PURIFICATION AND PARTIAL CHARACTERIZATION OF PYOVERDINE SYNTHESIZED BY PSEUDOMONAS PUTIDA

Urszula Jankiewicz¹, Olga Kuzawińska^2
1 Department of Biochemistry, Warsaw University of Life Sciences – SGGW, Poland
² Department of Biochemistry, Warsaw University of Life Sciences, Poland

ABSTRACT

A soil strain of Pseudomonas putida intensively secreted, under iron deficiency conditions, a single form of siderophore-pyoverdine to the medium. The siderophore was isolated from the culture medium and purified using chelating chromatography. Purified pyoverdine inhibited the growth of all studied Fusarium strains except for F. graminearum. The presence of pyoverdine stimulated the growth of some of the tested strains. Based on MS spectrum analysis it can be concluded that Pseudomonas putida produced a pyoverdine with low molecular mass of 800 Da.

Key words: siderophore, pyoverdine, Pseudomonas, biocontrol.

INTRODUCTION

Microorganisms can take up iron using two different pathways. The first, which takes place at iron concentrations above 1×10^-4 M is a low affinity system based on the diffusion of iron ions through biological membranes. The second is a high affinity system that has developed under iron deficiency conditions and consists of an iron chelator – a siderophore [23]. Siderophores bind Fe³⁺ ions and are defined as low molecular mass compounds produced by fungi and bacteria growing under iron limiting conditions. The expression of the biosynthesis of the siderophore and the remaining components is regulated by the concentration of iron in the environment [14,24]. The complexes Fe-siderophore are identified by specific receptors whose function is governed by TonB protein. Inside the cell, the iron ion (III) is reduced, the complex disintegrates and the siderophore can be used again. There is also second mechanism that leads to iron release from siderophores, which is mediated by Fe-siderophore complex hydrolysis [21].

Pyoverdine is the main siderophore produced by fluorescent Pseudomonas strains. Several other structurally divergent siderophores have also been described for this bacterial genus. These include pyochelin, pseudomonin and 2,6-pyridine dithiocarboxylic acid, which are usually produced in smaller amounts than pyoverdine and are repressed by this compound. The affinity of these compounds for iron is usually much lower [15,30]. A molecule of pyoverdine consists of three parts: 1) a conservative dihydroxyquinoline chromophore, 2) a side chain, usually a dicarboxylic acid or its amide, 3) a peptide chain with variable structure. So far several different structures of this siderophore that differ mainly in the amino acid sequence of the peptide chain, have been identified. These structural differences are used in the siderotyping of bacteria belonging to the genus Pseudomonas [17]. The peptide chain has two functions: it provides two ferric iron binding sites and is responsible for identifying the complex of siderophore and iron on the surface of the producing cell by specific receptors located in the outer membrane [5]. The diverse amino acid composition of the peptide chain is closely related to the second function – it ensures that a given pyoverdine-iron complex will be accessible only for the strain producing it, by virtue of specific receptors on the surface of the cell. The sequence of the peptide chain is usually strain-specific but can vary within a species [18].

The synthesis of pyoverdine by Pseudomonas bacteria in the rhizosphere is most likely one of the main factors responsible for the natural protection of plants. Bacteria of this genus that secrete pyoverdine, frequently inhibit the growth and development of plant pathogens belonging, amongst other, to the genera: Fusarium, Gaeumannomyces, Erwinia and Rhizozoctonia [2,3,13]. This phenomenon may be caused by the capture by pyoverdine, which is a particularly efficient siderophore, of ferric ions in the rhizosphere, which consequently limits the accessible amounts of this element for pathogens [4]. However, even though siderophores synthesized by microorganisms are one of the more intensely pursued studies, there continue to be controversies with regard to the role of pyoverdine in the antagonism of fluorescent Pseudomonas rhizosphere bacteria towards plant pathogens that affect the roots of plants. The results of some investigations point to the significant role of pyoverdine in both restricting the growth of  pathogenic fungi and bring about induced systemic resistance – ISR – in plants [12,20]. However, some other authors negate any relationship between the production of pyoverdine and the antagonistic behavior of bacteria towards fungal pathogens of plants [26,27]. Studies within the framework of biological control of pathogens by rhizosphere bacteria have an important practical aspect. Rhizosphere microorganisms that are not pathogenic for plants can be used as components of biopreparations. Although biopreparations are currently used in agriculture on a small scale, studies have shown them to be highly effective. Consequently, they can be used as an alternative to chemical plant protection agents whose use results in contamination of the environment.

The objective of these studies was characterization of the biological activity of purified pyoverdine synthesized by P. putida. The studied bacterial strain demonstrated under in vitro conditions strong antagonism towards certain fungal Fusarium strains pathogenic for plants. In order to purify pyoverdine released to the growth medium an appropriate procedure for the purification of the compound was elaborated. A further aim of these studies was to examine whether the isolated pyoverdine synthesized by Pseudomonas putida can be utilized by other bacterial isolates of the genus Pseudomonas.

MATERIAL AND METHODS

The biological material used in the experiments was a strain of Pseudomonas putida which secreted pyoverdine to the culture medium. The studied strain was isolated from the rhizosphere of wheat.

Moreover, the studies embraced two other P. putida (P1 and P2) strains and four different P. fluorescens strains (F1, F2, F3, F4) isolated from rhizosphere of wheat. The bacteria were stored on King B medium at 4ºC [11]. The species identity of the bacteria was established basing on morphological and biochemical characteristics. Genetic analyses were carried using the MP PCR (Melting Profiles PCR) technique and demonstrated the genetic separatedness of the individual bacterial strains within the species.

In experiments aimed at establishing the effect of pyoverdine on the growth of plant pathogens belonging to the Fusarium genus, the species Fusarium avenaceum, Fusarium culmorum, Fusarium graminearum, Fusarium solani, obtained from the collection of the Department of Plant Pathology, Faculty of Horticulture and Landscape Architecture, Warsaw University of Life Sciences, were employed. Cultures of the fungi were maintained on PDA medium at 4ºC.

Culture conditions
The bacteria were grown in SSM mineral medium [16]. The final pH of the medium was adjusted to 7.0 with 2 M solution of NaOH. All culture media were prepared using deionized water. All laboratory glass was kept in a 2 M HCl for 12 h and then rinsed several times with deionized water.  The bacteria were grown at 28ºC with shaking (120 rpm) for 72 h and then were spun down in Sigma centrifuge at 24000×g. The obtained supernatant was taken as a source of pyoverdine. Intensity of bacterial growth was monitored based on optical density of the media (OD 550 nm).

Detection of siderophores
The presence of siderophores was determined using the CAS method [28]. The presence of pyoverdine was determined photometrically by measuring absorbance at λ₄₀₅. The content of pyoverdine was calculated taking into account the value of the molar absorbance coefficient 20000 mol·cm^-1 [16].

Purification of pyoverdine
The chelating column used was Sepharose 4B activated with epichlorhydrin coupled with a ligand that chelates Cu²⁺ ions – diiminoacetic acid. The column was saturated with CuSO₄ solution in 20 mM HEPES buffer, pH 6.8. Following concentration, the preparation was applied to a copper-chelate column which was equilibrated with 20 mM HEPES buffer, pH 7,0 containing 100 mM NaCl. Elution was carried out using 20 mM Hepes buffer, pH 7,0.  Pyoverdine was eluted with 20 mM acetate buffer (pH 5.0) containing 100 mM NaCl. Fractions of 1.6 ml were collected. The thus obtained preparation was used to study the biological activity of pyoverdine against selected plant pathogens as well as to determine the cross uptake of the siderophore by individual Pseudomonas strains.

Effect of purified P. putida pyoverdine on fungal plant pathogens
The effect of purified pyoverdine on the growth of fungal pathogens of plants was studied on plates with King B medium as well as iron-free King B medium. The medium was depleted of iron ions using chloroform extraction in the presence of 8-hydroxychinoline. A filter paper disc saturated with 200 µl (0.2 mg of pyoverdine) purified and filter-sterilized P. putida pyoverdine solution was placed in the centre of a plate with solid medium on which a Fusarium fungus had been inoculated. Plates with the discs saturated with sterile deionized water but non containing pyoverdine were the control. The plates were incubated for 7 days at 28°C, after which the growth of the mycelium was evaluated. Evaluation of the degree of inhibition of the growth of the pathogen was conducted by measuring the radius of the mycelium growing under control conditions in the presence of disc saturated with sterile buffer.

The ability of other Pseudomonas strains to utilize pyoverdine produced by P. putida
Experiments aimed at identifying the ability of selected Pseudomonas strains to take purified pyoverdine from strain P. putida were carried out under sterile conditions, spreading the strains on KB medium supplemented with the iron chelator, 0.75 mM 1,10-phenanthroline. The pyoverdine solution used in the experiments was sterilized by filtration (0.22 µm filters). Filter paper discs previously saturated with an appropriate solution of purified pyoverdine (0.2 mg) from strains P. putida were placed on the surface of medium spread with bacteria. The growth of bacteria around the discs was determined after 48 hours of incubation at 28°C. The experiment was carried out using four different strains of the species P. fluorescens and two strains of the species P. putida.

Mass spectrometry
To prepare pyoverdine for MS, the concentrated pyoverdine-containing fractions following separation cooper-chelating chromatography were applied to C₁₈ column (Waters Symmetry 5 µm). Pyoverdine was eluted from the bed using a 0-70% gradient of acetonitrile with 0.1% trifluoroacetic acid [25]. Analysis was carried out using a mass spectrometer (Shimadzu MS 2010) and the electrospray ionization mass spectrometry (ESI/MS) technique after introducing the sample directly into the source of ions. The parameters used were: detector voltage 1.5 V; interphase voltage 5 kV; CDL voltage 100 V; CDL temperature 300°C; nebulizer gas flow rate 0.6 L·min^-1. The standard used was pyoverdine from standard strain P. fluorescens ATCC13525. Experiments involving the standard strain were conducted under identical conditions to those for the studied P. putida strain.

All results presented in this paper in the form of numerical values are means from three independent repetitions. The mean error, reflecting maximal deviation of the results of measurements from the mean, did not exceed 5%.

RESULTS AND DISCUSSION

The universal method for the detection of siderophores allowed detecting their presence in all the studied Pseudomonas strains used in the study. The ability of the studied strains to secrete siderophores was indicated by a change of the color of the medium around the colonies from blue to orange (Photo 1).

Culture fluid concentrated three-fold in a vacuum centrifuge was applied to a cooper-chelate chromatography column. In chelating chromatography pyoverdine was eluted from the column with 50 mM acetate buffer pH 5.0 containing 0.1 M NaCl (Fig. 1). The results of these experiments allow assuming that the studied P. putida strain synthesizes a one form of pyoverdine.

Xiao and Kisaalita [29] as a result of chelating chromatography obtained three purified isoforms of pyoverdine for a strain of P. fluorescens. One of those isoforms was also eluted with 50 mM acetate buffer (pH 5.0) supplemented with NaCl, as described by us. Gamalero et al. [8] also used chelating chromatography to purify siderophores synthesized two different P. fluorescens strains. They obtained a single peak with absorbance at 400 nm in the case of P. fluorescens Pf92. With a different strain, P. fluorescens BBc6, they obtained two peaks, indicating the presence of two isoforms of pyoverdine varying in their affinity for the chelating bed saturated with copper ions. In both cases siderophores were eluted from the column with acetate buffer supplemented with NaCl.

The pyoverdine purified in the studies was used to determine its antagonism with respect to several plant pathogens belonging to the genus Fusarium. The results of the present investigations revealed that pyoverdine synthesized by the bacterial strain can inhibit the growth of some fungi of the genus Fusarium. In our studies growth inhibition of F. solani, F. avenaceum and F. culmorum in the presence of pyoverdine was observed. The most susceptible of the mentioned plant pathogen species was F. solani, (Photo 2). No growth inhibition of F. graminearum in the presence of the siderophore under the experimental conditions employed was obtained. The experiments were carried out using KB medium (Photo 2a, b) as well as KB medium that had been previously depleted of iron (Photo 2c, d). In both cases similar dependencies were observed, but the growth inhibition of the plant pathogens was somewhat stronger on the iron-free medium. Inhibition of mycelium growth in the presence of pyoverdine should most probably be put down to the creation of conditions of severe iron starvation in the environment. The absence of iron, one of the elements indispensable for growth, in the culture medium, resulted in limited growth of the plant pathogens. The biological activity of pyoverdine synthesized by P. putida A was also observed in the studies of Boopathi and Sankara Rao [4]. Using a purified preparation of pyoverdine, they observed inhibited growth of the mycelium of F. oxysporum f.sp.ciceri and Helminthosporium oryzae. The purified compound did not cause growth inhibition of other Fusarium strains tested by the authors. Similar studies carried out by Gamalero et al. [8] revealed the strong antagonistic action of P. fluorescens pyoverdine against the cucurbis pathogen Heterobasidion annosum. On the other hand, Ahl et al. [1] did not observe growth inhibition of the mycelium of Thielaviopsis basicola, which causes black root rot, by the pyoverdine of P. fluorescens CHA0. An analysis of the literature data shows that the specificity of the antagonistic action of pyoverdine again fungal plant pathogens is not entirely characterized and requires further studies.

Studies were also carried out to determine the ability of P. fluorescens strains as well as other P. putida strains to utilize the purified pyoverdine Pseudomonas putida (Table 1). The electrophoregram illustrates the genetic distinctness of Pseudomonas bacteria belonging to the same species (Photo 3). None of the strains used in the experiment was able to grow on medium supplemented with the 0.75 M 1, 10-phenanthroline. However, the presence of pyoverdine stimulated the growth of some of the tested strains. The growth of bacteria around the area where the siderophores were applied to the medium points to the ability of strains P. fluorescens ATCC 13525, F1 as well as F2 to take up the pyoverdine-Fe complexes formed. In this case the growth of only the single studied P. putida strain was observed. The cross utilization of pyoverdines by different strains depends on the structure of the peptide part of the siderophore. The greater the similarity of the amino acid sequence of the peptide side chain of pyoverdine molecules synthesized by different strains, the greater the probability that these siderophores will be taken up interchangeably by these strains. However, the diversity of the peptide chain is closely related to one of its functions – it ensures that the complex pyoverdine-iron is accessible only for a strain producing a given pyoverdine, which is the result of a specific interaction between the complex and its receptor [9]. The observed growth of bacteria in the presence of exogenous pyoverdine on medium supplemented with iron chelator in concentration precluding their growth points to the utilization by a given bacterial strain of a Fe-siderophore of foreign origin. The possibility of taking up a foreign siderophore points to similarities in the structure of the pyoverdines produced by both strains. It is also possible that a strain taking up the molecules produced additional receptors on the outer membrane that are specific for the foreign pyoverdine. The scientific literature brings an increasing number of examples demonstrating the ability of Pseudomonas bacteria to use pyoverdine differing in the amino acid sequence of the peptide chain from own siderophore, mainly in the case of P. fluorescens and P. putida [10]. According to Meyer [19] there is a common to several pyoverdines fragment of the molecule that consists, for instance, of three amino acids of the peptide chain that is recognized by the receptor in the outer membrane of the bacterial cell. Molecules of pyoverdine containing such a fragment could be recognized by a receptor regardless of other structural characteristics.

Bultreys et al. [6] presented interesting results of their studies which indicate that also non-fluorescent strains of P. syringae can utilize the siderophores produced by fluorescent bacteria of this species. In the obtained mass spectrum of P. putida pyoverdine the presence of ion with mass to charge ratio (m/z) 401 was observed. This ion probably carries two elementary charges and can derive from a compound with molecular mass 800 Da. This can be confirmed by the appearance in the spectrum of ion with value m/z 801 – that is of a singly protonated molecular ion. In the case of the pyoverdine from the reference strain P. fluorescens ATCC 13525 the mass spectrum revealed a doubly protonated ion with mass to charge ratio m/z 567, as well as the occurrence of an ion with m/z 1132 and with a single charge. This indicates the presence of a compound with molecular mass 1131 Da. This indicates that P. putida synthesizes a siderophore with lower mass than that of the reference strain P. fluorescens ATCC 13525 (mass 1131 Da) used in the study. The similar value of the molecular weight of pyoverdine synthesis by ATTCC 13525 strain is presented in the study  Fuks at al. [7].

CONCLUSIONS

1. The studied strain of the soil bacteria P.  putida in the applied experimental conditions   synthetised only one  isoform of pyoverdine.

2. Pyoverdine demonstrated stronger antagonism towards pathogenic fungi on iron- deficient King B medium. In the conducted studies the Fusarium species being the most susceptible to the action of pyoverdine was F. solani.

3. The observed growth of bacteria in the presence of P.  putida pyoverdine on medium supplemented with iron chelator in concentration precluding their growth points to the utilization by a given bacterial strain of a siderophore of foreign origin.

4. As the analysis of MS spectrum has proved, the molecular weight of the pyoverdine synthesized by the described Pseudomonas is relatively low and amounts to 800 Da.

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ACKNOWLEDGEMENTS

The authors thank dr hab. W. Wakuliński of the Department of Plant Pathology, Faculty of Horticulture and Landscape Architecture, Warsaw University of Life Sciences for providing access to plant pathogen strains used in the study and for valuable information regarding their cultivation.
We also thank prof. M. Obiedzinski from the Division of Food Quality Evaluation, Faculty of Food Sciences, Warsaw University of Life Sciences for enabling the carrying out of MS analysis.

Accepted for print: 26.02.2009

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