Introduction

Almost all microorganisms require iron for their cellular processes. Iron is involved in several pivotal cellular processes, including amino acid synthesis, oxygen transport, respiration, nitrogen fixation, methanogenesis, the citric acid cycle, photosynthesis and DNA biosynthesis. Iron concentrations in the extracellular environment are low (10−18 M) and limited by the insolubility of Fe (OH)3. Microorganisms require micromolar concentrations of iron, but many environments, such surface ocean water, has concentrations ranging from 0.01 to 2 nM (Luther and Wu 1997). Iron can be present in its ferric (Fe+3) or ferrous (Fe+2) forms. The redox potential of the latter is suitable as a protein catalytic center (Krewulak and Vogel 2008). For pathogenic microorganisms, iron availability is even lower due to mammalian host proteins (heme, lactoferrin, transferrin and ferritin) that sequester iron from the plasma (Krewulak and Vogel 2008). In overcome the scarcity of iron, many organisms have developed different mechanisms for iron capture: siderophores, hemophores, ferric binding proteins and transferrin/lactoferrin receptors (Sandy and Butler 2009). These molecules and proteins work together to increase the iron acquisition capabilities of the microbial cell. Siderophores are small molecules of approximately 500–1200 Da in size which specifically bind ferric iron with high affinity, are excreted into the environment, thus increasing the bioavailability of this otherwise insoluble nutrient.

The production of siderophores by disease causing microbes and many terrestrial microorganisms is well studied. The prevalence of siderophore production within extreme environments or by microbes classified as “extremophiles” is less so. This review will briefly summarize iron acquisition and siderophores produced by both terrestrial and pathogenic microbes. A discussion of the siderophores synthesized by extremophiles (acidophiles, alkaliphiles, thermo/hyperthermophiles, psychrophiles, piezophiles etc.) or siderophores produced by microbes tolerant of extreme conditions (pH, salinity, temperature, pressure) will follow.

Acquisition and transport of ferric iron siderophore-mediated iron acquisition

The active transport of ferric iron across the cell (Fig. 1; Crosa et al. 2004), is due to the use of porins for the high molecular weight ferric-siderophore complex, and requires different transport proteins and receptors. Outer membrane receptors are responsible for of recognizing iron-bound siderophores outside the cell, and include FhuA, FecA and FepA found in Escherichia coli (Ferguson et al. 1998, 2000, 2002, 2001; Yue et al. 2003), and BtuB, FpvA and FptA found in Pseudomonas aeruginosa (Cobessi et al. 2005a, b; Cornelis et al. 2009). Their structure is composed of β-barrel and cork domains, (Krewulak and Vogel 2008) the former of which spans the membrane and helps to form a hollow space where the iron-sidrophore complex passes through to the periplasm, while the cork domain binds the iron-siderophore complex and brings it into the β-barrel.

Fig. 1
figure 1

Iron acquisition in a gram-negative bacterial cell. OM outer membrane, CM cytoplasmic membrane, PSBP periplasmic siderophore binding protein, PBP periplasmic binding protein. ABC transporter includes proteins ExbB and ExbD and help in ATP hydrolysis to obtain energy for active transport. Adapted from Krewulak and Vogel (2008)

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The outer membrane of gram-negative cells does not have a proton motive force that will provide the energy required for active transport, so that role is performed by the TonB, ExbB and ExbD proteins. The amino- and carboxy-terminal domains provide the energy required to transport ferri-siderophores across the outer membrane. The remaining proteins, ExbB and ExbD, assist TonB in energy transduction.

In the periplasm there are periplasmic siderophore binding proteins (PSBP) to transport ferric siderophores across the cytoplasmic membrane. The word siderophore is included in this term because there are about eight different clusters of periplasmic binding proteins (Crosa et al. 2004; Krewulak and Vogel 2008) that bind oligosaccharides, sugars, phosphate, amino acids (polar and non-polar), organic polyanions, peptides, iron complexes and other metals. FhuD, FbpA and BtuF are the most studied PSBPs and are associated with iron uptake metabolism and transport. Specific group-coordinating PSBPs will shuttle their corresponding siderophore through the periplasm until the PSBP-siderophore-iron complex reaches transporters on the cytoplasm.

The transporters are a class of ATP-binding cassettes (ABC transporter proteins). The best example known today is the FhuBDC. The complex couples ATP hydrolysis to transport the iron-siderophore through the cytoplasmic membrane into the cytoplasm. FhuD is the PSBP for hydroxamate siderophores, and well-studied due to its complete structure elucidation. FhuB and FhuC are ABC transporter proteins. Due to their transmembrane domains, FhuBC form channels where the siderophore-iron complex passes through. It also has two nucleotide binding domains that hydrolyze ATP. In brief, FhuD transfers the siderophore-iron complex to FhuB, the complex is translocated to the channel; in the meantime FhuC hydrolyses ATP. Finally the siderophore-iron complex is dissociated by an electron transfer to ferric iron (Fe+3), converting it to ferrous iron (Fe+2).

Ferrous iron uptake

The mechanism of ferrous iron intake, contrary to ferric iron acquisition, is not well understood. Ferrous iron solubility is higher at neutral pH compared to ferric iron and this facilitates transport across the membrane. Ferrous iron concentration is higher under anaerobic or reducing conditions. Two different systems for ferrous iron uptake have been described: Feo and Sit (Carpenter and Payne 2014; Hantke 1987; Weaver et al. 2013). The Feo and Sit operon systems, co-repressed by Fur and iron (II), code for membrane-bound and cytoplasmic proteins that participate in the active transport of ferrous iron. Both ferrous uptake systems described are required for pathogenicity (Boyer et al. 2002; Fisher et al. 2009; Tsolis et al. 1996).

Regulatory characteristics of siderophore production

Fur-mediated regulation of siderophore production

Bacteria utilize siderophores to obtain the iron necessary for their survival. However, iron is toxic and must be tightly regulated within the cell. The most studied iron regulation system is fur (for ferric uptake regulation) (Hantke 2004; Lorenzo et al. 2004). Figure 2 presents a general description of the regulatory system at high and low iron concentration, inducing transcription of siderophore synthesis proteins and transportation into the extracellular space for iron scavenging. At high iron concentrations, the Fur protein undergoes conformational changes, attaches to the fur box to hinder the transcription of siderophore genes. At low iron concentrations, no iron (II) is bounded to the Fur protein, allowing the transcription of siderophore-related genes.

Fig. 2
figure 2

Siderophore-mediated iron acquisition system regulated by the Fur protein. Adapted from Crosa et al. (2004)

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Siderophore regulation by quorum sensing

In addition to iron-dependent regulation, some bacteria regulate siderophore production by means of quorum sensing. Quorum sensing occurs in a cell density-dependent manner and helps regulate different functions in the cell, triggering the corresponding phenotype response. Different physiological conditions are affected by quorum sensing, including: biofilm formation (Christiaen et al. 2014; Kadirvel et al. 2014), swarming motility (Vasavi et al. 2014), bioluminescence (Packiavathy et al. 2013), antibiotic production and resistance (Fineran et al. 2005), production of pharmaceuticals (Raina et al. 2012) and toxins (Tal-Gan et al. 2013).

Due to some contradictory findings, it is unclear how quorum sensing regulates siderophore synthesis, therefore more research efforts need to address this lack of information. Siderophore production is significantly impacted by quorum sensing in some bacteria, especially pathogens. Stintzi et al. (1998) described that lasR mutants were affected in siderophore production, specifically pyoverdine in P. aeruginosa. Another siderophore produced by P. aeruginosa is pyochelin and it was not affected by the deficiency in the autoinducer production. In contrast, Burkholderia cepacia quorum sensing mutants tend to overproduce the siderophore ornibactin (Lewenza et al. 1999). Complementation restored siderophore production to original levels.

Siderophore coordination groups

Siderophore classification is based on certain functional groups that are involved in ferric iron coordination. Those groups include catechols, as in enterobactins; hydroxamates, as in desferrioxamines; and α-hydroxycarboxilates, as in achromobactins (Fig. 3). These functional groups are donors of three OO’ in order that six oxygen atoms coordinate the ferric iron. Mixed functional group siderophores, such as the siderophore aerobactin which contains two hydroxamates and one hydroxycarboxilic acid group, are also observed. A quintessential representative of a catecholate siderophore is enterobactin (Fig. 4) and is produced by E. coli and other enteric pathogens (Gehring et al. 1997). Salmochelin is a glucosylated form of enterobactin produced by Salmonella enterica and uropathogenic E. coli (Bister et al. 2004).

Fig. 3
figure 3

Siderophore functional groups: catechols (a), hydroxamates (b) and α-hydroxycarboxylates (c). Note the OO′ groups provided by the hydroxyl and carbonyl moieties. Adapted from Krewulak and Vogel (2008)

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Fig. 4
figure 4

Enterobactin (catechol, a), desferrioxamine E (cyclic hydroxamate, b), achromobactin (hydroxycarboxylate, c), vibrioferrin (hydroxycarboxylate, d), citrate (e), desferrioxamine B (linear, hydroxamate, f) and staphyloferrin A (hydroxycarboxylate, g) structures. Adapted from Sandy and Butler (2009)

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The best known example of hydroxamate siderophores are the ferrioxamines (so-called desferrioxamines when no ferric iron is coordinated). Their structural composition is alternating units of succinic acid and monohydroxylated diamine, either N-hydroxycadaverine or N-hydroxyputrescine. Some desferrioxamines are in cyclic form, such as desferrioxamine E (Fig. 4) (Ejje et al. 2013; Konetschny-Rapp et al. 1992), while others are linear, such as desferrioxamines B (Fig. 4) (Ejje et al. 2013; Martinez et al. 2001) and G (Bergeron et al. 1992; Ejje et al. 2013). More recently, di- and tri-hydroxamic acid siderophores (putrebactins, scabichelins and turgichelins) have been characterized from Shewanella putrefaciens, Streptomyces antibioticus, Streptomyces scabies and Streptomyces turgidiscabies (Kodani et al. 2013; Soe and Codd 2014).

Achromobactin is produced by Pseudomonas syringae pv. syringae (Berti and Thomas 2009) and is a tris-α-hydroxycarboxylate siderophore (Fig. 4). A second siderophore that falls in this group is vibrioferrin (Fig. 4), categorized as a bis-α-hydroxycarboxilic siderophore (Amin et al. 2009; Harris et al. 2007). Some siderophores use only citrate to coordinate ferric iron (Fig. 4). Rhizoferrin’s two coordinating groups are donated from two citrates as well as those from staphyloferrin A (Fig. 4) (Cotton et al. 2009; Drechsel et al. 1991; Harris et al. 2007; Meiwes et al. 1990).

There are many siderophores that contain mixed coordinating functional groups. This group of siderophores is composed by bidentate ligand and also amphiphilic siderophores. Examples are aerobactins, amphibactins, ochrobactins, marinobactins, aquachelins, lystabactins, rhodobactins and amychelins among others (Fig. 5; Dhungana et al. 2007; Neilands 1995; Seyedsayamdost et al. 2011; Zane and Butler 2013).

Fig. 5
figure 5

Some mixed functional groups siderophores: lystabactins (a–c), aerobactin (d), rhodobactin (e) and amychelin (f). Adapted from Sandy and Butler (2009), Seyedsayamdost et al. (2011) and Dhungana et al. (2007)

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Siderophores produced by extremophiles

Microorganisms living in extreme environmental conditions (high acidities, alkalinities, temperatures, or barometric pressures; the ability to grow in organic solvents; or low temperatures) are known as extremophiles. It is important to make the distinction between tolerant and extremophile microorganisms. Certain microbes isolated in non-extreme environments, can grow in specific extreme conditions [like high pressure, (Olsson-Francis et al. 2010) or high temperature and in the presence of organic solvents (Tang et al. 2009)], however surpassing their tolerance level could cause death of the microbe (Huidrom et al. 2011).

Microorganisms classified as acidophiles (living at pH < 4), alkaliphiles (pH > 8), thermophiles (from about 40 to 110 °C), piezophiles (high barometric pressures) and psychrophiles (−20–10 °C) depending on their extreme environment. Although these physiological classifications exist, it is important to note that a species could have two or more physiological requirements (for example: halophile and thermophile, alkaliphile and halophile, or piezophile and psychrophile). Extremophiles possess the required cellular machinery to survive such conditions (Calo et al. 2010; Doukyu and Ogino 2010; Eichler 2003; Georlette et al. 2003; Jarrell et al. 2011; Kogej et al. 2006; Scandurra et al. 1998).

Scarce information is available for siderophore-mediated iron acquisition in extremophiles (Ye et al. 2004) but some physiological groups (halophiles and thermophiles) have more available structural information than others (alkaliphiles, acidophiles, piezophiles and psychrophiles). Microbial Fe(III) reduction has been described in diverse conditions, including acidic (Kusel et al. 1999), alkaliphilic (McMillan et al. 2010; Ye et al. 2004), thermophilic (Liu et al. 1997; Zhang et al. 2013; Zhou et al. 2001), psychrophilic (Zhang et al. 1999) and halophilic (Emmerich et al. 2012; Handley and Lloyd 2013) environments. Depending on the metabolic requirements and environmental conditions, ferric iron could be an essential micronutrient (although in extreme acidic conditions ferrous, and not ferric, iron is available).

Siderophores produced by acidophiles

Iron bioavailability in acidic environments is high when comparing to neutral pH environments. At such environmental situations soluble ferric iron is at a concentration of 0.1 M (at pH 2) (Quatrini et al. 2007). In neutrophilic environments the ferric iron bioavailable concentration is at 10−18 M. Ferrous iron predominates in acidic conditions presenting risk for cellular toxicity due to the production of radicals. Therefore, acidic environments present two main challenges to microorganisms living in such conditions: (1) cellular iron homeostasis needs to be maintained to avoid toxic levels and (2) iron utilized as energy source and an important micronutrient for optimal cellular metabolic functions (Quatrini et al. 2007) regulated.

Microbial iron oxidation for the production of cellular energy tend to occurs frequently in acidic environments and several studies described the different mechanisms of iron acquisition and management of selected acidophiles (Bonnefoy and Holmes 2012). Osorio et al. took a bioinformatics approach for the study of iron transport proteins in three acidithiobacilli (Osorio et al. 2008). It was found that Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Acidithiobacillus caldus possess the corresponding genetic information to oxidize ferrous iron. The A. ferrooxidans Fur box regulator was studied by another group of scientists and it was determined to have the ability to complement Δfur E.coli (Quatrini et al. 2005). Other bioinformatic studies present valuable information regarding iron homeostasis in Ferroplasma acidarmanus (Potrykus et al. 2011). Genes responsible for iron metabolism were up-regulated when F. acidarmanus was grown in iron poor conditions.

There is some genomic computational analysis information available on siderophore-mediated iron acquisition (Osorio et al. 2008; Potrykus et al. 2011). A. ferrooxidans and A. thiooxidans have the genetic capability for citrate (a siderophore) production, a citrate efflux pump and a TonB-dependent Fe(III)-dicitrate transport systems (Osorio et al. 2008). Potrykus et al. (2011) determined that F. acidarmanus carries a gene for siderophore synthesis and internalization (ZP_05571308 and ZP_05571695, respectively). However, the type of siderophore produced by F. acidarmanus was not determined, leaving an open question regarding the structure of the siderophore produced.

Phylogenetic and diversity studies have been performed on acidic soil environments due to plant growth-promotion capabilities of secondary metabolite production of rhizobia. The studies focused on siderophore production and other growth-promoting metabolites produced by acidophilic rhizobacteria like Bacillus, but no structural characterization of the siderophore was performed (Yadav et al. 2011). In that study, researchers found that most of the isolates produced siderophores (31 out of 49 isolates). Similar results were obtained by studies done by Karagoz et al. (2012). The isolates were related to Pseudomonas putida (7), P. fluorescens (2), Rhizobium radiobacter (1), P. syringae (1) and Bacillus atrophaeus (1). An earlier report by Verma et al. measured siderophore production activity in acidic soil isolates related to P. fluorescens (Verma et al. 2007). No structural studies were carried on in order to characterize the siderophore(s) produced by the isolates, but it is likely that pyoverdine (produced by Pseudomonas species) is a candidate (Kalinowski et al. 2006).

Siderophores produced by alkaliphiles

Alkaliphilic microorganisms are capable of growth and reproduction at high pH environments, typically >8. At such environmental conditions, iron exists in its ferric form (Fe+3), reacting with oxygen to produce Fe (OH)3 and reducing its bioavailability. Alkaliphiles must overcome this physiological problem to survive, making siderophore-mediated iron acquisition a critical physiological step (McMillan et al. 2010; Sarethy et al. 2011). Several studies have examined alkaliphiles isolated from different environments that foster lower solubility and bioavailability to determine their physiological requirements (carbon and energy sources) and their possible utilization of certain metals (Blum et al. 1998; Luque-Almagro et al. 2005a, b; Wood and Kelly 1991; Ye et al. 2004).

Iron uptake by complexation with chelators has been studied previously in alkaline, anaerobic conditions (Ye et al. 2004). Additional strategies selected alkaliphiles use to obtain iron employ the interaction with cyanide (Luque-Almagro et al. 2005a, b, 2011). In these reports, researchers described the physiological characteristics of Pseudomonas pseudoalcaligenes CECT5344, especially its biodegradation potential by the bacterial utilization of ferrocyanide. The microorganism was able to synthesize siderophores but no chemical characterizations were performed. Other microorganisms, like Streptomyces, produce respiratory inhibitors with iron-chelating properties (Pick 2004). It was demonstrated in the report that the iron-bound antimycin A was utilized by the halotolerant alga, Dunaniella salina, as a possible iron-acquisition pathway. Although researchers do not classify antimycin A as a siderophore, its chemical structure resembles that of enterobactin (Fig. 6). These reports demonstrate the capacity of microorganisms to utilize xenosiderophores, or siderophores produced by other microorganisms. In another study by Gascoyne and co-workers, bacterial isolates were obtained from different sample types including chalk soils, cement work wastes, alkaline soils and soda lakes (Gascoyne et al. 1991a, b). Researchers demonstrated that isolates were capable of obtaining iron and gallium by a siderophore-mediated mechanisms. Assay-mediated structural characterization (Arnow and Atkin assays) confirmed hydroxamic and catecholic acids type siderophores.

Fig. 6
figure 6

Enterobactin and antimycin A chemical structures

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The studies presented here demonstrate the capabilities of alkaliphiles to thrive in low iron concentrations and their evolutionary responses to such environmental conditions that help them in their survival. However information regarding specific siderophore structures is scarce since most of the studies only determine siderophore presence via the chrome azurol sulfonate (CAS) assay. Future investigations of siderophores isolation and microbial ecology organisms in alkaline pH environments should focus not only on siderophore detection, but characterization, to better understand the specific mechanisms of iron uptake.

Siderophores produced at high-temperatures: thermophiles

High-temperature environments are widely spread on Earth, from thermal hot springs in Yellowstone National Park (Beam et al. 2014; Kozubal et al. 2013; Wu et al. 2013a) and Mexico (Brito et al. 2014), to deep-sea vents (Kaye et al. 2011; Pettit 2011), mines (Gounder et al. 2011) and volcanoes (Amaresan et al. 2014; Connell et al. 2009). A diverse population of organisms are found, representing the three branches of the tree of life: eukaryotes (Connell et al. 2009), archaea (Beam et al. 2014; Hedlund et al. 2013; Kozubal et al. 2013) and bacteria (Kaye et al. 2011; Temirov et al. 2003). New organisms and even phyla are being discovered, and their physiological characteristics determined, in such environmental conditions (Beam et al. 2014; Kozubal et al. 2013). For example, one possible mechanism for iron reduction in high-temperature is explained by methanogenic bacteria. In brief, ferric iron reduction decrease the reduction potential of the system allowing methanogenesis (which in return helps the methanogen to grow and in turn enhance ferric iron reduction) (Zhang et al. 2013).

Thermophiles are producers of a plethora of secondary metabolites like siderophores that could be key players for drug developments including antibiotics, antifungals, antibacterials and anticancer drugs (Pettit 2011), but little information about siderophore production and structural characterization is available (Temirov et al. 2003). Iron transport systems have been characterized in few thermophilic bacteria (Guerry et al. 1997). In some cases siderophore production assays are performed to characterize the isolates obtained, but no structural elucidation is attained (Sudek et al. 2009). The more common, siderophore-producing species present were Pseudomonas and Pseudoalteromonas, representing more than half of isolated microorganisms (41 and 26 %, respectively). The microorganisms withstood fluid temperatures up to 81 °C in basalt rock and iron mat samples in situ. Further in vitro studies of siderophore and metal chelation from volcanic rocks revealed the transfer of the iron and other metals from the rock matrix to the aqueous phase of glacial meltwater, increasing iron bioavailability (Bau et al. 2013). Therefore, it is not surprise to find metal oxidizing bacteria with siderophore-producing bacteria, creating a community where mutualism is the key for survival.

To the best of our knowledge, there are three reports that present the structural characterization of siderophores produced by thermophilic species. Machuca and co-workers purified and partially characterized a low-molecular mass component (530 Da) from the fungus Thermoascus aurantiacus grown at 48 °C and low-pH conditions (Machuca et al. 1999). X-ray fluorescence determined the presence of iron, calcium and magnesium in the purified metabolite samples, indicating potential metal chelation (main characteristic of siderophores). The following study investigated the siderophore production of the thermoresistant bacterium B. licheniformis VK21 (Temirov et al. 2003). The purified compound, SVK21 (Fig. 7), was analyzed with NMR spectroscopy and its structure was determined to be 2,3-dihydroxybenzoylglycyl-threonine, which after comparison with bacillibactin, it was suggested to be a fragment of the latter. The final study elucidated the structure and biosynthesis of fuscachelins (Fig. 8), produced by the moderate thermophile Thermobifida fusca, (Dimise et al. 2008). These few studies show that there is still more to investigate to unveil the relationship between secondary metabolites and important biogeochemical processes.

Fig. 7
figure 7

SVK21 siderophore produced by B. licheniformis VK21

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Fig. 8
figure 8

Fuscachelins produced by the thermophile T. fusca

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Siderophores produced by psychrophilic microorganisms

Low-temperature environments represent one of the most hostile living conditions. For some time they were thought to be inhabitable (Horowitz et al. 1972). An example of such extreme environmental conditions (average temperature of −22 °C and winter temperatures as low as −60 °C) is the McMurdo Dry Valleys in Antarctica (Friedmann et al. 1987; Friedmann and Ocampo 1976; Siebert et al. 1996). In spite of the hostile conditions, living organisms’ presence was confirmed and it was demonstrated that they thrived in those environments (Friedmann and Ocampo 1976). Even the production of siderophores was described in such environment (Siebert et al. 1996) suggesting the presence of iron acquisition systems. Iron acquisition studies have been done on psychrophilic microorganisms (e.g., Oleispira antarctica) based on genome sequencing (Kube et al. 2013). Siderophore biosynthesis genes were also described in the microorganism and the genes were expressed during iron-limiting conditions. No structural characterization of the particular siderophore was done.

Bioprospecting studies have been done in low-temperature environments with the aim of isolating plant growth-promoting bacteria (Balcazar et al. 2015; Yadav et al. 2015a). In those studies siderophore production experiments were performed but no structural characterizations were done. Since isolates were related to Pseudomonas sp., and other species members of siderophore-producing phyla (Actinobacteria, Firmicutes, β-Proteobacteria and γ-Proteobacteria), this suggest that these microorganisms could produce pyoverdines, pyochelins, bacillibactins, yersiniabactins and aerobactins.

Another study presented the results for diversity and functional annotation of psychrotrophic bacteria (Yadav et al. 2015b). The samples were obtained from soil and water from a cold desert and generated 325 bacterial isolates. Siderophore producers were identified by the typical CAS assay revealing 29 strains. As in previous reports discussed here, no structural characterizations were made. However, siderophore producers were related to Bacillus, Sanguibacter, Arthrobacter and other species for which siderophore structures are known (e.g., bacillibactin, sanguibactin, pyoverdine and pyochelin).

Studies of single species siderophore production are found in literature. One report characterized a bacterium, Pseudomonas sp. PGERs17 (MTCC 9000), isolated from the northern Indian Himalayas (Mishra et al. 2008). Siderophore production was confirmed by CAS assay at as low as 4 °C, but higher siderophore production values were recorded at room temperature. No structural characterization of the siderophores produced was achieved, suggesting only pyoverdines and pyochelins as potential siderophore candidates in this microorganism. Ren et al. studied the bacterium Bacillus sp. PZ-1 (Ren et al. 2015). Siderophore production was confirmed but no structural characterization studies were performed.

The lack of information regarding structure of siderophores may be due to the difficulty to grow psychrophilic microorganisms. But the presence of iron acquisition systems and confirmation of siderophore production genes must be the motivation to engage in such studies to increase important scientific knowledge.

Siderophores produced by halophiles

The halophilic environments are those found in the oceans, salterns, desserts and soda (hypersaline) lakes and the microbial richness and diversity that thrives in them has been described (Bamforth 1984; Crognale et al. 2013; Emmerich et al. 2012; Ghozlan and Deif 2006; Pandit et al. 2015; Sunagawa et al. 2015). Halophilic microorganisms were found in the rhizosphere of halotolerant plants. Some bacterial species are unable to produce siderophores (Tipre et al. 2015) but others are capable of such mechanism (Sahay et al. 2012). In the latter report, researchers found that 21 % of the hypersaline-lake isolated bacteria produce siderophores. No structural characterizations were made to elucidate the chemical structure of those siderophores produced. After 16S rRNA gene sequencing, the siderophore-producing microorganisms were related to bacterial species of which some siderophore structures are known (bacillibactin, marinobactins, amphibactins, sodachelins and halochelins) (Figueroa et al. 2015; Gledhill et al. 2004; Martinez and Butler 2007; Richards et al. 2006, 2007; Woo and Kim 2008). Other siderophore-producing microorganisms have been isolated from marsh water and soil samples but they failed to determine structure of siderophores (Malviya et al. 2014) or the presence of siderophore-mediated iron reduction (Rossello-Mora et al. 1995). Several published studies isolated siderophore producers from soda, or hypersaline, lakes (Figueroa et al. 2015, 2012; Ramadoss et al. 2013; Richards et al. 2006, 2007; Serrano Figueroa 2015). Chemical structure characterizations were performed by mass spectrometry and fatty acid methyl ester, and siderophores were classified as amphiphilic siderophores: sodachelins and halochelins (Fig. 9). Ramadoss and co-workers found that a total of three isolates (SL3, SL32 and PU62), out of 84, from different hypersaline lakes of India produced siderophores. No chemical structure studies were done but phylogenetic analysis determined that SL3 was related to Halobacillus; SL32 to Bacillus pumilus; and PU62 to Bacillus halodenitrificans. Known siderophore production studies have been done on Halobacillus species to the best of our knowledge, however Bacillus species produce bacillibactin and we could infer that SL32 and PU62 may produce this siderophore. Additional reports described studies done for siderophore production with desert-isolated Kocuria turfanensis 2M4 and Bacillus licheniformis A2 bacteria (Goswami et al. 2014a, b) but in both reports no structural characterization was presented. Therefore, we suggest bacillibactin as the siderophore produced by B. licheniformis A2 but further confirmation is needed. A more recent study confirmed the production of siderophores by another desert-isolated bacterium, B. cereus brm, however no chemical characterizations were performed (Vishal and Manuel 2015).

Fig. 9
figure 9

Halochelins (a, b) and sodachelins (c) produced by Halomonas sp. SL01 (a, b) and SL28 (c)

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Iron acquisition studies have linked siderophores to proper and optimal cell development of halophiles (Anderson et al. 2011; Buyer et al. 1991; Hopkinson and Morel 2009; Wilhelm et al. 1998). Every year halophilic, siderophore-producing microorganisms are characterized by different research groups. Most of the siderophores produced by halophiles are classified as amphiphiles, meaning that the same molecule will have polar and non-polar properties. The polar headgroup is the iron binding site and it is attached to one or two of a series of fatty acids (Gauglitz et al. 2012; Ito and Butler 2005; Martin et al. 2006; Martinez et al. 2003, 2000; Owen et al. 2005). Figure 10 shows selected amphiphilic siderophore structures presenting the structural feature of α-hydroxycarboxylic acid moiety, usually in the form of β-hydroxyaspartic acid or citric acid, conferring photochemical reactivity to the molecule as demonstrated previously (Barbeau et al. 2003, 2002; Butler et al. 2001; Butler and Theisen 2010).

Fig. 10
figure 10

Suites of marine amphiphilic siderophores structures characterized: marinobactins (a), aquachelins (b), amphibactins (c), loihichelins (d), moanachelins (e) and synechobactins (f). Adapted from Sandy and Butler (2009)

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As observed from the review in siderophore-producing halophiles there is considerable information available about marine amphiphilic siderophores. However, little information is published regarding soda, hypersaline lakes and deserts. It is important to ensure the studies of these environments and look for secondary metabolite production, specifically siderophore production, and their relationship to iron acquisition and biogeochemical pathways.

Siderophores produced by piezophiles

Piezophilic microorganisms (previously known as barophiles) grow and reproduce under high hydrostatic, or barometric, pressures (>1100 atm) (Deming and Colwell 1981). Typically that level of pressurization can be found in deep-sea environments (due to the pressure of the water column), under the basaltic sea floor (caused by both the water column and Earth’s crust weight) and underground (subsurface) environments. In addition to hydrostatic pressure, most of the temperatures are below 4 °C, and up to 400 °C nearby hydrothermal vents presenting a double challenge for microorganisms living in such conditions. It was thought by most scientists that life could not withstand the conditions present in these environments until independent studies by Certes and Regnard in 1884 stated the contrary. Posterior studies presented the evidence of life at depths greater than 10,000 m under sea level (Zobell and Morita 1957). Organisms living under the influence of high pressures evolved membranes with modified fatty acyl moieties in the phospholipids, making the membrane more fluid (Abe 2013). During the early times of deep-sea studies (piezobiology), a main objective was always present in the experimental design: to keep high hydrostatic pressures when sampling deep-sea water and sediments, avoiding decompression that may kill obligate piezophiles. Nowadays scientists have developed modern sampling systems that solve the decompression problem (Fang et al. 2010). With the assistance of robots and submarines sediments can be sampled and novel piezophilic species isolated to lead to an understanding of nutritional and physical requirements for their optimal cellular function (Bale et al. 1997; Nogi et al. 1998). In addition, with modern molecular biology techniques in non-cultivable approaches can be used to describe the microbial population living under the influence of high pressures (Fleming et al. 2013; Jorgensen et al. 2013; Wang et al. 2008).

Siderophore production and iron reduction are closely linked in microorganisms (Adams et al. 1992) by the effects of some physical and physicochemical conditions in the latter. Nutrient availability could also exert an effect on the viable cell count of these microorganisms (Wirsen and Molyneaux 1999). Microbial iron reduction and oxidation processes have been described in the literature. Model simulations proved that increasing the hydrostatic pressure on organisms causes a decrease of the energy yield of cellular reactions like oxygen reduction (Fang et al. 2010). In contrast, ferric iron reduction free energy increases as the pressure increases. Several studies have demonstrated that microorganisms belonging to the domains Bacteria and Archaea possess the required genetic machinery (Fleming et al. 2013; Jorgensen et al. 2013; Wang et al. 2008) and other investigations presented in vitro evidence of microbial iron reduction and oxidation (Picard et al. 2014; Wu et al. 2013b). Wang and co-workers isolated an iron-reducing bacterium, Shewanella piezotolerans WP3 and determined by its genome sequencing the presence of mtr-omc gene cluster involved in metal reduction in most Shewanella species. The group of Jorgensen and co-workers discovered that members of the deep sea archaeal group constitute up to 50 % of the microbial population in certain sediment horizons. Also that group of investigators determined that there were significant variations in iron oxide and dissolved iron levels, implying redox reactions occurring in the microscopic community.

Not much information is available regarding siderophore-mediated iron acquisition and it is limited to simple, but significant, siderophore production experiments. The first evidence of potential siderophore production by genome analysis of the deep-sea bacterium, Pseudomonas sp. 10B238, confirmed the presence of non-ribosomal peptide synthetases genes (Pan and Hu 2015). Additional genome studies have confirmed heterologous expression of hydrothermal deep-sea metagenomic DNA in E. coli, producing the siderophore avaroferrin (Fig. 11) and putrebactin (Fujita and Sakai 2014). The only direct siderophore isolation and structure identification was reported from the deep-sea microorganism Streptomyces olivaceus FXJ8.012 which produces tetroazolemycins A and B (Fig. 11; Liu et al. 2013).

Fig. 11
figure 11

Additional siderophores produced by piezophilic microbes: avaroferrin (a), tetroazolemycin A (b) and tetroazolemycin B (c)

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In another study, arsenic-rich groundwater samples demonstrated the presence of siderophore-producing microorganisms (Sarkar et al. 2013). Siderophore structural studies were not done in this study but we can infer that ochrobactins (Martin et al. 2006) and alcaligin (Li et al. 2013) may be the ones produced since bacterial species corresponding to these siderophores were isolated and identified through 16S rRNA analysis. Since scarce siderophore structures were directly linked to siderophore production, there is a need to elucidate novel siderophore from piezophilic isolates. In this manner we may be able to link iron reduction and acquisition in these microorganisms with their siderophore production, and understand their role in deep-ocean and sediment biogeochemical processes.

Concluding remarks

Observing Fig. 10, we can conclude there might be certain levels of hydrophilicity or hydrophobicity in amphiphilic siderophores due to some molecular characteristics (length of fatty acyl moieties and headgroup). A clear example of this is the hydrophobicity of amphibactins (Gledhill et al. 2004; Vraspir et al. 2011), moanachelins (Gauglitz and Butler 2013) and ochrobactins (Gauglitz et al. 2012) contrasting to the hydrophilic lohichelins (Homann et al. 2009). This affects their extraction: amphibactins, moanachelins and ochrobactins are mainly obtained from cell pellet extracts, contrasting to the supernatant extracts that contain loihichelins. Marinobactins have been the most studied of the amphiphilic siderophores and have different degrees of hydrophobicity and hydrophilicity (Martinez and Butler 2007; Xu et al. 2002). Membrane partitioning studies were also done with ochrobactins giving similar results to those of the marinobactins (Martin et al. 2006). Aquachelins are quite hydrophilic especially aquachelins I and J isolated from Halomonas meridiana (Vraspir et al. 2011). Synechococcus sp. produces synechobactins (Fig. 10) which are related to the siderophore schizokinen (Ito and Butler 2005). Other siderophores can be taken up by another type of microorganism and then undergo changes in their structure. In Vibrio harveyi, enterobactin undergoes a chemical modification (addition of fatty acids moieties with different levels of saturation and hydroxylation) to transform it into an amphi-enterobactin (Zane et al. 2014). The relevance of amphiphilic siderophores chemical characteristics determine their extraction process, important in the study of new family members.

Another property of amphiphilic siderophores is the capacity of the molecules to self-assemble in micelles and vesicles upon iron coordination (Bednarova et al. 2008; Luo et al. 2002; Martinez et al. 2000; Owen et al. 2007, 2005, 2008). The science behind this phenomenon is based on the critical micelle concentration (cmc) of the amphiphile molecule. When the concentration of the amphiphilic siderophore is over the cmc and the siderophore is not bound to iron, micelle formation occurs. Adding ferric iron (~1 Eq.) to the solution causes micelle size reduction, but if the iron concentration increases (>1 Eq.) a micelle-to-vesicle transition happens (Owen et al. 2005). A brief description of this process appears in Fig. 12.

Fig. 12
figure 12

Micelle and vesicle formation on amphiphilic siderophores due to critical micelle concentration (cmc) and ferric iron chelation

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Iron is ubiquitous in the environment and required by most living organisms. It is not bioavailable due to its poor solubility in water, but microorganisms have developed iron acquisition systems to overcome this problem. These acquisition systems help regulate a balanced iron concentration across the cell. Siderophore production and respective receptors and transport proteins are part of that system and help obtain iron from the extracellular environment in iron-limiting conditions. In general, three different functional groups (hydroxamates, catacholates and hydroxycarboxylates) are found in the siderophore molecule and assist in ferric iron coordination. Different structures are found in diverse environments including extreme environments. Extremophiles evolved to overcome the hostile conditions in such environments ensuring their survival and have developed mechanisms for iron acquisition. The physiological requirements for extremophiles are so complex and interweaved that a single extremophilic species could possess characteristics from two or more physiological groups (e.g., thermoacidophilic bacteria, haloalkaliphilic archaea etc.). Halophiles synthesize a variety of siderophores and some information is available regarding their structural composition. These structures contrast with the siderophore production and structural characterization studies from other physiological requirements groups (like piezophiles and acidophiles). Siderophores help maintain the equilibrium of iron in extreme environments providing physiological niches for extremophiles. More studies on extremophiles and siderophore production should be performed along characterization studies to elucidate siderophore chemical structures.