Monoclonal Antibodies to Ferric Pseudobactin, the Siderophore of Plant Growth-Promoting Pseudomonas putida B10

Monoclonal antibodies to ferric pseudobactin, the siderophore (microbial iron transport agent) of plant growth-promoting Pseudomonas putida B10, have been developed. Three immunoglobulin G subclass 1-type monoclonal antibodies have been characterized. Each antibody appears to be unique on the basis of their reactions with ferric pseudobactin and with culture supernatants from other pseudomonads. None of the three cross-reacts with ferric pseudobactin-type siderophores produced by seven other pseudomonads. However, P. aeruginosa ATCC 15692 and P. fluorescens ATCC 17400 produced relatively high-molecular-mass compounds (mass greater than approximately 30,000 daltons) that did react with the antibodies. The compound from P. aeruginosa was not iron regulated, while the compound from P. fluorescens was produced only under iron-limiting conditions. A competitive assay using these antibodies has a detection limit of 5 x 10 mol of ferric pseudobactin. This is, to our knowledge, the first report of monoclonal antibodies reactive with siderophores.     

Multiple outer membrane receptors for uptake of ferric pseudobactins inPseudomonas putida WCS358

Under iron limitationPseudomonas putida WCS358 produces a fluorescent siderophore, pseudobactin 358, which, after complexing iron, is transported back into the cell via the specific outer membrane receptor PupA. In addition, this strain has the capacity to take up iron via a large variety of siderophores produced by other fluorescent pseudomonads. Putative receptor genes for such siderophores were identified in the chromosome of strain WCS358 by PCR using primers matching two domains conserved in four ferric pseudobactin receptors, including PupA. Eleven amplification products within the expected size range were obtained. Sequence analysis confirmed that the products were derived from genes encoding outer membrane receptors. Two complete receptor genes were isolated from a genomic library ofP. putida WCS358. Both protein products are involved in the transport of a limited number of specific ferric pseudobactins. These results indicate that the ability ofP. putida WCS358 to exploit many different heterologous pseudobactins is related to the presence of multiple outer membrane receptor proteins.     

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.     

Rhizosphere competence of fluorescent Pseudomonas sp. B24 genetically modified to utilise additional ferric siderophores

Abstract: The ability to utilise additional siderophores may increase the ecological fitness of biocontrol inoculants of Pseudomonas in the rhizosphere. Plasmid pCUP2 carries a copy of the gene pbu A coding for the membrane receptor of ferric pseudobactin M114. Pseudomonas sp. B24Rif containing pCUP2 can utilise ferric pseudobactin of P. fluorescens M114 in addition to its own siderophore. A larger fraction of the culturable resident fluorescent pseudomonads in the rhizosphere of sugarbeet grown in a low-iron sandy loam soil could supply siderophore-complexed iron to B24Rif(pCUP2) rather than to B24Rif. However, B24Rif and B24Rif(pCUP2) were found at similar population levels in the rhizosphere for 21 days after their inoculation on seeds. A total of 25 of 43 isolates of resident fluorescent Pseudomonas unable to cross-feed iron to B24Rif could cross-feed B24Rif(pCUP2) and they were subdivided into seven different strains by arbitrary-primed PCR fingerprinting. The siderophores produced by 11 of them were typed by HPLC and they were similar to pseudobactin M114. However, the ability to utilise ferric pseudobactin M114 did not improve the ecological fitness of B24Rif in the rhizosphere of sugarbeet although a larger fraction of the culturable resident fluorescent pseudomonads could supply pseudobactin M114-complexed iron to B24Rif(pCUP2) than to B24Rif.     

Multiple outer membrane receptors for uptake of ferric pseudobactins inPseudomonas putida WCS358

Under iron limitationPseudomonas putida WCS358 produces a fluorescent siderophore, pseudobactin 358, which, after complexing iron, is transported back into the cell via the specific outer membrane receptor PupA. In addition, this strain has the capacity to take up iron via a large variety of siderophores produced by other fluorescent pseudomonads. Putative receptor genes for such siderophores were identified in the chromosome of strain WCS358 by PCR using primers matching two domains conserved in four ferric pseudobactin receptors, including PupA. Eleven amplification products within the expected size range were obtained. Sequence analysis confirmed that the products were derived from genes encoding outer membrane receptors. Two complete receptor genes were isolated from a genomic library ofP. putida WCS358. Both protein products are involved in the transport of a limited number of specific ferric pseudobactins. These results indicate that the ability ofP. putida WCS358 to exploit many different heterologous pseudobactins is related to the presence of multiple outer membrane receptor proteins.     

Siderophore Utilization by Bradyrhizobium japonicum

Bradyrhizobium japonicum USDA 110 and 61A152 can utilize the hydroxamate-type siderophores ferrichrome and rhodotorulate, in addition to ferric citrate, to overcome iron starvation. These strains can also utilize the pyoverdin-type siderophore pseudobactin St3. The ability to utilize another organism's siderophores may confer a selective advantage in the rhizosphere.     

Iron transport-mediated antagonism between plant growth-promoting and plant-deleterious Pseudomonas strains

Both plant growth-promoting Pseudomonas B10 and its yellow-green, fluorescent iron transport agent (siderophore) pseudobactin enhance potato growth and biologically control certain soil-borne fungal diseases in part by depriving specific root-colonizing endemic microorganisms including phytopathogens of iron(III), thus inhibiting their growth. The present study examines this mode of iron deprivation. The growth inhibition of certain bean-deleterious fluorescent pseudomonads by specific bean-beneficial fluorescent pseudomonads is due in part to the inability of susceptible strains to utilize siderophores from beneficial strains to transport iron(III). Conversely, deleterious strains which were able to utilize siderophores from beneficial strains were not inhibited. The ability of a given pseudomonad to utilize another pseudomonad's siderophore may depend upon its possessing a specific outer membrane receptor protein for that pseudomonad's ferric siderophore. Siderophore-mediated competition for iron in microbial systems appears to be a widespread phenomenon.     

Structure of pseudobactin A214, a siderophore from a bean-deleterious Pseudomonas

Bean-deleterious Pseudomonas A214 produced the extracellular yellow-green, fluorescent siderophore [microbial iron(III) transport agent] pseudobactin A214 under iron-limiting conditions. Pseudobactin A214 has a molecular formula of C46H64N13O22 and a molecular mass of 1151 g/mol. Pseudobactin A214 contained an N-blocked linear octapeptide with the amino acid sequence Ser-Ala-Gly-Ser-Ala-threo-beta-OH-Asp-L-allo-Thr-N delta-OH-Orn with a yellow-green, fluorescent quinoline derivative attached via an amide bond to the amino terminus. A succinamide group was linked to carbon 3 of the quinoline derivative. Sequencing was accomplished by two-dimensional NMR spectroscopy and by Edman degradation of smaller peptides obtained from partial acid hydrolysis. Since pseudobactin A214 was not affected by nonspecific proteolytic enzymes, it might contain D-amino acids. The three bidentate iron-(III)-chelating groups consisted of a 1,2-dihydroxy aromatic group in the quinoline chromophore, an alpha-hydroxy acid group present as beta-hydroxyaspartic acid, and a hydroxamate group derived from N delta-acetyl-N delta-hydroxyornithine. The chemical structure of pseudobactin A214 is remarkably similar to those of pseudobactin and pseudobactin 7SR1, the siderophores of plant growth promoting and plant-deleterious Pseudomonas B10 and Pseudomonas 7SR1, respectively.     

A Method for Detection of Pseudobactin, the Siderophore Produced by a Plant-Growth-Promoting Pseudomonas Strain, in the Barley Rhizosphere

Detection in the rhizosphere of the siderophore produced by an inoculated microorganism is critical to determining the role of microbial iron chelators on plant growth promotion. We previously reported the development of monoclonal antibodies (MAb) to ferric pseudobactin, the siderophore of plant-growth-promoting Pseudomonas strain B10. One of these MAb reacted less strongly to pseudobactin than to ferric pseudobactin. The MAb reacted to Al(III), Cr(III), Cu(II), and Mn(II) complexes of pseudobactin at a level similar to the level at which it reacted to ferric pseudobactin and reacted less to the Zn(II) complex, but these metals would make up only a small fraction of chelated pseudobactin in soil on the basis of relative abundance of metals and relative binding constants. Fourteen-day-old barley plants grown in limed and autoclaved soil were inoculated with 10⁹ CFU of Pseudomonas strain Sm1-3, a strain of Pseudomonas B10 Rif^r Nal^r selected for enhanced colonization, and sampled 3 days later. Extraction and analysis of the roots and surrounding soil using the MAb in an immunoassay indicated a concentration of 3.5 × 10^-10 mol of ferric pseudobacting g^-1 (wet weight). This is the first direct measurement of a pseudobactin siderophore in soil or rhizosphere samples.     

Synthesis of optically pure chrysobactin and immunoassay development

Chrysobactin (-N-(2,3-dihydroxybenzoyl)-d-lysyl-l-serine), a siderophore that is essential for systemic virulence by plant pathogenic Erwinia chrysanthemi, was synthesized with high diastereomeric purity. Chrysobactin was prepared by coupling the N-hydroxysuccinimide ester of -N-(2,3-dibenzyloxybenzoyl)--N-Cbz-d-lysine with l-serine benzyl ester followed by deprotection via hydrogenolysis. Optically pure chrysobactin was obtained with 98% overall yield. A monoclonal antibody to ferric chrysobactin was developed and characterized as IgM. The antibody reacts with chrysobactin, ferric chrysobactin and less strongly with ferric dihydroxybenzoic acid. The antibody reacts weakly with the siderophores ferrichrome, A, ferric pseudobactin and ferric rhodotorulic acid. This antibody was used in a competitive immunoassay to detect ferric chrysobactin at 10^–8 to 10^–10 mol. This immunoassay may provide a useful method for the detection of chrysobactin in plant samples.     

Purification, characterization, and structure of pseudobactin 589 A, a siderophore from a plant growth promoting Pseudomonas

Under conditions of low-iron stress the plant growth promoting bacterium Pseudomonas putida 589 (DSM 50202) produced a yellow-green fluorescent iron-binding peptide siderophore, which was designated pseudobactin 589 A and had an affinity constant toward Fe3+ of 10(25) at pH 7. Protonated pseudobactin 589 A had the molecular formula C54H78O26N15 and a nominal mass spectral molecular mass of 1353 g/mol. Its structure was determined by a combination of nuclear magnetic resonance, fast atom bombardment mass spectrometry, and Edman degradation. Pseudobactin 589 A consisted of a nonapeptide with the amino acid sequence L-Asp-L-Lys-(D)-beta-OH-Asp-D(L)-Ser-L-Thr-D-Ala-D-Glu-L(D)-Ser-L-N delta-OH- Orn, in which lysine was amide bonded via the carboxy and the N epsilon-amino groups. A quinoline-derived chromophore was connected via an amide bond to the alpha-amino nitrogen of aspartic acid and an L-malamide residue was attached to the chromophore. The three bidentate Fe3+ binding ligands consisted of an o-dihydroxy aromatic group from the quinoline derivative, beta-hydroxyaspartic acid, and an internally cyclized N delta-hydroxyornithine. The structure of pseudobactin 589 A is unique but strikingly similar to that of other pseudobactin-type siderophores from other plant growth promoting and plant deleterious pseudomonads.     

A simple assay for fluorescent siderophores produced by Pseudomonas species and an efficient isolation of pseudobactin

Several iron binding metabolites (siderophores) of Pseudomonas fluorescens B10 (JL-3133) have been detected using C₁₈ reverse phase HPLC coupled with photodiode array detection methods. This analysis utilized a volatile mobile phase of 90% 20 mm NH₄HCO₃/10% MeOH, pH 6.5. It has been shown to be applicable to other P. fluorescens strains for the detection of related metabolites. Direct scale-up of the analytical HPLC conditions allowed for the efficient preparative isolation of pseudobactin, the principle siderophore produced by P. fluorescens B10 (JL-3133).     

Siderophore-bound iron in the peribacteriod space of soybean root nodules

Water-soluble, non-leghemoglobin iron (125 µmol kg^-1 wet weight nodule) is found in extracts of soybean root nodules. This iron is probably confined to the peribacteroid space of the symbiosome, where its estimated concentration is 0.5 – 2.5 mM. This iron is bound by siderophores (compounds binding ferric iron strongly) which are different for each of the three strains of Bradyrhizobium japonicum with which the plants were inoculated. One of these, that from nodules inoculated with strain CC 705, is tentatively identified as a member of the pseudobactin family of siderophores. Leghemoglobin is present in only very small amounts in the peribacteroid space of symbiosomes isolated from soybean root nodules, and may be absent from the peribacteroid space of the intact nodule.     

Characterization of Fluorescent Siderophore-Mediated Iron Uptake in Pseudomonas sp. Strain M114: Evidence for the Existence of an Additional Ferric Siderophore Receptor

In Pseudomonas sp. strain M114, the outer membrane receptor for ferric pseudobactin M114 was shown to transport ferric pseudobactins B10 and A225, in addition to its own. The gene encoding this receptor, which was previously cloned on pCUP3, was localized by Tn5 mutagenesis to a region comprising >1.6 kb of M114 DNA. A mutant (strain M114R1) lacking this receptor was then created by a marker exchange technique. Characterization of this mutant by using purified pseudobactin M114 in radiolabeled ferric iron uptake studies confirmed that it was completely unable to utilize this siderophore for acquisition of iron. In addition, it lacked an outer membrane protein band of 89 kDa when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As a result, growth of the mutant was severely restricted under low-iron conditions. However, this phenotype was reversed in the presence of another fluorescent siderophore (pseudobactin MT3A) from Pseudomonas sp. strain MT3A, suggesting the presence of a second receptor in strain M114. Furthermore, wild-type Pseudomonas sp. strain B24 was not able to utilize ferric pseudobactin MT3A, and this phenotype was not reversed upon expression of the M114 receptor encoded on pCUP3. However, a cosmid clone (pMS1047) that enabled strain B24 to utilize ferric pseudobactin MT3A was isolated from an M114 gene bank. Radiolabel transport assays with purified pseudobactin MT3A confirmed this event. Plasmid pMS1047 was shown to encode an outer membrane protein of 81 kDa in strain B24 under iron-limiting conditions; this protein corresponds to a similar protein in strain M114.     

Differential Siderophore Utilization and Iron Uptake by Soil and Rhizosphere Bacteria

The differential availabilities of the hydroxamate siderophores ferrioxamine B (FOB) and ferrichrome (FC) and the pseudobactin siderophores St3, 7NSK₂, and WCS 358 as sources of Fe for soil and rhizosphere bacteria were studied. About 20% of the total bacterial CFU from the rhizospheres of four plant species were able to use FOB as the sole Fe source in an Fe-deficient medium, while about 12, 10, 2, and > 1% were able to use FC and pseudobactins 7NSK₂, St3, and WCS 358, respectively. Of the 165 colonies isolated from plates containing pseudobactins, 64 were able to use the pseudobactin on which they were isolated as the sole Fe source in pure culture. Cross-feeding tests showed that almost all of these 64 strains were also able to use at least one of the other siderophores studied (pseudobactin, FOB, or FC). Pseudomonas putida StS2, Pseudomonas maltophilia 7NM1, and Vibrio fluvialis WS1, which were originally isolated on pseudobactins St3, 7NSK₂, and WCS 358, respectively, were selected for their ability to grow with pseudobactin St3 as the sole Fe source. They incorporated ⁵⁵Fe³⁺ mediated by pseudobactin St3 at various rates (71.5, 4, and 23 pmol/min/mg [dry weight] of cells, respectively). Similarly, P. putida St3 was shown to incorporate ⁵⁵Fe³⁺ mediated by FOB and FC. We suggest that the ability of bacteria to utilize a large variety of siderophores confers an ecological advantage.