An iron fist in a velvet glove: The cooperation of a novel pyoverdine from Pseudomonas donghuensis P482 (PVDP482) with 7-hydroxytropolone is pivotal for its antibacterial activity.

doi:10.21203/rs.3.rs-3227966/v1

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An iron fist in a velvet glove: The cooperation of a novel pyoverdine from Pseudomonas donghuensis P482 (PVD_P482) with 7-hydroxytropolone is pivotal for its antibacterial activity.

https://doi.org/10.21203/rs.3.rs-3227966/v1

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Pseudomonas donghuensis P482 exhibits broad antimicrobial activity against phytopathogens, including the soft rot bacteria of the Dickeya genus. Here, we report that under limited nutrient availability, the antibacterial activity of P. donghuensis P482 against Dickeya solani requires the reciprocal action of two iron scavengers: 7-hydroxytropolone (7-HT) and a newly characterised pyoverdine (PVD_P482) and is quenched in the iron-augmented environment. Further, we show that the biosynthesis of pyoverdine and 7-HT is metabolically coordinated, and the functional BV82_4709 gene involved in 7-HT synthesis is pivotal for expressing the BV82_3755 gene, essential for pyoverdine biosynthesis and vice versa. The synthesis of both scavengers is under the control of Gac/Rsm, but only PVD is controlled by Fur. The isoelectric focusing profile of the P482 siderophore differs from that of the other Pseudomonas spp. tested. This finding led to the unveiling of the chemical structure of the new pyoverdine PVD_P482. To summarise, the antibacterial activity of P. donghuensis P482 is attributed to 7-HT and PVD_P482, varies depending on the nutrient and iron availability, highlighting the importance of these factors in the competition between P482 and D. solani.

General Microbiology

pyoverdine

7-hydroxytropolone

nutrient availability

iron ions

antimicrobial activity

Pseudomonas spp.

Dickeya solani

The complex network of microorganisms around the plant roots is critical in plant health and development. In the rhizosphere, considered a hot spot of microbial activity (Walker et al., 2003; Raaijmakers et al., 2009), broad inter- and intraspecies interactions occur (Mendes et al., 2013), by which the microorganisms compete for nutrients and niches. One of the factors limiting microbial growth, apart from a carbon source, is iron. In organism-define quantities, this element is essential for most (micro)organisms (Cornelis and Matthijs, 2002; Roche et al., 2013). Iron is ubiquitous in the soil environment, however, mostly in a ferric state (Fe (III)). Fe(III) iron forms less soluble salts, which precipitate in soil, limiting its availability. With the rhizosphere’s low Fe(III) concentration, the microorganisms and the roots compete for this scarce resource (Vansuyt et al., 2007; Butaite et al., 2017). Although essential for the vital function of bacterial cells, iron might also be detrimental to them by generating highly toxic reactive oxygen species (Cornelis et al., 2022). Therefore, bacteria developed tight mechanisms regulating iron uptake, transport, and storage to maintain iron homeostasis in the cell (Andrews et al., 2003; Wandersman and Delepelaire, 2004). The low molecular weight compounds, known as siderophores, are the most common iron scavengers that bacteria produce to acquire Fe(III) ions from the environment. Siderophores vary in terms of their chemical structure and their iron binding capacity (Cezard et al., 2015; Butaite et al., 2018). A single bacterial cell may produce several types of siderophores, with some strains being able to intercept iron-scavenging molecules produced by other microorganisms. The synthesis of siderophores is regulated by a global ferric uptake regulatory protein, Fur, commonly found in the bacterial world (Touati, 2000). The Fur protein is a transcriptional regulator which negatively regulates the synthesis of siderophores under an abundance of Fe(II) ions (Troxell and Hassan, 2013).

Fluorescent pseudomonads exploit the efficient iron uptake and transport system, which, combined with their metabolic versatility, makes them active players in plant-related environments (Raaijmakers et al., 2009; Cornelis, 2010; Gu et al., 2020; Kramer et al., 2020). They are readily isolated from the rhizosphere and possess properties beneficial for the host plants (Mazurier et al., 2009; Ramette et al., 2011; Krzyzanowska et al., 2012; Calderon et al., 2015; Zboralski et al., 2020; Costa-Gutierrez et al., 2022). The two key regulatory pathways known to regulate the synthesis of siderophores in pseudomonads are the one based on the ferric uptake regulatory protein (Fur) and the Gac/Rsm signal transduction system (Yu et al., 2014; Cezard et al., 2015). It acts as a negative transcriptional regulator, preventing the synthesis of siderophores in the presence of Fe(II) ions (Hassan and Troxell, 2013). The Gac/Rsm signal transduction system acts as the principal global regulatory mechanism that controls the expression of ca. 10% of genes in P. protegens, P. chlororaphis, P. fluorescens, and possibly other species (Heeb and Haas, 2001; Ferreiro and Gallegos, 2021). This system controls major phenotype traits of plant-associated pseudomonads such as biofilm formation, motility (Zboralski and Filion, 2020), and the production of secondary metabolites crucial in rhizosphere competition: siderophores, 2,4-DAPG, phenazines, hydrogen cyanide, and cyclic lipopeptides (Blumer and Haas, 2000; Heeb and Haas, 2001; Raaijmakers et al., 2006; Mavrodi et al., 2007; Yu et al., 2014).

In our former studies, we isolated from the tomato rhizosphere an isolate P482, further characterised as Pseudomonas donghuensis (Krzyzanowska et al., 2012; Krzyzanowska et al., 2016). P482 exhibits a broad spectrum of inhibitory activity towards bacterial plant pathogens (Pseudomonas syringae and the members of the Soft Rot Pectobacteriaceae) (Golanowska et al., 2012; Krzyzanowska et al., 2012). Furthermore, the volatile organic compounds produced by this strain are responsible for the growth inhibition of the fungal pathogens Rhizoctonia solani, Fusarium culmorum, Verticillium dahliae, and oomycete Pythium ultimum (Ossowicki et al., 2017).

Pseudomonas donghuensis produces two major iron scavengers: pyoverdine (PVD) (Krzyzanowska et al., 2016) and 7-hydroxytropolone (7-HT) (Jiang et al., 2016). In P. donghuensis SVPB6, it was shown that the expression of the biosynthetic genes for 7-HT is positively regulated through the Gac/Rsm pathway (Yu et al., 2014; Chen et al., 2018; Muzio et al., 2020), which yet does not affect the biosynthesis of PVD (Muzio et al., 2020)

We previously reported that 7-HT is involved in the antibacterial activity of P. donghuensis P482 against D. solani on LB agar (Krzyzanowska et al., 2016), with a limited contribution of PVD to the antibacterial effect on this medium. However, PVD was found important for antibiosis against this pathogen on an M9 medium with either glycerol or glucose as the sole carbon source, with up to no contribution of 7-HT to the antibacterial effect observed on glycerol (Matuszewska et al., 2021)

Within this study, we investigate the complex interplay between 7-hydroxytropolone (7-HT) and P482 pyoverdine (PVD_P482) in the context of their contribution to the P. donghuensis P482 antibacterial activity towards Dickeya solani under varying nutritional and ferric iron availability. Moreover, we determined the structure of a novel pyoverdine (PVD_P482) produced by P. donghuensis P482.

Strains and culture conditions

The strains used in the study are listed in Table 1. Unless otherwise stated, P. donghuensis P482, HYS^T, and the P482 mutants were routinely grown in Miller’s Lysogeny Broth (LB, Novagen, Germany) or on LB solidified with 1.5% agar (LB-agar, Novagen, Germany). Pseudomonas spp. and Dickeya solani IFB102 (referred to as Ds) were cultured at 28°C, and Escherichia coli ST18 at 37°C. For the P482 mutants: KN1009 and KN3755 defective in pyoverdine synthesis (referred to as PVD_A and PVD_B, respectively), KN4706 and KN4709 defective in 7-hydroxytropolone production (referred to as: 7-HT_A and 7-HT_B), KN4243 defective in emrA gene (later referred to as EmrA), KN3318 defective in gacA gene (later referred to as GacA), KN4870 defective in fur gene (later referred to as Fur) the medium was supplemented with 30 µg ml^− 1 of kanamycin.

Table 1

Bacterial strains and plasmids used in the study.
Strain	Origin/features	References
Pseudomonas spp.
P. donghuensis HYS^T	Donghu Lake (China)	(Gao et al., 2012)
P. donghuensis P482	The rhizosphere of tomato (Poland)	(Krzyzanowska et al., 2014)
P. entomophila L48^T	Drosophila melanogaster (France, Guadeloupe)	(Mulet et al., 2012)
P. putida KT2440	Soil (Japan), a derivative of meta-toluate degrading strain (mt-2) lacking the TOL plasmid (pWW0)	(Bagdasarian et al., 1981; Regenhardt et al., 2002)
P. vranovensis DSM16006^T	Soil contaminated with nitroaromatics (Czech Republic)	(Tvrzova et al., 2006)
P. protegens Pf-5	Root surface of cotton	(Paulsen et al., 2005)
Plant pathogen
Dickeya solani IFB0102	Potato plant (Poland), a strain belonging to the Pectobacteriaceae (SRP) family	(Sławiak et al., 2009)
Genetically modified strains
Escherichia coli ST18	Donor strain for biparental mating; pro thi hsdR + Tp^R Sm^R; chromosome::RP4-2 Tc::Mu-Kan::Tn7/λpir ΔhemA	(Thoma and Schobert, 2009)
PVD_A (KN1009)	P. donghuensis P482 mutants carrying an insertion of pKNOCK-Km suicide vector in the locus BV82_1009, pvdL gene relevant to the production of putative NRPS for chromophore synthesis (BV82_1009::pKNOCK-Km)	(Krzyzanowska et al., 2016)
GacA (KN3318 )	P. donghuensis P482 mutants carrying an insertion of pKNOCK-Km suicide vector in the locus BV82_3318, the gacA gene relevant to the synthesis of GacA protein, a response regulator of the two-component GacS/GacA system (BV82_3318::pKNOCK-Km)	(Krzyzanowska et al., 2016)
PVD_B (KN3755)	P. donghuensis P482 mutants carrying an insertion of pKNOCK-Km suicide vector in the locus BV82_3755, partially similar to the PvdD pyoverdine synthase, a single thioesterase “release domain” (BV82_3755::pKNOCK-Km)	(Krzyzanowska et al., 2016)
7-HT_A (KN4706)	P. donghuensis P482 mutants carrying an insertion of pKNOCK-Km suicide vector in the locus BV82_4706 encoding the HpcH/HpaI aldolase/citrate lyase family protein (BV82_4706::pKNOCK-Km)	(Krzyzanowska et al., 2016)
7-HT_B (KN4709)	P. donghuensis P482 mutants carrying an insertion of pKNOCK-Km suicide vector in the locus BV82_4709 relevant to the synthesis of the acyl-CoA dehydrogenase, C-terminal domain protein (BV82_4709::pKNOCK-Km)	(Krzyzanowska et al., 2016)
EmrA (KN4243)	P. donghuensis P482 mutants carrying an insertion of pKNOCK-Km suicide vector in the locus BV82_4243, the emrA gene relevant to the synthesis of the efflux transporter, RND family, MFP subunit (HlyD_D23 domain containing) (BV82_4243::pKNOCK-Km)	(Matuszewska et al., 2021)
Fur (KN4870)	P. donghuensis P482 mutants carrying an insertion of pKNOCK-Km suicide vector in the locus BV82_4870, the fur gene encoding a global Fur regulator (BV82_4780::pKNOCK-Km)	This study
Plasmids
pKNOCK	Km^R; suicide vector for site-directed mutagenesis; insertion occurs via a single crossing-over event	(Alexeyev, 1999)
pKN4870	Km^R; pKNOCK-Km bearing a 529 bp fragment of BV82_4870 (primers F_ XbaI_P482_4870_F / R_ KpnI_P482_4870_R) in the XbaI-KpnI cloning site	This study

For the growth of the auxotrophic strain E. coli ST18, the medium was supplemented with 50 µg ml^− 1 of 5-aminolevulinic acid (5-ALA, Sigma-Aldrich, USA). When necessary, the medium was supplemented with kanamycin (30 µg ml^− 1) or streptomycin (50 µg ml^− 1), or both.

Direct antibacterial activity

For the antimicrobial activity assays, the bacterial strains were taken directly from 36 h cultures growing on either LB agar (rich medium), a nutrient-limited and iron-restricted medium CAA (CasAmino Acid) solidified with 1.5% agar (Casamino acids 5 g l^− l; K₂HPO₄ 0.9 g l^− l; MgSO₄·7H₂O; 0.25 g 1^− 1)(Cornelis et al., 1992) or succinate minimal medium agar (SMM - K₂HPO₄, 6.0 g l^− 1; KH₂PO₄, 3.0 g l^− 1; (NH₄)₂SO₄, 1.0 g l^− 1; MgSO₄.7H₂O, 0.2 g l^− 1; succinic acid, 4.0 g l^− 1) (nutrient-poor and iron-limited conditions) (Meyer and Abdallah, 1978). To assess the role of iron scavengers in the antibiosis activity, the medium was supplemented with FeCl₃ (Fe(III)) to a final concentration of 10 or 100 µM. The concentration of iron ions used in this study was adjusted in a preliminary study according to a literature search showing the universal working concentration of Fe(II) and Fe(III) to obtain non-toxic, iron-abundant media for pseudomonads ranges from 10 to 100 µM (Meyer and Abdallah, 1978; Neilands, 1984; Manninen and Mattila-Sandholm, 1994). These two types of iron ions are commonly used since they vary in their availability for the bacteria, Fe(II) ions are readily available for the uptake, while the poorly soluble Fe(III) require the aid of iron chelators or siderophores.

Inhibition of D. solani IFB0102 was examined according to (Krzyzanowska et al., 2016) with some modifications. Briefly, the suspension of the pathogen in the given medium, with turbidity adjusted to 4 McFarland (McF) units, was spread with a swab soaked in the bacterial suspension on the surface of the LB agar, CAA agar, or the solidified SMM. Two microliters of the suspensions of the tested strains, prepared in the given medium adjusted to 12 McF units, were spotted on the surface of the pre-inoculated media. The samples were incubated at 28 ℃ for 24 h. The diameter of the growth inhibition zones was measured. All results were reported as the percentage of positive control (P482) of the pathogen (Ds) growth inhibition. All experiments were repeated three times using the same setup (n = 4), with three technical replicates each.

Allele exchange mutagenesis

The P482 mutant KN4870 (later referred to as Fur) was obtained as described previously (Krzyzanowska et al., 2016) with the primer pair listed in Table S1 and the annealing conditions listed in Table S2

. Briefly, the amplicon (529 bp), being a fragment of the gene to be inactivated (the locus BV82_4870 encoding Fur family transcriptional regulator), was cloned into XbaI/KpnI cloning site of pKNOCK-Km suicide vector (Alexeyev, 1999). The 5-ALA auxotrophic E. coli ST18 (Thoma and Schobert, 2009) donor was transformed with the obtained construct (pKN4870). The resulting E. coli [pKN4870] strain was used to transfer the vector by biparental mating with recipient P. donghuensis strain P482. First, the transconjugants were screened on the LB-agar supplemented with kanamycin to select the P482 pKNOCK vector recipients but without 5-ALA to inhibit the growth of the ST18 donor. Next, the transconjugants were screened for the presence of the pKNOCK-Km insert with colony PCR using the pKNOCK-Km backbone primers (Supplementary Data Table S1 and S2). The insertion in the correct locus was then confirmed by sequencing the transconjugant genome fragment, performed at the Institute of Biophysics and Biochemistry (IBB PAS, Warsaw, Poland).

Iron-scavenging analyses

Analysis of absorbance maxima of cell-free supernatants

Cell-free supernatants of P482 cultures in CAA, collected at five different time points (6, 12, 18, 24, and 48h of growth), were analysed for the presence of the UV absorbance maxima characteristic for 7-HT and PVD. Before the analysis, the bacterial cultures were grown in 200 ml of CAA medium in Erlenmeyer flasks, rinsed with 20% HCl to remove iron traits, and washed with double distilled water. Then, the cells were removed from the medium by centrifugation of 1.5 ml aliquot (at 3500 × g for 3 min.). The absorption spectra (λ range of 300–500 nm) were measured every 0.5 nm using the Epoch 2 Microplate Spectrophotometer (Biotek, USA) in 96-well microplates. The experiment was replicated three times using the same setup.

Estimation of iron chelation kinetics with CAS solution

The kinetics of iron chelation by bacterial strains was estimated based on the rate of discolouration of the CAS solution (for 100 ml: 10 mM CTAB, 6 ml; ddH₂O, 45 ml; 1 mM FeCl₃ in 10 mM HCl, 1.5 ml; 2 mM Chrome Azurol S (Riedel-de Haen, NC, USA), 7.5 ml; 0.5 M MOPS, pH 6.8, 20 ml). The solution was filter-sterilised (0.22 µm cellulose acetate syringe filters, VWR) kept at 4 ℃ prior to use and with the cell-free culture supernatants (Arora and Verma, 2017). The cell-free supernatants were obtained by centrifugation (at 3500 × g for 3 min.) of 1.5 ml aliquots of bacterial cultures at 6, 12, 18, 24 and 48 h culturing. The supernatants were diluted 10-fold in water, and 100 µl of each was mixed with the CAS solution at the ratio 1:1. Immediately upon preparation, the discolouration of the blue CAS solution was measured at 630 nm within 20 min. The data were expressed as percentage iron scavenger units according to the formula (Eq. 1) (Payne, 1993):

Equation 1. Calculating the kinetics of iron chelation

$$\frac{\left(\text{A}\text{r}-\text{A}\text{s}\right) \text{x} 100}{\text{A}\text{r}}$$

where: Ar = absorbance of the reference (CAS solution and water), As = absorbance of the sample (CAS solution and cell-free supernatant). The experiment was performed three times independently using the same setups.

Isoelectric focusing of the siderophores (IEF)

Analysis of the siderophores produced by the tested strains of Pseudomonas spp. was performed from 48-h bacterial liquid cultures in CAA as described by (Koedam et al., 1994), with modifications. Briefly, the cell-free supernatants were concentrated 20-fold by lyophilisation, and the total protein concentration in the samples was measured using the Pierce™ BCA Protein Assay Kit (Thermo Fisher, USA). Protein content was used as a parameter to normalise the volume of the samples applied on the strips (ReadyStrip IPG Strips 11 cm, BIO-RAD, USA). The separation was preceded by 18-h passive rehydration of the strips at 4 ℃. The single mixture components for the IEF and the reaction conditions are listed in Supplementary Table S3. Next, siderophores were separated in a 3–10 pH gradient using the PROTEAN IEF Cell Bio-Rad device (Bio-Rad, USA). The conditions of IEF are provided in Supplementary Table S4. Immediately after IEF separation, fluorescent siderophores’ profiles were visualised under UV light (365 nm), and the non-fluorescent siderophores were detected by covering the strips with CAS solution mixed with 1% agar and monitoring the appearance of discoloured areas.

RNA extraction, cDNA synthesis, and reverse transcription-quantitative PCR (RT-qPCR)

Primer design

Primers (Supplementary Table S1) for the RT-qPCR expression study were designed with PerlPrimer (Marshall, 2004) and Primer3 (Untergasser et al., 2012) tools and tested for their specificity in silico with the use of the BLAST tool as described before (Matuszewska et al., 2021).

Culture preparation and RNA extraction

For the total RNA extraction, bacteria were grown in the CAA liquid medium. Briefly, the bacterial suspension of P482 and its selected mutants (PVD_B, GacA, 7-HT_B, and Fur) in CAA medium at the optical density of 2 McF (ca. 6×10⁸ cfu ml^− 1) served as the inoculum (1:100) of 500 ml of fresh CAA. The cultures were grown at 28 ℃ with shaking (130 rpm). The samples of bacterial culture of P482 not exceeding 10⁹ cfu ml^− 1 were harvested every hour between the 6th - and 12th hour of growth. In the case of PVD_B, GacA, 7-HT_B, and Fur, the bacterial culture samples not exceeding 10⁹ cfu ml^− 1 were harvested only at a single time point after 12 hours of growth. After centrifugation (10 000×g for 2 min), the bacterial pellet was suspended in 0.5 ml of fixRNA solution (the RAN stabilising agent) (Amplicon, Poland). Three biological replicates for each tested condition were prepared.

RNA isolation followed by DNA removal, RNA quantification, normalisation, and the statistical analyses of the results obtained were all outsourced to Amplicon (Wroclaw, Poland) (https://en.amplicon.pl/). Briefly, RNA was isolated from the bacterial cultures using GeneMATRIX Universal RNA Purification Kit (EURx, Gdansk, Poland). The RNA was purified from the residual genomic DNA (gDNA) using DNAse I and the GeneMATRIX Universal RNA Purification Kit (EURx, Gdansk, Poland) according to the manufacturer’s protocol. The RNA concentration was measured using a NanoDrop spectrophotometer (Thermo Scientific, USA). The lack of gDNA contamination was confirmed by real-time PCR with the BV82_4709 gene as the target and the purified RNA as a template. cDNA was synthesised with the NG dART RT kit (EURx, Gdansk, Poland). The reaction was prepared in 10 µl volume, with the maximum recommended quantity of input RNA.

The qPCR reactions were performed on the Bio-Rad CFX96TouchTM device (Bio-Rad, USA) with the attached HOT FIREPol® SolisGreen qPCR Mix, previously preparing cDNA and gDNA calibration curves consisting of six points. The initial cDNA solution was used to prepare the calibration curve for the rpoD, gyrB, BV82_3755, and BV82_4709 genes. All points of the calibration curves were obtained with 4-fold serial dilutions. The reactions of amplification were prepared in a 15 µl volume. Five µl of 8-fold diluted cDNA was added to the reaction. The expression level was calculated by the Pfaffl method (Pfaffl and Hageleit, 2001). The statistical analysis of the results obtained was performed using the Q Dixon test (Dean and Dixon, 1951) to identify and reject the outliers. The expression levels for each sample transcript were scaled down to obtain an average relative expression level in the reference group equal to 1 (100%). The mean, standard deviation, and standard error were calibrated in a test group for each transcript. The Shapiro-Wilk test (Shapiro and Wilk, 1965) was performed to analyse the normality and the distribution for individual transcripts (p < 0.005). The mean expression values obtained for each tested gene and the reference genes were subjected to a two-direction comparison with Student’s t-test with correction for multiple testing (Student, 1908).

Determining genes involved in the synthesis of pyoverdine and prediction of the amino acid sequence of pyoverdine in silico

Genes related to the biosynthesis of pyoverdine in strain P482 were identified with the help of antiSMASH (Blin et al., 2019), eggNOG-mapper (Huerta-Cepas et al., 2019), and literature data (Ringel and Brüser, 2018). The number of modules encoded by the NRPS-type genes responsible for the peptide chain of pyoverdine was determined using antiSMASH. The in silico prediction of the amino acid sequence of pyoverdine synthesised by P482 was performed in parallel by NRPSpredictor2 (Röttig et al., 2011) (http://nrps.informatik.uni-tuebingen.de/) and the PKS/NRPS Analysis Website (Bachmann and Ravel, 2009) (http://nrps.igs.umaryland.edu/). Both prediction tools rely on the sequence of eight residues of the binding pocket residues determining the substrate specificity of adenylation domains present in each NRPS module.

Purification and characterisation of P482 pyoverdine (Pvd_P482)

Pyoverdine was purified from 200 ml 48-h culture of the P482 strain grown in CAA at 28 ⁰C. The cells were removed by centrifugation and the supernatant was filter-sterilised on PES membrane (Nalgene™ Rapid-Flow™ Sterile Disposable Filter Units). The supernatant was lyophilised and resuspended in 20 ml of water. The suspension was filtered through filtration units with MWCO 5000 Da (Concentrators, Spin 5K MWCO, 4 ml, Agilent Technologies).

Pyoverdine was purified by using semi-preparative HPLC system (flow rate 5 mL min^− 1, 223 nm) with C8 column (Phenomenex Luna 100 Å 10 µm 250x10 mm), and gradient 0–10% B in 10 min; 10–30% B in 60 min, where A: 0.1% TFA/Mili-Q water, and B: 0.1% TFA/acetonitrile. After each purification step, all fractions were checked by MALDI-TOF mass spectrometer using DHB as a sample matrix and RF-HPLC (flow rate 1 ml min^− 1, 190–800 nm PDA detector) with Phenomenex Proteo column (90 Å 4 µm 250x4.6 mm) in gradient 0-100% B, where A: 0.1% TFA/Mili-Q water and B: 0.1% TFA in 80% acetonitrile (in Milli-Q water). After each step, all fractions containing desirable concentrations and mass were lyophilised. Purification steps were repeated until a final purified fraction was obtained.

NMR measurements

The NMR spectra of pyoverdine were measured on a Bruker AVANCE III 700 MHz spectrometer at 295 K. The following 2D NMR spectra were recorded: DQF-COSY, TOCSY (80 ms), ROESY (150 ms and 300 ms), and ¹H -¹³C HSQC. The sample was dissolved in H₂O/D₂O (9:1 v/v). Proton signals were assigned based on the TOSCY spectrum.

Analysis of pyoverdine using mass spectrometry

Mass spectra were registered with a MALDI TOF/TOF 5800 Sciex spectrometer. Sample analysis was performed in a dihydroxybenzoic acid matrix (DHB, final concentration 10 mg ml^− 1) dissolved in 50% acetonitrile with 0.1% trifluoroacetic acid (TFA). One µl of the matrix was mixed with 1 µl of pyoverdine solution in water directly at the MALDI target. After crystallisation, spectra were recorded in the reflector positive ionisation mode. The fragmentation spectrum was recorded in MS/MS 1KV mode for the ion corresponding to the pyoverdine dimer. The system was controlled by the Series Explorer software. Electrospray ionisation mass spectra and MS/MS analysis were performed on a Triple TOF 5600 + Sciex mass spectrometer connected to the microLC200 Eksigent chromatography system. The chromatographic separation of the preparation was carried out on a ChromXP C18 column 0.3x150 mm with a particle size of 0.3 µm in a gradient of 8% B for 1 min, 8–40% B 1–26 minutes, 98% B 26–29 min and 8% B 29–30 min. The flow was kept at 5 µl min^− 1, and the injection was 5 µL of the preparation. The system was controlled by the Analyst 1.7 TF software.

Statistical analyses

Statistical analyses were performed with R version 3.5.1 (R Team, 2018) using the RStudio (RStudio, 2017). The antagonism of P. donghuensis against D. solani was calculated as a percentage of growth inhibition by wild-type P. donghuensis P482. All the statistical tests on this data were performed using the Kruskal-Wallis test. For the post hoc test, Fisher’s least significant difference criterium with “BH” correction method was applied. The analysis was performed with the Kruskal function implemented in the “agricolae” package (de Mendiburu, 2014).

To calculate the iron-binding velocity based on liquid CAS medium discolouration, the absorbance was converted into discolouration value by subtracting absorbance in t_n from absorbance in t₀. Then the discolouration was normalised to an optical density of the culture. This way, the relative iron-binding value was obtained (Eq. 2)

Equation 2. Calculating relative iron-binding from discolouration of CAS medium

$$F=\frac{\left(xA600{(t}_{0}\right)-xA600\left({t}_{n}\right))- (\left(ncA600{(t}_{0}\right)-ncA600\left({t}_{n}\right))}{OD600}$$

F- relative iron binding; x- each sample; t_n – timepoint; t₀ - time zero; nc - negative control (no supernatant added); A600–600nm absorbance; OD600 – optical density of the culture

The average discolouration curve was calculated for each supernatant, discarding outliers (the repetitions were the A600(t₀) differed from negative control). To obtain a linear model of discolouration needed to calculate the initial iron-binding velocity, six different data transformations were made (Table S5), and the linear models of these models were calculated. The transformation with the best-fitted model (highest R² value) was selected for analysis.

The selected transformation was used to transform and get the linear model for the replicates and calculate the initial iron-binding velocity (Eq. 3). The models with R² < 0.8 were excluded for poor fitting and, therefore, poor prediction power. The initial iron-binding velocity was calculated as a percentage of the activity of wild-type P. donghuensis P482, then compared between samples and tested using Kruskal-Wallis statistical test. A nonparametric test was selected for the unequal number of replicates, excluding poorly fitted models.

Equation 3. Calculating initial iron-binding velocity

$${{V}_{0}=F}_{{m}_{{t}_{1}}}-{F}_{{m}_{{t}_{0}}}$$

V₀ -initial iron binding; F_m - relative iron-binding calculated from the best-fitted model; t₁ - time after 1 minute; t₀ - time zero

The growth inhibition of plant pathogenic D. solani by P. donghuensis P482 depends on two iron scavengers: PVD and 7-HT.

To understand the impact of the iron scavengers on the antibacterial activity of P. donghuensis P482 against Ds, antibiosis assays were performed on LB-agar (full medium), CAA (amino acids medium) and SMM (a single organic acid medium) (Fig. 1). HYS^T, a type strain for the species (Gao et al., 2015), was included in these analyses (Supplementary Figure S1). The results obtained for the wild-type (WT) strains (P482 and HYS^T) showed the impact of nutrient availability and iron ions on the antibacterial activity against Ds. In LB, the amount of iron ions is high enough (c.a. 4.3 µM of Fe (Cunrath et al., 2016)) to limit the production of PVD, and the antibacterial activity of P482 and HYS^T might be attributed only to 7-HT (Yu et al., 2014; Krzyzanowska et al., 2016). A similar role of 7-HT was also observed for HYS^T and SVBP6 antifungal activity, where 7-HT played a crucial role (Muzio et al., 2020; Tao et al., 2020). Jiang et al. (2016) showed that 7-HT can form a complex with ferric chloride (Fe(III)) in the stoichiometry of 2:1 (Jiang et al., 2016) and deprive the environment of iron. Bacteria from the Dickeya genus required iron to grow, especially during growth on and inside plants and during infections, as demonstrated for D. dadantii 3937 (Expert et al., 1996; Franza et al., 2005). Therefore, the observed Ds growth inhibition might result from iron depleting by 7-HT of P. donghuensis.

To verify the role of iron scavengers of P482 in antibacterial activity, we used previously obtained P482 mutants affected in the iron scavengers synthesis or transport: PVD_A and PVD_B (KN1009, KN3755) and 7-HT_A and 7-HT_B (KN4706, KN4709); GacA affected in Gac/Rsm regulatory pathway and involved in the 7-HT synthesis; and the EmrA (KN4243) mutant affected in potential antimicrobial(s) transportation out of the cell, the EmrA protein (Matuszewska et al., 2021) (Table 1). The results obtained in LB for the mutants affected in PVD synthesis were similar to that of WT, and the mutants affected in 7-HT synthesis confirmed the previous findings (Krzyzanowska et al., 2016), in which the antibacterial activity of strains unable to produce 7-HT was highly diminished (Fig. 1A). Furthermore, GacA, involved in the regulation of 7-HT synthesis (Chen et al., 2018) but not in the synthesis of pyoverdine (Muzio et al., 2020), lost its ability to inhibit the growth of Ds, indicating the importance of the Gac-Rsm regulatory network for the antibacterial activity of P482. Significantly, in the case of EmrA, the antibacterial activity towards Ds was reduced to c.a. 70% of WT, confirming its role in the potential secretion of the antimicrobial(s).

Adding 10 µM FeCl₃ to the rich medium affected the antibacterial activity of WT (up to c.a. 60% of WT in unsupplemented LB) and PVD_AB mutants (to c.a. 50%) (Fig. 1A). In the presence of 100 µM Fe(III), a decrease in the antibacterial activity of these mutants was observed (up to c.a. 25% of the WT activity), although, under this condition, we expected that the antibacterial activity of WT and the PVD_AB mutants would be maintained by 7-HT, as observed for the unsupplemented medium (Fig. 1A). Thus, the remaining PVD mutants’ antibacterial activity is either attributed to 7-HT or another unidentified factor (Matuszewska et al., 2021). The supplementation of LB-agar with the increasing Fe(III) concentration had no impact on GacA, 7-HT_A, and EmrA mutants, except 7-HT_B, which showed a slight increase of the antibacterial activity compared to that observed on unsupplemented LB-agar.

Due to their metabolic plasticity, Pseudomonas spp. can thrive in various environmental niches (Raaijmakers et al., 2008; Loper et al., 2012; Bai et al., 2015; Zboralski and Filion, 2020; Getzke et al., 2023) using available sources of carbon and nitrogen to establish and outcompete competitors via production of antimicrobials (Duffy and Defago, 1999). Therefore, we verified how nutrient and iron limitation (amino acids as the source of carbon and nitrogen) influences the antibacterial capacity of P482 and its mutants. We also examined the roles that 7-HT and PVD play in this process. The antibiosis on CAA (nutrient-limited and iron-restricted medium (Cunrath et al., 2016)) indicated that the PVD mutants demonstrated c.a. 60 and 45% of the WT activity for PVD_A and PVD_B, respectively (Fig. 1B). EmrA presented c.a. 40% of the WT activity, and the remaining mutants showed no antibacterial activity. Interestingly, when 10 µM Fe(III) was added to the medium, WT showed more than 90% of its activity compared to unsupplemented CAA, and the PVD mutants showed even higher antibacterial activity than on the unsupplemented CAA (about 62% and 70% for PVD_A and BVD_B, respectively). C.a. 60% of the WT activity was also observed for EmrA. These results indicate that in iron-restricted conditions, low concentrations of exogenous iron ions in the medium caused the increase in antibacterial activity of PVD mutants, plausibly resulting from iron chelation by 7-HT and/or the activity of another unidentified antimicrobial factor (Matuszewska et al., 2021). A further increase in Fe(III) concentration caused a decrease in antimicrobial activity: for WT to 50%, for both PVD_AB and EmrA to 20% and 16%, respectively. These results confirmed that 7-HT is a primary antimicrobial produced by P482 with antibacterial activity (Fig. 1B).

Contrasting results were observed on SMM (Fig. 1C). Succinic acid is one of the organic acids in the root exudates in many plants and is a preferred carbon source utilised by pseudomonads in the plant rhizosphere (Kamilova et al., 2006). The SMM containing succinate as a sole carbon source is a nutrient-poor and iron-restricted (Cunrath et al., 2016) medium, described and used as a preferred medium for promoting siderophore biosynthesis in P. fluorescens by Meyer and Abdallah (1978). Interestingly on SMM, PVD mutants lost their antibacterial activity against Ds entirely, unlike the 7-HT mutants, which retained at least c.a. 60% of the WT activity. Thus, we might assume that under this condition, the function of 7-HT in PVD mutants is inhibited, or 7-HT is not synthesised. In addition, for EmrA and GacA, a significant reduction of the antagonistic activity (on average to about 40% of WT) was observed. This indicates that access to iron via the PVD iron acquisition system is essential for antibacterial activity under nutrient-poor conditions. Thus, PVD is a key antimicrobial under these conditions. These results contrast with these of Matuszewska et al. (2021), in which other single carbon sources, such as glucose or glycerol, were used in the antibiosis assay. The PVD mutants showed significantly lowered antibacterial activity against Ds, regardless of the carbon source used; however, they still were able to inhibit Ds growth. At the same time, 7-HT mutants remained partially active on glucose and restored the mutation effect on glycerol (Matuszewska et al., 2021).

Supplementation of SMM with 10 µM of Fe(III) lowered the antibacterial activity of the WT strain to c.a. 70% of its activity on unsupplemented SMM (Fig. 1C). Although SMM supplementation with 10 µM Fe(III) had a limited effect on the antibacterial properties of the remaining mutants compared to their activity on unsupplemented SMM, an increase in the Fe(III) concentration to 100 µM resulted in approx. 70% reduction of the WT ability to inhibit the growth of Ds and its decline by the other mutants tested. We can assume that iron availability under the nutrient-limited condition determines the antibacterial potential of P482 based on the cooperation of PVD and 7-HT. However, the production of both iron scavengers might be regulated by the concentration of iron in the medium (Jiang et al., 2016).

Together, these results confirmed that in the nutrient-rich (LB-agar) environment, the antibacterial activity of P482 relies on the 7-HT activity. However, under nutrient-poor conditions (SMM), PVD is pivotal.

Evidence of reciprocal synthesis of the P482 PVD and 7-HT

Presence of 7-HT and PVD in the cell-free culture supernatants

As the antimicrobial potential of P. donghuensis P482 requires both types of iron scavengers: PVD and 7-HT, we investigated their production and secretion over time in P482 and its mutants via monitoring the UV absorption (Fig. 2, Supplementary Figure S2) of the cell-free supernatants collected from the bacterial cultures in CAA. For this analysis, the Fur (KN4870) insertional mutant was included to get insight into the role of the ferric uptake regulator, the Fur protein, in synthesising pyoverdine and 7-HT by P482.

During the first six hours of the strains’ growth in the iron-restricted CAA medium, we observed a slight increase of absorbance at the values expected for pyoverdine (405 nm) (Jiang et al., 2016; Muzio et al., 2020) for WT, Fur and 7-HT_A; however, no characteristic peaks for 7-HT (327 and 393 nm, (Jiang et al., 2016)) were noted (Supplementary Figure S2). The results indicated that these two mutants produce PVD under the tested conditions from the very beginning of the culturing. Interestingly, Jiang et al. (2016) reported that the production of 7-HT, likewise pyoverdine in HYS^T, also starts at 6 hours of culture (Jiang et al., 2016). Since 12-h of growth, the peaks characteristic for 7-HT were observed for P482 WT, the Fur and EmrA mutants with the stabilised value in 24-h of growth (Fig. 2). However, as expected, these characteristic peaks were absent in GacA and the mutants of 7-HT. Moreover, these peaks were also undetectable for PVD mutants PVD_A and PVD_B, indicating that 7-HT biosynthesis is related to pyoverdine synthesis. The peak characteristic for PVD is superimposed on that of 7-HT in the region around 405 nm (Jiang et al., 2016; Muzio et al., 2020) in P482. Therefore, it is impossible to cleanly indicate its presence in the tested mutants. However, the lack of a 405 nm peak in PVD_A and PVD_B and its presence in Fur and GacA are evident. After 24-h of bacteria culturing, the pyoverdine peak is slightly shifted towards 415 nm wavelength in these two mutants. A tiny peak of 415 nm was also observed in 7-HT mutants; although the analysis was not quantitative, we can assume that the PVD production in these two mutants is highly reduced.

Together, these results indicate that the production of P482 PVD is related to the synthesis of 7-HT, as the PVD mutants are also affected in the synthesis of 7-HT. Likewise, in the case of the 7-HT mutants, the synthesis of P482 PVD is highly diminished. Thus, we can presume that the synthesis of these two compounds is mutually dependent. Furthermore, these results support the role of both iron scavengers in the antibacterial activity of P482.

Expression of the genes involved in PVD and 7-HT syntheses

The timing of iron scavengers’ synthesis showed that they are produced after 6 hours of the P482 WT culture in CAA (Fig. 2). However, the peaks characteristic for 7-HT were undetectable in PVD mutants. Therefore, we verified the expression level of the genes essential for synthesising both iron scavengers, targeting the BV82_3755 gene for PVD and BV82_4709 for 7-HT, during the first 12 hours of growth. The RT-qPCR analysis revealed an increase in the expression level of both genes between 6 and 7 hours of growth in CAA. The expression of BV82_4709 increased over the time of the experiment (up to 12-h of culturing), yet in the case of the BV82_3755 gene, we observed a sudden drop in the expression level at the 9 h of culture, followed by a slow increase over the next 3 hours. The expression level of BV82_4709 is higher than that of BV82_3755; however, the increase in expression of both genes over time is consistent with the synthesis of both iron scavengers (Figs. 2 and 3A). These results confirmed that both iron scavengers are produced reciprocally under tested conditions. It might be possible that pyoverdine synthesis is essential to start the synthesis of 7-HT. Therefore, we analysed the expression level of these two genes in the genetic background of the selected P482 mutants:7-HT_B, GacA, and Fur in the case of BV82_3755, and PVD_B, GacA, and Fur for BV82_4709 (Fig. 3B and C). The expression of BV82_3755 was significantly decreased in the genetic background of 7-HT_B, and GacA mutants and meaningly increased in the Fur mutant (Fig. 3B).

Surprisingly, the GacA impairment decreased the expression of BV82_3755; thus, the Gac/Rsm pathway also seems involved in the pyoverdine synthesis regulation. The Gac-RsmA pathway is involved in the 7-HT synthesis regulation (Yu et al., 2014), so the lack of the functional GacA might indirectly affect pyoverdine synthesis via the lack of 7-HT production (Fig. 3C). Consistent with this hypothesis, the expression of BV82_3755 was highly reduced in the 7-HT deficient genetic background. These results show that the functional BV82_4709 gene (relevant to synthesising the acyl-CoA dehydrogenase) (Krzyzanowska et al., 2016) is also essential for pyoverdine synthesis.

In the case of GacA, the expression level of BV82_4709 is decreased (Fig. 3C), confirming the importance of the Gac/Rsm regulatory pathway in the 7-HT synthesis (Agaras et al., 2018). At the same time, we discovered that the functional BV82_3755 gene is essential for the expression of BV82_4709 and, consequently, the synthesis of 7-HT (Fig. 3C). This result aligns with the UV absorption analysis indicating the absence of 7-HT peaks in the PVD_B mutant. Likewise, these findings indicate that the functional BV82_3755 is crucial for 7-HT synthesis.

These results indicate that synthesising one of the iron scavengers is necessary for synthesising the other (Fig. 3). It was reported earlier that siderophores such as pyoverdine secreted by Pseudomonas aeruginosa might impact the synthesis of the virulence factors such as exotoxin, endoprotease, and pyoverdine itself (Lamont et al., 2002), indicating its regulatory role. Moreover, it was also suggested that siderophores of P. aeruginosa: PVD and pyochelin might act as the sensing molecules in like manner in quorum sensing mechanisms, employing iron content in the environment as the signal to the lifestyle change. So that, it was especially essential that synthesising both siderophores requires the presence of the ferric-siderophore complex and is linked with the positive autoregulation loop (Cunrath et al., 2020). However, in the case of PVD and 7-HT in P. donghuensis, the common synthesis mechanism of these two compounds must be further elucidated.

Interestingly, in the genetic background of fur defective mutant, the expression of BV82_4709 is of the same level as in the WT. Therefore, we might conclude that, unlike pyoverdine synthesis (Cornelis et al., 2022), the synthesis of 7-HT is not under the control of the Fur global regulator. This result is consistent with the genomic study performed for P. donghuensis SVBP6 (Agaras et al., 2018), in which no fur-box was found near the 7-HT operon. To the best of our knowledge, we are the first to show experimentally the lack of a role for the Fur regulator in 7-HT synthesis.

Secretion of PVD and 7-HT results in rapid binding of iron by P482

To verify the iron-binding velocity of P482 and its selected mutants, we assessed the kinetics of iron chelation of the cell-free culture supernatants from the CAA medium by measuring the rate of discolouration of the CAS blue solution. During the first 6 hours, all mutants, except for PVD_B, showed decreased iron-binding velocity compared to the WT strain (from c.a. 75–50% of the WT capacity) (Supplementary Figure S2) and PVD_B showed similar to the WT rate of iron chelation. Over the next 6 hours, the maximal iron-binding velocity of the tested mutants changed (at 12 hours of culturing) and dropped to c.a. 10–20% compared to WT. However, Fur reverted to c.a. 80% of P482 within the next 6 hours of growth (18 h of culturing). Nevertheless, the maximal iron-binding rate of other mutants remained very low (10–20% of the WT iron-binding velocity) during the entire experiment (up to 48 h). Interestingly, the mutation in the emrA gene impacts the iron-binding capacity.

Together, these results indicated that both iron scavengers are needed for the effective iron-binding by P482, and the disruption of the biosynthesis pathway of one (PVD or 7-HT) affects the global iron-binding kinetics in P482.

P. donghuensis produces a novel pyoverdine (PVD₄₈₂)

The IEF revealed differences in the fluorescent and non-fluorescent iron-chelator profiles of P482, its mutants, and other Pseudomonas spp. tested

Since pseudomonads produce various types of siderophores (Bultreys, 2007; Matthijs et al., 2009), we explored the profile of fluorescent and non-fluorescent siderophores produced by P482 and its selected mutants and compared them with the siderophores’ profiles of other plant-associated Pseudomonas spp.: P. putida KT2440, P. protegens Pf-5, P. entomophila L48, and the sibling strain of P482, P. donghuensis HYS^T, (Table 1). These results confirmed the presence of siderophores - the fluorescent pyoverdine(s) and the non-fluorescent ones in the case of P. donghuensis strains, P482 and HYS^T, and the mutants: GacA, 7-HT_A, 7-HT_B, and Fur (Supplementary Figure S3). None of the siderophore types was observed in PVD deficient mutant PVD_B (Supplementary Figure S3), as no fluorescence nor CAS agar discolouration were observed for this mutant. Unexpectedly, a faint fluorescent signal was observed in the case of EmrA (Supplementary Figure S3A), yet no CAS agar discolouration was observed in the overlayer assay (Figure S3B). The results obtained for GacA agree with those obtained from the analysis of the UV spectra of culture supernatants (Fig. 2), where the pick of PVD was observed but not with that of the expression level of the BV82_3755 gene in the GacA genetic background.

In the case of other Pseudomonas spp., we observed both types of siderophores, fluorescent and non-fluorescent; however, with different pI (isoelectric points). Interestingly, for L48 (P. enthomophila), while the fluorescence was observed, we did not observe discolouration of the CAS agar. However, the siderophores produced by L48 have already been well-characterised, and PVD produced by this strain was unique among 450 different Pseudomonas spp. (Matthijs et al., 2009). The IEF results obtained in our study differ from that obtained by Matthijs et al. (2009); considering these differences, it is worth noticing that although both groups used IFE to determine the siderophores profile, there were technical differences in the experimental setups, e.g., purified pyoverdines (Matthijs et al., 2009) versus culture supernatants (this study).

Together, the results of IEF revealed that P482 and HYS^T produce fluorescent siderophores with similar pI, yet visibly distinct from those produced by P. putida KT2440, P. entomophila L48, and P. protegens Pf5. The mutants PVD_B and EmrA, affected in the synthesis of PVD or its secretion (respectively), did not discolour the CAS agar and thus did not produce siderophores.

It was already noticed that among already structurally described pyoverdines of Pseudomonas spp., several could not be distinguished due to the identical IEF profiles and the way of iron uptake (Meyer et al., 2008). Here, we tested only three other Pseudomonas sp.; therefore, to better understand the P. donghuensis PVD character, we performed in silico analysis of the genes involved in its biosynthesis, followed by NMR and mass spectrometry.

In silico prediction of the amino acid chain of P482 PVD reveals a 7 aa peptide

Using in silico tools, we predicted the amino acid sequence of PVD synthesised by P482 and located other non-NRPS type genes involved in synthesising this siderophore in the genome of P482. Our earlier study reported that strain P482 harbours two gene clusters encoding non-ribosomal peptide synthases: one cluster with a single NRPS-encoding gene (BV82_1009) and the second with three adjacent genes of this type (BV85_3755, BV82_3756, BV82_3757) (Krzyzanowska et al., 2016). Furthermore, allele exchange mutagenesis of BV82_1009 and BV82_3755 led to a non-fluorescent phenotype, indicating that those genes are involved in synthesising a fluorescent siderophore-pyoverdine (Krzyzanowska et al., 2016). Therefore, in this study, we explored in silico the genome of P482 to locate other non-NRPS type genes involved in synthesising pyoverdine and predict the amino acid sequence of P482 PVD (PVD_P482). Non-ribosomal peptide synthases (NRPSs) are modular enzymes in which each subsequent module is responsible for incorporating single amino acids during the elongation of the synthesised non-ribosomal peptide. A module consists of three core domains enabling the elongation process: peptidyl carrier protein (PCP), adenylation domain (A), and condensation domain (C). Additional domains may also be present, responsible for the release of the polypeptide product (thioesterase, TE, or reductase) or its modification (e.g., epimerisation, lipid chain attachment, cyclisation) (reviewed in (Hur et al., 2012)).

Three NRPS-like genes of P482: BV82_1009, BV85_3755, BV82_3756, and BV82_3757 were analysed regarding protein modules they encode, and the predicted amino acids incorporated by these modules into the polypeptide chain of pyoverdine (Fig. 5). BV82_1009 was found to encode three NRPS modules, one with an epimerisation domain. The amino acid incorporated by the first module was unanimously predicted to be gluconic acid. The prediction for two subsequent amino acids was ambiguous or unavailable. Additionally, BV82_1009 encodes an N-terminal CAL domain presumably responsible for the acylation of ferribactin – the precursor of pyoverdine (Hohlneicher et al., 2001). The presence of the CAL domain followed by gluconic acid indicates that BV82_1009 is a pvdL. Inline, the same gene name was suggested by the eggNOG-mapper (Table 2). PvdL synthesises the conserved N-terminal tripeptide of ferribactin and introduces myristic- or myristoleic acid residue to the free amino group of the first amino acid of ferribactin. The three amino acids incorporated by PvdL, L-glutamic acid, D-tyrosine, L-2,4-diaminobutyrate, are conserved for ferribactins of fluorescent pseudomonads, the latter two amino acids further converted into the chromophore (Ringel and Brüser, 2018). This allowed speculation that the unidentified amino acids incorporated into ferribactin by PvdL encoded by BV82_1009 are D-tyrosine and L-2,4-diaminobutyrate, respectively. BV82_1016, a gene located near BV82_1009, was found to encode L-2,4-diaminobutyrate synthase PvdH.

Table 2

Genes related to pyoverdine synthesis in strain P. donghuensis P482.
Locus	Annotation	Prot. size (aa)	Proposed gene name ^A	Putative role in pyoverdine synthesis ^B
BV82_1008	RNA polymerase sigma factor, sigma-70 family protein	176	pvdS	sigma factor required for the expression of pyoverdine biosynthesis genes and other, often virulence-related genes
BV82_1009	D-alanine–poly(phosphoribitol) ligase, subunit 1	4320	pvdL	assembly of ferribactin, an initially acylated peptide precursor of pyoverdine; the first module of PvdL incorporates either a myristic- or myristoleic acid side-chain as the first building block of ferribactin
BV82_1016	2,4-diaminobutyrate 4-transaminase family protein	470	pvdH	Synthesis of L-2,4-diaminobutyrate (L-Dab), one of the three first amino acids in ferribactin incorporated by PvdL, from L-aspartate β-semialdehyde
BV82_1017	putative pvdJ/PvdD/PvdP-like protein	522	pvdP	maturation of pyoverdine chromophore
BV82_1018	efflux transporter, outer membrane factor (OMF) lipo, NodT family protein	466	opmQ	transport of mature pyoverdine to the extracellular medium
BV82_1021	RNA polymerase sigma factor, sigma-70 family protein	159	fpvI	sigma factor required for the expression of genes encoding the outer membrane pyoverdine receptor/importer FpvA
BV82_1169	fecR family protein	333	fpvR	anti-sigma factor binding to PvdS and FpvI, resulting in their inactivation
BV82_2211	transporter-associated domain protein	446	pvdQ	deacylation of ferribactin; additional role in quorum sensing – cleavage of certain N-acyl homoserine lactones
BV82_3747	membrane dipeptidase family protein	453	pvdM	Takes part in the modification of side chains in ferribactin to succinamide
BV82_3748	aminotransferase class-V family protein	423	pvdN	modification of glutamic acid within the tree initial aa in ferribactin
BV82_3749	formylglycine-generating sulfatase enzyme family protein	284	pvdO	maturation of pyoverdine chromophore
BV82_3751	pyridine nucleotide-disulfide oxidoreductase family protein	446	pvdA	produces L-N5-formyl-N5-hydroxy ornithine (L-fOHOrn) from L-ornithine by hydroxylation
BV82_3752	Cyclic peptide transporter	549	pvdE	transport of acylated ferribactin from the cytoplasm to the periplasm
BV82_3755	non-ribosomal peptide synthase	2846	n.d.	assembly of ferribactin - an initially acylated peptide precursor of pyoverdine
BV82_3756	non-ribosomal peptide synthase	2618	n.d.	assembly of ferribactin - an initially acylated peptide precursor of pyoverdine
BV82_3757	D-alanine–poly(phosphoribitol) ligase, subunit 1	3683	pvdI	assembly of ferribactin - an initially acylated peptide precursor of pyoverdine

While the N-terminal part of pyoverdine precursor ferribactin is relatively conserved, the rest of the side chain is more strain-specific, and so are the related NRPSs. The cluster comprising BV85_3755, BV82_3756, and BV82_3757 in P482 encodes seven NRPS modules, three containing epimerisation domains and a single thioesterase’ release domain’ encoded by BV82_ 3755 – the last of the transcribed genes (Fig. 5). According to NRPSpredictor2 the sequence of peptide synthesised by the NRPS complex encoded by the three genes was Asp-?-?-Glu-Ser-Asp-Lys, with the question marks symbolising the lack of unambiguous identification. A complete prediction, not entirely consistent with that obtained by NRPSpredictor2, was provided by the PKS/NRPS analysis website: Asp-Arg-OHOrn-Gln-Ser-Asp-OHOrn (with OHOrn standing for hydroxy ornithine) (Fig. 5). In relation to the latter prediction, the genome of P482 harbours a gene (BV82_3751) encoding PvdA, which produces L-N5-formyl-N5-hydroxy ornithine (L-fOHOrn) from L-ornithine by hydroxylation.

Apart from the genes mentioned above, more those related to pyoverdine synthesis were identified in the genome of P482. Among them there were pvdP (BV82_1017) and pvdO (BV82_3749) involved in the maturation of the pyoverdine chromophore (Ringel and Brüser, 2018), other genes related to modification of ferribactin: pvdQ (BV82_2211), pvdM (BV82_3747) and pvdN (BV82_3748), transport-related genes opmQ (BV82_1018) and pvdE (BV82_3752), and regulatory genes pvdS (BV82_1008), fvrI (BV82_1021) and fpvR (BV82_1169). The summary of the genes involved in pyoverdine synthesis is presented in Table 2.

The chemical structure of P482 pyoverdine (PVD_P482)

The chemical structure of PVD_P482 was analysed using ¹H and ¹³C NMR spectroscopy. The chemical shifts of the proton resonances for PVD_P482 at 295 K are listed in Supplementary Table S6, and the diagnostic region in the TOCSY spectra of PVD_P482 are presented in Supplementary Figure S4). We identified hydrogen nuclei resonances of the chromophore, succinate, and peptidic chain of PVD_P482. The sequential assignment of the molecule was confirmed according to Hα(i)-NH(i + 1) correlations in the ROESY spectrum.

The NMR spectra were confirmed by mass spectrometry analysis. The mass analysis of HPLC purified P482 pyoverdine showed the presence of two major ions [M + 2H⁺]²⁺ 639.7876 and [M + 3H⁺]³⁺ 426.8642 on ESI spectra with the mass of 1277.5659 (Fig. 6A). One major signal in MALDI ionisation is 1278.2 [M + H⁺]⁺, corresponding to mass 1277.2 of the compound (Fig. 6B, C). The 1260.2 m/z ion visible in the MALDI spectrum differs from the main signal by 18, corresponding to the loss of water molecule. Successive lower masses observed on the spectrum correspond to further defragmentation reactions of the molecule taking place during ionisation. Ions higher than the main signal corresponds to adducts with ions such as, e.g., Na⁺, as exemplified by the signal 1300.2 m/z [M + Na⁺]⁺. Checking the MALDI spectrum in the range of higher masses revealed a small signal of m/z 2555.9. The performed fragmentation of this signal (MALDI TOF/TOF) gave a response in the form of an ion corresponding precisely to the PVD_P482 mass, which indicates the possibility of the interaction of two molecules and the formation of homodimeric forms (Fig. 6D). To confirm the isolated PVD_P482 structure, the ESI MS / MS fragmentation analysis was performed for the doubly charged 639.7876 precursor ion. It was possible to identify both series of ions (b and y) on the spectrum characteristic for fragmentation by collision-induced dissociation (CID). The difference in masses revealed the presence of two modified ornithines in the compound structure, characteristic of most pyoverdines. One is the cyclo N5 -hydroxy ornithine (cOHOnr) form located at the C-terminus of the pyoverdine chain, confirmed by the presence of related characteristic ions: m/z 131, 84 (-CO). The other is the formyl derivative, N5-formyl-N5 -hydroxy ornithine (fOHOrn) (Fig. 7A), the third amino acid in the sequence, confirmed by ions m/z 131, 114 (-NH₃), 113 (-H₂O), and 86 (-NH₃, - CO). The chromophore (1S)-5-amino-2,3-dihydro8,9-dihydroxy-1H-pyrimido[1,2-a]quinoline-1-carboxylic acid is linked with succinic acid. The fragmentation spectrum shows the m / z 101.0619 ions corresponding to the loss of the succinic acid residue, 259.1248 to the loss of the chromophore structure, and the m / z signal 360.1721 related to the loss of the entire construct (chromophore + succinic acid). The chemical structure of pyoverdine produced by P482, PVD_P482, was established as chromophore group (Chr), N-formyl-N-hydroxyornithine (FoOHOrn), O-formylserine (FoSer), and N-hydroxy-cyclo-ornithine (cOHOrn) (Fig. 7B). Considering all mass spectrometry data, the sequence of isolated P482 pyoverdine is as follows: Chr-Asp-Arg-FoOHOrn-Gln-FoSer-Asp-cOHOrn. Comparing the proposed sequence to the classification presented by Cezard et al. (Cezard et al., 2015), it is most similar to pyoverdine isolated from Pseudomonas sp. (strain LBSA1), where the sequence is as follows: X-Asp-Arg-AcOHOrn-Lys-Ser-Asp-cOHOrn. In our case, the X residue is a chromophore group, the first ornithine in the sequence has a modification in the form of formylation, the lysine residue is replaced by a Gln residue, and the serine residue has a modification in the form of formylation. Thus, PVD_P482 can be classified as class II pyoverdine (PvdII)(Cezard et al., 2015).

In conclusion, the study provides valuable insights into the role of iron scavengers, specifically PVD and 7-HT, in the antibacterial activity of P. donghuensis P482 against the plant pathogenic bacterium D. solani. The results indicate that the antibacterial activity of P482 is influenced by nutrient availability, iron concentration and depends on the reciprocal synthesis of PVD and 7-HT. In nutrient-rich and nutrient-limited conditions, the antibacterial activity relies on 7-HT, while PVD plays a pivotal role under nutrient-poor conditions. The study also highlights the importance of the EmrA protein in the secretion of antimicrobials. Iron supplementation experiments demonstrated the dependence of antibacterial activity on the concentration of iron ions available in the environment and the interplay between PVD and 7-HT. The reciprocal synthesis of PVD and 7-HT was confirmed through gene expression and UV absorption analysis of culture supernatants. Furthermore, the study reveals the impact of PVD and 7-HT on iron chelation. Overall, these findings enhance our understanding of the antimicrobial mechanisms employed by P. donghuensis P482 and the role of iron scavengers in its antibacterial activity against plant pathogens. The analysis of PVD_P482 chemical structure confirmed its classification as a novel representative within the class II pyoverdine category.

Authors Contributions

Conceptualisation, S.J and M.J.; methodology, M.J., M.M., T.M., W. Ż, M.T., D.K, and P.C.; formal analysis, S.J., T.M., D.K; investigation, M.J., M.M., M.B. M.P. W.Ż. M.T. P.C. and D.K.; resources, S.J.; data curation, S.J. and R.C.; writing—original draft preparation, S.J., R.C.; visualisation, S.J., T.M., M.J.; supervision, S.J.and M.J.; project administration, S.J.; funding acquisition, S.J. All authors have read and agreed to the published version of the manuscript.

Fundings

This study was supported by the Polish Ministry of Education and Science (Ministerstwo Edukacji i Nauki) 531-N105-D786-21 (S.J) and, in terms of the gene expression study, by the Polish National Science Centre (Narodowe Centrum Nauki, Polska) research grant OPUS13 no. 2017/25/B/NZ9/00513 (S.J.).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Conflict of Interest

The authors declare no conflict of interest.

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An iron fist in a velvet glove: The cooperation of a novel pyoverdine from Pseudomonas donghuensis P482 (PVD_P482) with 7-hydroxytropolone is pivotal for its antibacterial activity.

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Strains and culture conditions

Direct antibacterial activity

Allele exchange mutagenesis

Iron-scavenging analyses

Analysis of absorbance maxima of cell-free supernatants

Estimation of iron chelation kinetics with CAS solution

Isoelectric focusing of the siderophores (IEF)

RNA extraction, cDNA synthesis, and reverse transcription-quantitative PCR (RT-qPCR)

Primer design

Culture preparation and RNA extraction

Purification and characterisation of P482 pyoverdine (Pvd_P482)

NMR measurements

Analysis of pyoverdine using mass spectrometry

Statistical analyses

Results and Discussion

Evidence of reciprocal synthesis of the P482 PVD and 7-HT

Presence of 7-HT and PVD in the cell-free culture supernatants

Expression of the genes involved in PVD and 7-HT syntheses

Secretion of PVD and 7-HT results in rapid binding of iron by P482

The chemical structure of P482 pyoverdine (PVD_P482)

Summary and conclusions

Declarations

References

Supplementary Files

Status:

Version 1

An iron fist in a velvet glove: The cooperation of a novel pyoverdine from Pseudomonas donghuensis P482 (PVDP482) with 7-hydroxytropolone is pivotal for its antibacterial activity.

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Strains and culture conditions

Direct antibacterial activity

Allele exchange mutagenesis

Iron-scavenging analyses

Analysis of absorbance maxima of cell-free supernatants

Estimation of iron chelation kinetics with CAS solution

Isoelectric focusing of the siderophores (IEF)

RNA extraction, cDNA synthesis, and reverse transcription-quantitative PCR (RT-qPCR)

Primer design

Culture preparation and RNA extraction

Purification and characterisation of P482 pyoverdine (PvdP482)

NMR measurements

Analysis of pyoverdine using mass spectrometry

Statistical analyses

Results and Discussion

Evidence of reciprocal synthesis of the P482 PVD and 7-HT

Presence of 7-HT and PVD in the cell-free culture supernatants

Expression of the genes involved in PVD and 7-HT syntheses

Secretion of PVD and 7-HT results in rapid binding of iron by P482

The chemical structure of P482 pyoverdine (PVDP482)

Summary and conclusions

Declarations

References

Supplementary Files

Status:

Version 1

An iron fist in a velvet glove: The cooperation of a novel pyoverdine from Pseudomonas donghuensis P482 (PVD_P482) with 7-hydroxytropolone is pivotal for its antibacterial activity.

Purification and characterisation of P482 pyoverdine (Pvd_P482)

The chemical structure of P482 pyoverdine (PVD_P482)