A suite of citrate-derived siderophores from a marine Vibrio species isolated following the Deepwater Horizon oil spill

Nearly all microbes require iron for growth. The low concentration of iron found in the ocean makes iron acquisition a particularly difficult task. In response to these low iron conditions, many bacteria produce low-molecular-weight iron-binding molecules called siderophores to aid in iron uptake. We report herein the isolation and structural characterization of a suite of amphiphilic siderophores called the ochrobactins-OH, which are produced by a Vibrio species isolated from the Gulf of Mexico after the 2010 Deepwater Horizon oil spill. The citrate-based ochrobactins-OH are derivatives of aerobactin, replacing the acetyl groups with fatty acid appendages ranging in size from C8 to C12, and are distinctly different from the ochrobactins in that the fatty acid appendages are hydroxylated rather than unsaturated. The discovery of the marine amphiphilic ochrobactin-OH suite of siderophores increases the geographic and phylogenetic diversity of siderophore-producing bacteria.     

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.     

The role of siderophores in iron acquisition by photosynthetic marine microorganisms

The photosynthetic picocyanobacteria and eukaryotic microorganisms that inhabit the open ocean must be able to supply iron for their photosynthetic and respiratory needs from the subnanomolar concentrations available in seawater. Neither group appears to produce siderophores, although some coastal cyanobacteria do. This is interpreted as an adaptation to the dilute oceanic environment rather than a phylogenetic constraint, since there are cases in which related taxa from different environments have the capacity to produce siderophores. Most photosynthetic marine microorganisms are presumably, however, capable of accessing iron from strong chelates since the majority of dissolved iron in seawater is complexed by organic ligands, including siderophores. Rather than direct internalization of siderophores and other iron chelates, marine organisms primarily appear to use uptake pathways that involve a reduction step to free bound iron, closely coupled with transport into the cell.     

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.     

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.     

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.     

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.     

Siderophore production by Salmonella typhi

This study was initiated to determine the mechanism of iron-uptake in Salmonella typhi. When stressed for iron, microorganisms produce siderophores to obtain the necessary nutrient. Generally two types of siderophores exist: the phenolate-type predominantly produced by bacteria and the hydroxamate-type commonly secreted by fungi. Results of this investigation showed that S. typhi produced siderophores of the phenolate-type since culture supernatant of the organism grown under iron-deprivation supported the growth of the phenolate-dependent auxotroph. The culture supernatant when extracted for phenolate siderophores, also supported the growth of the phenolate auxotroph but not the hydroxamate auxotroph. Production of phenolate-type siderophores were further confirmed using biochemical assays. These results showed that S. typhi utilized the high-affinity iron transport system to obtain the necessary iron.     

产嗜铁素砷抗性微生物在砷污染环境中的作用

在砷污染环境中,许多微生物进化出了砷抗性,对地球环境中砷的命运起着决定性的作用。其次,由于自然条件下铁有效浓度低,微生物一般会表达嗜铁素,协助微生物吸收铁。嗜铁素除了与铁结合外,还可与多种金属离子形成稳定的复合物,促进环境中砷酸盐的溶解和亚砷酸盐的氧化。最后,产嗜铁素微生物有促进植物生长和促进或减弱植物吸收砷的可能性。因此,产嗜铁素砷抗性微生物可能具有在砷污染环境的修复中发挥作用的潜力。     

Siderocalin/Lcn2/NGAL/24p3 Does Not Drive Apoptosis Through Gentisic Acid Mediated Iron Withdrawal in Hematopoietic Cell Lines

Siderocalin (also lipocalin 2, NGAL or 24p3) binds iron as complexes with specific siderophores, which are low molecular weight, ferric ion-specific chelators. In innate immunity, siderocalin slows the growth of infecting bacteria by sequestering bacterial ferric siderophores. Siderocalin also binds simple catechols, which can serve as siderophores in the damaged urinary tract. Siderocalin has also been proposed to alter cellular iron trafficking, for instance, driving apoptosis through iron efflux via BOCT. An endogenous siderophore composed of gentisic acid (2,5-dihydroxybenzoic acid) substituents was proposed to mediate cellular efflux. However, binding studies reported herein contradict the proposal that gentisic acid forms high-affinity ternary complexes with siderocalin and iron, or that gentisic acid can serve as an endogenous siderophore at neutral pH. We also demonstrate that siderocalin does not induce cellular iron efflux or stimulate apoptosis, questioning the role siderocalin plays in modulating iron metabolism.     

Siderophores of Marinobacter aquaeolei: petrobactin and its sulfonated derivatives

Siderophores are low molecular weight, high-affinity iron(III) ligands, produced by bacteria to solubilize and promote iron uptake under low iron conditions. Two prominent structural features characterize the majority of the marine siderophores discovered so far: (1) a predominance of suites of amphiphilic siderophores composed of an iron(III)-binding headgroup that is appended by one or two of a series of fatty acids and (2) a prevalence of siderophores that contain α-hydroxycarboxylic acid moieties (e.g., β-hydroxyaspartic acid or citric acid) which are photoreactive when coordinated to Fe(III). Variation of the fatty acid chain length affects the relative amphiphilicity within a suite of siderophores. Catecholate sulfonation is another structural variation that would affect the hydrophilicity of a siderophore. In addition to a review of the marine amphiphilic siderophores, we report the production of petrobactin disulfonate by Marinobacter aquaeolei VT8. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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 identification of siderophores produced by cyanobacteria

Cyanobacteria are one of the most successful and oldest forms of life that are present on Earth. They are prokaryotic photoautotrophic microorganisms that colonize so diverse environments as soil, seawater, and freshwater, but also stones, plants, or extreme habitats such as snow and ice as well as hot springs. This diversity in the type of environment they live in requires a successful adaptation to completely different conditions. For this reason, cyanobacteria form a wide range of different secondary metabolites. In particular, the cyanobacteria living in both freshwater and sea produce many metabolites that have biological activity. In this review, we focus on metabolites called siderophores, which are low molecular weight chemical compounds specifically binding iron ions. They have a relatively low molecular weight and are produced by bacteria and also by fungi. The main role of siderophores is to obtain iron from the environment and to create a soluble complex available to microbial cells. Siderophores play an important role in microbial ecology; for example, in agriculture they support the growth of many plants and increase their production by increasing the availability of Fe in plants. The aim of this review is to demonstrate the modern use of physico-chemical methods for the detection of siderophores in cyanobacteria and the use of these methods for the detection and characterization of the siderophore-producing microorganisms. Using high-performance liquid chromatography-mass spectrometry (LC-MS), it is possible not only to discover new chemical structures but also to identify potential interactions between microorganisms. Based on tandem mass spectrometry (MS/MS) analyses, previous siderophore knowledge can be used to interpret MS/MS data to examine both known and new siderophores.     

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。     

Iron transporters in marine prokaryotic genomes and metagenomes

In the pelagic environment, iron is a scarce but essential micronutrient. The iron acquisition capabilities of selected marine bacteria have been investigated, but the recent proliferation of marine prokaryotic genomes and metagenomes offers a more comprehensive picture of microbial iron uptake pathways in the ocean. Searching these data sets, we were able to identify uptake mechanisms for Fe(3+), Fe(2+) and iron chelates (e.g. siderophore and haem iron complexes). Transport of iron chelates is accomplished by TonB-dependent transporters (TBDTs). After clustering the TBDTs from marine prokaryotic genomes, we identified TBDT clusters for the transport of hydroxamate and catecholate siderophore iron complexes and haem using gene neighbourhood analysis and co-clustering of TBDTs of known function. The genomes also contained two classes of siderophore biosynthesis genes: NRPS (non-ribosomal peptide synthase) genes and NIS (NRPS Independent Siderophore) genes. The most common iron transporters, in both the genomes and metagenomes, were Fe(3+) ABC transporters. Iron uptake-related TBDTs and siderophore biosynthesis genes were less common in pelagic marine metagenomes relative to the genomic data set, in part because Pelagibacter ubique and Prochlorococcus species, which almost entirely lacked these Fe uptake systems, dominate the metagenomes. Our results are largely consistent with current knowledge of iron speciation in the ocean, but suggest that in certain niches the ability to acquire siderophores and/or haem iron chelates is beneficial.     

New roles for bacterial siderophores in metal transport and tolerance

Siderophores are chelators with extremely strong affinity for ferric iron and are best known for their capacity to feed microorganisms with this metal. Despite their preference for iron, they can also chelate numerous other metals with variable affinities. There is also increasing evidence that metals other than iron can activate the production of siderophores by bacteria, thereby implicating siderophores in the homeostasis of metals other than iron and especially heavy metal tolerance. This article considers this new concept that siderophores play a role in protecting bacteria against metal toxicity and discusses the possible contribution of these chelators to the transport of biological relevant metals in addition to iron.     

Analysis of the global ocean sampling (GOS) project for trends in iron uptake by surface ocean microbes

Microbial metagenomes are DNA samples of the most abundant, and therefore most successful organisms at the sampling time and location for a given cell size range. The study of microbial communities via their DNA content has revolutionized our understanding of microbial ecology and evolution. Iron availability is a critical resource that limits microbial communities' growth in many oceanic areas. Here, we built a database of 2319 sequences, corresponding to 140 gene families of iron metabolism with a large phylogenetic spread, to explore the microbial strategies of iron acquisition in the ocean's bacterial community. We estimate iron metabolism strategies from metagenome gene content and investigate whether their prevalence varies with dissolved iron concentrations obtained from a biogeochemical model. We show significant quantitative and qualitative variations in iron metabolism pathways, with a higher proportion of iron metabolism genes in low iron environments. We found a striking difference between coastal and open ocean sites regarding Fe(2+) versus Fe(3+) uptake gene prevalence. We also show that non-specific siderophore uptake increases in low iron open ocean environments, suggesting bacteria may acquire iron from natural siderophore-like organic complexes. Despite the lack of knowledge of iron uptake mechanisms in most marine microorganisms, our approach provides insights into how the iron metabolic pathways of microbial communities may vary with seawater iron concentrations.     

Isolation of bacteria producing siderophores under alkaline conditions

Summary The isolation of bacteria producing siderophores under alkaline conditions is reported. Enrichment cultures initiated with samples from a number of alkaline environmental sources yielded 80 isolates. From this group selections were made on the basis of growth at high pH and the gallium-binding capacity of the siderophores. It was found that some isolates grew well and high concentrations of siderophore were detected whereas others grew well in the presence of much lower concentrations of siderophore. The effect of iron, gallium and aluminium on growth and siderophore production in batch culture was investigated for six isolates. The presence of iron greatly decreased the siderophore concentration in these cultures, whereas the response to added gallium or aluminium was dependent upon the isolate. Offsprint requests to: D. J. Gascoyne     

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     

Iron uptake and metabolism in the rhizobia/legume symbioses

Iron-containing proteins figure prominently in the nitrogen-fixing symbioses between bacteria of the genera Azorhizovium, Bradyrhizobium and Rhizobium and their respective plant hosts. Although iron is abundant in the soil, the acquisition of iron is problematic due to its low solubility at biological pH under aerobic conditions. The study of iron acquisition as it pertains to these economically important symbioses is directed at answering three questions: 1) how do rhizobial cells acquire iron as free-living microorganisms where they must compete for this nutrient with other soil inhabitants 2) how do the plant hosts acquire enough iron for the symbiosis and 3) how do rhizobia acquire iron as symbionts? Production and/or utilization of ferric-specific ligands (siderophores) has now been documented in the laboratory for a number of rhizobial species, but there is limited information on whether production and/or untilization occurs either in the soil or in planta. Studies with rhizobial mutants which can no longer produce and/or utilize siderophores should address whether siderophores contribute to functional symbioses. In addition, the ability to produce and/or utilize siderophores may affect the outcome of both interstrain and interspecific competition in the rhizosphere and in bulk soil. Some progress has been made at documenting the effects of iron deficiency on nodule development. Studies are also underway to determine whether, in addition to its central structural role, iron may also play a regulatory role in the symbioses. This review is an attempt to give an overview of the field, and hopefully will stimulate further research on the iron nutrition of these symbioses which account for such a significant proportion of the world's biologically fixed nitrogen.