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.     

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.     

Role of Molybdate and Other Transition Metals in the Accumulation of Protochelin by Azotobacter vinelandii

Both molybdate and iron are metals that are required by the obligately aerobic organism Azotobacter vinelandii to survive in the nutrient-limited conditions of its natural soil environment. Previous studies have shown that a high concentration of molybdate (1 mM) affects the formation of A. vinelandii siderophores such that the tricatecholate protochelin is formed to the exclusion of the other catecholate siderophores, azotochelin and aminochelin. It has been shown previously that molybdate combines readily with catecholates and interferes with siderophore function. In this study, we found that the manner in which each catecholate siderophore interacted with molybdate was consistent with the structure and binding potential of the siderophore. The affinity that each siderophore had for molybdate was high enough that stable molybdo-siderophore complexes were formed but low enough that the complexes were readily destabilized by Fe³⁺. Thus, competition between Fe³⁺ and molybdate did not appear to be the primary cause of protochelin accumulation; in addition, we determined that protochelin accumulated in the presence of vanadate, tungstate, Zn²⁺, and Mn²⁺. We found that all five of these metal ions partially inhibited uptake of ⁵⁵Fe-protochelin and ⁵⁵Fe-azotochelin complexes. Also, each of these metal ions partially inhibited the activity of ferric reductase, an enzyme important in the deferration of ferric siderophores. Our results suggest that protochelin accumulates in the presence of molybdate because protochelin uptake and conversion into its component parts, azotochelin and aminochelin, are inhibited by interference with ferric reductase.     

Effect of Exogenous Siderophores on Iron Uptake Activity of Marine Bacteria under Iron-Limited Conditions

More than 60% of species examined from a total of 421 strains of heterotrophic marine bacteria which were isolated from marine sponges and seawater were observed to have no detectable siderophore production even when Fe(III) was present in the culture medium at a concentration of 1.0 pM. The growth of one such non-siderophore-producing strain, alpha proteobacterium V0210, was stimulated under iron-limited conditions with the addition of an isolated exogenous siderophore, N,N′-bis (2,3-dihydroxybenzoyl)-O-serylserine from a Vibrio sp. Growth was also stimulated by the addition of three exogenous siderophore extracts from siderophore-producing bacteria. Radioisotope studies using ⁵⁹Fe showed that the iron uptake ability of V0210 increased only with the addition of exogenous siderophores. Biosynthesis of a hydroxamate siderophore by V0210 was shown by paper electrophoresis and chemical assays for the detection of hydroxamates and catechols. An 85-kDa iron-regulated outer membrane protein was induced only under iron-limited conditions in the presence of exogenous siderophores. This is the first report of bacterial iron uptake through an induced siderophore in response to exogenous siderophores. Our results suggest that siderophores are necessary signaling compounds for growth and for iron uptake by some non-siderophore-producing marine bacteria under iron-limited conditions.     

Catecholate Siderophores Protect Bacteria from Pyochelin Toxicity

Background

Bacteria produce small molecule iron chelators, known as siderophores, to facilitate the acquisition of iron from the environment. The synthesis of more than one siderophore and the production of multiple siderophore uptake systems by a single bacterial species are common place. The selective advantages conferred by the multiplicity of siderophore synthesis remains poorly understood. However, there is growing evidence suggesting that siderophores may have other physiological roles besides their involvement in iron acquisition.

Methods and Principal Findings

Here we provide the first report that pyochelin displays antibiotic activity against some bacterial strains. Observation of differential sensitivity to pyochelin against a panel of bacteria provided the first indications that catecholate siderophores, produced by some bacteria, may have roles other than iron acquisition. A pattern emerged where only those strains able to make catecholate-type siderophores were resistant to pyochelin. We were able to associate pyochelin resistance to catecholate production by showing that pyochelin-resistant Escherichia coli became sensitive when biosynthesis of its catecholate siderophore enterobactin was impaired. As expected, supplementation with enterobactin conferred pyochelin resistance to the entE mutant. We observed that pyochelin-induced growth inhibition was independent of iron availability and was prevented by addition of the reducing agent ascorbic acid or by anaerobic incubation. Addition of pyochelin to E. coli increased the levels of reactive oxygen species (ROS) while addition of ascorbic acid or enterobactin reduced them. In contrast, addition of the carboxylate-type siderophore, citrate, did not prevent pyochelin-induced ROS increases and their associated toxicity.

Conclusions

We have shown that the catecholate siderophore enterobactin protects E. coli against the toxic effects of pyochelin by reducing ROS. Thus, it appears that catecholate siderophores can behave as protectors of oxidative stress. These results support the idea that siderophores can have physiological roles aside from those in iron acquisition.     

Social evolution of toxic metal bioremediation in Pseudomonas aeruginosa

Bacteria are often iron-limited, and hence produce extracellular iron-scavenging siderophores. A crucial feature of siderophore production is that it can be an altruistic behaviour (individually costly but benefitting neighbouring cells), thus siderophore producers can be invaded by non-producing social ‘cheats’. Recent studies have shown that siderophores can also bind other heavy metals (such as Cu and Zn), but in this case siderophore chelation actually reduces metal uptake by bacteria. These complexes reduce heavy metal toxicity, hence siderophore production may contribute to toxic metal bioremediation. Here, we show that siderophore production in the context of bioremediation is also an altruistic trait and can be exploited by cheating phenotypes in the opportunistic pathogen Pseudomonas aeruginosa. Specifically, we show that in toxic copper concentrations (i) siderophore non-producers evolve de novo and reach high frequencies, and (ii) producing strains are fitter than isogenic non-producing strains in monoculture, and vice versa in co-culture. Moreover, we show that the evolutionary effect copper has on reducing siderophore production is greater than the reduction observed under iron-limited conditions. We discuss the relevance of these results to the evolution of siderophore production in natural communities and heavy metal bioremediation.     

Vanadium Requirements and Uptake Kinetics in the Dinitrogen-Fixing Bacterium Azotobacter vinelandii

Vanadium is a cofactor in the alternative V-nitrogenase that is expressed by some N₂-fixing bacteria when Mo is not available. We investigated the V requirements, the kinetics of V uptake, and the production of catechol compounds across a range of concentrations of vanadium in diazotrophic cultures of the soil bacterium Azotobacter vinelandii. In strain CA11.70, a mutant that expresses only the V-nitrogenase, V concentrations in the medium between 10⁻⁸ and 10⁻⁶ M sustain maximum growth rates; they are limiting below this range and toxic above. A. vinelandii excretes in its growth medium micromolar concentrations of the catechol siderophores azotochelin and protochelin, which bind the vanadate oxoanion. The production of catechols increases when V concentrations become toxic. Short-term uptake experiments with the radioactive isotope ⁴⁹V show that bacteria take up the V-catechol complexes through a regulated transport system(s), which shuts down at high V concentrations. The modulation of the excretion of catechols and of the uptake of the V-catechol complexes allows A. vinelandii to precisely manage its V homeostasis over a range of V concentrations, from limiting to toxic.     

Siderophore-mediated iron uptake in two clades of Marinobacter spp. associated with phytoplankton: the role of light

Iron is an essential element for oceanic microbial life but its low bioavailability limits microorganisms in large areas of the oceans. To acquire this metal many marine bacteria produce organic chelates that bind and transport iron (siderophores). We have previously shown that algal-associated heterotrophic bacteria belonging to the γ-proteobacterial Marinobacter genus release the siderophore vibrioferrin (VF). The iron-VF complex was shown to be both far more photolabile than all previously examined photolabile siderophores and to generate a photoproduct incapable of re-chelating the released iron. Thus, the photo-generated iron was shown to be highly bioavailable both to the producing bacterium and its algal partner. In exchange, we proposed that algal cells produced dissolved organic matter that helped support bacterial growth and ultimately fueled the biosynthesis of VF through a light-dependent “carbon for iron mutualism”. While our knowledge of the importance of light to phototrophs is vast, there are almost no studies that examine the effects of light on microbial heterotrophs. Here, we characterize iron uptake mechanisms in “algal-associated” VF-producers. Fe uptake by a VF knock-out mutant mimics the wild-type strain and demonstrates the versatility of iron uptake mechanisms in Marinobacter VF-producers. We also show that VF-producers selectively regulate a subset of their siderophore-dependent iron uptake genes in response to light exposure. The regulation of iron uptake and transport genes by light is consistent with the light driven algal–bacterial “carbon for iron mutualism” hypothesis in the marine environment.     

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.     

A rapid and sensitive paper electrophoresis assay for the detection of microbial siderophores elicited in solid-plating culture

A rapid and sensitive assay for the detection of microbial siderophores (iron-binding compounds) is described. Nine representative fungal and bacterial cultures including Ustilago sphaerogena, Penicillium sp., Fusarium roseum, Rhodotorula pilimanae, Bacillus subtilis W 23, Bacillus subtilis W 168, Bacillus megaterium, Azotobacter vinelandii OP, and Escherichia coli B, were nutritionally stressed for iron by sequential transfers on iron-deficient solid-plating media. In response to Fe-stress conditions, the microorganisms excreted siderophore compounds into the extracellular solid culture medium. The solid agar matrix effectively concentrated and restricted the migration of the siderophore compounds to the region immediately adjacent to colonial growth. Agar-block samples from this region were removed and placed at the origin of an electrophoresis paper strip. The resultant absorbed material from the agar-block sample was subjected to high-voltage paper electrophoresis which separated the siderophore compounds by size and molecular net charge. Phenolic acid (“catechol”)-type siderophores were detected by fluorescence under uv light. Hydroxamic acid-type siderophores were visualized by spraying the electrophoretogram with ferric iron solution.     

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.     

Detection of photoactive siderophore biosynthetic genes in the marine environment

Iron is an essential element for oceanic microbial life but its low bioavailability limits microorganisms in large areas of the oceans. To acquire this metal many marine bacteria produce organic chelates that bind and transport iron (siderophores). While it has been hypothesized that the global production of siderophores by heterotrophic bacteria and some cyanobacteria constitutes the bulk of organic ligands binding iron in the ocean because stability constants of siderophores and these organic ligands are similar, and because ligand concentrations rise sharply in response to iron fertilization events, direct evidence for this proposal is lacking. This lack is due to the difficulty in characterizing these ligands due both to their extremely low concentrations and their highly heterogeneous nature. The situation for characterizing photoactive siderophores in situ is more problematic because of their expected short lifetimes in the photic zone. An alternative approach is to make use of high sensitivity molecular technology (qPCR) to search for siderophore biosynthesis genes related to the production of photoactive siderophores. In this way one can access their “biochemical potential” and utilize this information as a proxy for the presence of these siderophores in the marine environment. Here we show, using qPCR primers designed to detect biosynthetic genes for the siderophores vibrioferrin, petrobactin and aerobactin that such genes are widespread and based on their abundance, the “biochemical potential” for photoactive siderophore production is significant. Concurrently we also briefly examine the microbial biodiversity responsible for such production as a function of depth and location across a North Atlantic transect.     

A comparative study of siderophore production by fungi from marine and terrestrial habitats

Siderophore producing potential of 20 fungal isolates (same 10 species from each marine and terrestrial habitat) were examined and compared. Except marine Aspergillus flavus, all isolates produced siderophores as evidenced by positive reaction in FeCl₃ test, CAS assay and CAS agar plate test. The results indicated widespread occurrence of siderophores in both the habitats. Examination of the chemical nature of siderophores revealed that mucoraceous fungi produced carboxylate, while others produced hydroxamate siderophores. Thus, the nature of siderophore was found to be independent of habitat. Among all the isolates, Cunninghamella elegans (marine form) was maximum siderophore producer (1987.5 μg/ml) followed by terrestrial form of C. elegans (1248.75 μg/ml). There was no marked variation in siderophore concentration of Penicillium funiculosum strains. Comparison of quantification of siderophore production between marine and terrestrial revealed that four terrestrial isolates (Aspergillus niger, Aspergillus ochraceous, Penicillium chrysogenum, Penicillium citrinum) were ahead in siderophore production, while, the other four marine isolates (Aspergillus versicolor, C. elegans, Rhizopus sp., Syncephalastrum racemosum) were found to be more potent siderophore producers, indicating that they were equally competent.     

Potential of Siderophore Production by Bacteria Isolated from Heavy Metal: Polluted and Rhizosphere Soils

Recently, heavy metals have been shown to have a stimulating effect on siderophore biosynthesis in various bacteria. In addition, several studies have found that siderophore production is greater in bacteria isolated from soil near plant roots. The aim of this study was to compare the production of siderophores by bacterial strains isolated from heavy metal-contaminated and uncontaminated soils. Chrome azurol sulphonate was used to detect siderophore secretion by several bacterial strains isolated from heavy metal-contaminated and rhizosphere-uncontaminated soils with both a qualitative disc diffusion method and a quantitative ultraviolet spectrophotometric method. Siderophore production by rhizosphere bacteria was significantly greater than by bacteria isolated from contaminated soil. The Pearson’s correlation test indicated a positive correlation between the amount of siderophore produced by bacteria isolated from the rhizosphere using the quantitative and qualitative detection methods and the amount of heavy metal in the soil. However, a significant negative correlation was observed between the amount of siderophore produced by bacteria isolated from heavy metal-contaminated soil and the amount of heavy metal (r value of ?0.775, P < 0.001).     

Pseudomonas fluorescens Pirates both Ferrioxamine and Ferricoelichelin Siderophores from Streptomyces ambofaciens

Iron is essential in many biological processes. However, its bioavailability is reduced in aerobic environments, such as soil. To overcome this limitation, microorganisms have developed different strategies, such as iron chelation by siderophores. Some bacteria have even gained the ability to detect and utilize xenosiderophores, i.e., siderophores produced by other organisms. We illustrate an example of such an interaction between two soil bacteria, Pseudomonas fluorescens strain BBc6R8 and Streptomyces ambofaciens ATCC 23877, which produce the siderophores pyoverdine and enantiopyochelin and the siderophores desferrioxamines B and E and coelichelin, respectively. During pairwise cultures on iron-limiting agar medium, no induction of siderophore synthesis by P. fluorescens BBc6R8 was observed in the presence of S. ambofaciens ATCC 23877. Cocultures with a Streptomyces mutant strain that produced either coelichelin or desferrioxamines, as well as culture in a medium supplemented with desferrioxamine B, resulted in the absence of pyoverdine production; however, culture with a double mutant deficient in desferrioxamines and coelichelin production did not. This strongly suggests that P. fluorescens BBbc6R8 utilizes the ferrioxamines and ferricoelichelin produced by S. ambofaciens as xenosiderophores and therefore no longer activates the production of its own siderophores. A screening of a library of P. fluorescens BBc6R8 mutants highlighted the involvement of the TonB-dependent receptor FoxA in this process: the expression of foxA and genes involved in the regulation of its biosynthesis was induced in the presence of S. ambofaciens. In a competitive environment, such as soil, siderophore piracy could well be one of the driving forces that determine the outcome of microbial competition.     

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.     

Siderophore-Mediated Iron Sequestering by Shewanella putrefaciens

The iron-sequestering abilities of 51 strains of Shewanella putrefaciens isolated from different sources (fish, water, and warm-blooded animals) were assessed. Thirty strains (60%) produced siderophores in heat-sterilized fish juice as determined by the chrome-azurol-S assay. All cultures were negative for the catechol-type siderophore, whereas 24 of the 30 siderophore-producing strains tested positive in the Csáky test, indicating the production of siderophores of the hydroxamate type. Siderophore-producing S. putrefaciens could to some degree cross-feed on the siderophores of other S. putrefaciens strains and on compounds produced by an Aeromonas salmonicida strain under iron-limited conditions. The siderophores of S. putrefaciens were not sufficiently strong to inhibit growth of other bacteria under iron-restricted conditions. However, siderophore-producing Pseudomonas bacteria were always inhibitory to S. putrefaciens under iron-limited conditions. Growth of siderophore-producing strains under iron-limited conditions induced the formation of one major new outer membrane protein of approximately 72 kDa. Two outer membrane proteins of approximately 53 and 23 kDa were not seen when iron was restricted.     

Hydroxamate-siderophore production and utilization by marine eubacteria

Siderophore (iron-binding chelator) production was examined in 30 strains of open ocean bacteria from the generaVibrio, Alteromonas, Alcaligenes, Pseudomonas, andPhotobacterium. The results showed that hydroxamate-type siderophore production was widely distributed in various marine species, except for isolates ofAlteromonas macleodii andV. nereis. In all cases, the ability to produce siderophores was under the control of iron levels in the medium and satisfied the iron requirements of the siderophore bioassay organism. On the basis of chemical assay and bacterial bioassays, none of the examined isolates produced phenolate-type siderophores. Several isolates produces siderophores that were neither hydroxamatenor phenolate-type siderophores. Some strains such asAlteromonas communis produce siderophores that could be used by many other isolates. In contrast, the siderophore produced byAlcaligenes venustus had little cross-strain utilization. These findings suggest that the ability to produce siderophores may be common to open ocean bacteria.     

Development and Application of an Assay for Uranyl Complexation by Fungal Metabolites, Including Siderophores

An assay to detect UO₂²⁺ complexation was developed based on the chrome azurol S (CAS) assay for siderophores (B. Schwyn and J. B. Neilands, Anal. Biochem. 160:47-56, 1987) and was used to investigate the ability of fungal metabolites to complex actinides. In this assay the discoloration of two dyed agars (one containing a CAS-Fe³⁺ dye and the other containing a CAS-UO₂²⁺ dye) caused by ligands was quantified. The assay was tested by using the siderophore desferrioxamine B (DFO), and the results showed that there was a regular, reproducible relationship between discoloration and the amount of siderophore added. The ratio of the discoloration on the CAS-UO₂²⁺ agar to the discoloration on the CAS-Fe³⁺ agar was independent of the amount of siderophore added. A total of 113 fungi and yeasts were isolated from three soil samples taken from the Peak District National Park. The fungi were screened for the production of UO₂²⁺ chelators by using the CAS-based assay and were also tested specifically for hydroxamate siderophore production by using the hydroxamate siderophore auxotroph Aureobacterium flavescens JG-9. This organism is highly sensitive to the presence of hydroxamate siderophores. However, the CAS-based assay was found to be less sensitive than the A. flavescens JG-9 assay. No significant difference between the results for each site for the two tests was found. Three isolates were selected for further study and were identified as two Pencillium species and a Mucor species. Our results show that the new assay can be effectively used to screen fungi for the production of UO₂²⁺ chelating ligands. We suggest that hydroxamate siderophores can be produced by mucoraceous fungi.