected and the diversity of pyoverdines further investigated. Recent examples are the originally named P. fluorescens Pf-5 and P. fluorescens CHA0, which were proposed to belong to a new species, Pseudomonas protegens (Ramette et al. 2011). Interestingly, the pyoverdines produced by these two strains are almost identical and differ from the pyoverdines produced by other P. fluorescens strains. They share the same amino acid sequence in the peptide chain, but the sixth residue is d-Ala in Pf-5, and l-Ala in CHA0 (Hartney et al. 2013). In this study, we define the structure of an unusual pyoverdine produced by P. putida W15Oct28, isolated from the water surface of the Woluwe river in Belgium (Pirnay et al. 2005) by combining the data of a series of chemical analyses and a bioinformatic interpretation of the biosynthetic genes. Furthermore, we review all the pyoverdine structures produced by P. putida both from the literature and made a comparison of bioinformatic analysis of the whole genome sequenced strains. The correlation of the pyoverdine structures with the phylogeny of the producing strain by whole genome comparison is further validated, which confirms the potential of using pyoverdine as taxonomic and phylogenetic markers to distinguish P. putida subspecies (Meyer et al. 2007).
Materials and methods
Large scale pyoverdine purification
Pseudomonas putida W15Oct28 was grown at 28 °C in 1 l of iron-poor CAA medium (BactoCasamino Acid, BD, 5 g l−1; K2HPO4 1.18 g l−1; MgSO4·7H2O 0.25 g l−1) in 5 l Erlenmeyer flasks, at a shaking speed of 160 rpm for 48 h. Bacterial cells were removed by centrifugation at 10,000×g during 15 min. After filtration the supernatant was passed on a C-18 column that was activated with methanol and washed with distilled water. Elution was done with acetonitrile/H2O (70/30 %). Samples were lyophilized after most of the acetonitrile was evaporated (Matthijs et al. 2009).
High-performance liquid chromatography (HPLC), gas chromatography (GC) and mass spectrometry (MS)
HPLC analysis was carried out with an Agilent 1100 Series HPLC System with auto-sampler and degasser UV detector and thermostated column compartment with an operating temperature range from ambient to 105 °C. Preparative-scale purification was done on Gilson 712 semi-preparative HPLC system with a 322 pump, an UV–Vis 156 detector, a manual injector and 206 Fraction Collector. ESI–LC/MS was done by means of a Waters 600E HPLC Pump, a Waters 2487 Dual Absorbance Wavelength Detector and a Fisons VG II Quattro Mass Spectrometer (ESP ionisation). Operating temperature is from ambient to 80 °C. The mobile phase consists of a water/acetonitrile/TFA mixture with a gradient going from a mixture water/AcN (97:3) containing 0.1 % TFA to a mixture water/AcN (0:100) containing 0.1 % TFA in 30 min followed by 10 min isocratic run at these conditions, and with a flow rate of 20 ml min−1. High resolution MS and collision-induced dissociation tandem fragmentation MS were done on a QTof Micro MS (positive ion mode). Under standard measurement conditions the sample was dissolved in CH3CN/H2O (1:1) containing 0.1 % TFA. GC/MS spectra were recorded on a Trace MS Plus (Thermo). Separation was done on a J&W Scientific DBxXLB (30 m, 0.25 mm ID, 0.25 μm film thickness).
Amino acid analysis
Partial hydrolysis was realized by subjecting 4.2 mg of pure pyoverdine sample to 1.5 ml of 6 M HCl at 110 °C for 2 h, followed by ESI–LC/MS analysis. Full hydrolysis was done by using 4.8 mg of pyoverdine pure sample in 1.5 ml of 6 M HCl at 110 °C for 24 h. The amino acids were derivatized as N-TFA-n-butylesters according to Leimer et al. (1977). More precisely, the HCl solution was evaporated by gas N2, then n-butanol·3 N HCl is added and incubated at 100 °C for 15 min for esterification. After allowing the flask to cool down, the n-butanol·3 N HCl is removed by evaporation. Two ml of dichloromethane is added to azeotrope any remaining water and then removed by evaporation. Two ml of dichloromethane and 1 ml of TFAA are added to acylate the sample at 150 °C for 5 min; the derivatized sample is kept at 4 °C for GC/MS analysis.
Genome sequencing and bioinformatic analysis of the pyoverdine biosynthetic genes
The genome of P. putida W15Oct28 was sequenced at the VIB nucleomics core by the IlluminaMiseq system. The library was constructed using the Nextera kit, read length as 150 bp paired end. The coverage is about 62 times, and the sequencing reads were assembled by Velvet (Zerbino and Birney 2008), with 99 contigs representing the draft genome. Each contig was uploaded to antiSMASH to detect the pyoverdine biosynthetic genes cluster. The pyoverdine biosynthesis genes clusters were annotated using the RAST website (Aziz et al. 2008). To predict the substrate of the different adenylation domains, antiSMASH gives 4 predictions based on NRPSPredictor2 SVM, Stachelhaus code, Minowa, and consensus. When the 4 predictions are identical, then the proposed activated amino acid is accepted. When the 4 predictions are different, the amino acids sequence of the adenylation domain will be further analyzed by PKS–NRPS to compare with the Stachelhaus code. The three dimensional model of pyoverdine–Fe3+ complex is generated by drawing the peptide structure in the program Molecular Operating Environment (MOE) (2012). The complexation is proposed, not based on any experimental results. Restrictions are applied and the structure is energy minimized with the MMFF94x force field to obtain the presented structure. All structures were visualized using Pymol 22.214.171.124.
Characterization of the α-ketoglutaric acid side chain, dihidropyoverdinechromophore and aspartic acid as the first amino acid of the peptide chain
Signal assignment of the different pyoverdine fragments
Dihydropyoverdine chromophore signature
[kgl + dihydropyoverdinechromophore + Asp]-CO2-H2O
kgl + dihydropyoverdinechromophore + Asp
[kgl + dihydropyoverdinechromophore + Asp + Ala]-CO2-H2O
kgl + dihydropyoverdinechromophore + Asp + Ala
[kgl + dihydropyoverdinechromophore + Asp + Ala + AOHOrn]-CO2-H2O
[kgl + dihydropyoverdinechromophore + Asp + Ala + AOHOrn]-CH3
kgl + dihydropyoverdinechromophore + Asp + Ala + AOHOrn
[pyoverdine + 2H]2+
pyoverdine-CO2-H2O-[Orn + Ser]-OHAsp-Hse-Ser-Hse
pyoverdine-CO2-H2O-[Orn + Ser]-OHAsp-Hse-Ser
pyoverdine-[kgl + dihydropyoverdinechromophore + Asp]
pyoverdine-CO2-H2O-[Orn + Ser]-OHAsp-Hse
pyoverdine-CO2-H2O-[Orn + Ser]-OHAsp
pyoverdine-CO2-H2O-[Orn + Ser]
Determination of the amino acid composition of the peptide chain
Pyoverdine biosynthesis genes clusters of P. putida W15Oct28
Pyoverdines structure of P. putida strains and confirmation of the usefulness of pyoverdines as phylogenetic markers
In contrast to strains of the species P. syringae which all produce the same pyoverdine, most representatives of fluorescent Pseudomonas species produce several types of pyoverdines. In the well-studied species P. aeruginosa, three types of pyoverdines were determined and are associated with three types of receptors, respectively (Cornelis et al. 1989; Meyer et al. 1997; de Chial et al. 2003; Smith et al. 2005; Bodilis et al. 2009). In the case of the species P. fluorescens and P. putida, a large number of pyoverdines structures was reported (Table 2). With the structure of the P. putida W15Oct28 pyoverdine in hand, we decided to compare the different known P. putida pyoverdines. Furthermore, the pyoverdine structures of available whole genome sequenced P. putida strains were predicted by bioinformatics analysis (Table 2). Altogether, the pyoverdines can be divided into several groups, which provide more information for siderotyping of P. putida strains and support the proposal to use pyoverdines as phylogenetic markers for fluorescent Pseudomonas strains, especially within the species P. putida (Meyer et al. 2007). Thirteen genomes of P. putida strains, two genomes of Pseudomonas sp. strains (UW4 and GM78), the genome of P. entomophila L48 (a close relative of P. putida), and the genome of A. vinelandii DJ were analyzed by antiSMASH to predict the amino acid composition of the peptide chains of their respective pyoverdines. The pyoverdine of P. putida KT2440 is predicted to be the same as reported by Matthijs et al. (2009), with the third amino acid being a hydroxyl-aspartic acid in d-configuration. The pyoverdines of P. putida KT2440, P. putida BIRD-1, and P. putida S12 were predicted to be identical by antiSMASH and correspond to the pyoverdine of P. putida G4R (Salah-el-Din et al. 1997). The pyoverdine of P. putida GB-1 was predicted to be similar to the pyoverdine of P. putida L1, CFBP2461, and WCS358, with the fifth allothreonine amino acid missing. The pyoverdine of P. putida S16 was predicted to belong to this type, but with four amino acids missing (from 5th to 8th position, aThr-Ala-Thr-Lys). The third major type of pyoverdines produced by P. putida strains share similar structure with the pyoverdine produced by P. putida PutC. The pyoverdines of P. putida SJTE-1 and P. putida F1 have the same amino acid sequence and configuration as the pyoverdine of P. putida PutC. The modification of the second amino acid (OHbutOHOrn) is very rare among the pyoverdine structures, thus the precise structure of pyoverdines produced by SJTE-1 and F1 should be further confirmed by MS. There are more pyoverdines produced by genome-sequenced P. putida strains which are assumed to belong to this group, however, with one or more amino acid missing (P. putida ND-6, B6-2, and LS46).There exists a bias for the amino acid substrates incorporated in pyoverdines as compared to lipopeptides, which are also synthesized by NRPS, an observation made by Caboche et al. (2010) who found that different classes of bioactive peptides have a preference for certain amino acids. For example, leucine, isoleucine, proline, tryptophan, cysteine, and methionine are commonly found in lipopeptides, but have never been described to be present in pyoverdines. Valine and histidine were reported to be included in the structure of some pyoverdines, but with really rare records. Some non-proteinogenic amino acids including diamino-butanoic acid (Dab), ornithine, homoserine, and citrulline were also found in pyoverdines. As discussed before, the structural and genetic information of azotobactin D gave crucial clue of the existence of homoserines in the pyoverdine produced by P. putida W15Oct28. When analyzing the genomes of P. entomophila L48 and Pseudomonas sp. UW4, the third adenylation domains of the pyoverdine peptide synthetases of both strains gave no clear annotation, neither by antiSMASH nor by PKS–NRPS websites. The signature residues of these two adenylation domains are quite similar (DSAAIAEV for P. entomophila L48, and DSALIAEV for Pseudomonas sp. UW4). Adenylation domains with similar Stachelhaus codes were found inthe Norine and SBSPKS databases (Caboche et al. 2008; Anand et al. 2010), which are the tenth adenylation domain of bacitracin synthetase and the fourth adenylation domain of bleomycin synthetase both of them activating histidine as substrate. Only one pyoverdine containing histidine was reported with the sequence: (d)Ser-(l)eLys-(l)OHHis-(d)aThr-(l)Ser-(l)cOHOrn, produced by both P. fluorescens 9AW and P. putida 9BW (Budzikiewicz et al. 1997). The pyoverdine of Pseudomonas sp. UW4 is designated as the same as the pyoverdine produced by both P. fluorescens 9AW and P. putida 9BW by bioinformatics analysis. Strain UW4 was reassigned as being a Pseudomonas sp. strain which belongs to the fluorescens group, jessenii subgroup recently after being first designated as a P. putida strain (Duan et al. 2013). This result may suggest us to reinvestigate the taxonomy of both P. fluorescens 9AW and P. putida 9BW. The pyoverdine of P. entomophila L48 is predicted to be different from the structure reported by Matthijs et al. (2009) and in which the second amino acid still remains undetermined.
This study shows the power of the combination of analytical techniques, bioinformatic analysis and literature survey to determine the structure of a novel pyoverdine with interesting features. It contains a dihydropyoverdine type of chromophore and has an unusually long peptide chain containing two residues of homoserine, an amino acid rarely found in pyoverdines. This study also confirms the diversity of pyoverdines structures within the species P. putida.