Rhizosphere interactions and siderophores

Certain plant growth-promoting pseudomonads inhibit deleterious and pathogenic rhizosphere bacteria and fungi by producing siderophores. Properties of a siderophore transport system which might provide a competitive advantage under iron stress conditions include ability to utilize other organisms' siderophores, higher Fe(III) stability constant, faster kinetics of dissolution of Fe(III) minerals, more efficient transport system, and resistance to degradation. In order to determine the concentration and localization of siderophores in the rhizosphere monoclonal antibodies (Mabs) to ferric pseudobactin, the siderophore of Pseudomonas putida B10, have been developed. Several Mabs cross reacted differently with various pseudobactins. A growth medium has been developed for the study for siderophore-mediated rhizosphere interactions in the laboratory.     

Growth promoting influence of siderophore-producing Pseudomonas strains GRP3A and PRS9 in maize (Zea mays L.) under iron limiting conditions

Maize seeds were bacterized with siderophore-producing pseudomonads with the goal to develop a system suitable for better iron uptake under iron-stressed conditions. Siderophore production was compared in fluorescent Pseudomonas spp. GRP3A, PRS9 and P. chlororaphis ATCC 9446 in standard succinate (SSM) and citrate (SCM) media. Succinate was better suited for siderophore production, however, deferration of media resulted in increased siderophore production in all the strains. Maximum siderophore level (216.23 microg/ml) was observed in strain PRS9 in deferrated SSM after 72 h of incubation. Strains GRP3A and PRS9 were used for plant growth promotion experiments. Strains GRP3A and PRS9 were also antagonistic against the phytopathogens, Colletotrichum dematium, Rhizoctonia solani and Sclerotium rolfsii. Bacterization of maize seeds with strains GRP3A and PRS9 showed significant increase in germination percentage and plant growth. Maximum shoot and root length and dry weight were observed with 10 microM Fe3+ along with bacterial inoculants suggesting application of siderophore producing plant growth promoting rhizobacterial strains in crop productivity in calcareous soil system.     

Characterization of Pyoverdin(pss), the Fluorescent Siderophore Produced by Pseudomonas syringae pv. syringae

Pseudomonas syringae pv. syringae B301D produces a yellow-green, fluorescent siderophore, pyoverdin(pss), in large quantities under iron-limited growth conditions. Maximum yields of pyoverdin(pss) of approximately 50 mug/ml occurred after 24 h of incubation in a deferrated synthetic medium. Increasing increments of Fe(III) coordinately repressed siderophore production until repression was complete at concentrations of >/= 10 muM. Pyoverdin(pss) was isolated, chemically characterized, and found to resemble previously characterized pyoverdins in spectral traits (absorbance maxima of 365 and 410 nm for pyoverdin(pss) and its ferric chelate, respectively), size (1,175 molecular weight), and amino acid composition. Nevertheless, pyoverdin(pss) was structurally unique since amino acid analysis of reductive hydrolysates yielded beta-hydroxyaspartic acid, serine, threonine, and lysine in a 2:2:2:1 ratio. Pyoverdin(pss) exhibited a relatively high affinity constant for Fe(III), with values of 10 at pH 7.0 and 10 at pH 10.0. Iron uptake assays with [Fe]pyoverdin(pss) demonstrated rapid active uptake of Fe(III) by P. syringae pv. syringae B301D, while no uptake was observed for a mutant strain unable to acquire Fe(III) from ferric pyoverdin(pss). The chemical and biological properties of pyoverdin(pss) are discussed in relation to virulence and iron uptake during plant pathogenesis.     

Bacterial growth stimulation with exogenous siderophore and synthetic N-acyl homoserine lactone autoinducers under iron-limited and low-nutrient conditions

The growth of marine bacteria under iron-limited conditions was investigated. Neither siderophore production nor bacterial growth was detected for Pelagiobacter sp. strain V0110 when Fe(III) was present in the culture medium at a concentration of <1.0 microM. However, the growth of V0110 was strongly stimulated by the presence of trace amounts of exogenous siderophore from an alpha proteobacterium, V0902, and 1 nM N-acyl-octanoylhomoserine lactone (C(8)-HSL), which is known as a quorum-sensing chemical signal. Even though the iron-binding functionality of a hydroxamate siderophore was undetected in the supernatant of V0902, a hydroxamate siderophore was detected in the supernatant of V0110 under the above conditions. These results indicated that hydroxamate siderophore biosynthesis by V0110 began in response to the exogenous siderophore from V0902 when in the presence of C(8)-HSL; however, C(8)-HSL production by V0110 and V0902 was not detected. Direct interaction between V0902 and V0110 through siderophore from V0902 was observed in the dialyzing culture. Similar stimulated growth by exogenous siderophore and HSL was also observed in other non-siderophore-producing bacteria isolated from marine sponges and seawater. The requirement of an exogenous siderophore and an HSL for heterologous siderophore production indicated the possibility that cell-cell communication between different species was occurring.     

Combat of iron-deprivation through a plant growth promoting fluorescent Pseudomonas strain GRP3A in mung bean (Vigna radiata L. Wilzeck)

Microorganisms and plants sustain themselves under iron-deprived conditions by releasing siderophores. Among others, fluorescent pseudomonads are known to exert extensive biocontrol action against soil and root borne phytopathogens through release of antimicrobials and siderophores. In this study, production and regulation of siderophores by fluorescent Pseudomonas strain GRP3A was studied. Among various media tested, standard succinate medium (SSM) promoted maximum siderophore production of 56.59 mg l(-1). There were low levels of siderophore in complex media like King's B medium, trypticase soya medium and nutrient medium (41.27, 29.86 and 27.63 mg l(-1)), respectively. In defferrated SSM, siderophore level was quantified to be 68.74 mg l(-1). Supplementation with iron (FeCl3) resulted in decreased siderophore levels depending on concentration. Siderophore production was promoted by Zn2+ (78.94 mg l(-1)), Cu2+ (68.80 mg l(-1)) whereas Co2+ (57.33 mg l(-1)) and Fe3+ reduced siderophore production (37.44 mg l(-1) as compared to control (55.97 mg l(-1)). Strain GRP3A showed plant growth promotion under iron limited conditions.     

Metal regulation of siderophore synthesis in Pseudomonas aeruginosa and functional effects of siderophore-metal complexes.

Pseudomonas aeruginosa synthesizes two siderophores, pyochelin and pyoverdin, characterized by widely different structures, physicochemical properties, and affinities for Fe(III). Titration experiments showed that pyochelin, which is endowed with a relatively low affinity for Fe(III), binds other transition metals, such as Cu(II), Co(II), Mo(VI), and Ni(II), with appreciable affinity. In line with these observations, Fe(III) and Co(II) at 10 microM or Mo(VI), Ni(II), and Cu(II) at 100 microM repressed pyochelin synthesis and reduced expression of iron-regulated outer membrane proteins of 75, 68, and 14 kDa. In contrast, pyoverdin synthesis and expression of the 80-kDa receptor protein were affected only by Fe(III). All of the metals tested, except Mo(VI), significantly promoted P. aeruginosa growth in metal-poor medium; Mo(VI), Ni(II), and Co(II) were more efficient as pyochelin complexes than the free metal ions and the siderophore. The observed correlation between the affinity of pyochelin for Fe(III), Co(II), and Mo(VI) and the functional effects of these metals indicates that pyochelin may play a role in their delivery to P. aeruginosa.     

Metal regulation of siderophore synthesis in Pseudomonas aeruginosa and functional effects of siderophore-metal complexes.

Pseudomonas aeruginosa synthesizes two siderophores, pyochelin and pyoverdin, characterized by widely different structures, physicochemical properties, and affinities for Fe(III). Titration experiments showed that pyochelin, which is endowed with a relatively low affinity for Fe(III), binds other transition metals, such as Cu(II), Co(II), Mo(VI), and Ni(II), with appreciable affinity. In line with these observations, Fe(III) and Co(II) at 10 microM or Mo(VI), Ni(II), and Cu(II) at 100 microM repressed pyochelin synthesis and reduced expression of iron-regulated outer membrane proteins of 75, 68, and 14 kDa. In contrast, pyoverdin synthesis and expression of the 80-kDa receptor protein were affected only by Fe(III). All of the metals tested, except Mo(VI), significantly promoted P. aeruginosa growth in metal-poor medium; Mo(VI), Ni(II), and Co(II) were more efficient as pyochelin complexes than the free metal ions and the siderophore. The observed correlation between the affinity of pyochelin for Fe(III), Co(II), and Mo(VI) and the functional effects of these metals indicates that pyochelin may play a role in their delivery to P. aeruginosa.     

Siderophore production by the magnetic bacterium Magnetospirillum magneticum AMB-1

Siderophore production by the magnetic bacterium Magnetospirillum magneticum AMB-1 is elicited by sufficient iron rather than by iron starvation. In order to clarify this unusual pattern, siderophore production was monitored in parallel to iron assimilation using the chrome azurol sulfonate assay and the ferrozine method respectively. Iron concentration lowered approximately five times less than its initial concentration only within 4 h post-inoculation, rendering the medium iron deficient. A concentration of at least 6 microM Fe(3+) is required to initiate siderophore production. The propensity of M. magneticum AMB-1 for the assimilation of large amounts of iron accounts for the rapid depletion of iron in the medium, thereby triggering siderophore excretion. M. magneticum AMB-1 produces both hydroxamate and catechol siderophores.     

Chemical characterization and quantification of siderophores produced by marine and terrestrial aspergilli

Ten aspergilli (five each from marine and terrestrial habitats) were screened for siderophore production. All test isolates produced siderophores as indicated by a positive reaction in the FeCl(3) test, chrome azurol sulphonate assay, and chrome azurol sulphonate agar plate test. Further, the test isolates were compared for their siderophore production potential and chemical characteristics. Examination of the chemical nature of the siderophores revealed that all test isolates produced hydroxamate siderophores that were trihydroxamate hexadentates. Wide-spread occurrence of siderophores in marine isolates indicate their functional role in maintaining overall productivity of coastal waters. Among all test aspergilli, marine Aspergillus versicolor was found to be the largest siderophore producer (182.5 microg/mL desferrioxamine mesylate equivalent), least siderophore production was recorded in a marine strain of Aspergillus niger (3.5 microg/mL desferrioxamine mesylate equivalent).     

Studies of iron-uptake mechanisms in two bacterial species of the shewanella genus adapted to middle-range (Shewanella putrefaciens) or antarctic (Shewanella gelidimarina) temperatures

Iron(III)-uptake mechanisms in bacteria indigenous to the Antarctic, which is the most Fe-deficient continent on Earth, have not been extensively studied. The cold-adapted, Antarctic bacterium, Shewanella gelidimarina, does not produce detectable levels of the siderophore, putrebactin, in the supernatant of Fe(III)-deprived cultures. This is distinct from the putrebactin-producing bacterium from the same genus, Shewanella putrefaciens, which is adapted to middle-range temperatures. The production of putrebactin by S. putrefaciens is optimal, when the pH value of the medium is 7.0. According to the strong positive response from whole cells in the Chrome Azurol S (CAS) agar diffusion assay, Shewanella gelidimarina appears to produce cell-associated siderophores. In the RP-HPLC trace of an Fe(III)-loaded extract from the cell-associated components of S. gelidimarina cultured in media with [Fe(III)] ca. 0 microM, a peak appears at [MeCN] ca. 77%, which decreases in intensity in a parallel experiment in which [Fe(III)] ca. 5 microM, and is barely detectable in Fe(III)-replete media ([Fe(III)] ca. 20 microM). The Fe(III)-dependence of this peak suggests that the attendant species, which is significantly more hydrophobic than putrebactin (RP-HPLC elution: [MeCN] ca. 14%), is associated with Fe(III)-management in S. gelidimarina. This study highlights the diversity in Fe(III)-uptake mechanisms in Shewanella species adapted to different environmental and thermal niches.     

Ferric reductase, superoxide dismutase and alkaline phosphatase activities in siderophore producing fungi

Enzymes associated with release of iron from internalized ferrated siderophore (ferrisiderophore reductase), with damage to the cell at high iron concentration (superoxide dismutase) and siderophore synthesis (alkaline phosphatase), were examined in 3 test fungi viz., Aspergillus sp. ABp4, Aureobasidium pullulans and Rhizopus sp. Extracellular ferrisiderophore reductase activity was present in all the three fungi, but Aureobasidium pullulans, that showed the highest activity (84.3 microM min(-1)), was the only one to produce intra-cellular ferric reductase (147.9 microM min(-1)). Superoxide dismutase was produced by Aureobasidium pullulans and Rhizopus sp., but not by Aspergillus sp. ABp4, that showed intra-cellular enzyme activity in case of ferric reductase and alkaline phosphatase. Maximum SOD activity was seen in Aureobasidium pullulans both extra-cellularly (93.83 ng ml(-1)) and intra-cellularly (57.14 ng ml(-1)). All the test fungi examined, produced intra-cellular alkaline phosphatase. There was no extracellular alkaline phosphatase. Among the three fungi, Aureobasidium pullulans showed highest alkaline phosphatase activity (129.9 microM min(-1)) and Aspergillus sp. ABp4 the least (76.4 microM min(-1)).     

Characterization of fluorescent and nonfluorescent peptide siderophores produced by Pseudomonas syringae strains and their potential use in strain identification

Nonfluorescent highly virulent strains of Pseudomonas syringae pv. aptata isolated in different European countries and in Uruguay produce a nonfluorescent peptide siderophore, the production of which is iron repressed and specific to these strains. The amino acid composition of this siderophore is identical to that of the dominant fluorescent peptide siderophore produced by fluorescent P. syringae strains, and the molecular masses of the respective Fe(III) chelates are 1,177 and 1,175 atomic mass units. The unchelated nonfluorescent siderophore is converted into the fluorescent siderophore at pH 10, and colors and spectral characteristics of the unchelated siderophores and of the Fe(III)-chelates in acidic conditions are similar to those of dihydropyoverdins and pyoverdins, respectively. The nonfluorescent siderophore is used by fluorescent and nonfluorescent P. syringae strains. These results and additional mass spectrometry data strongly suggest the presence of a pyoverdin chromophore in the fluorescent siderophore and a dihydropyoverdin chromophore in the nonfluorescent siderophore, which are both ligated to a succinamide residue. When chelated, the siderophores behave differently from typical pyoverdins and dihydropyoverdins in neutral and alkaline conditions, apparently because of the ionization occurring around pH 4.5 of carboxylic acids present in beta-hydroxyaspartic acid residues of the peptide chains. These differences can be detected visually by pH-dependent changes of the chelate colors and spectrophotochemically. These characteristics and the electrophoretic behavior of the unchelated and chelated siderophores offer new tools to discriminate between saprophytic fluorescent Pseudomonas species and fluorescent P. syringae and P. viridiflava strains and to distinguish between the two siderovars in P. syringae pv. aptata.     

bvg Repression of alcaligin synthesis in Bordetella bronchiseptica is associated with phylogenetic lineage.

Recent studies have shown that Bordetella bronchiseptica utilizes a siderophore-mediated transport system for acquisition of iron from the host iron-binding proteins lactoferrin and transferrin. We recently identified the B. bronchiseptica siderophore as alcaligin, which is also produced by B. pertussis. Alcaligin production by B. bronchiseptica is repressed by exogenous iron, a phenotype of other microbes that produce siderophores. In this study, we report that alcaligin production by B. bronchiseptica RB50 and GP1SN was repressed by the Bordetella global virulence regulator, bvg, in addition to being Fe repressed. Modulation of bvg locus expression with 50 mM MgSO4 or inactivation of bvg by deletion allowed strain RB50 to produce alcaligin. In modulated organisms, siderophore production remained Fe repressed. These observations contrasted with our previous data indicating that alcaligin production by B. bronchiseptica MBORD846 and B. pertussis was repressed by Fe but bvg independent. Despite bvg repression of alcaligin production, strain RB50 was still able to acquire Fe from purified alcaligin, suggesting that expression of the bacterial alcaligin receptor was not repressed by bvg. We tested 114 B. bronchiseptica strains and found that bvg repression of alcaligin production was strongly associated with Bordetella phylogenetic lineage and with host species from which the organisms were isolated.     

Characterization of Pyoverdinpss, the Fluorescent Siderophore Produced by Pseudomonas syringae pv. syringae

Pseudomonas syringae pv. syringae B301D produces a yellow-green, fluorescent siderophore, pyoverdin_pss, in large quantities under iron-limited growth conditions. Maximum yields of pyoverdin_pss of approximately 50 μg/ml occurred after 24 h of incubation in a deferrated synthetic medium. Increasing increments of Fe(III) coordinately repressed siderophore production until repression was complete at concentrations of ≥ 10 μM. Pyoverdin_pss was isolated, chemically characterized, and found to resemble previously characterized pyoverdins in spectral traits (absorbance maxima of 365 and 410 nm for pyoverdin_pss and its ferric chelate, respectively), size (1,175 molecular weight), and amino acid composition. Nevertheless, pyoverdin_pss was structurally unique since amino acid analysis of reductive hydrolysates yielded β-hydroxyaspartic acid, serine, threonine, and lysine in a 2:2:2:1 ratio. Pyoverdin_pss exhibited a relatively high affinity constant for Fe(III), with values of 10²⁵ at pH 7.0 and 10³² at pH 10.0. Iron uptake assays with [⁵⁵Fe]pyoverdin_pss demonstrated rapid active uptake of ⁵⁵Fe(III) by P. syringae pv. syringae B301D, while no uptake was observed for a mutant strain unable to acquire Fe(III) from ferric pyoverdin_pss. The chemical and biological properties of pyoverdin_pss are discussed in relation to virulence and iron uptake during plant pathogenesis.     

Increase in spermine content coordinated with siderophore production in Paracoccus denitrificans.

Spermine is present in relatively low amounts in Paracoccus denitrificans cultured aerobically in an ammonium succinate minimal salts medium supplemented with 50 microM iron(III). However, in iron-deprived cultures [minimal salts medium containing 0.5 microM iron(III)], spermine content increases by an order of magnitude in coordination with the well-known responses to iron derivation, e.g., derepression of siderophore synthesis and siderophore excretion. When iron-deprived cultures exhibiting both high spermine content and strong siderophore production are reseeded into fresh minimal salts medium containing 50 microM iron[III], both siderophore production and spermine content fall rapidly. Five hours after iron supplementation, spermine is below limits of detection. These results suggest a specific role for spermine in the response of P. denitrificans to low-iron stress.     

Characterization of Fluorescent and Nonfluorescent Peptide Siderophores Produced by Pseudomonas syringae Strains and Their Potential Use in Strain Identification

Nonfluorescent highly virulent strains of Pseudomonas syringae pv. aptata isolated in different European countries and in Uruguay produce a nonfluorescent peptide siderophore, the production of which is iron repressed and specific to these strains. The amino acid composition of this siderophore is identical to that of the dominant fluorescent peptide siderophore produced by fluorescent P. syringae strains, and the molecular masses of the respective Fe(III) chelates are 1,177 and 1,175 atomic mass units. The unchelated nonfluorescent siderophore is converted into the fluorescent siderophore at pH 10, and colors and spectral characteristics of the unchelated siderophores and of the Fe(III)-chelates in acidic conditions are similar to those of dihydropyoverdins and pyoverdins, respectively. The nonfluorescent siderophore is used by fluorescent and nonfluorescent P. syringae strains. These results and additional mass spectrometry data strongly suggest the presence of a pyoverdin chromophore in the fluorescent siderophore and a dihydropyoverdin chromophore in the nonfluorescent siderophore, which are both ligated to a succinamide residue. When chelated, the siderophores behave differently from typical pyoverdins and dihydropyoverdins in neutral and alkaline conditions, apparently because of the ionization occurring around pH 4.5 of carboxylic acids present in β-hydroxyaspartic acid residues of the peptide chains. These differences can be detected visually by pH-dependent changes of the chelate colors and spectrophotochemically. These characteristics and the electrophoretic behavior of the unchelated and chelated siderophores offer new tools to discriminate between saprophytic fluorescent Pseudomonas species and fluorescent P. syringae and P. viridiflava strains and to distinguish between the two siderovars in P. syringae pv. aptata.     

Effect of Ferric Iron on Siderophore Production and Pyrene Degradation by Pseudomonas fluorescens 29L

The effect of ferric iron [Fe(III)] on pyrene degradation and siderophore production was studied in Pseudomonas fluorescens 29L. In the presence of 0.5 muM of Fe(III) and 50 mg of pyrene per liter of medium as a carbon source, 2.2 mg of pyrene was degraded per liter of medium per day and 25.3 muM of 2,3-DHBA (2,3-dihydroxybenzoic acid) equivalent of siderophores was produced per day. However, the pyrene degradation rate was 1.3 times higher and no siderophores were produced with the addition of 1 muM of Fe(III). Similar trends were seen with 50 mg of succinate per liter of medium as a carbon source, although the growth of strain 29L and the succinate degradation rate were higher. In the absence of siderophore production, pyrene and succinate continued to be biodegraded. This indicates that Fe(III) and not siderophore production affects the hydrocarbon degradation rate. Only 18% of strain 29L mutants capable of growth on pyrene produced siderophores, while among the mutants capable of growth on succinate, only 10% produced siderophores. This indicates that siderophores are not required for pyrene biodegradation. Fe(III) enhances pyrene degradation in Pseudomonas fluorescens 29L but it may be utilized by mechanisms other than siderophores.     

Exchange of iron by gallium in siderophores

Siderophores are iron transport compounds produced by numerous microorganisms and which strongly chelate Fe(III), but not Fe(II). Other trivalent metals, such as Al(III), Cr(III), or Ga(III), are not capable of significantly displacing iron from siderophores. However, I demonstrate here that Ga(III) can effectively displace iron under reducing conditions. With ascorbate as reductant and ferrozine as Fe(II) trapping agent, the kinetics of reductive displacement of iron by Ga(III) were followed spectroscopically by the increase of absorbance at 562 nm due to formation of the Fe(II)-ferrozine complex. No significant reduction of siderophore occurred in the absence of Ga(III). With excess Ga(III), the displacement was quantitative and very rapid. The rate of metal exchange was pseudo first order with respect to Ga(III) concentration and highly pH dependent, suggesting that siderophore ligands are displaced from the iron in a concerted mechanism by Ga(III) and protonation to expose the Fe(III) to reduction by ascorbate. Reaction rates were dependent upon the structure of the siderophore, being greatest for ferric rhodotorulic acid and slowest for ferrichrome A at pH 5.4. The pH profile for ferric rhodotorulic acid was unusual in that it showed a maximum at pH 6.5, while all other siderophores examined showed an increase in rate as pH was lowered from 7.0. The physiological significance of this reaction to the clinical use of gallium is discussed.     

Hydroxamate siderophore effect on growth of enterococci]

In eleven strains of genus Enterococcus relation between hydroxamate siderophore production and growth in iron-restricted liquid medium was investigated. Fe(III) deficiency caused moderate decrease of growth rate: the exponential growth phase was prolonged and yield of bacterial mass was lower. The hydroxamate siderophore was produced by all strains but occurred at different phase of growth. Only part of strains showed classical pattern of growth kinetic and siderophore synthesis.     

Chemical properties and NMR spectroscopic identification of certain fungal siderophores

Siderophores of six fungi viz. Aspergillus sp. ABp4, Aureobacidium pullulans, Penicillium oxalicum, P. chrysosporium, Mycotypha africana and Syncephalastrum racemosum were examined for their (1) electrophoretic mobilities to determine the acidic, basic or neutral charge; (2) Fe (III) binding nature viz., mono-, di-, or trihydroxamate; (3) amino acid composition; and (4) NMR (nuclear magnetic resonance) spectroscopy to determine their structure. Electrophoretic mobilities of siderophores of 3 fungi (P. oxalicum, P. chrysosporium, and M, africana) exhibited net basic charge, siderophores of 2 fungi (Aspergillus sp. ABp4 and S. racemosum) were acidic and 1 fungus (A. pullullans) was neutral. Electrophoresis of ferrated siderophore at pH 2 and colour of the spots indicated that siderophores of Aspergillus sp. ABp4 and P. oxalicum and A. pullulans were trihydroxamates, whereas siderophore of P. chrysosporium was dihydroxamate. Amino acid composition of siderophores purified by XAD-2 column chromatography, revealed the presence of asparagine, histidine, and proline in Aspergillus sp. ABp4, serine and alanine in P. chrysosporium, and valine in M. africana. The structure of purified siderophores as revealed by NMR spectroscopy identified siderophore of AB - 2670 (A. pullulans) as asperchrome F1, and AB-513 (M. africana) as rhizoferrin. The peak obtained for siderophore AB-5 (Aspergillus sp. ABp4) did not show resemblance to any known siderophore, therefore may be an exception.