Structure of pseudobactin 7SR1, a siderophore from a plant-deleterious Pseudomonas

When grown in iron-limiting culture medium, sugar beet deleterious Pseudomonas 7SR1 produced extra-cellularly the yellow-green, fluorescent siderophore pseudobactin 7SR1. Pseudobactin 7SR1 had a molecular formula of C46H63N13O23 and a molecular mass of 1166 g/mol. Pseudobactin 7SR1 contained a cyclic octapeptide with the amino acid sequence L-Ala-Gly-Ser-Ser-threo-beta-OH-Asp-Thr-Ser-N delta-OH-Orn. Since pseudobactin 7SR1 was not affected by nonspecific enzymes, it might contain D-amino acids. A yellow-green, fluorescent quinoline derivative is postulated to be attached via an ester bond to the serine residue following the glycine. A malamide group was attached to carbon 3 of the quinoline derivative. The three bidentate iron(III)-chelating groups consisted of an alpha-hydroxy acid group derived from beta-hydroxyaspartic acid, an omicron-dihydroxy aromatic group derived from the yellow-green, fluorescent chromophore, and a hydroxamate group derived from N delta-acetyl-N delta-hydroxyornithine. The chemical structure of pseudobactin 7SR1 is remarkably similar to that of pseudobactin, the siderophore of plant growth promoting Pseudomonas B10.     

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.     

Cloning of the gene coding for the outer membrane receptor protein for ferric pseudobactin, a siderophore from a plant growth-promoting Pseudomonas strain

Plant growth-promoting Pseudomonas B10 produces its yellow-green, fluorescent siderophore (microbial iron transport agent) pseudobactin under iron-limiting conditions. A structural gene encoding the 85,000-Da putative outer membrane receptor protein for ferric pseudobactin was identified in a gene bank from Pseudomonas B10 prepared with the broad host-range conjugative cosmid cloning vector pLAFR1. Transposon Tn5 mutagenesis of recombinant plasmid pJLM300 localized the functional gene to a region of approximately 2.4 kilobases consistent with the apparent molecular weight of the receptor protein. Mobilization of pJLM300 into Pseudomonas A124 and A225, whose growth was inhibited by Pseudomonas B10 or pseudobactin, rendered these strains no longer susceptible to iron starvation by pseudobactin because they were now able to transport ferric pseudobactin. Pseudobactin biosynthetic genes flanked this receptor gene on both sides and were on separate operons. Transposon Tn5 insertion mutants of Pseudomonas B10 lacking this receptor protein were generated by a marker exchange technique and were defective in ferric pseudobactin transport. Such mutants could be complemented in trans by pJLM300. The production of pseudobactin, the receptor protein, and four other outer membrane proteins in Pseudomonas B10 was coordinately regulated by the level of intracellular iron.     

Amplification of the groESL operon in Pseudomonas putida increases siderophore gene promoter activity

Molecular Genetics and Genomics - Pseudobactin 358 is the yellow-green fluorescent siderophore [microbial iron(III) transport agent] produced by Pseudomonas putida WCS358 under iron-limiting...     

Cloning of genes involved in the biosynthesis of pseudobactin, a high-affinity iron transport agent of a plant growth-promoting Pseudomonas strain

A gene bank of DNA from plant growth-promoting Pseudomonas sp. strain B10 was constructed using the broad host-range conjugative cosmid pLAFR1. The recombinant cosmids contained insert DNA averaging 21.5 kilobase pairs in length. Nonfluorescent mutants of Pseudomonas sp. strain B10 were obtained by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, or UV light and were defective in the biosynthesis of its yellow-green, fluorescent siderophore (microbial iron transport agent) pseudobactin. No yellow-green, fluorescent mutants defective in the production of pseudobactin were identified. Nonfluorescent mutants were individually complemented by mating the gene bank en masse and identifying fluorescent transconjugants. Eight recombinant cosmids were sufficient to complement 154 nonfluorescent mutants. The pattern of complementation suggests that a minimum of 12 genes arranged in four gene clusters is required for the biosynthesis of pseudobactin. This minimum number of genes seems reasonable considering the structural complexity of pseudobactin.     

Antagonistic Effect of Nonpathogenic Fusarium oxysporum Fo47 and Pseudobactin 358 upon Pathogenic Fusarium oxysporum f. sp. dianthi

Pseudobactin production by Pseudomonas putida WCS358 significantly improves biological control of fusarium wilt caused by nonpathogenic Fusarium oxysporum Fo47b10 (P. Lemanceau, P. A. H. M. Bakker, W. J. de Kogel, C. Alabouvette, and B. Schippers, Appl. Environ. Microbiol. 58:2978-2982, 1992). The antagonistic effect of Fo47b10 and purified pseudobactin 358 was studied by using an in vitro bioassay. This bioassay allows studies on interactions among nonpathogenic F. oxysporum Fo47b10, pathogenic F. oxysporum f. sp. dianthi WCS816, and purified pseudobactin 358, the fluorescent siderophore produced by P. putida WCS358. Both nonpathogenic and pathogenic F. oxysporum reduced each other's growth when grown together. However, in these coinoculation experiments, pathogenic F. oxysporum WCS816 was relatively more inhibited in its growth than nonpathogenic F. oxysporum Fo47b10. The antagonism of nonpathogenic F. oxysporum against pathogenic F. oxysporum strongly depends on the ratio of nonpathogenic to pathogenic F. oxysporum densities: the higher this ratio, the stronger the antagonism. This fungal antagonism appears to be mainly associated with the competition for glucose. Pseudobactin 358 reduced the growth of both F. oxysporum strains, whereas ferric pseudobactin 358 did not; antagonism by pseudobactin 358 was then related to competition for iron. However, the pathogenic F. oxysporum strain was more sensitive to this antagonism than the nonpathogenic strain. Pseudobactin 358 reduced the efficiency of glucose metabolism by the fungi. These results suggest that pseudobactin 358 increases the intensity of the antagonism of nonpathogenic F. oxysporum Fo47b10 against pathogenic F. oxysporum WCS816 by making WCS816 more sensitive to the glucose competition by Fo47b10.     

Can the peptide chain of a pyoverdin be bound by an ester bond to the chromophore?--The old problem of pseudobactin 7SR1

The structure which had been proposed for the pyoverdin named pseudobactin 7SR1 (Yang and Leong, 1984) differed from those of all other pyoverdins investigated so far: its peptide chain was supposedly linked to the chromophore not by an amide bond originating from its N-terminal amino acid, but rather by an ester bond involving one of the three Ser. It will be shown that the peptide chain of pseudobactin 7SR1 is actually bound to the chromophore amidically by its N-terminal Ser and that it comprises a cyclodepsipeptidic substructure with an ester bond between the C-terminal Thr and the OH-group of the second Ser in the chain.     

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.     

Effects of iron(III) analogs on growth and pseudobactin synthesis in a chromiumtolerantPseudomonas isolate

Summary The growth and siderophore production of a fluorescentPseudomonas species isolated from soil contaminated with chromium was found to be influenced by the presence of trivalent cations. Overproduction of pseudobactin occurred when the isolate was grown in media containing 1 mM Cr(III) under ironlimited conditions but not when Fe(III) was added at 10 M Pseudobactin synthesis was derepressed in iron-limited cultures containing 1 mM Sc(III) or Y(III), examples of group III-B elements. We found that AI(III), Ga(III) or In(III), representative metals from group III-A, repressed synthesis of pseudobactin under iron-deficient conditions. Analogs of Fe(III) were found to inhibit growth of thePseudomonas isolate in iron-limited media and the trivalent metals listed in order of decreasing toxicity were as follows: Ga > In > Sc > Cr > Y > Al. The inhibition of growth by 1 mM In(III), Sc(III) and Ga(III) was greater during iron-limited growth than in media containing 10 M Fe(III). These data show that, although the metal analogs of Fe(III) have similar chemical and physical characteristics, the physiological response of the fluorescent pseudomonad when grown in the presence of these metals varied markedly.     

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.     

Defined media for optimal pyoverdine production by Pseudomonas fluorescens 2-79

Pseudomonas fluorescens strain 2-79 (NRRL-15132) produces a fluorescent yellow-green pyoverdine when cultured on Fe(III)-poor medium. When cultured on Fe(III)-rich medium, strain 2-79 produces an antibiotic, phenazine 1-carboxylic acid, which is effective in suppressing plant fungal diseases such as take-all of wheat. A 2³ factorial design was used to examine pyoverdine production as a function of the presence or absence of Bacto casamino acids, purines-pyrimidines and vitamins in an iron-deficient medium. Amino acids were found to be an important factor (P=0.0002). A Plackett-Burman design was used to identity eight amino acids, out of the 19 present in casamino acids, that were responsible for the increased pyoverdine production: methionine, valine, isoleucine, tyrosine, proline, phenylalanine, glutamic acid, and glycine. Biomass was enhanced only by glutamic acid. Correspondence to: W. S. Kisaalita     

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.