Siderophore production in Azotobacter vinelandii in response to Fe‐, Mo‐ and V‐limitation

Azotobacter vinelandii is a terrestrial diazotroph well studied for its siderophore production capacity and its role as a model nitrogen fixer. In addition to Fe, A. vinelandii siderophores are used for the acquisition of the nitrogenase co‐factors Mo and V. However, regulation of siderophore production by Mo‐ and V‐limitation has been difficult to confirm and knowledge of the full suite of siderophores synthesized by this organism has only recently become available. Using this new information, we conducted an extensive study of siderophore production in N₂‐fixing A. vinelandii under a variety of trace metal conditions. Our results show that under Fe‐limitation the production of all siderophores increases, while under Mo‐limitation only catechol siderophore production is increased, with the strongest response seen in protochelin. We also find that the newly discovered A. vinelandii siderophore vibrioferrin is almost completely repressed under Mo‐ and V‐limitation. An examination of the potential nitrogen ‘cost’ of siderophore production reveals that investments in siderophore N can represent as much as 35% of fixed N, with substantial differences between cultures using the Mo‐ as opposed to the less efficient V‐nitrogenase.     

The Siderophore Metabolome of Azotobacter vinelandii

In this study, we performed a detailed characterization of the siderophore metabolome, or “chelome,” of the agriculturally important and widely studied model organism Azotobacter vinelandii. Using a new high-resolution liquid chromatography-mass spectrometry (LC-MS) approach, we found over 35 metal-binding secondary metabolites, indicative of a vast chelome in A. vinelandii. These include vibrioferrin, a siderophore previously observed only in marine bacteria. Quantitative analyses of siderophore production during diazotrophic growth with different sources and availabilities of Fe showed that, under all tested conditions, vibrioferrin was present at the highest concentration of all siderophores and suggested new roles for vibrioferrin in the soil environment. Bioinformatic searches confirmed the capacity for vibrioferrin production in Azotobacter spp. and other bacteria spanning multiple phyla, habitats, and lifestyles. Moreover, our studies revealed a large number of previously unreported derivatives of all known A. vinelandii siderophores and rationalized their origins based on genomic analyses, with implications for siderophore diversity and evolution. Together, these insights provide clues as to why A. vinelandii harbors multiple siderophore biosynthesis gene clusters. Coupled with the growing evidence for alternative functions of siderophores, the vast chelome in A. vinelandii may be explained by multiple, disparate evolutionary pressures that act on siderophore production.     

Azotobacter vinelandii strains of disparate origin produce azotobactin siderophores with identical structures

Summary The yellow green fluorescent siderophore, azotobactin, was purified from cultures of twoAzotobacter vinelandii strains. Structural analysis of azotobactin from the North AmericanA. vinelandii strains O and its capsule negative variant UW (also called OP) revealed that both strains produced azotobactins with identical structures. Moreover, azotobactin produced by these two strains was structurally identical to azotobactin D, the fluorescent siderophore previously isolated from the EuropeanA. vinelandii strain D (CCM 289). Unlike strains of fluorescentPseudomonas which produce structurally diverse pyoverdins, strains ofA. vinelandii of disparate origin produced azotobactins of identical structure. Lactonization of azotobactin did not interfere with the ability of this compound to function as a siderophore.     

Azotobacter vinelandii gene clusters for two types of peptidic and catechol siderophores produced in response to molybdenum

Aim: To characterize the complementary production of two types of siderophores in Azotobacter vinelandii. Methods and Results: In an iron‐insufficient environment, nitrogen‐fixing A. vinelandii produces peptidic (azotobactin) and catechol siderophores for iron uptake to be used as a nitrogenase cofactor. Molybdenum, another nitrogenase cofactor, was also found to affect the production level of siderophores. Wild‐type cells excreted azotobactin into molybdenum‐supplemented and iron‐insufficient medium, although catechol siderophores predominate in molybdenum‐free environments. Two gene clusters were identified to be involved in the production of azotobactin and catechol siderophores through gene annotation and disruption. Azotobactin‐deficient mutant cells produced catechol siderophores under the molybdenum‐supplemented and iron‐insufficient conditions, whereas catechol siderophore–deficient mutant cells extracellularly secreted excess azotobactin under iron‐deficient condition independent of the concentration of molybdenum. This evidence suggests that a complementary siderophore production system exists in A. vinelandii. Conclusions: Molybdenum was found to regulate the production level of two types of siderophores. Azotobacter vinelandii cells are equipped with a complementary production system for nitrogen fixation in response to a limited quantity of metals. Significance and Impact of the Study: This is the first study identifying A. vinelandii gene clusters for the biosynthesis of two types of siderophores and clarifying the relationship between them.     

Isolation and partial characterization of siderophore mutants ofAzotobacter vinelandii

Azotobacter vinelandii was mutagenized with ethyl methanesulfonate, and colonies that did not produce the fluorescent yellow-green pigment that is characteristic of the wild type were selected. All 32 stable nonfluorescent mutants failed to secrete the siderophore azotobactin and were also impaired to some extent in the production of the second majorA. vinelandii siderophore, azotochelin. Mutants also showed differences in their capacity to grow on medium supplemented with either 200 M bipyridyl or 200 M Fe (III). In the absence of iron, an 84-kilodalton outer membrane protein, which is a major derepressed component, was missing in some of the mutants. Thus, siderophore production inA. vinelandii appears to be a highly integrated system in which the syntheses of azotobactin and azotochelin are functionally coupled.     

The DNA gyrase inhibitors,nalidixic acid and oxolinic acid,prevent iron-mediated repression of catechol siderophore synthesis inAzotobacter vinelandii

Summary Low concentrations of nalidixic acid and oxolinic acid that were just inhibitory toAzotobacter vinelandii growth promoted the production of the catechol siderophores azotochelin and aminochelin, in the presence of normally repressive concentrations of Fe³⁺. There was a limited effect on the pyoverdin siderophore, azotobactin, where low concentrations of Fe³⁺ were rendered less repressive, but the repression by higher concentrations of Fe³⁺ was normal. These drugs did not induce high-molecular-mass iron-repressible outer-membrane proteins and similar effects on the regulation of catechol siderophore synthesis were not produced by novobiocin, coumermycin, or ethidium bromide. The timing of nalidixic acid and Fe³⁺ addition to iron-limited cells was critical. Nalidixic acid had to be added before iron-repression of catechol siderophore synthesis and before the onset of iron-sufficient growth. Continued production of the catechol siderophores, however, was not due to interference with normal iron uptake. These data indicated that nalidixic acid prevented normal iron-repression of catechol siderophore synthesis but could not reverse iron repression once it had ocurred. The possible roles of DNA gyrase activity in the regulation of catechol siderophore synthesis is discussed.     

Production of the triacetecholate siderophore protochelin by Azotobacter vinelandii

Azotobacter vinelandii grown in iron-limited medium containing 1 m molybdate released the catecholate siderophores azotochelin and aminochelin [bis(2,3-dihydroxybenzoyl-lysine) and 2,3-dihydroxybenzoyl-putrescine, respectively] into the culture fluid. However these catecholates were not observed when the medium contained 1 mm molybdate, but were replaced by another catecholate compound. The appearance of this new compound was not an artifact of extraction of the catecholates from the culture fluid in the presence of high molybdate. Full and partial acid hydrolysis and fast atom bombardment mass spectroscopy showed that the new compound was the tricatecholate protochelin, a product of the condensation of azotochelin and aminochelin. The production of protochelin was iron-repressible and protochelin very rapidly decolorized the Chrome Azurol-S assay. Protochelin promoted the growth of the siderophore-deficient A. vinelandii strain P100 under iron-restricted conditions and promoted ⁵⁵Fe uptake into iron-limited cells, confirming that protochelin can be used as a siderophore by A. vinelandii.     

Characterization of the pyoverdines ofAzotobacter vinelandii ATCC 12 837 with regard to heterogeneity

Summary Azotobacter vinelandii strain ATCC 12 837 produces peptide siderophores of the general class known as pyoverdines. In the past, it was assumed that a single well-defined pyoverdine was produced by each parent microorganism. However, there are a number of reports of incompletely characterized pyoverdines that demonstrate heterogeneity in pyoverdine preparations obtained from a single organism, but the nature of this phenomena has not been explained. This study shows thatA. vinelandii does indeed produce more than one pyoverdine and that these compounds differ in their peptide components. The metabolism of these siderophores suggests that only one of them is a true siderophore while the others are metabolic byproducts. It was demonstrated that this phenomenon is likely due to intrinsic limitations of the synthetase complex involved in the biosynthesis of these compounds. Characterization of two of the major pyoverdines produced demonstrated that they are novel compounds, although they belonged to theAzotobacter-type family of pyoverdines.     

Stimulation of Agrobacterium tumefaciens Growth by Azotobacter vinelandii Ferrisiderophores

Azotobacter vinelandii stimulated the growth of Agrobacterium tumefaciens H2, H23, H24, H27, and ATCC 15955 on media containing insoluble iron sources. The Azotobacter vinelandii siderophores appeared to promote Agrobacterium tumefaciens growth by solubilizing mineral iron, and the ferrisiderophores so formed then acted as iron sources for Agrobacterium tumefaciens. Agrobactin, the Agrobacterium siderophore, appeared to be inefficient in solubilizing mineral iron directly.     

Plant Growth Promoting Characterization of Indigenous Azotobacteria Isolated from Soils in Iran

It has been well known that the bacteria of the genus Azotobacter, in addition to the beneficial N₂-fixing activity, are able to improve plant growth by a number of direct and indirect mechanisms. To identify this potential in indigenous azotobacteria, the efficiency of 17 isolates of Azotobacter from the rhizosphere of wheat and barley plants cultivated in salt- and/or drought-affected soils in Iran were evaluated for their ability to dissolve inorganic and organic phosphates, siderophore secretion, indole acetic acid (IAA) production; and protease, chitinase, and ACC deaminase (ACCD) activities. First, they were biochemically characterized and one isolate (strain) was identified by 16S rDNA sequencing. Eight isolates were designated as Azotobacter vinelandii and the remaining isolates were identified as A. chroococcum. All isolates hydrolyzed the organic and inorganic phosphate compounds and effectively produced IAA. Fifteen isolates produced siderophore, but only one isolate showed protease activity which is being reported for the first time in relation to Azotobacter. None of the 17 isolates was capable of producing ACCD or chitinase. However, polymerase chain reaction amplification of the ACCD coding genes, by the use of the gene-specific primers, indicated that not all contain the ACCD gene. The standard screening methods with slight modifications, especially in the case of ACCD assay, were applied. The results showed that the use of specific screening methods, modified according to bacterial nutritional requirements, are the efficient methods for precise evaluation of the plant growth promoting rhizobacteria activity.     

The stability of the molybdenum-azotochelin complex and its effect on siderophore production in Azotobacter vinelandii

 Azotobacter vinelandii produces five siderophores with different metal binding properties, depending on the concentrations of Fe(III) and molybdate in the growth medium. The three lower protonation constants of the unusual bis(catecholamide) siderophore azotochelin (L) were determined by a simultaneous spectrophotometric and potentiometric titration as log K ₅=3.65(5), log K ₄=7.41(3) and log K ₃=8.54(4). The metal-ligand equilibrium constant for [MoO₂(L)]^3– was obtained from analysis of the absorbance concentration data: at 20  °C and pH 6.6, log K _eq=4(1). Based on an average log K _a value of 12.1 for the two basic phenolic oxygens of azotochelin, the equilibrium formation constant was converted into the conventional formation constant K _f(MoL) = [MoO₂L^{3 –}]/[MoO₂ ²⁺][L^{5 –}] = 10³⁵ M^–1. To assess the influence of molybdenum-siderophore interactions on metal uptake in A. vinelandii, the dose-response effect of molybdate in the growth medium on siderophore biosynthesis was followed by UV-vis spectroscopy and HPLC. It could be shown that the formation of molybdenum siderophore complexes clearly reduces the concentration of free siderophores available for iron solubilization. Furthermore, in media with initial molybdate concentrations up to 100 μM, the molybdenum azotochelin complex is the predominant molybdenum species, suggesting that azotochelin might also possess sequestering activity towards molybdenum. Even higher molybdate levels result in a complete repression of the synthesis of the tetradentate siderophore azotochelin, while they initiate the alternative release of the more efficient iron chelator, the hexadentate siderophore protochelin. Received: 20 April 1998 / Accepted: 29 June 1998     

Molecular and bioengineering strategies to improve alginate and polydydroxyalkanoate production by <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis>

Several aspects of alginate and PHB synthesis in Azotobacter vinelandii at a molecular level have been elucidated in articles published during the last ten years. It is now clear that alginate and PHB synthesis are under a very complex genetic control. Genetic modification of A. vinelandii has produced a number of very interesting mutants which have particular traits for alginate production. One of these mutants has been shown to produce the alginate with the highest mean molecular mass so far reported. Recent work has also shed light on the factors determining molecular mass distribution; the most important of these being identified as; dissolved oxygen tension and specific growth rate. The use of specific mutants has been very useful for the correct analysis and interpretation of the factors affecting polymerization. Recent scale-up/down work on alginate production has shown that oxygen limitation is crucial for producing alginate of high molecular mass, a condition which is optimized in shake flasks and which can now be reproduced in stirred fermenters. It is clear that the phenotypes of mutants grown on plates are not necessarily reproducible when the strains are tested in lab or bench scale fermenters. In the case of PHB, A. vinelandii has shown itself able to produce relatively large amounts of this polymer of high molecular weight on cheap substrates, even allowing for simple extraction processes. The development of fermentation strategies has also shown promising results in terms of improving productivity. The understanding of the regulatory mechanisms involved in the control of PHB synthesis, and of its metabolic relationships, has increased considerably, making way for new potential strategies for the further improvement of PHB production. Overall, the use of a multidisciplinary approach, integrating molecular and bioengineering aspects is a necessity for optimizing alginate and PHB production in A. vinelandii.     

Diazotrophic mixed culture ofAzotobacter vinelandii andRhodobacter capsulatus

The question, whetherAzotobacter vinelandii can provide fixed N for the growth of other organisms, was studied with mixed cultures ofA. vinelandii andRhodobacter capsulatus, grown with aeration in the light. N₂-fixation byR. capsulatus was prevented by growing the cultures on either mannitol, glycerol or ethanol, which cannot be used by this organism. In the course of growth with mannitol, cell numbers of both organisms increased largely in parallel and attained a maximal ratio of about oneA. vinelandii per tenR. capsulatus. Prolonged growth of mixed cultures with mannitol did not lead to an adaptation ofR. capsulatus to this compound. After growth on either one of the three alcohols, mixed cultures exhibited almost twice as high protein levels as pure cultures ofA. vinelandii. Up to 80% of the protein of mixed cultures was incorporated intoR. capsulatus. The results suggest thatA. vinelandii provided an organic N-source for the growth ofR. capsulatus.     

Isolation and characterization of siderophore from cowpeaRhizobium (peanut isolate)

In an iron-depleted broth culture of cowpeaRhizobium (a peanut isolate), phenolate type of compounds were detected. Chemical characterization showed the presence of 2,3-dihydroxy benzoic acid (DHBA) and 3,4-DHBA in the siderophore extract. Lysine and alanine were identified as conjugated amino acids of the siderophore. Maximum concentration of the siderophore in the culture supernatant was found after 24 h of growth. The compounds in the extracted siderophore induced growth ofRhizobium in a medium containing EDTA. Addition of lysine and alanine in the growth medium (20 mM each) led to a fourfold increase in siderophore production.     

Iron-dependent growth of and siderophore production by two heterotrophic bacteria isolated from brackish water of the southern Baltic Sea

Iron is indispensable to the growth and metabolism of all marine organisms, including bacteria. In this work, we investigated and compared the influence of iron(III) concentration on the growth of and siderophore production by two heterotrophic bacteria – Micrococcus luteus and Bacillus silvestris.Our results showed that the iron concentration strongly influences the growth of both species. The growth curves were different for each iron concentration and each strain. M. luteus grew more rapidly than B. silvestris, but produced a roughly four times smaller quantity of siderophores. Both M. luteus and B. silvestris secreted hydroxamate-type siderophores and α-keto/α-hydroxy acids, but did not produce catecholates.This paper is probably the first to report on siderophore production by B. silvestris and M. luteus isolated from seawater. Moreover, the influence of different iron concentrations on the growth of and siderophore production in these bacteria has been documented. This provides further evidence indicating iron bioavailability as the actual reason for siderophore release by biota.     

Isolation and characterisation of catechol-like siderophore from cowpea Rhizobium RA-1

Cowpea Rhizobium RA-1 produced a catechol-like siderophore. Secondary hydroxamic acids were not detected. Bioassay of the siderophore exhibited a distinct zone of growth of cowpea rhizobia. One litre of culture filtrate gave 6.2 mg of catechol-like siderophore. Glycine and threonine were detected in the siderophore. Maximum production of siderophore was found at 36 h of growth of cowpea Rhizobium RA-1.Abbreviations 2,3-DHBA 2,3-dihydroxy benzoic acid - EDTA ethylenediamine tetraacetic acid     

Specific substrates for isolation and differentiation of Azotobacter vinelandii

Summary Resorcinol, ethylene glycol and glutaric acid have been found to be specific substrates for the enrichment of Azotobacter vinelandii in the presence of other Azotobacter species. The three compounds may also be used for species differentiation in the genus Azotobacter. In contrast to other reports strains of A. chroococcum and A. beijerinckii have been isolated which are able to use L-rhamnose for growth.Conditions for the production of a yellow pigment and green fluorescence by A. vinelandii from non-aromatic substrates on agar-plates have been tested.The production of water soluble yellow pigments in benzoate containing media cannot be used as a criterium for the presence of A. vinelandii. The pigment seems to be -hydroxy-muconic semialdehyde, the splitting product resulting from metacleavage of catechol. It may be formed by all Azotobacter species that can metabolize aromatic substrates.     

Xanthoferrin,the α‐hydroxycarboxylate‐type siderophore of Xanthomonas campestris pv. campestris,is required for optimum virulence and growth inside cabbage

Xanthomonas campestris pv. campestris causes black rot, a serious disease of crucifers. Xanthomonads encode a siderophore biosynthesis and uptake gene cluster xss (Xanthomonas siderophore synthesis) involved in the production of a vibrioferrin‐type siderophore. However, little is known about the role of the siderophore in the iron uptake and virulence of X. campestris pv. campestris. In this study, we show that X. campestris pv. campestris produces an α‐hydroxycarboxylate‐type siderophore (named xanthoferrin), which is required for growth under low‐iron conditions and for optimum virulence. A mutation in the siderophore synthesis xssA gene causes deficiency in siderophore production and growth under low‐iron conditions. In contrast, the siderophore utilization ΔxsuA mutant is able to produce siderophore, but exhibits a defect in the utilization of the siderophore–iron complex. Our radiolabelled iron uptake studies confirm that the ΔxssA and ΔxsuA mutants exhibit defects in ferric iron (Fe³⁺) uptake. The ΔxssA mutant is able to utilize and transport the exogenous xanthoferrin–Fe³⁺ complex; in contrast, the siderophore utilization or uptake mutant ΔxsuA exhibits defects in siderophore uptake. Expression analysis of the xss operon using a chromosomal gusA fusion indicates that the xss operon is expressed during in planta growth and under low‐iron conditions. Furthermore, exogenous iron supplementation in cabbage leaves rescues the in planta growth deficiency of ΔxssA and ΔxsuA mutants. Our study reveals that the siderophore xanthoferrin is an important virulence factor of X. campestris pv. campestris which promotes in planta growth by the sequestration of Fe³⁺.     

Response of the cyanobacterium,Oscillatoria tenuis,to low iron environments: the effect on growth rate and evidence for siderophore production

Cyanobacteria vary in their ability to grow in media contaning low amounts of biologically available iron. Some strains, such as Oscillatoria tenuis, are well adapted to thrive in low-iron environments. We investigated the mechanism of iron scavenging in O. tenuis and found that this cyanobacterium has a siderophore-mediated iron transport system that differs significantly from the traditional hydroxamate-siderophore transport system reported from other cyanobacteria. Unlike other cyanobacteria, this strain produces two types of siderophores, a hydroxamate-type siderophore and a catechol-type siderophore. Production of these two siderophores is expressed at two different iron levels in the medium, suggesting two different iron regulated uptake systems. We compared the production of each siderophore with the growth rate of the culture and found that the production of the catechol siderophore enhances the growth rate of the cyanobacterium, whereas the cells maintain lower than maximal growth rates when only the hydroxamate-type siderophore is being produced.Abbreviation EDDA ethylene diamine di-(o-hydroxyphenylacetic acid)     

Isolation and partial characterization of catechol-type siderophore fromPseudomonas stutzeri RC 7

Pseudomonas stutzeri RC 7 grown under iron-deficient conditions produced catecholtype siderophore, which was identified to be arginine conjugate of 2,3-dihydroxy-benzoic acid. Hydroxamic acids were not detected. The concentration of siderophore in the culture supernatant was maximal after 24 h of growth. Addition of iron to the medium increased bacterial growth but repressed the production of siderophore.