Hydroxamate Production as a High Affinity Iron Acquisition Mechanism in Paracoccidioides Spp

Iron is a micronutrient required by almost all living organisms, including fungi. Although this metal is abundant, its bioavailability is low either in aerobic environments or within mammalian hosts. As a consequence, pathogenic microorganisms evolved high affinity iron acquisition mechanisms which include the production and uptake of siderophores. Here we investigated the utilization of these molecules by species of the Paracoccidioides genus, the causative agents of a systemic mycosis. It was demonstrated that iron starvation induces the expression of Paracoccidioides ortholog genes for siderophore biosynthesis and transport. Reversed-phase HPLC analysis revealed that the fungus produces and secretes coprogen B, which generates dimerumic acid as a breakdown product. Ferricrocin and ferrichrome C were detected in Paracoccidioides as the intracellular produced siderophores. Moreover, the fungus is also able to grow in presence of siderophores as the only iron sources, demonstrating that beyond producing, Paracoccidioides is also able to utilize siderophores for growth, including the xenosiderophore ferrioxamine. Exposure to exogenous ferrioxamine and dimerumic acid increased fungus survival during co-cultivation with macrophages indicating that these molecules play a role during host-pathogen interaction. Furthermore, cross-feeding experiments revealed that Paracoccidioides siderophores promotes growth of Aspergillus nidulans strain unable to produce these iron chelators. Together, these data denote that synthesis and utilization of siderophores is a mechanism used by Paracoccidioides to surpass iron limitation. As iron paucity is found within the host, siderophore production may be related to fungus pathogenicity.     

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 (59)Fe 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.     

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.     

Petrobactin,a siderophore produced by Alteromonas,mediates community iron acquisition in the global ocean

It is now widely accepted that siderophores play a role in marine iron biogeochemical cycling. However, the mechanisms by which siderophores affect the availability of iron from specific sources and the resulting significance of these processes on iron biogeochemical cycling as a whole have remained largely untested. In this study, we develop a model system for testing the effects of siderophore production on iron bioavailability using the marine copiotroph Alteromonas macleodii ATCC 27126. Through the generation of the knockout cell line ΔasbB::km^r, which lacks siderophore biosynthetic capabilities, we demonstrate that the production of the siderophore petrobactin enables the acquisition of iron from mineral sources and weaker iron-ligand complexes. Notably, the utilization of lithogenic iron, such as that from atmospheric dust, indicates a significant role for siderophores in the incorporation of new iron into marine systems. We have also detected petrobactin, a photoreactive siderophore, directly from seawater in the mid-latitudes of the North Pacific and have identified the biosynthetic pathway for petrobactin in bacterial metagenome-assembled genomes widely distributed across the global ocean. Together, these results improve our mechanistic understanding of the role of siderophore production in iron biogeochemical cycling in the marine environment wherein iron speciation, bioavailability, and residence time can be directly influenced by microbial activities.Subject terms: Biogeochemistry, Marine microbiology     

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.     

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.     

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)     

Utilization of microbial siderophores in iron acquisition by oat

Iron uptake by oat (Avena sativa cv Victory) was examined under hydroponic chemical conditions that required direct utilization of microbial siderophores for iron transport. Measurements of iron uptake rates by excised roots from the hydroxamate siderophores, ferrichrome, ferrichrome A, coprogen, ferrioxamine B (FOB), and rhodotorulic acid (RA) showed all five of the siderophores supplied iron, but that FOB and RA were preferentially utilized. FOB-mediated iron uptake increased four-fold when roots were preconditioned to iron stress and involved an active, iron-stress induced transport system that was inhibited by 5 millimolar sodium azide or 0.5 millimolar dinitrophenol. Kinetic studies indicated partial saturation with an apparent K_m of 5 micromolar when FOB was supplied at 0.1 to 50 micromolar concentrations. Whole plant experiments confirmed that 5 micromolar FOB was sufficient for plant growth. Siderophore-mediated iron transport was inhibited by Cr-ferrichrome, an analog of ferrated siderophore. Our results confirm the existence of a microbial siderophore iron transport system in oat which functions within the physiological concentrations produced and used by soil microorganisms.     

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.     

Ecology of siderophores with special reference to the fungi

Ecology of siderophores, as described in the present review, analyzes the factors that allow the production and function of siderophores under various environmental conditions. Microorganisms that excrete siderophores are able to grow in natural low-iron environments by extracting residual iron from insoluble iron hydroxides, protein-bound iron or from other iron chelates. Compared to the predominantly mobile bacteria, the fungi represent mostly immobile microorganisms that rely on local nutrient concentrations. Feeding the immobile is a general strategy of fungi and plants, which depend on the local nutrient resources. This also applies to iron nutrition, which can be improved by excretion of siderophores. Most fungi produce a variety of different siderophores, which cover a wide range of physico-chemical properties in order to overcome adverse local conditions of iron solubility. Resource zones will be temporally and spatially dynamic which eventually results in conidiospore production, transport to new places and outgrow of mycelia from conidiospores. Typically, extracellular and intracellular siderophores exist in fungi which function either in transport or storage of ferric iron. Consequently, extracelluar and intracellular reduction of siderophores may occur depending on the fungal strain, although in most fungi transport of the intact siderophore iron complex has been observed. Regulation of siderophore biosynthesis is essential in fungi and allows an economic use of siderophores and metabolic resources. Finally, the chemical stability of fungal siderophores is an important aspect of microbial life in soil and in the rhizosphere. Thus, insolubility of iron in the environment is counteracted by dissolution and chelation through organic acids and siderophores by various fungi.     

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.     

Fate of ferrisiderophores after import across bacterial outer membranes: different iron release strategies are observed in the cytoplasm or periplasm depending on the siderophore pathways

Siderophore production and utilization is one of the major strategies deployed by bacteria to get access to iron, a key nutrient for bacterial growth. The biological function of siderophores is to solubilize iron in the bacterial environment and to shuttle it back to the cytoplasm of the microorganisms. This uptake process for Gram-negative species involves TonB-dependent transporters for translocation across the outer membranes. In Escherichia coli and many other Gram-negative bacteria, ABC transporters associated with periplasmic binding proteins import ferrisiderophores across cytoplasmic membranes. Recent data reveal that in some siderophore pathways, this step can also be carried out by proton-motive force-dependent permeases, for example the ferrichrome and ferripyochelin pathways in Pseudomonas aeruginosa. Iron is then released from the siderophores in the bacterial cytoplasm by different enzymatic mechanisms depending on the nature of the siderophore. Another strategy has been reported for the pyoverdine pathway in P. aeruginosa: iron is released from the siderophore in the periplasm and only siderophore-free iron is transported into the cytoplasm by an ABC transporter having two atypical periplasmic binding proteins. This review presents recent findings concerning both ferrisiderophore and siderophore-free iron transport across bacterial cytoplasmic membranes and considers current knowledge about the mechanisms involved in iron release from siderophores.     

Iron Uptake in Ustilago maydis: Tracking the Iron Path

In this study, we monitored and compared the uptake of iron in the fungus Ustilago maydis by using biomimetic siderophore analogs of ferrichrome, the fungal native siderophore, and ferrioxamine B (FOB), a xenosiderophore. Ferrichrome-iron was taken up at a higher rate than FOB-iron. Unlike ferrichrome-mediated uptake, FOB-mediated iron transport involved an extracellular reduction mechanism. By using fluorescently labeled siderophore analogs, we monitored the time course, as well as the localization, of iron uptake processes within the fungal cells. A fluorescently labeled ferrichrome analog, B9-lissamine rhodamine B, which does not exhibit fluorescence quenching upon iron binding, was used to monitor the entry of the compounds into the fungal cells. The fluorescence was found intracellularly 4 h after the application and later was found concentrated in two to three vesicles within each cell. The fluorescence of the fluorescently labeled FOB analog CAT18, which is quenched by iron, was visualized around the cell membrane after 4 h of incubation with the ferrated (nonfluorescent) compounds. This fluorescence intensity increased with time, demonstrating fungal iron uptake from the siderophores, which remained extracellular. We here introduce the use of fluorescent biomimetic siderophores as tools to directly track and discriminate between different pathways of iron uptake in cells.     

Iron gathering of opportunistic pathogenic fungi. A mini review

Iron is an essential nutrient for most organisms because it serves as a catalytic cofactor in oxidation-reduction reactions. Iron is rather unavailable because it occurs in its insoluble ferric form in oxides and hydroxides, while in serum of mammalian hosts is highly bound to carrier proteins such as transferrin, so the free iron concentration is extremely low insufficient for microbial growth. Therefore, many organisms have developed different iron-scavenging systems for solubilizing ferric iron and transporting it into cells across the fungal membrane. There are three major mechanisms by which fungi can obtain iron from the host: (a) utilization of a high affinity iron permease to transport iron intracellularly, (b) production and secretion of low molecular weight iron-specific chelators (siderophores), (c) utilization of a hem oxygenase to acquire iron from hemin. Patients with elevated levels of available serum iron treated with iron chelator, deferoxamine to remedy iron overload conditions have an increased susceptibility of invasive zygomycosis. Presumably deferoxamine predisposes patients to Zygomycetes infections by acting as a siderophore]. The frequency of zygomycosis is increasing in recent years and these infections respond very poorly to currently available antifungal agents, so new approaches to develop strategies to prevent and treat zygomycosis are urgently needed. Siderophores and iron-transport proteins have been suggested to function as virulence factors because the acquisition of iron is a crucial pathogenetic event. Biosynthesis and uptake of siderophores represent possible targets for antifungal therapy.     

Cybernetic modeling of iron-limited growth and siderophore production

The cybernetic modeling framework developed by Ramkrishna and co-workers has been applied to a case of bacterial metabolite production, namely the production of siderophores (iron-chelating agents) associated with iron-limiting fermentation conditions. Experimental growth data showed that, even though final biomass levels were controlled by exhaustion of the carbon source, iron-limiting conditions also affected the biomass yield. A structured model which includes the process of an iron-limiting energy resource production was able to quantitatively account for this apparent dual-substrate limitation over a wide range of batch and continuous operating conditions. The experiments data also showed quite large difference in iron uptake over the wide range of operating condition and iron levels investigated. The inclusion in the model of the processes of low and high (siderophore-mediated) affinity iron transport, and siderophore production led to simulation results that were in good quantitative agreement with the siderophore, medium and cell iron levels, in both batch and steady-state continuous culture operating conditions.     

Recent achievements on siderophore production and application

Iron is the most abundant chemical element on Earth but its most common oxidation state is Fe(III) which presents a very low solubility under physiological conditions. During evolution, micro-organisms have developed sound strategies to acquire iron from both the environment and superior organisms, including direct uptake of iron ions from exogenous iron/heme sources and the synthesis of specialized Fe(III) chelators called siderophores. The present review paper aims at presenting and discussing the latest achievements in siderophore isolation and production, as well as novel applications of these molecules in therapies against iron-related diseases and in vaccines, and their application as antimicrobial agents and biosensors.     

Diversity of siderophore-mediated iron uptake systems in fluorescent pseudomonads: not only pyoverdines

Fluorescent pseudomonads are gamma-proteobacteria known for their capacity to colonize various ecological niches. This adaptability is reflected by their sophisticated and diverse iron uptake systems. The majority of fluorescent pseudomonads produce complex peptidic siderophores called pyoverdines or pseudobactins, which are very efficient iron scavengers. A tremendous variety of pyoverdines has been observed, each species producing a different pyoverdine. This variety can be used as an interesting tool to study the diversity and taxonomy of fluorescent pseudomonads. Other siderophores, including newly described ones, are also produced by pseudomonads, sometimes endowed with interesting properties in addition to iron scavenging, such as formation of complexes with other metals or antimicrobial activity. Factors other than iron limitation, and different regulatory proteins also seem to influence the production of siderophores in pseudomonads and are reviewed here as well. Another peculiarity of pseudomonads is their ability to use a large number of heterologous siderophores via different TonB-dependent receptors. A first genomic analysis of receptors in four different fluorescent pseudomonads suggests that their siderophore ligand repertoire is likely to overlap, and that not all receptors recognize siderophores as ligands.     

Mechanisms of siderophore iron transport in enteric bacteria.

Uptake of 55Fe- and 3H-labeled siderophores and their chronic analogues have been studied in Salmonella typhimurium LT-2 and Escherichia coli K-12. In S. typhimurium LT-2, at least two different mechanisms for siderophore iron transport may be operative. Uptake of 55Fe- and 3H-labeled ferrichrome and kinetically inert lambda-cis-chromic [3H]deferriferrichrome by the S. typhimurium LT-2 enb7 mutant, which is defective in the production of its native siderophore, enterobactin, appears to occur by two concurrent mechanisms. The first mechanism is postulated to involve either rapid uptake of iron released from the ferric complex by cellular reduction without penetration of the complex or ligand or dissociation of the complex and simultaneous uptake of both ligand and iron coupled with simultaneous expulsion of the ligand. The second mechanism appears to consist of slower uptake of the intact ferric complex.     

铁限制条件下东海原甲藻分泌铁载体

在铁限制条件下,进行东海原甲藻分泌铁载体的动态研究。对藻类在富铁与缺铁条件下生长状况、生长过程中分泌铁载体的情况以及海藻接种量对铁载体分泌的影响进行了连续观测,结果表明:东海原甲藻在缺铁条件下生长状况远不如在富铁条件下;随着藻类的生长,分泌铁载体不断增多,达指数生长期时,其分泌量也达到了最大值,之后藻类的生长和铁载体分泌都呈现下降趋势;高接种量东海原甲藻能分泌较多的铁载体,并在较短时间到达峰值。     

Genome-wide screen for genes with effects on distinct iron uptake activities in Saccharomyces cerevisiae

We screened a collection of 4847 haploid knockout strains (EUROSCARF collection) of Saccharomyces cerevisiae for iron uptake from the siderophore ferrioxamine B (FOB). A large number of mutants showed altered uptake activities, and a few turned yellow when grown on agar plates with added FOB, indicating increased intracellular accumulation of undissociated siderophores. A subset consisting of 197 knockouts with altered uptake was examined further for regulated activities that mediate cellular uptake of iron from other siderophores or from iron salts. Hierarchical clustering analysis grouped the data according to iron sources and according to mutant categories. In the first analysis, siderophores grouped together with the exception of enterobactin, which grouped with iron salts, suggesting a reductive pathway of iron uptake for this siderophore. Mutant groupings included three categories: (i) high-FOB uptake, high reductase, low-ferrous transport; (ii) isolated high- or low-FOB transport; and (iii) induction of all activities. Mutants with statistically altered uptake activities included genes encoding proteins with predominant localization in the secretory pathway, nucleus, and mitochondria. Measurements of different iron-uptake activities in the yeast knockout collection make possible distinctions between genes with general effects on iron metabolism and those with pathway-specific effects.