Structural characterization of amphiphilic siderophores produced by a soda lake isolate, Halomonas sp. SL01, reveals cysteine-, phenylalanine- and proline-containing head groups Authors: Luis O'mar Figueroa, Benjamin Schwarz, & Abgail M. Richards NOTICE: The final publication is available at Springer via http://dx.doi.org/10.1007/s00792-015-0790-x. Figueroa LO, Schwarz B, Richards AM, "Structural characterization of amphiphilic siderophores produced by a soda lake isolate, Halomonas sp. SL01, reveals cysteine-, phenylalanine- and proline- containing head groups," Extremophiles Nov 2015 19(6):1183–1192.] Made available through Montana State University’s ScholarWorks scholarworks.montana.edu - - - acid, are required for the catecholate siderophores to coor- dinate iron; carboxylate siderophore must have α-hydroxy donor groups (Butler and Theisen 2010; Patus and Abdal- lah 2000). Some of siderophores wil possess only one of the classes of iron-coordinating groups while others may utilize multiple ones. For example, aerobactin, produced by E. coli, contains both hydroxamic and hydroxycarbox- ylic coordinating groups (Gauglitz et al. 2012; Valdebenito et al. 2006). Amphiphilic siderophores are stil another class of iron-chelating molecules that are set apart by the com- bination of a polar amino acid head group and an aliphatic faty acid tail atached to the N-terminus of the head group, lending amphiphilic characteristics. Like non-amphiph- ilic siderophores, amphiphilic siderophores may contain a variety of iron-coordinating moieties such as catechols, α-hydroxycarboxylic acid and hydroxamic acids within the polar head group. Citrate and hydroxyaspartic acid have also been found as part of amphiphilic siderophores struc- ture (Sandy and Butler 2009). Examples of the variability found in functional groups of amphiphilic siderophores are ochrobactins (Martin et al. 2006), synechobactins (Ito and Butler 2005) and petrobactins (Homann et al. 2009a). To date, most of the amphiphilic siderophores come from marine microorganisms, such as Marinobacter sp. and Vibrio sp. (Amin et al. 2012; Butler and Theisen 2010; Sandy and Butler 2009; Sandy et al. 2010). In this report, we describe two distinct families of amphiphilic siderophores, both pro- duced by an isolate from Soap Lake located in Washington State, Halomonas sp. SL01. Soap Lake is a meromictic lake with a pH of 9.8 and a dissolved solid concentration rang- ing from 140 g/L in the monimolimnion layer to 14 g/L in the mixolimnion layer (Edmondson and Anderson 1965; Sorokin et al. 2007). The microbial population in Soap Lake is diverse and includes phylogenetic groups α-, β-, and γ-Proteobacteria, Acidobacteria, Verucomicrobia, Synecho- coccus, Actinobacteria and Thermotogales, among others (Dirnitriu et al. 2008). Also, Fe(II) reduction has been char- acterized on diferent isolates including Bacilus sp. (Polock et al. 2007) and our siderophore-producing halophile Halo- monas sp. SL01 (VanEngelen et al. 2008). It is implied then that this reduction process is due to siderophore production by this microorganism. The main objective of this report is to determine if the hypersaline bacterial isolate, Halomonas sp. SL01, produces siderophores and if so, to identify the chemi- cal structure of the molecule or molecules. Methods Growth medium The growth media used in al the experiments were an arti- where Halomonas sp. SL01 was isolated. The media were prepared with 50.0 g/L sodium chloride (Fisher), 1.12 g/L sodium borate, 1.0 g/L ammonium chloride, 0.06 g/L cal- cium chloride, 0.05 g/L magnesium chloride hexahydrate, 0.85 g/L sodium nitrate, 0.50 g/L potassium phosphate monobasic, 0.01 g/L potassium chloride, 0.25 g/L yeast extract and sodium pyruvate (5.0 g/L, Fisher) as the car- bon source. The media were treated with Chelex (Sigma Aldrich) resin according to the manufacturer’s instructions to reduce iron and glassware was acid washed. Al media were filter sterilized with 0.22 µm polyethylene sulfonate (PES, Nalgene) membrane filters and pH was adjusted to 9.0 with 10 N NaOH (Fisher). Siderophore detection in solution by CAS assay Stock solutions were prepared in advance: (1) 10 mM HDTMA (Fisher); (2) iron solution: 1 mM FeCl3•6H2O (Acros) in 10 mM HCl; (3) 2 mM aqueous chrome azurol sulfonate (CAS, Sigma) and (4) 0.2 M 5-sulfosalicylic acid (Fisher). Then, the CAS assay solution was prepared by adding 6 mL of 10 mM HDTMA in a volumetric flask (100 mL) diluted with a bit of water. In that same flask, 1.5 mL iron solution and 7.5 mL of 2 mM CAS solu- tion were added. Then, in a separate flask, 4.307 g anhy- drous piperazine (Acros) was dissolved in some water and 6.25 mL of 12 M HCl was added. This bufer was rinsed into the volumetric flask with HDTMA, CAS and iron solu- tion and volume completed with nanopure water. To determine the presence and concentration of sidero- phores in solution, a 500 µL aliquot of media supernatant (previously centrifuged at 12,000×g for 5 min, Eppendorf 5417C) plus 500 µL CAS assay solution were added to a cuvete. Then, 20 µL of 5-sulfosalicylic acid (or to a final concentration of 4 mM) was added. Absorbance of the solu- tion at 630 nm was measured after equilibrium was reached (30 min-2 h, but no longer than 6 h). Concentrations were determined based on ferioxamine B standard curve. Growth culture preparation and sampling To prepare the growth cultures, a frozen stock of Halo- monas sp. SL01 was inoculated in 100 mL SLM. The ster- ile control was 50 mL of SLM. Starter culture and control were placed in the shaker incubator (140 rpm at room tem- perature or 37 °C; Infors HT Ecotron) and optical densi- ties (OD, at 600 nm) and CAS assays (at 630 nm) were done once a day withdrawing 1 mL aliquots. Once the CAS absorbance read about 0.100, 1 mL aliquots were trans- fered into 3, 1 L baffled flasks with 400 mL SLM. A sterile control flask was also prepared for the study. Flasks were incubated at previous conditions and OD and CAS read- ings were taken twice a day until a maximum siderophore production was detected. Finaly, the media were ultra-cen- trifuged (Sorval Instruments RC5C; GSA rotor) at 6238 xg for 20 min at 4° C. SLM and CAS plates To check for culture purity, SLM plates were streaked. To prepare the plates, Noble agar (30 g/L, Difco) was dis- solved and sterilized by autoclaving in 500 mL nanopure water; the solution was placed in a water bath at 60 °C. Soap Lake media were prepared as previously mentioned, pH adjusted and filter sterilized with 0.22 µm membrane filter and placed in a water bath for 3 h. When solutions were at temperature, 500 mL of SLM was poured in the Noble agar and alowed to mix by magnetic stiring. Plates were poured and solidified overnight. When growth culture was in stationary phase and siderophore production was detected, streaks of SLM plates were done. CAS plates were prepared in a similar way to SLM plates. A CAS/HDTMA mix was prepared by first mixing chrome azurol sulfonate (605 mg), water (500 mL) and 1 mM FeCl3 in 10 mM HCl (100 mL) together (Solution A). HDTMA (729 mg) was dissolved in water (400 mL, Solution B). Solution A was added to B, providing gentle stiring. The mixture was sterilized and placed in a water bath. Noble agar solution was prepared as previously men- tioned and sterilized (placed in water bath). SLM (400 mL) was filter sterilized and placed in a water bath. SLM and CAS/HDTMA solutions were mixed with the Noble agar solution and plates were poured and solidified overnight. Streak plates were done as a double verification for sidero- phore production and culture purity. Siderophore extraction, purification and lyophilization A C2 column (Varian) was used to remove siderophores from cel-free supernatant. Each column was conditioned with methanol (Fisher) and nanopure water according to the manufacturer’s instructions. To extract siderophores from the supernatant, a 50 mL volume was run through the column. After loading the column with culture super- natant, the column was filed with nanopure water and this phase was colected. After this, the column was filed with methanol; this phase was colected in a second tube. Then, the column was filed with a second portion of nanopure water and this was eluted and discarded. This process was repeated until al supernatant had passed through the col- umn. Al methanol extracts and first colected nanopure water were pooled. Extracts were concentrated by evapora- tion centrifugation (at 50 °C, Labconco) for 2 h, stored at 4 °C and purified by HPLC (Dionex). To purify the extracts obtained from Halomonas sp. SL01, an HPLC method was created. Briefly, two mobile phases were used: (A) water with 0.01 % (v/v) trifluoro- acetic acid (TFA, Fisher) and (B) acetonitrile (Fisher) with 0.01 % (v/v) TFA. The mobile phase gradient was 10–70 % (v/v) B for 63 min. A C4 reverse phase column (4.6 mm ID X 250 mm, Grace) was used. UV detection wavelength was set to 220 nm. Siderophore fractions were colected, frozen, lyophilized overnight and stored (4 °C) for future tests. Mass spectrometry (MS) analysis To confirm siderophore presence, lyophilized samples were dissolved in nanopure water and the CAS assay was per- formed. Siderophore-positive samples were analyzed on an Agilent 6538 QTOF LC/MS with electro spray ionization (ESI) to determine their chemical structure. Samples were further purified using a 10–100 % (v/v) acetonitrile gradi- ent for 10 min on the LC before entering the MS. Acidified water with 0.1 % (v/v) formic acid was used as aqueous bufer. Samples were analyzed in positive mode and with a fragmentation voltage of 150 V. MS/MS analysis was done on the same equipment with a constant stream of directly infused sample administered with a syringe pump. A target ion was selected from the MS analysis and fragmentation voltage was ramped in cycle to provide a progression of fragments for each sample (Tables 2, 3). Data analysis was done using the Bruker’s DataAnalysis software. Faty acid methyl ester (FAME) To determine aliphatic tail structure, faty acid methyl ester (FAME) analyses were done by MIDI Labs, Inc. Lyophi- lized siderophore samples were dissolved in nanopure water and analyzed through FAME in a four-step reaction process: (1) saponification, (2) methylation, (3) extrac- tion and (4) wash for sample clean up. Four reagents were prepared to help cleaved the tail from the siderophore and they were particularly related to each reaction step in FAME. Reagent 1 (for saponification) was made from 45 g NaOH, 150 mL methanol and 150 mL distiled water. Rea- gent 2 (for methylation) was made with 325 mL certified 6.0 N HCl and 275 mL methyl alcohol. The faty acid was poorly soluble in the aqueous phase at this point. Reagent 3 (for extraction) was made of 200 mL hexane and 200 mL methyl tert-butyl ether. This reagent extracted the faty acid tails into the organic phase for use with the gas chromato- graph (GC). Reagent 4 (for sample clean up) was made of 10.0 g NaOH dissolved in 900 mL distiled water. Sample processing to prepare GC ready extracts was made folowing the 4 steps mentioned previously. Briefly, for saponification, 1 mL of reagent 1 was added to the siderophore samples. Tubes were sealed and vortexed (5–10 s) and heated in a boiling water bath for 5 min, at which time the tubes were vortexed again and placed in the bath for an additional 25 min. In the methylation reac- tion step, 2 mL of reagent 2 was added. The tubes were capped and vortexed and tubes were heated for 10 ± 1 min at 80 ± 1 °C and after this samples were cooled at room temperature. For the extraction step, 1.25 mL of reagent 3 was added, tubes recapped and tumbled in a clinical rota- tor for 10 min. The aqueous phase was discarded. To clean up the sample, about 3 mL of reagent 4 was added to the organic phase; the tubes were tumbled for 5 min. After that, approximately 2/3 of the organic phase was then analyzed in the GC. Gas chromatography (GC) After FAME, samples were analyzed in a gas chroma- tograph (Agilent Technologies 5890, 6890 and 6850) with Sherlock MIS Software. A 25 mm × 0.2 mm phenyl methyl silicone fused silica capilary column (Ultra 2) was used. The method increased temperature from 170 °C to 270 °C at 5 °C/min. To ensure column cleaning and life span, a 300 °C balistic temperature increase was held for 2 min after each sample was analyzed. A flame ionization detector was also employed to provide good sensitivity. Hydrogen was the carier gas, nitrogen the “make up” gas and air was used to support the flame. GC calibration was done using a standard with mixtures of straight chain satu- rated faty acids from 9 to 20 carbons in length (9:0–20:0) and 5 hydroxy acids. Results Siderophore production in soap lake media and purification Strain SL01 from the genus Halomonas sp. grew very wel in Soap Lake media. The conditions to which it was sub- jected were, as previously mentioned, 5.0 % (w/v) NaCl concentration, room temperature (25 °C) and pH 9.0. As shown in Fig. 1, a lag phase was absent. In contrast, a delay was detected for siderophore production. Production of siderophores detected by the chrome azurol sulfonate (CAS) assay was stable after 76 h of growth and harvest- ing was done for eventual purification. A maximum sidero- phore production was detected (38.1 µM). Optical density reached a maximum of 0.625, decreased and increased again up to 0.530. Purification of the lipid extract of the media via reverse phase chromatography yielded at least 19 unique features (Online Resource 1). Peaks B, C, D, E and F yielded iron- chelating species by CAS analysis and were considered candidate siderophores. Peaks A and H were also colected and examined but were not active for iron chelation while peak G was not soluble in water and could not be analyzed. Table 1 presents a summary of the iron-chelating activities of the coresponding peaks revealing siderophore presence in B–F. Fractions active in iron chelation were further ana- lyzed to determine siderophore structure via mass spec- trometry and faty acid methyl ester. Structural analysis of siderophores by mass spectrometry and faty acid methyl ester (FAME) To study siderophore structure, a QTOF LC/MS tandem MS/MS method was developed. Initial results showed two diferent siderophore families produced by Halomonas sp. SL01. For al siderophore samples, both a “y” series, breaking at the peptide bond and including the c-terminus, and a “b” series, breaking at the peptide bond and including the N-terminus, were observed in the fragmentation spec- tra. Three siderophore fractions (B, C and E) have nearly identical “y” fragmentation paterns. This sharing of frag- ments suggested a common amino acid composition in the head group. The “b” fragmentation series within this group included the same peak spacing but was shifted by a diferent constant mass for each sample, suggesting that the distinguishing feature is the length and/or structure of 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 0.000 0.100 0.200 0.300 0.400 0.500 0.600 0.700 0.800 0 20 40 60 80 100 120 140 160 Co nce ntr ati on (µ M) Opt ica l D ens ity (6 00 nm ) Time (h) Optical Density Control Optical Density CAS Negative Siderophore Production Fig. 1 Growth and siderophore production of Halomonas sp. SL01at 5 % NaCl soap lake media and room temperature conditions. Gray circles with solid line represent growth in terms of optical density; gray circles with dashed line represent optical density control; black triangles with solid line represent siderophore concentration in solu- tion; and black triangles with doted line represent siderophore con- centration negative control. Time, in hours, is on the x axis, optical density is on the primary y axis and concentration is on the secondary y axis. Values are average ± SD the hydrophobic tail atached at the N-terminus. Fractions D and F also shared a common “y” fragmentation series, while the “b” series was distinguished by a constant mass shift again suggesting common amino acid sequence in the head group and difering tails. The fragmentation paterns in both the “y” and “b” series were almost entirely unique between the D and F set and the B, C & E set. This sug- gests that Halomonas sp. SL01 produces distinct two fami- lies of amphiphilic siderophores for which we propose the name, halochelins. For Halochelins B, C and E, molecular weights were calculated to be 1091.39 atomic mass units (amu), 1135.42 amu and 1119.43 amu, respectively (Online Resources 2 through 4, respectively). Parent ions showed adducts to potassium (M + K) providing the apparent total mass for each molecule (refer to Table 1). The coresponding amino acid residue sequence for the polar head group of these molecules was found to be (N- to C-terminus): phe- nylalanine (Phe), threonine (Thr), cysteine (Cys), arginine (Arg), glutamine (Gln), threo-β-OH-asp (Thr-β-OH-asp) and D–N–OH-ornithine (D–N–OH-orn). Accounting for the constant mass shift in the “b” series, these fragments also confirmed the proposed amino acid sequence. Table 2 shows a summary of fragmentation found with mass spec- trometry studies. An anticipated molecular diference in the hydropho- bic tail could be calculated from the constant mass shifts in the “b” series fragments within a family. Halochelin B total apparent mass was 1052 amu and Halochelin C mass was 1096 amu. The mass diference between them was 44 amu, which suggested an additional (–CH2CH2OH–) group in Halochelin C presumably in the hydrophobic tail. By comparing Halochelins B and E, a 28 amu difer- ence was found. This suggested an additional (–CH2CH2–) group for Halochelin E. A 16 amu diference was found between Halochelins C and E that suggests to an additional hydroxyl group (–OH) for the former. The structural groups described above were associated with faty acid tail difer- ences for the respective siderophores. One fragment, which was observed at low abundance, for each siderophore also suggested the approximate molecular mass for the faty acid tails: 155 amu for Halochelin B, 199 amu for Halo- chelin C and 183 amu for Halochelin E (Table 2). For more detailed information about fragments generated, please refer to Online Resources 2 through 4. Faty acid methyl ester (FAME) analysis was used via gas chromatography (GC) to confirm faty acid tail pres- ence and structure in each siderophore. Various faty acid GC fractions were present in the lyophilized sample for Halochelins B and C (analyzed in the same fraction). At retention times, 1.2238 and 2.0545 min GC fractions were identified by the Sherlock MIS Software to be related to Halochelins B and C, respectively (Online Resource 5). Halochelin B faty acid fraction (retention time 1.2238 min) revealed a faty acid with structure 10:0, suggesting that the aliphatic tail was composed of a 10 carbon chain with no double bonds or side groups (Online Resource 6). Halochelin C showed a faty acid fraction (retention time 2.0545 min) with a 12:0 3OH structure, which suggested a 12 carbon chain, no double bonds and a hydroxyl (–OH) side group on the third α carbon position. The diference in atomic mass from FAME revealed an additional 44 amu to Halochelin C that corelated with MS data. Halochelin E HPLC lyophilized fraction was also analyzed and the GC showed, at a retention time 1.6482 min, a faty acid with structure 12:0 (Online Resources 7 and 8). This suggested a 12-carbon chain, with no double bonds or side group sub- stitutions. The atomic mass diference between Halochelins E and B was 28 amu, corelating with the findings of MS Table 1 HPLC fractions with siderophore detection and absence as per the CAS assay a Symbols “+” and “−“means siderophore detection and absence, respectively b Symbol “−“means that fractions were not analyzed in mass spec- trometry due to negative CAS assay result (no siderophore detected) Fraction Siderophore presence by CAS assaya Apparent mass (amu)b A – – B + 1052.45 C + 1096.45 D + 989.4 E + 1080.48 F + 1017.38 G – – H – – Control – – Table 2 Mass spectrometry patern fragmentation for Halochelins B, C and E produced by Halomonas sp. SL01 “y” fragments break at the peptide bond and including the c-terminus. “b” series fragments break the peptide bond and include the n-termi- nus Fragments (C- to N-terminus) y (amu) B b (amu) C b (amu) E b (amu) Halochelins 131 921 965 949 262 790 834 818 390 662 706 690 546 506 550 534 649 403 447 431 750 302 346 330 897 155 199 183 data. For gas chromatograms and retention times for faty acid fractions, refer to Online Resources 5 through 8. A detailed and complete siderophore structure is presented in Fig. 2 demonstrating similarities with other marine amphi- philic siderophores, such as the marinobactins. Halochelin D and F molecular weights were 1079.45 and 1107.48 amu, respectively (Online Resources 9 and 10). Parent ions detected by mass spectrometry were atached to hydrogen (M + H) and hydrated with 5 molecules of water providing the apparent total mass for each molecule (refer to Table 1). The common “y” fragments suggested that, like halochelin B, C and E, the polar head group is conserved. The coresponding amino acid sequence for the polar head group of these amphiphilic siderophore molecules was determined as: proline (Pro), arginine (Arg), serine (Ser), threo-β-OH-asp (Thr-β-OH-asp), threonine (Thr), serine (Ser) and D–N–OH-ornithine (D–N–OH-orn). The “b” fragmentation patern was conserved but shifted by 28 amu suggesting diferences in an N-terminal hydrophobic tail as seen with the B, C and E family. These fragments also con- firmed the amino acid residue sequence. Table 3 shows a summary of fragmentation found with mass spectrometry studies for Halochelin D and F. As was done with the first suite of halochelins (B, C and E), fragments for each siderophore molecule were com- pared and mass diferences were obtained. Halochelin D total apparent mass was 989 amu and Halochelin F mass was 1017 amu. The mass diference between them was 28 amu, which suggested an additional (–CH2CH2–) group in Halochelin F. This 28 amu diference was maintained across the entire “b” series. To analyze faty acid tail structure of Halochelin D and F, FAME with gas chromatography (GC) analysis was performed. The 1.9840 min GC fraction was identified by the Sherlock MIS Software to be the Halochelin D faty acid (Online Resources 11 and 12). The fraction analysis revealed a faty acid with structure 12:0 3OH suggesting that the aliphatic tail was composed of a 12 carbon chain, no double bonds and a hydroxyl (–OH) side group on the third α carbon. Aliphatic tail fragment masses for Haloche- lin C and D were the same (199 amu) providing the possi- bility that tail structure was also conserved. For Halochelin F HPLC lyophilized fraction at retention time 2.663 min, a faty acid with structure 14:0 3OH was detected (Online Resource 13 and 14). That suggested a saturated faty acid tail with a 14-carbon chain and a hydroxyl (–OH) side group on the third α carbon. The atomic mass diference between Halochelins D and F was 28 amu, corelating with the findings of MS data. A detailed and complete sidero- phore structure is presented in Fig. 3 demonstrating similar structures to previously reported amphiphilic siderophores (Martinez and Butler 2007; Vraspir et al. 2011). Discussion The siderophores produced by Halomonas sp. SL01 were found to be amphiphilic and we propose the names of Halochelins B, C, D, E and F. A slight delay in siderophore production relative to cel growth was detected in cultures, suggesting that siderophore production was enhanced as cel growth induced iron limitation. Similar results were presented in a report that studied erythrobactin, a Fig. 2 Molecular structure of halochelins B, C and E, produced by Halomonas sp. SL01. On top, the polar head group is presented with the amino acid sequence. On the botom, the “R” represents the ali- phatic tail (faty acids) atached to it Table 3 Mass spectrometry patern fragmentation of the Halochelins D and F produced by Halomonas sp. SL01 “y” fragments break at the peptide bond and including the c-terminus. “b” series fragments break the peptide bond and include the n-termi- nus Fragments (C- to N-terminus) y (amu) D b (amu) F b (amu) Halochelins 131 858 886 218 771 799 319 670 698 450 539 567 537 452 480 693 296 324 790 199 227 hydroxamate-type siderophore produced by the actinomy- cete S. erythraea (Crosa 2004; Oliveira et al. 2006). Sidero- phore production was detected after 24 h of growth. This trend in growth was also found in physiological data about Halomonas sp. SL01 and SL28, in which the microorgan- isms start to grow and after a time period (usualy 24 h) produce siderophores. Bertrand and co-workers (2009) also presented data that show similar paterns in sidero- phore production and microorganism growth at diferent media pH. The siderophore production delay should be further investigated to determine what celular processes, at the genetic or molecular levels, controled it and what ecological or evolutionary importance has. This approach may determine if there are feritins, bacterioferitins or Dps proteins in Halomonas sp SL01 that may serve as iron res- ervoirs (Andrews et al. 2003), therefore, explaining sidero- phore production delay. Halomonas sp. SL01 was found to produce two diferent suites of siderophores, each found to contain two distinct amino acid head groups. These amino acid head groups, in addition to the ability to chelate iron, possess polar prop- erties due to the amino acid composition. Fragmentation paterns found in the MS analysis were conserved within each family, suggesting the common headgroup. The pres- ence of proline, phenylalanine and cysteine was surprising, but previous research by other groups has demonstrated siderophores that contain these amino acids: amonabactins (Telford and Raymond 1997), pyochelins (Cox et al. 1981; Liu and Shokrani 1978; Quadri et al. 1999), yersiniabactins (Heesemann et al. 1993; Suo et al. 1999) and thioquinolo- bactins (Mathijs et al. 2007). The incorporation of serine, arginine, threonine, glutamine, thr-β-OH-asp and cyclized ornithine is common for amphiphilic siderophores (Dhun- gana et al. 2007; Homann et al. 2009b; Martinez and Butler 2007; Rosconi et al. 2013; Vraspir et al. 2011). In contrast to the aforementioned studies, our present report describes the first cysteine-, phenylalanine- and proline-containing amphiphilic siderophores produced by a halophile isolated from a soda lake. As mentioned previously, Halomonas sp. SL01was iso- lated from Soap Lake, WA (a hypersaline lake). The CAS assay detected siderophore production by the halophile, demonstrating iron binding activity (Schwyn and Neilands 1987). The chrome azurol sulfonate weakly binds feric iron and transfer of the cation occurs in the presence of high affinity molecules (iron chelating), like siderophores. Mass spectrometry experiments confirmed amphiphilic structure and showed that halochelins are related, but diferent, to previously characterized siderophores from non-hyper- saline/alkaline environments (Martinez and Butler 2007; Vraspir et al. 2011). Because of this affinity of halochelins to feric iron as demonstrated by the CAS assay (McMil- lan et al. 2010; Neilands 1976; Schwyn and Neilands 1987; Zheng and Nolan 2012), it is possible that halochelins pro- duced by Halomonas sp. SL01 play a role in the bioavaila- bility of iron within Soap Lake. Soap Lake is a meromictic soda lake, containing three layers, the mixolimnion, chem- ocline and monimolimnion layers, which are permanently stratified and do not physicaly mix. Halomonas sp. SL01 was isolated from the upper layer (mixolimnion) which has more exposure to light and oxygen compared to the lower layers. Also, the mixolimnion has a lower dissolved solid concentration (about 14 g/L) compared to the monimolim- nion (140 g/L), but high when compared to other lake types that intermix their layers (Edmondson and Anderson 1965; Sorokin et al. 2007). Soda lakes dissolved solids values are higher compared to hydrochemical studies of freshwa- ter lakes (Conzonno and Ulibarena 2010; Karatayev et al. 2008). Karatayev and co-workers measured average dis- solved solids at 119 mg/L in 550 lake in Belarus and Conz- onno and Ulibarena determined the value at 10 g/L in Lago Grande, Argentina. These high dissolved solids in soda lakes could provide iron hydroxides that are not soluble in aerobic, alkaline environments (Duckworth et al. 2009) but alowing haloalkaliphilic microorganisms to use sidero- phores to obtain feric iron. Evidence of carbon, sulfur and nitrogen biogeochemistry in soda lakes has been described and reviewed (Sorokin et al. 2014); however, information on iron cycling is limited (Emmerich et al. 2012). Emmerich and co-workers studied the mineralogy, geo- chemistry and microbial ecology of Lake Kasin (South- ern Russia). Feric oxides content in lake sediments were reported to be 1.13 % (w/w) and a comparison of feric reducing microorganisms most probable number (MPN) counts against feric oxidizing microorganisms values Fig. 3 Halochelins D and F, produced by Halomonas sp. SL01. On top, the polar head group is presented with the amino acid sequence. On the botom, the “R” represents the aliphatic tail (faty acids) atached to it revealed similar numbers. However, the research was done on sediments (no pelagic lake waters) and no information on siderophore-mediated iron acquisition was linked to fer- ric iron cycling. Bacteria and Archaea isolates (from Lake Kasin sediment samples) sequencing data revealed the pres- ence of potential feric iron-reducing microorganisms, but siderophore-producing organisms were not identified. The marine amphiphilic siderophores, such as the aquachelins, are photoreactive and this may play an important role in biotic and abiotic feric iron cycling (Barbeau et al. 2001). Photochemical reactivity causes a cleavage on aquachelin, separating it from its faty acid moiety and reduces fer- ric iron to ferous iron. However, the polar head group of aquachelin remains active and continues to facilitate feric iron chelation. Therefore, photochemical reactivity as an abiotic process likely contributes in feric iron cycling in pelagic ocean waters providing reduced iron to organisms. How halochelins in pelagic hypersaline lake waters func- tion or contribute to iron cycling is to be investigated, but photoreactivity may play a significant role based on their structural similarity to the aquachelins and other photoreac- tive amphiphilic siderophores. Another question lies in the relevance, importance or function that the faty acyl moieties themselves serve within amphiphilic siderophores. The main hypothesis is that ali- phatic tails may provide siderophores the ability to interact with the cel membrane, alowing exposure of the molecule to the extracelular environment without totaly releasing the molecule to the extracelular environment. Diferent difusion limitation or prevention mechanisms utilized are: (1) acylated siderophores that anchor in the cel membrane; (2) polysaccharide- or matrix-mediated protection of the siderophore in a sheltered environment, limiting sidero- phore release; or (3) siderophore piracy and utilization of siderophores from various bacteria (Martinez and Butler 2007; Stintzi et al. 2000). Siderophores with smal peptide head groups and longer faty acid tails (>C15) would be more likely associated with the cel (Martinez and Butler 2007) as shown with the siderophore marinobactin F, which has those characteristics. In the case of marinobactin F, the supernatant concentration of this molecule was low, but halos present (due to the difusion of the siderophore within the solid medium surounding bacterial colonies) on inocu- lated CAS assay plates suggested the possibility that the amphiphile could remain closely associated with the Mar- inobacter sp cels. Another study presented by the same group shows how marinobactin E in its deferated form has more affinity for L-α-dimyristoylphosphatidilcholine vesicles than its ferated counterpart (Xu et al. 2002). It is apparent the importance for the bacterial cel to prevent siderophore difusion in pelagic ocean waters to optimize iron acquisition in such diluted environments. To confirm this importance in hypersaline lakes, it would be required to assess halochelins membrane interactions and affinity with the coresponding experiments. In addition to Halomonas sp. SL01, other microorgan- isms from Bacteria and Archea produce diverse sidero- phore suites, the composition of which may be in response to diferent environmental conditions. Valdebenito and co- workers (2006) described how neutral or alkaline media afected siderophore type produced by E. coli Nissle 1917. Enterobactin and aerobactin were produced at higher con- centrations in more acidic media compared to salmoche- lin and yersiniabactin. Pseudomonas aeruginosa produces both pyoverdine and pyochelin siderophores and each one acts in diferent ways that contribute to pathogenicity. Pyo- verdine helps in biofilm formation (Banin et al. 2005); meanwhile, pyochelin helps in host immune response eva- sion by not binding to siderocalins (proteins that hi-jack siderophores) in mammals (Abergel et al. 2006). In this case, pyochelin stil provides iron to Pseudomonas aerugi- nosa alowing its growth and survival in spite of the host’s innate immunity by means of restricting iron. Some bacte- ria wil produce the hydrophilic form of the siderophore but others, due to a change in hydrophilicity, incorporate faty acids to them (Martin et al. 2006). This wil provide an amphiphilicity to the molecule and helps in the organism’s survival. Additional studies regarding halochelin synthesis and the distribution of siderophore type produced by SL01 in response to a variety of environmental conditions could provide insight into that organism’s adaptions to changing the environmental conditions. Previous microbiological data in soda lakes, or hyper- saline lakes, have shown a diverse microbial population and examples of these environments are Sambhar Lake in India, Soap Lake in WA, USA and Mierlei Lake in Romania (Dirnitriu et al. 2008; Sahay et al. 2012). Simi- lar hypersaline study sites include salt marshes and salterns (Ghozlan et al. 2006). Bacterial phyla observed in these extreme environments are Proteobacteria, Firmicutes and Actinobacteria and diferent Halomonas-related species (Halomonas campisalis, H. titanicae, H. taeanensis and H. elongata among others) have been isolated, sequenced and identified (Sorokin et al. 2014). Halobacteriacea and Methanomicrobia are archaea phyla identified in hypersa- line environments and haloarchaeal species (Haloferax vol- cani, Halobacterium sp., Halogeometricum borinquense, Haloarcula and Halorubrum alkaliphilum among others) have been isolated, identified and sequenced (Anderson et al. 2011; Ghozlan et al. 2006; Sorokin et al. 2014). Dif- ferent studies provided information on siderophore-pro- ducing microorganisms. Halomonas campisalis is one of the siderophore-producing microorganisms in Sambhar Lake (Sahay et al. 2012) and the presence of siderophore synthesis genes has been described in H. borinquense; however, the siderophore structural analyses have been performed (Anderson et al. 2011). Another study by Buyer and co-workers (1991) described aerobactin production by a halophilic pseudomonad. Amphiphilic siderophores have been mostly isolated and described from marine envi- ronments: marinobactins, aquachelins, ochrobactins, loi- hichelins, amphibactins and synechobactins (Gauglitz and Butler 2013; Homann et al. 2009a, b; Vraspir et al. 2011). With the present report, it is demonstrated that amphiphilic siderophores are synthesized by microorganisms in other extreme environments, like soda lakes, which difer from those previously identified in marine waters. Structural resemblance is conserved among marine and hypersaline amphiphilic siderophores. Conclusion Prior research to date has suggested the siderophore pro- duction potential in hypersaline lakes but until now, no spe- cific identification or structural identification or structural characterization of siderophores from these environments has been made. This report presents and describes one of the first molecular structural characterizations of amphi- philic siderophores (halochelins) produced by a hypersaline lake isolate, Halomonas sp. SL01. These findings present unique amino acid residues in amphiphilic siderophores. Proline, cysteine and phenylalanine amino acid-containing head groups were discovered in these siderophores. The presence of α-hydroxycarboxylates and hydroxamates contained within these amino acids could possibly par- ticipate as iron-coordinating groups. Future studies should determine the synthesis pathways of these molecules, and examine which and how many, non-ribosomal peptidyl synthetases (NRPSs, responsible for siderophore synthe- sis) are involved in halochelin synthesis. Determination of the transport of iron into the cels via these siderophores, such as which proteins are involved in siderophore active transport (TonB-like transporter proteins), receptor proteins and periplasmic binding proteins, may yield insight into the specificity of these siderophores for an individual species or show that these siderophores could be universaly shared by multiple organisms. Comparisons between synthesis and uptake mechanisms of other species within the genus could provide more details in how the amphiphilic siderophore system evolved in other Halomonas species and describe its ecological role in diverse environments. Acknowledgments Mass spectrometry data was done with the assis- tance of Dr. Jonathan Hilmer from the Mass Spectrometry Facility in the Department of Chemistry and Biochemistry, Montana State Uni- versity-Bozeman. Special thanks to Ann Wilis for HPLC support. 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