Microbial siderophore-based iron assimilation and therapeutic applications

Siderophores are structurally diverse, complex natural products that bind metals with extraordinary specificity and affinity. The acquisition of iron is critical for the survival and virulence of many pathogenic microbes and diverse strategies have evolved to synthesize, import and utilize iron. There has been a substantial increase of known siderophore scaffolds isolated and characterized in the past decade and the corresponding biosynthetic gene clusters have provided insight into the varied pathways involved in siderophore biosynthesis, delivery and utilization. Additionally, therapeutic applications of siderophores and related compounds are actively being developed. The study of biosynthetic pathways to natural siderophores augments the understanding of the complex mechanisms of bacterial iron acquisition, and enables a complimentary approach to address virulence through the interruption of siderophore biosynthesis or utilization by targeting the key enzymes to the siderophore pathways.     

Iron and siderophores in fungal–host interactions

Most fungi and bacteria express specific mechanisms for the acquisition of iron from the hosts they infect for their own survival. This is primarily because iron plays a key catalytic role in various vital cellular reactions in conjunction with the fact that iron is not freely available in these environments due to host sequestration. High-affinity iron uptake systems, such as siderophore-mediated iron uptake and reductive iron assimilation, enable fungi to acquire limited iron from animal or plant hosts. Regulating iron uptake is crucial to maintain iron homeostasis, a state necessary to avoid iron-induced toxicity from iron abundance, while simultaneously supplying iron required for biochemical demand. Siderophores play diverse roles in fungal–host interactions, many of which have been principally delineated from gene deletions in non-ribosomal peptide synthetases, enzymes required for siderophore biosynthesis. These analyses have demonstrated that siderophores are required for virulence, resistance to oxidative stress, asexual/sexual development, iron storage, and protection against iron-induced toxicity in some fungal organisms. In this review, the strategies fungi employ to obtain iron, siderophore biosynthesis, and the regulatory mechanisms governing iron homeostasis will be discussed with an emphasis on siderophore function and relevance for fungal organisms in their interactions with their hosts.     

Assembly of aryl-capped siderophores by modular peptide synthetases and polyketide synthases

Bacterial siderophores assist pathogens in iron acquisition inside their hosts. They are often essential for achieving a successful infection, and their biosynthesis represents an attractive antibiotic target. Recently, several siderophore biosynthetic loci have been identified, and in vitro studies have advanced our knowledge of the biosynthesis of aryl-capped peptide and peptide–polyketide siderophores from Mycobacterium spp., Pseudomonas spp., Yersinia spp. and other bacteria. These studies also provided insights into the assembly of related siderophores and many secondary metabolites of medical relevance. Assembly of aryl-capped peptide and peptide–polyketide siderophores involves non-ribosomal peptide synthetase, polyketide synthase and non-ribosomal-peptide polyketide hybrid subunits. Analysis of these subunits suggests that their domains and modules are functionally and structurally independent. It appears that nature has selected a set of functional domains and modules that can be rearranged in different order and combinations to biosynthesize different products. Although much remains to be learned about modular synthetases and synthases, it is already possible to conceive strategies to engineer these enzymes to generate novel products.     

Elucidation of the complete ferrichrome A biosynthetic pathway in Ustilago maydis

Iron is an important element for many essential processes in living organisms. To acquire iron, the basidiomycete Ustilago maydis synthesizes the iron‐chelating siderophores ferrichrome and ferrichrome A. The chemical structures of these siderophores have been elucidated long time ago but so far only two enzymes involved in their biosynthesis have been described. Sid1, an ornithine monoxygenase, is needed for the biosynthesis of both siderophores, and Sid2, a non‐ribosomal peptide synthetase (NRPS), is involved in ferrichrome generation. In this work we identified four novel enzymes, Fer3, Fer4, Fer5 and Hcs1, involved in ferrichrome A biosynthesis in U. maydis. By HPLC‐MS analysis of siderophore accumulation in culture supernatants of deletion strains, we show that Fer3, an NRPS, Fer4, an enoyl‐coenzyme A (CoA)‐hydratase, and Fer5, an acylase, are required for ferrichrome A production. We demonstrate by conditional expression of the hydroxymethyl glutaryl (HMG)‐CoA synthase Hcs1 in U. maydis that HMG‐CoA is an essential precursor for ferrichrome A. In addition, we heterologously expressed and purified Hcs1, Fer4 and Fer5, and demonstrated the enzymatic activities by in vitro experiments. Thus, we describe the first complete fungal siderophore biosynthetic pathway by functionally characterizing four novel genes responsible for ferrichrome A biosynthesis in U. maydis.     

Pyoverdine biosynthesis and secretion in Pseudomonas aeruginosa: implications for metal homeostasis

Pyoverdines are siderophores produced by fluorescent Pseudomonads to acquire iron. At least 60 different pyoverdines produced by diverse strains have been chemically characterized. They all consist of a dihydroquinoline‐type chromophore linked to a peptide. These peptides are of various lengths and the sequences are strain specific. Pyoverdine biosynthesis in Pseudomonas aeruginosa and fluorescent Pseudomonads is a complex process involving at least 12 different proteins, starting in the cytoplasm and ending in the periplasm. The cellular localization of pyoverdine precursors was recently shown to be consistent with their biosynthetic enzymes. In the cytoplasm, pyoverdine appears to be assembled at the inner membrane and particularly at the old cell pole of the bacterium. Mature pyoverdine is uniformly distributed throughout the periplasm, like the periplasmic enzyme PvdQ. Secretion of pyoverdine involves a recently identified ATP‐dependent efflux pump, PvdRT‐OpmQ. This efflux system does not only secrete newly synthesized pyoverdine but also pyoverdine that already transported iron into the bacterial periplasm and any pyoverdine–metal complex other than ferri‐pyoverdine present in the periplasm. This review considers how these new insights into pyoverdine biosynthesis and secretion contribute to our understanding of the role of pyoverdine in iron and metal homeostasis in fluorescent Pseudomonads.     

Iron transport in the genus Marinobacter

Marinobacter belong to the class of Gammaproteobacteria and these motile, halophilic or halotolerent bacteria are widely distributed throughout the world’s oceans having been isolated from a wide variety of marine environments. They have also been identified as members of the bacterial flora associated with other marine organisms. Here, using a combination of natural products chemistry and genomic analysis, we assess the nature of the siderophores produced by this genus and their potential relationship to phylogeny and lifestyle/ecological niche of this diverse group of organisms. Our analysis shows a wide level of diversity in siderophore based iron uptake systems among this genus with three general strategies: (1) production and utilization of native siderophores in addition to utilization of a variety of exogenous ones, (2) production and utilization of native siderophores only, (3) lack of siderophore production but utilization of exogenous ones. They all share the presence of at least one siderophore-independent iron uptake ABC transport systems of the FbpABC iron metal type and lack the ability for direct transport of ferrous iron. Siderophore production and utilization can be correlated with phylogeny and thus it forms a type of chemotaxonomic marker for this genus.     

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.     

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.     

Siderophore Cross-Utilization Amongst Rhizospheric Bacteria and the Role of Their Differential Affinities for Fe3+ on Growth Stimulation Under Iron-Limited Conditions

The majority of bacteria isolated from rhizospheres of Arachis hypogea (Groundnut) and Vigna radiata (Mung bean) predominantly produced catechol-type siderophores except for a few fluorescent pseudomonads that produced hydroxamates in addition to catecholates. The rhizospheric isolates differed in their ability to cross-utilize siderophores produced by other rhizospheric isolates (heterologous); some were highly proficient at utilizing heterologous siderophores, while others were poor cross-utilizers. Isolate G9, which utilized hydroxamate as well as catecholate siderophores, was found to be an efficient siderophore cross-utilizer, while isolates G2 and G6 were poor-utilizers of catecholate and non-utilizers of hydroxamate siderophores. Growth stimulation of two isolates G9 and G6 was seen when grown in the presence of externally supplied heterologous siderophores, which they cross-utilized. The iron-regulated outer membrane protein (IROMP) profiles differed for the most cross-utilizer and the least cross-utilizer strains, but in both the cases no new outer membrane proteins (OMP) were induced in response to the exogenous siderophores supplied. The growth of the organisms in the presence of heterologous siderophores that they failed to cross-utilize led to growth inhibition in the case of isolate G9. This appears to be due to a lower affinity of the siderophore of G9 as compared to the exogenously supplied G6 siderophore. A simple method was devised to measure relative affinities of respective siderophores for iron based on CAS solution decolorization by the siderophore preparations. The effect on the growth of the differential affinities of the siderophores for iron and the interactions of the organisms through cross-utilization is also discussed.     

Gene cluster for ferric iron uptake in Agrobacterium tumefaciens MAFF301001

Agrobacterium tumefaciens harboring a Ti plasmid causes crown gall disease in dicot plants by transferring its T-DNA into plant chromosomes. Iron acquisition plays an important role for pathogenicity in animal pathogens and several phytopathogens and for growth in the rhizosphere and on plant surfaces. Under iron-limiting condition, bacteria produce various iron-chelating agents called siderophores. Agrobacterium strains have the diversity in producing siderophores and a certain strain produces a typical catechol-type siderophore, called agrobactin, although its biosynthesis genes have not been analyzed yet. Here we describe the cloning and characterization of a functional gene cluster involved in ferric iron uptake in A. tumefaciens strain MAFF301001. Four complete open reading frames (ORFs) were found in 5-kb region of a genomic library clone 1A3. We named these genes agb, after agrobactin. agbC, agbE, agbB and agbA genes were identified in this order, and narrow intergenic spaces suggested that these genes constitute an operon. Predicted agb gene products and their phylogenetic analysis showed sequence similarity with enzymes which are involved in ferric iron uptake in other bacteria. Southern hybridization analysis clearly indicated the location of agb genes on the linear chromosome in strain MAFF301001 but the complete lack in another A. tumefaciens strain C58. Mutation analysis of agbB revealed that it is essential for growth and production of catechol compounds in iron-limiting medium.     

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.     

Genetics and assembly line enzymology of siderophore biosynthesis in bacteria.

The regulatory logic of siderophore biosynthetic genes in bacteria involves the universal repressor Fur, which acts together with iron as a negative regulator. However in other bacteria, in addition to the Fur-mediated mechanism of regulation, there is a concurrent positive regulation of iron transport and siderophore biosynthetic genes that occurs under conditions of iron deprivation. Despite these regulatory differences the mechanisms of siderophore biosynthesis follow the same fundamental enzymatic logic, which involves a series of elongating acyl-S-enzyme intermediates on multimodular protein assembly lines: nonribosomal peptide synthetases (NRPS). A substantial variety of siderophore structures are produced from similar NRPS assembly lines, and variation can come in the choice of the phenolic acid selected as the N-cap, the tailoring of amino acid residues during chain elongation, the mode of chain termination, and the nature of the capturing nucleophile of the siderophore acyl chain being released. Of course the specific parts that get assembled in a given bacterium may reflect a combination of the inventory of biosynthetic and tailoring gene clusters available. This modular assembly logic can account for all known siderophores. The ability to mix and match domains within modules and to swap modules themselves is likely to be an ongoing process in combinatorial biosynthesis. NRPS evolution will try out new combinations of chain initiation, elongation and tailoring, and termination steps, possibly by genetic exchange with other microorganisms and/or within the same bacterium, to create new variants of iron-chelating siderophores that can fit a particular niche for the producer bacterium.     

Genetics and Assembly Line Enzymology of Siderophore Biosynthesis in Bacteria

The regulatory logic of siderophore biosynthetic genes in bacteria involves the universal repressor Fur, which acts together with iron as a negative regulator. However in other bacteria, in addition to the Fur-mediated mechanism of regulation, there is a concurrent positive regulation of iron transport and siderophore biosynthetic genes that occurs under conditions of iron deprivation. Despite these regulatory differences the mechanisms of siderophore biosynthesis follow the same fundamental enzymatic logic, which involves a series of elongating acyl-S-enzyme intermediates on multimodular protein assembly lines: nonribosomal peptide synthetases (NRPS). A substantial variety of siderophore structures are produced from similar NRPS assembly lines, and variation can come in the choice of the phenolic acid selected as the N-cap, the tailoring of amino acid residues during chain elongation, the mode of chain termination, and the nature of the capturing nucleophile of the siderophore acyl chain being released. Of course the specific parts that get assembled in a given bacterium may reflect a combination of the inventory of biosynthetic and tailoring gene clusters available. This modular assembly logic can account for all known siderophores. The ability to mix and match domains within modules and to swap modules themselves is likely to be an ongoing process in combinatorial biosynthesis. NRPS evolution will try out new combinations of chain initiation, elongation and tailoring, and termination steps, possibly by genetic exchange with other microorganisms and/or within the same bacterium, to create new variants of iron-chelating siderophores that can fit a particular niche for the producer bacterium.     

Small-molecule inhibition of siderophore biosynthesis in Mycobacterium tuberculosis and Yersinia pestis

Mycobacterium tuberculosis and Yersinia pestis, the causative agents of tuberculosis and plague, respectively, are pathogens with serious ongoing impact on global public health and potential use as agents of bioterrorism. Both pathogens have iron acquisition systems based on siderophores, secreted iron-chelating compounds with extremely high Fe3+ affinity. Several lines of evidence suggest that siderophores have a critical role in bacterial iron acquisition inside the human host, where the free iron concentration is well below that required for bacterial growth and virulence. Thus, siderophore biosynthesis is an attractive target in the development of new antibiotics to treat tuberculosis and plague. In particular, such drugs, alone or as part of combination therapies, could provide a valuable new line of defense against intractable multiple-drug-resistant infections. Here, we report the design, synthesis and biological evaluation of a mechanism-based inhibitor of domain salicylation enzymes required for siderophore biosynthesis in M. tuberculosis and Y. pestis. This new antibiotic inhibits siderophore biosynthesis and growth of M. tuberculosis and Y. pestis under iron-limiting conditions.     

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 Two-Component Regulators GacS and GacA Positively Regulate a Nonfluorescent Siderophore through the Gac/Rsm Signaling Cascade in High-Siderophore-Yielding Pseudomonas sp. Strain HYS

Siderophores, which are produced to overcome iron deficiency, are believed to be closely related to the adaptability of bacteria. The high-siderophore-yielding Pseudomonas sp. strain HYS simultaneously secretes the fluorescent siderophore pyoverdine and another nonfluorescent siderophore that is a major contributor to the high siderophore yield. Transposon mutagenesis revealed siderophore-related genes, including the two-component regulators GacS/GacA and a special cluster containing four open reading frames (the nfs cluster). Deletion mutations of these genes abolished nonfluorescent-siderophore production, and expression of the nfs cluster depended on gacA, indicating that gacS-gacA may control the nonfluorescent siderophore through regulation of the nfs cluster. Furthermore, regulation of the nonfluorescent siderophore by GacS/GacA involved the Gac/Rsm pathway. In contrast, inactivation of GacS/GacA led to upregulation of the fluorescent pyoverdine. The two siderophores were secreted under different iron conditions, probably because of differential effects of GacS/GacA. The global GacS/GacA regulatory system may control iron uptake by modulating siderophore production and may enable bacteria to adapt to changing iron environments.     

Siderophore production by actinobacteria

Produced by bacteria, fungi and plants, siderophores are low-molecular-weight chelating agents (200–2,000 Da) to facilitate uptake of iron (Fe). They play an important role in extracellular Fe solubilization from minerals to make it available to microorganisms. Siderophores have various chemical structures and form a family of at least 500 different compounds. Some antibiotics (i.e., albomycins, ferrimycins, danomycins, salmycins, and tetracyclines) can bind Fe and some siderophores showed diverse biological activities. Functions and applications of siderophores derived from actinobacteria were reviewed to better understand the diverse metabolites.