Keywords

15.1 Introduction

Since the past six decades, various studies have been focused on some 500 small low-molecular-mass (≤10 kDa) molecules called the siderophores which are secreted by both plants and microbes into the rhizosphere (Hider and Kong 2010; Ahmed and Holmström 2014; Johnstone and Nolana 2015). The bacterial siderophores possess higher affinity for metal ions (especially ferric ions or Fe+3) as compared to phytosiderophores (mainly mugineic acid) and are often present in lower concentrations (Kraemer 2004; Kraemer et al. 2006; Glick 2012). Many physiochemical factors such as ligand-binding sites onto metal ion, denticity, pH, redox, etc. govern the metal-binding ability of these molecules (Akafia et al. 2014). Their role as metal scavengers in the rhizosphere is very well known specially for iron ions (Aznar and Dellagi 2015). Other pertinent functions of siderophores include their antibiotic activity against many resistant bacterial strains and potential superbugs as sideromycins (Braun et al. 2009). They possess strong affinity for non-iron metal ions such as copper, manganese, molybdenum, vanadium, zinc, etc. (Hood and Skar 2012). Siderophores from bacterial strains bind to Zn to form zincophores or tsinkophores (Prentice et al. 2007), Pseudomonad strains show affinity for Mn (Harrington et al. 2012; Duckworth et al. 2014), and several other microbial siderophores attract Mo and Vn to form stable complexes (Deicke et al. 2013). Methanobactin is a copper-binding compound (CBC) or chalkophores (Kenney and Rosenzweig 2012), and other well-known copper siderophores include coproporphyrin and yersiniabactin (Chaturvedi et al. 2014). Enterobactin, yersiniabactin and aerobactin possess the capacity to form gold nanoparticles (Wyatt et al. 2014). Their active role as transporters of non-metal moieties such as boron and silicon and signalling molecules in plant defence mechanisms and oxidative stress is also well documented now (Chaturvedi and Henderson 2014; Butler and Theisen 2010; Nadal-Jimenez et al. 2012). Stable non-metal-siderophore complexes exist due to marine siderophores such as vibrioferrein from Marinobacter spp. (Amin et al. 2007). Citrate and catecholate siderophores interact and bind to boron to form strong signalling and sensing molecules (Sandy and Butler 2009). Additionally, they have gained relevance as therapeutic and diagnostic molecules in medical science (Ali and Vidhale 2013). Antioxidant and hormonal signalling cascades indicate the role of iron siderophores in various spheres of biology (Aznar et al. 2015). More recently, siderocalin, a mammalian siderophore-binding protein from the lipocalin family, specifically binding to actinide and lanthanide complexes, has been discovered (Allred et al. 2015). Despite the mind-boggling diversity on display across the microbial, plant and mammalian spheres, this review focuses on the most pertinent and applicative aspects of microbial siderophores in agriculture, therapeutics, etc.

15.2 Biosynthesis, Classification and Functional Diversity

Siderophores are synthesized by several bacteria and show significant variation in structures. These are mainly classified on the basis of characterization of functional or coordinating groups that bind with Fe3+ ions. The most important and commonly occurring groups include catecholates, hydroxamates and carboxylates (Ali and Vidhale 2013; Sah and Singh 2015; Gupta et al. 2015) (Fig. 15.1). A very small group of siderophores include pyoverdines, which are also termed as mixed ligands. They constitute the fourth class of siderophores which have functional groups that are classified in chemically distinct classes. Numerous types of siderophores have been identified employing latest techniques of spectrophotometry, mass spectrometry, acid hydrolysis, electrophoretic mobility, proton NMR (nuclear magnetic resonance) spectroscopy and biological activities (Sah and Singh 2015; Kurth et al. 2016) (Fig. 15.2).

Fig. 15.1
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Classification of microbial siderophores: an outline

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Fig. 15.2
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Therapeutic approaches and applications

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15.2.1 Biosynthesis

Microbial siderophore synthesis takes place through two pathways: non-ribosomal peptide synthetases (NRPSs) multienzyme dependent and NRPS independent (Sah and Singh 2015). NRPS-dependent biosynthesis involves the enzyme ATP pyrophosphate for the formation of hydroxamate siderophores (Lautru and Challis 2004). NRPSs are enzymes with large subunits which catalyse non-ribosomal peptide (NRP) synthesis by incorporating one amino acid per unit into the peptide chain. For instance, NRPSs synthesize the chromophores and the peptide chains of the microbial siderophore pyoverdine (Mossialos et al. 2002; Crosa and Walsh 2002).

Peptide synthetase is a multicomplex enzyme that produces peptide products without RNA template. 2, 3-dihydroxybenzoic acid (DHBA) is one of the precursor compounds of siderophores, which is synthesized from chorismate through the sequential action of a series of enzymes (Farrell et al. 1990). For instance, in anguibactin, the coordinating bonds are synthesized by molecular oxygen from various groups such as diphenoxylate group, hydroxamate group, imidazole group and thiazoline group. The structure of anguibactin is completed by two molecules of anguibactin, metal ion and solvent each. Anguibactin retrobiosynthesis, {(−N-hydroxy-N)- [2-(2,3-dihydroxyphenyl) thiazolin-4-yl] carboxyl} involving histamine, indicates the presence of 2,3-dihydroxybenzoic acid (DHBA), L-cysteine and N-hydroxy-histamine. In retrobiosynthesis of vibriobactin in V.cholerae, N1-(2,3-dihydroxybenzoyl)-N5,N9-bis[2- (2,3-dihydroxyphenyl)-5-methyloxazolinyl-4-carboxamido] norspermidine shows that it is comprised of DHBA, L-threonine and unusual polyamine norspermidine [bis (3-aminopropyl) amine] (Keating and Walsh 1999; Yamamoto et al. 1991).

15.2.2 Catecholate Siderophores

Siderophores belonging to the catecholate category have 2, 3-dihydroxybenzoate (DHB) or phenolate chelating groups as functional moieties (Table 15.1). They are also termed as pyrocatechols or 1, 2-dihydroxybenzene [C6H4(OH)2] (Cornish and Page 1998; Wittmann et al. 2001). Every catecholate group bestows two oxygen atoms to chelate with Fe ions by forming bidentate ligand complexes. As a result of this, a hexadentate octahedral complex is formed (Ali and Vidhale 2013). Catecholates are naturally occurring colourless compounds and are present as trace amounts in environment. They are composed of three isomeric benzenediols which make them an orthoisomeric molecule. One of the most important catecholate widely characterized is enterobactin or enterochelin. It is a prototype of catecholate siderophore and has a cyclic trimester coordinating group (2,3-dyhydroxyserine). It has been reported to be produced by Salmonella typhimurium and Klebsiella pneumoniae (Ali and Vidhale 2013; Achard et al. 2013).

Table 15.1 Siderophores in plant roles and associated chemical moieties
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15.2.3 Hydroxamate Siderophores

The most commonly occurring group of siderophores is the hydroxamate type, which is made up of C(=O) N-(OH) R. Here, R is an amino acid or its derivative that is primarily released by bacteria (Renshaw et al. 2002). Hydroxamate siderophores contain a fixed constancy ratio of 1:1 with Fe (III), which is in close proximity to that of the Fe (III)-EDTA complex (Mosa et al. 2016). On the basis of the side chain of the hydroxamate functional group, the hydroxamate siderophores are divided into three categories, i.e. ferrioxamines, ferrichrome and aerobactin (Winkelmann 2007). Ferrioxamines is linear in structure and its molecular formula is C25H48N6O. The ferrichromes are cyclic in structure, made of two unpredictable amino acids (alanine, glycine or serine), three N-acyl-N-hydroxyl-L-ornithine and a glycine connected by peptide bonds (Ali et al. 2011). Aerobactin is the third type of hydroxamate siderophore with a molecular formula of C22H36N4O13 (Neilands 1995). It is found in E. coli, Pseudomonas, K. pneumoniae, A. aerogenes and other bacteria (Buyer et al. 1991).

15.2.4 Carboxylate Siderophores

A recent group of siderophores have been identified, whose members neither exhibit hydromate nor 2, 3-dihydroxybenzoate (DHB) chelating groups (Table 15.1.). The chelation in this category of siderophores is done by carboxylate or hydroxyl carboxylate groups (Sah and Singh 2015; Schwyn and Neilands 1987). One of the most important carboxylate siderophore was also isolated from Rhizobium meliloti strain DM4. Rhizobactin is an aminopoly (carboxylic acid) which has hydroxycarboxyl and ethylenediamine dicarboxyl moieties or coordinating groups (Bergeron et al. 2014). Another imperative member of carboxylate siderophores is staphyloferrin A which is synthesized by Staphylococcus hyicus DSM20459. This siderophore consists of two citric acid residues and one D-ornithine residue, which bind by two amide bonds (Ali and Vidhale 2013). Rhizoferrin is another carboxylate siderophore synthesized by fungi belonging to zygomycetes family (Holinsworth and Martin 2009; Al-Fakih 2014).

15.2.5 Mixed Siderophores

Mixed siderophores possess a minimum of two different Fe-binding ligands (Aznar and Dellagi 2015). Mixed ligands are those siderophores which are derived from ornithine (pyoverdines), lysine (mycobactin) and histamine (anguibactin) (Sah and Singh 2015). Pyoverdine is the ornithine derivative type of mixed siderophore which is also known as pseudobactin. It is actively produced by Pseudomonas species (Meneely and Lamb 2007). Mycobactin is the lysine derivative type of mixed siderophores. It is produced by Mycobacterium tuberculosis and Mycobacterium smegmatis (Varma and Podila 2005). Anguibactin is the histamine derivative type of mixed siderophore. It is synthesized by marine pathogen Vibrio anguillarum (Naka et al. 2013). These diverse classes have been elaborated with their applications in plants in the table given below.

15.3 Siderophore-Mediated Responses Against Various Abiotic Stresses

Phytoremediation is today acknowledged as the most accepted green technology which is an effective in situ method for removal/treatment of heavy metals (Gratão et al. 2005). Rhizosphere is the region of soil and root interface and has an important role in the phytoremediation of various pollutants most importantly the heavy metals. This rhizospheric region is an extremely microbial active region due to the presence of siderophore-producing bacteria (SPB) (Rajkumar et al. 2010). These bacteria are reported to improve the phytoremediation process by increasing the mobility and bioavailability of heavy metals through their various secretions such as chelating compounds, phosphate-solubilizing complexes, production of phytohormones, changing redox state, etc. (Ma et al. 2011). Most common metals like Cd, Ni, Cu, Pb and Zn and actinides like U(IV), Th(IV) and Pu(IV) are found to be highly solubilized and bioavailable in the presence of siderophores (Schalk et al. 2011). But, the ability of siderophores in increasing the phytoremediation mainly depends upon their ligand specificity or selectivity to form a stable metal-siderophore complex (Braud et al. 2006, 2007). The siderophore-producing bacteria which are resistant to metal play a vital role in growth and endurance of plants by providing necessary nutrients (e.g. iron) to plants which grow in contaminated soils. Increased growth in presence of siderophore-producing bacteria will further improve the efficiency of phytoremediation process (Braud et al. 2009; He and Yang 2007; Rajkumar et al. 2010).

Recent advances indicate incorporating the siderophore-producing genes from bacterial and fungal genomes into the plant genomes or direct application of isolated siderophores onto the plant. Many studies have come forward to support the active production of siderophores by root-dwelling bacterial strains to overcome metal stress. Iron-phytosiderophore complexes and their transporters were found to be present in high concentrations in the root extracts of transgenic Petunia hybrida plants grown in iron deficient highly alkaline soils through the electrospray ionization-Fourier transform-ion cyclotron resonance mass spectrometry (Murata et al. 2015). Plants possess the ability to produce multiple siderophores such as enterobactin, which further facilitates E. coli colonization and commensalism in inducing stress tolerance (Searle et al. 2015). cDNA of ferritin siderophores from chickpea plants exposed to extreme dehydration and high salt stress showed immense induction of stress signals, which suggested a strong iron buffering role in the soil medium (Parveen et al. 2016). Systematic DNA analysis of siderophore producing bacteria Klebsiella sp. D5A genome and identification of its genes contributing to plant growth and stress management resulted in an increase in salt tolerance and wide pH adaptability. It became evident that they had well-defined roles to play under extreme environmental conditions (Liu et al. 2016). Soil- borne Cd-resistant bacterium Enterobacter sp. strain EG16 was found to produce multiple siderophores and plant hormone indole-3-acetic acid (IAA), both of which promote plant growth. The isolated extracts from bacteria were applied to plants which showed 31% Cd accumulation as compared to controls which made the bacterial strain a very apt instrument for inducing assisted phytoremediation through (Chen et al. 2016).

15.4 Diagnostic and Therapeutic Values

Most of the siderophores are reported to have major role in virulence by acting as iron scavengers, and these ferrisiderophores reenter the bacterial cells by means of specific cell surface receptors (Lamont et al. 2002). Convergence of sensitive technologies leads to siderophore neutralization by mammals and their re-consumption by bacterial pathogens (Aznar et al. 2015). Similarly, the hosts have also developed certain important cell conversion and siderophore-based iron delivery methods which are of great interest for diagnostic and therapeutic studies. There are different possible methods for exploitation of iron requirement which ultimately effect multiplication of pathogens and development of virulence (Aznr and Dellagi 2015). In recent past, the usage of various natural and synthetic compounds for effective treatment of iron-dependent infections and others had become popular. However, the use of bacterial siderophores against pathogen inhibition, removal of transuranic elements and against malaria has also emerged as a potential strategy (Beneduzi et al. 2012). These siderophores can adopt different mechanisms by which they can cut the supply of iron which effects the pathogen development and multiplication by either acting as “Trojan horse” toxins or by inhibiting siderophore synthesis pathway through the formation of siderophore-antibiotic conjugates. Application of siderophores in conception of “Trojan horse” makes them to act as intermediates which assist the uptake of antibiotics in the cells. The other ways include either the depletion of iron by application of siderophores which cannot be consumed as a source of iron by the pathogens or inhibition of siderophore utilization endogenously (Miethke and Marahiel 2007; Ahmed and Holmström 2014). However, all of the three mechanisms act differently for different pathogens. Different studies on the role of siderophores in biocontrol methods of pathogen development are available, e.g. siderophore secreted by Bacillus subtilis effectively controls the growth in Fusarium oxysporum, which causes the Fusarium wilting of pepper (Yu et al. 2011). Similarly, siderophores produced from Azadirachta indica effectively chelates Fe (III) from the soil which later affects the growth of various fungal pathogens negatively (Verma et al. 2011). The siderophore-triggered immunity is regulated by MYB72 gene, which imbalances the metal homeostasis and is required along with MYB10 to combat with deficiency of iron (Palmer et al. 2013). It was also reported that the dual function of NAGL (neutrophil gelatinase-associated lipocalin) can be used to kill cancer cells, by declining the supply of iron and increased efflux of iron leading to cell death due to inactivation of major oxidative enzymes (Tang et al. 2016). Similarly, the main causative agent of tuberculosis, i.e. Mycobacterium tuberculosis, secretes siderophores like mycobactin and carboxymycobactin. It was reported by Jones et al. (2014) that M. tuberculosis reuses its siderophores to effectively use the iron source. When this process is disordered, accumulation of siderophores in intracellular spaces was observed which later harms and detoxifies M. tuberculosis. These siderophores are poisonous and hamper the capacity of recycling of iron and the use of haeme as iron source. Thus, the enhancement of siderophore recycling can be used for development of one of the major pathogenic bacteria causing tuberculosis. The antibacterial property of siderophores was observed with the use of gallium to quench iron-scavenging siderophores in pathogenic bacteria Pseudomonas aeruginosa. It was observed that in gallium-mediated siderophore quenching is able to resist the bacterial growth and restrict virulence development (Ross-Gillespie et al. 2014).

15.5 Current Relevance and Future Prospects

Microbial siderophores synthesized and secreted by bacterial pathogenic strains such as Aerobacter, E. coli, Enterobacter, Pseudomonas sp. Salmonella, Vibrio, Yersinia, etc. acquire metal ions from the surrounding plant rhizospheric environment and end up generating several defence responses against fungal and bacterial pathogens and oxidative damage as well. However, more investigation is needed for getting a clear idea for the metal-siderophore interaction phenomenon. Metal scavenging is a competitive soil phenomenon for the diverse class of compounds, and many side benefits of prime agricultural and plant stress physiology regulation emerge. Not only this, these versatile agents have been studied as a special case of coordination chemistry in the living systems using techniques like NMR and X-ray crystallography. Advanced investigation has substantiated their role in phytoremediation, therapy against many contagious human diseases and improvising agents in imaging techniques such as MRI. In the coming times, the use of siderophores in immediate sensor-based technologies to curb spread of epidemics holds a lot of promise. Hence, microbial siderophores could be the next “wonder drugs” and “new age agricultural wizards” of our era. But, despite seeing siderophores in this new light of information and facts, we need to find out better ways of isolating them, applying them to living systems, inducing them in transgenic organisms and making all this cost-effective as well. A very interesting fact being that unlike other signalling molecules, siderophores are short-lived and don’t persist after triggering plant immunity. It will be amazing to discover the involvement of the lipocalin family in siderophore activation and a siderocalin-like response system in plants as in the case of mammals. Thus, the question of the role of such proteins in siderophore-mediated immunity remains to be addressed. If we are able to ace up research at the genetic level and crack the molecular mechanisms that bestow precision and versatility to siderophores, this could lead to better crop management strategies and extensive bio-patenting of siderophores to be used as novel therapeutic agents for fortifying both plant and human health care.