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.     

Essential metals for nitrogen fixation in a free-living N₂-fixing bacterium: chelation, homeostasis and high use efficiency

Biological nitrogen fixation, the main source of new nitrogen to the Earth's ecosystems, is catalysed by the enzyme nitrogenase. There are three nitrogenase isoenzymes: the Mo‐nitrogenase, the V‐nitrogenase and the Fe‐only nitrogenase. All three types require iron, and two of them also require Mo or V. Metal bioavailability has been shown to limit nitrogen fixation in natural and managed ecosystems. Here, we report the results of a study on the metal (Mo, V, Fe) requirements of Azotobacter vinelandii, a common model soil diazotroph. In the growth medium of A. vinelandii, metals are bound to strong complexing agents (metallophores) excreted by the bacterium. The uptake rates of the metallophore complexes are regulated to meet the bacterial metal requirement for diazotrophy. Under metal‐replete conditions Mo, but not V or Fe, is stored intracellularly. Under conditions of metal limitation, intracellular metals are used with remarkable efficiency, with essentially all the cellular Mo and V allocated to the nitrogenase enzymes. While the Mo‐nitrogenase, which is the most efficient, is used preferentially, all three nitrogenases contribute to N₂ fixation in the same culture under metal limitation. We conclude that A. vinelandii is well adapted to fix nitrogen in metal‐limited soil environments.     

Multiple roles of siderophores in free-living nitrogen-fixing bacteria

Free-living nitrogen-fixing bacteria in soils need to tightly regulate their uptake of metals in order to acquire essential metals (such as the nitrogenase metal cofactors Fe, Mo and V) while excluding toxic ones (such as W). They need to do this in a soil environment where metal speciation, and thus metal bioavailability, is dependent on a variety of factors such as organic matter content, mineralogical composition, and pH. Azotobacter vinelandii, a ubiquitous gram-negative soil diazotroph, excretes in its external medium catechol compounds, previously identified as siderophores, that bind a variety of metals in addition to iron. At low concentrations, complexes of essential metals (Fe, Mo, V) with siderophores are taken up by the bacteria through specialized transport systems. The specificity and regulation of these transport systems are such that siderophore binding of excess Mo, V or W effectively detoxifies these metals at high concentrations. In the topsoil (leaf litter layer), where metals are primarily bound to plant-derived organic matter, siderophores extract essential metals from natural ligands and deliver them to the bacteria. This process appears to be a key component of a mutualistic relationship between trees and soil diazotrophs, where tree-produced leaf litter provides a living environment rich in organic matter and micronutrients for nitrogen-fixing bacteria, which in turn supply new nitrogen to the ecosystem.     

The purple non‐sulfur bacterium Rhodopseudomonas palustris produces novel petrobactin‐related siderophores under aerobic and anaerobic conditions

Many bacteria produce siderophores to bind and take up Fe(III), an essential trace metal with extremely low solubility in oxygenated environments at circumneutral pH. The purple non‐sulfur bacterium Rhodopseudomonas palustris str. CGA009 is a metabolically versatile model organism with high iron requirements that is able to grow under aerobic and anaerobic conditions. Siderophore biosynthesis has been predicted by genomic analysis, however, siderophore structures were not identified. Here, we elucidate the structure of two novel siderophores from R. palustris: rhodopetrobactin A and B. Rhodopetrobactins are structural analogues of the known siderophore petrobactin in which the Fe chelating moieties are conserved, including two 3,4‐dihydroxybenzoate and a citrate substructure. In the place of two spermidine linker groups in petrobactin, rhodopetrobactins contain two 4,4′‐diaminodibutylamine groups of which one or both are acetylated at the central amine. We analyse siderophore production under different growth modes and show that rhodopetrobactins are produced in response to Fe limitation under aerobic as well as under anaerobic conditions. Evaluation of the chemical characteristics of rhodopetrobactins indicates that they are well suited to support Fe acquisition under variable oxygen and light conditions.     

SIDEROPHORE‐INDEPENDENT IRON UPTAKE BY IRON‐LIMITED CELLS OF THE CYANOBACTERIUM ANABAENA FLOS‐AQUAE1

Iron acquisition by iron‐limited cyanobacteria is typically considered to be mediated mainly by siderophores, iron‐chelating molecules released by iron‐limited cyanobacteria into the environment. In this set of experiments, iron uptake by iron‐limited cells of the cyanobacterium Anabaena flos‐aquae (L.) Bory was investigated in cells resuspended in siderophore‐free medium. Removal of siderophores decreased iron‐uptake rates by ～60% compared to siderophore‐replete conditions; however, substantial rates of iron uptake remained. In the absence of siderophores, Fe(III) uptake was much more rapid from a weaker synthetic chelator [N‐(2‐hydroxyethyl)ethylenediamine‐N,N′,N′‐triacetic acid (HEDTA); log K_cond = 28.64 for Fe(III)HEDTA(OH)^?] than from a very strong chelator [N,N′‐bis(2‐hydroxybenzyl)‐ethylenediamine‐N,N′‐diacetic acid (HBED); log K_cond = 31.40 for Fe(III)HBED^?], and increasing chelator:Fe(III) ratios decreased the Fe(III)‐uptake rate; these results were evident in both short‐term (4 h; absence of siderophores) and long‐term (116 h; presence of siderophores) experiments. However, free (nonchelated) Fe(III) provided the most rapid iron uptake in siderophore‐free conditions. The results of the short‐term experiments are consistent with an Fe(III)‐binding/uptake mechanism associated with the cyanobacterial outer membrane that operates independently of extracellular siderophores. Iron uptake was inhibited by temperature‐shock treatments of the cells and by metabolically compromising the cells with diphenyleneiodonium; this finding indicates that the process is dependent on active metabolism to operate and is not simply a passive Fe(III)‐binding mechanism. Overall, these results point to an important, siderophore‐independent iron‐acquisition mechanism by iron‐limited cyanobacterial cells.     

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.     

Multiple modes of iron uptake by the filamentous,siderophore‐producing cyanobacterium,Anabaena sp. PCC 7120

Iron is a member of a small group of nutrients that limits aquatic primary production. Mechanisms for utilizing iron have to be efficient and adapted according to the ecological niche. In respect to iron acquisition cyanobacteria, prokaryotic oxygen evolving photosynthetic organisms can be divided into siderophore‐ and non‐siderophore‐producing strains. The results presented in this paper suggest that the situation is far more complex. To understand the bioavailability of different iron substrates and the advantages of various uptake strategies, we examined iron uptake mechanisms in the siderophore‐producing cyanobacterium Anabaena sp. PCC 7120. Comparison of the uptake of iron complexed with exogenous (desferrioxamine B, DFB) or to self‐secreted (schizokinen) siderophores by Anabaena sp. revealed that uptake of the endogenous produced siderophore complexed to iron is more efficient. In addition, Anabaena sp. is able to take up dissolved, ferric iron hydroxide species (Fe′) via a reductive mechanism. Thus, Anabaena sp. exhibits both, siderophore‐ and non‐siderophore‐mediated iron uptake. While assimilation of Fe′ and FeDFB are not induced by iron starvation, FeSchizokinen uptake rates increase with increasing iron starvation. Consequently, we suggest that Fe′ reduction and uptake is advantageous for low‐density cultures, while at higher densities siderophore uptake is preferred.     

Reconstructing the evolutionary history of nitrogenases: Evidence for ancestral molybdenum‐cofactor utilization

The nitrogenase metalloenzyme family, essential for supplying fixed nitrogen to the biosphere, is one of life's key biogeochemical innovations. The three forms of nitrogenase differ in their metal dependence, each binding either a FeMo‐, FeV‐, or FeFe‐cofactor where the reduction of dinitrogen takes place. The history of nitrogenase metal dependence has been of particular interest due to the possible implication that ancient marine metal availabilities have significantly constrained nitrogenase evolution over geologic time. Here, we reconstructed the evolutionary history of nitrogenases, and combined phylogenetic reconstruction, ancestral sequence inference, and structural homology modeling to evaluate the potential metal dependence of ancient nitrogenases. We find that active‐site sequence features can reliably distinguish extant Mo‐nitrogenases from V‐ and Fe‐nitrogenases and that inferred ancestral sequences at the deepest nodes of the phylogeny suggest these ancient proteins most resemble modern Mo‐nitrogenases. Taxa representing early‐branching nitrogenase lineages lack one or more biosynthetic nifE and nifN genes that both contribute to the assembly of the FeMo‐cofactor in studied organisms, suggesting that early Mo‐nitrogenases may have utilized an alternate and/or simplified pathway for cofactor biosynthesis. Our results underscore the profound impacts that protein‐level innovations likely had on shaping global biogeochemical cycles throughout the Precambrian, in contrast to organism‐level innovations that characterize the Phanerozoic Eon.     

Bioavailability of mineral-associated trace metals as cofactors for nitrogen fixation by Azotobacter vinelandii

Life on Earth depends on N₂-fixing microbes to make ammonia from atmospheric N₂ gas by the nitrogenase enzyme. Most nitrogenases use Mo as a cofactor; however, V and Fe are also possible. N₂ fixation was once believed to have evolved during the Archean-Proterozoic times using Fe as a cofactor. However, δ¹⁵N values of paleo-ocean sediments suggest Mo and V cofactors despite their low concentrations in the paleo-oceans. This apparent paradox is based on an untested assumption that only soluble metals are bioavailable. In this study, laboratory experiments were performed to test the bioavailability of mineral-associated trace metals to a model N₂-fixing bacterium Azotobacter vinelandii. N₂ fixation was observed when Mo in molybdenite, V in cavansite, and Fe in ferrihydrite were used as the sole sources of cofactors, but the rate of N₂ fixation was greatly reduced. A physical separation between minerals and cells further reduced the rate of N₂ fixation. Biochemical assays detected five siderophores, including aminochelin, azotochelin, azotobactin, protochelin, and vibrioferrin, as possible chelators to extract metals from minerals. The results of this study demonstrate that mineral-associated trace metals are bioavailable as cofactors of nitrogenases to support N₂ fixation in those environments that lack soluble trace metals and may offer a partial answer to the paradox.     

Metal limitation of cyanobacterial N2 fixation and implications for the Precambrian nitrogen cycle

Nitrogen fixation is a critical part of the global nitrogen cycle, replacing biologically available reduced nitrogen lost by denitrification. The redox‐sensitive trace metals Fe and Mo are key components of the primary nitrogenase enzyme used by cyanobacteria (and other prokaryotes) to fix atmospheric N₂ into bioessential compounds. Progressive oxygenation of the Earth's atmosphere has forced changes in the redox state of the oceans through geologic time, from anoxic Fe‐enriched waters in the Archean to partially sulfidic deep waters by the mid‐Proterozoic. This development of ocean redox chemistry during the Precambrian led to fluctuations in Fe and Mo availability that could have significantly impacted the ability of prokaryotes to fix nitrogen. It has been suggested that metal limitation of nitrogen fixation and nitrate assimilation, along with increased rates of denitrification, could have resulted in globally reduced rates of primary production and nitrogen‐starved oceans through much of the Proterozoic. To test the first part of this hypothesis, we grew N₂‐fixing cyanobacteria in cultures with metal concentrations reflecting an anoxic Archean ocean (high Fe, low Mo), a sulfidic Proterozoic ocean (low Fe, moderate Mo), and an oxic Phanerozoic ocean (low Fe, high Mo). We measured low rates of cellular N₂ fixation under [Fe] and [Mo] estimated for the Archean ocean. With decreased [Fe] and higher [Mo] representing sulfidic Proterozoic conditions, N₂ fixation, growth, and biomass C:N were similar to those observed with metal concentrations of the fully oxygenated oceans that likely developed in the Phanerozoic. Our results raise the possibility that an initial rise in atmospheric oxygen could actually have enhanced nitrogen fixation rates to near modern marine levels, providing that phosphate was available and rising O₂ levels did not markedly inhibit nitrogenase activity.     

Pseudomonas protegens Pf‐5 favours self‐produced siderophore over free‐loading in interspecies competition for iron

Many microorganisms compete for extracellular iron using strain‐specific chelators known as siderophores. The ferric‐siderophore complex limits local access to iron because import requires a suitable cognate receptor. Interestingly, many species carry receptors that enable ‘cross‐feeding’ on heterologous siderophores made by neighboring organisms, although little is known about how this ubiquitous behaviour is regulated. Here, we investigated the soil bacterium Pseudomonas protegens Pf‐5, a strain remarkable for its ability to use dozens of heterologous siderophores. We characterized the expression of six pyoverdine‐type (PVD) siderophore receptors in response to their cognate PVD. In general, we found expression is tightly regulated to reflect availability of their cognate PVD. In contrast, Pf‐5 continues to secrete its own primary siderophore, PVD_Pf‐5, despite the capability and opportunity to cross‐feed. We demonstrate that this strategy is beneficial in co‐culture with a competing PVD_PAO1‐producer, P. aeruginosa PAO1. Although Pf‐5 can cross‐feed on PVD_PAO1, production of PVD_Pf‐5 is required to maintain a competitive advantage. We attribute this to an antagonistic effect of PVD_Pf‐5 on the growth of PAO1, presumably through limiting access to iron. Our results demonstrate the benefits of excluding competitors out‐weigh the incentives associated with a free‐loader lifestyle for Pf‐5.     

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.     

Mechanisms of iron acquisition from siderophores by microorganisms and plants

Most bacteria, fungi, and some plants respond to Fe stress by the induction of high-affinity Fe transport systems that utilize biosyrthetic chelates called siderophores. To competitively acquire Fe, some microbes have transport systems that enable them to use other siderophore types in addition to their own. Bacteria such as Escherichia coli achieve this ability by using a combination of separate siderophore receptors and transporters, whereas other microbial species, such as Streptomyces pilosus, use a low specificity, high-affinity transport system that recognizes more than one siderophore type. By either strategy, such versatility may provide an advantage under Fe-limiting conditions; allowing use of siderophores produced at another organism's expense, or Fe acquisition from siderophores that could otherwise sequester Fe in an unavailable form.Plants that use microbial siderophores may also be more Fe efficient by virtue of their ability to use a variety of Fe sources under different soil conditions. Results of our research examining Fe transport by oat indicate parity in plant and microbial requirements for Fe and suggest that siderophores produced by root-colonizing microbes may provide Fe to plants that can use the predominant siderophore types. In conjunction with transport mechanisms, ecological and soil chemical factors can influence the efficacy of siderophores and phytosiderophores. A model presented here attempts to incorporate these factors to predict conditions that may govern competition for Fe in the plant rhizosphere. Possibly such competition has been a factor in the evolution of broad transport capabilities for different siderophores by microorganisms and plants.     

106 Nitrogen Fixation Behavior of a Terrestrial Heterocystous Cyanobacterium,Nostoc Flagelliforme,as Affected by Available Nutrients in Liquid Suspension Culture

Nostoc flagelliforme is an edible terrestrial cyanobacterium of great economic value which is often distributed in arid or semi‐arid steppes in many parts of the world. The relationship between N2‐fixing capacity and nutrient availability of N. flagelliforme under various growth conditions was investigated. Phosphorus had a profound effect on nitrogenase activity (NA) of the alga over the concentration range of 2.3 μM–0.46 mM inorganic phosphorus (Pi) in the cultures, with a maximal NA at 23 μM Pi. The optimal temperature for NA was determined by the Arrhenius equation to be approximately 25°C. However, the response of NA to different phosphorus concentrations was not affected by temperature variation. Micro‐nutrients in the media also affected NA of the alga. A 2‐fold strength of both B and Ca (relative to the standard BG110 medium) significantly enhanced NA. In addition, simultaneous enrichments with B, Ca, Fe and Mo exerted marked beneficial effects on NA, and such effects were presumably in an interactive and synergistic manner. In contrast, removal of Mo gave rise to a drastic decrease in growth rate, while the NA was only moderately decreased. NA in the Mo‐, Co‐, and Fe‐free cultures was respectively inhibited, as compared to cultures devoid of Ca, Fe, Mg, Mn, Zn and Cu, respectively. Results of the present study suggested that N. flagelliforme could be an important contributor of nitrogen to the terrestrial ecosystem because of its strong nitrogen‐fixing ability even in an infertile environment.     

Diel variation of the cellular carbon to nitrogen ratio of Chlorella autotrophica (Chlorophyta) growing in phosphorus‐ and nitrogen‐limited continuous cultures

We investigated the relationship between daily growth rates and diel variation of carbon (C) metabolism and C to nitrogen (N) ratio under P‐ and N‐limitation in the green algae Chlorella autotrophica. To do this, continuous cultures of C. autotrophica were maintained in a cyclostat culture system under 14:10 light:dark cycle over a series of P‐ and N‐limited growth rates. Cell abundance, together with cell size, as reflected by side scatter signal from flow cytometric analysis demonstrated a synchronized diel pattern with cell division occurring at night. Under either type of nutrient limitation, the cellular C:N ratio increased through the light period and decreased through the dark period over all growth rates, indicating a higher diel variation of C metabolism than that of N. Daily average cellular C:N ratios were higher at lower dilution rates under both types of nutrient limitation but cell enlargement was only observed at lower dilution rates under P‐limitation. Carbon specific growth rates during the dark period positively correlated with cellular daily growth rates (dilution rates), with net loss of C during night at the lowest growth rates under N‐limitation. Under P‐limitation, dark C specific growth rates were close to zero at low dilution rates but also exhibited an increasing trend at high dilution rates. In general, diel variations of cellular C:N were low when dark C specific growth rates were high. This result indicated that the fast growing cells performed dark C assimilation at high rates, hence diminished the uncoupling of C and N metabolism at night.     

The path of electron transfer to nitrogenase in a phototrophic alpha‐proteobacterium

The phototrophic alpha‐proteobacterium, Rhodopseudomonas palustris, is a model for studies of regulatory and physiological parameters that control the activity of nitrogenase. This enzyme produces the energy‐rich compound H₂, in addition to converting N₂ gas to NH₃. Nitrogenase is an ATP‐requiring enzyme that uses large amounts of reducing power, but the electron transfer pathway to nitrogenase in R. palustris was incompletely known. Here, we show that the ferredoxin, Fer1, is the primary but not sole electron carrier protein encoded by R. palustris that serves as an electron donor to nitrogenase. A flavodoxin, FldA, is also an important electron donor, especially under iron limitation. We present a model where the electron bifurcating complex, FixABCX, can reduce both ferredoxin and flavodoxin to transfer electrons to nitrogenase, and we present bioinformatic evidence that FixABCX and Fer1 form a conserved electron transfer pathway to nitrogenase in nitrogen‐fixing proteobacteria. These results may be useful in the design of strategies to reroute electrons generated during metabolism of organic compounds to nitrogenase to achieve maximal activity.     

Siderophores mediate reduced and increased uptake of cadmium by Streptomyces tendae F4 and sunflower (Helianthus annuus), respectively

Aims: As a toxic metal, cadmium (Cd) affects microbial and plant metabolic processes, thereby potentially reducing the efficiency of microbe or plant‐mediated remediation of Cd‐polluted soil. The role of siderophores produced by Streptomyces tendae F4 in the uptake of Cd by bacteria and plant was investigated to gain insight into the influence of siderophores on Cd availability to micro‐organisms and plants. Methods and Results: The bacterium was cultured under siderophore‐inducing conditions in the presence of Cd. The kinetics of siderophore production and identification of the siderophores and their metal‐bound forms were performed using electrospray ionization mass spectrometry. Inductively coupled plasma spectroscopy was used to measure iron (Fe) and Cd contents in the bacterium and in sunflower plant grown in Cd‐amended soil. Siderophores significantly reduced the Cd uptake by the bacterium, while supplying it with iron. Bacterial culture filtrates containing three hydroxamate siderophores secreted by S. tendae F4 significantly promoted plant growth and enhanced uptake of Cd and Fe by the plant, relative to the control. Furthermore, application of siderophores caused slightly more Cd, but similar Fe uptake, compared with EDTA. Bioinoculation with Streptomyces caused a dramatic increase in plant Fe content, but resulted only in slight increase in plant Cd content. Conclusion: It is concluded that siderophores can help reduce toxic metal uptake in bacteria, while simultaneously facilitating the uptake of such metals by plants. Also, EDTA is not superior to hydroxamate siderophores in terms of metal solubilization for plant uptake. Significance and Impact of the Study: The study showed that microbial processes could indirectly influence the availability and amount of toxic metals taken up from the rhizosphere of plants. Furthermore, although EDTA is used for chelator‐enhanced phytoremediation, microbial siderophores would be ideal for this purpose.