Microorganisms, media and culture conditions
As a result of a literature survey previously performed for siderophore producer bacterial strains (Ferreira et al. 2019b) and taking into to account the selection criteria presented above, the following microorganisms were chosen to be used in the present work: Azotobacter vinelandii Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM) 2289; Bacillus megaterium American Type Culture Collection (ATCC) 19213; Bacillus subtilis DSM 10; Pantoea allii DSM 25133; and Rhizobium radiobacter DSM 30205. The original strains were obtained from DSM, Germany, or ATCC, U.S.A. All these bacteria belong to Risk 1 group, according to the U.S. Department of Health and Human Services, Centers for Disease Control, and National Institutes of Health (2009).
The microorganisms were maintained in minimal medium (MM) agar, except A. vinelandii, which was maintained in Burk´s medium (BM) agar, at 4 °C. MM agar contained per litre: 10 g glucose, 1.47 g glutamic acid, 3.0 g potassium hydrogenophosphate (K2HPO4), 1.0 g potassium dihydrogenophosphate (KH2PO4), 0.5 g ammonium chloride (NH4Cl), 0.1 g ammonium nitrate (NH4NO3), 0.1 g sodium sulphate (Na2SO4), 10 mg magnesium sulphate heptahydrate (MgSO4·7H2O), 1 mg magnesium sulphate tetrahydrate (MnSO4·4H2O), 0.5 mg calcium chloride (CaCl2) and 20 g agar. BM agar was prepared as previously described (HiMedia Laboratories 2015) replacing sucrose by glucose; the medium contained per litre: 10 g glucose, 0.8 g K2HPO4, 0.2 g KH2PO4, 0.20 g MgSO4·7H2O, 0.253 mg sodium molybdate, 0.13 g calcium sulphate and 20 g agar. The final pH of the media was set to 7.0 ± 0.1. For iron-replete media, 29 mg of iron(III) chloride (FeCl3) was also added.
All reagents used were obtained from Merck (Darmstadt, Germany), Panreac (Barcelona, Spain), Sigma-Aldrich (St. Louis, Missouri, EUA) or BD Difco (Waltham, Massachusetts, EUA). Inorganic chemicals were pro-analysis grade while organic chemicals were Ph. Eur. grade.
In order to avoid iron contamination (on iron-deficient-cultures), all glassware was soaked in 10% nitric acid, overnight and, subsequently, washed with deionized water prior to use.
All pre-starter cultures, with exception of A. vinelandii, were prepared by inoculating the bacteria in 20 mL of iron-replete MM broth in 100 mL Erlenmeyer flasks. Cells were incubated at 30 °C, for 8 h, in an orbital shaker at 150 rpm. Starter cultures were prepared by inoculating 40 mL of iron-replete MM broth, in 100 mL Erlenmeyer flasks, with an appropriate volume of the pre-starter cultures, and then incubated overnight to an OD600 ≈ 2.0, under the same conditions described for the pre-starter cultures. Cells, in exponential phase of growth, were harvested by centrifugation (3000×g, 10 to 30 min), rinsed and suspended in iron-deficient media. Next, cultures were obtained by inoculating the cells in 400 mL of iron-deficient MM broth (initial OD600 ≈ 0.1), in 1 L Erlenmeyer flasks. Cells were incubated under the same conditions as described above. For A. vinelandii, there were some differences in the protocol: BM broth was used instead of MM broth and only a starter culture was grown, for 24 h, due to the slower growth rate of the bacterium.
Determination of siderophore production
Due to its easiness and ability for a high and sensitive detection of siderophores (Schwyn and Neilands 1987), the chrome azurol S (CAS) method was used (Alexander and Zuberer 1991) in the estimation of the bacterial siderophore production. Therefore, samples were taken and cells were pelleted by centrifugation (3000×g, 10 to 30 min). The supernatant was carefully removed and, subsequently, filtered through a 0.45 μm pore size filter and immediately stored at − 20 °C until siderophore determination.
CAS method consists in mixing 1.0 mL of filtrate, properly diluted with deionized water, with 1 mL of CAS assay solution, prepared as described by Alexander and Zuberer (1991). One reference solution was also prepared by mixing 1.0 mL of CAS solution with 1.0 mL of deionized water. A zero-absorbance solution was also prepared by mixing 1.0 mL of CAS solution with 1.0 mL of 100 µmol L−1 desferrioxamine mesylate salt (desferal). The solutions were left to reach chemical equilibrium at room temperature in the dark for 24 h. Next, absorbance was read at 630 nm. A calibration curve was performed by plotting the ratio A/Aref versus the concentration of desferal; where, A is the standard solution absorbance at 630 nm and Aref stands for absorbance of the reference at 630 nm. Siderophore production is expressed as µmol L−1 desferal equivalent. For each culture, samples were read in triplicate and repeated independently at least two times (that is, two independent cultures read in triplicate).
Siderophore qualification
The type of siderophore present in each bacterial medium was characterized using Arnow’s and the Csaky’s methods for catecholates and hydroxamates, respectively (Payne 1994).
Arnow method is based on the reaction between catechol and nitrite-molybdate reagent, in acidic conditions, originating a yellow colour. The colour changes to an intense orange-red in alkaline conditions. For this purpose, 1.0 mL of culture filtrate was combined with 1.0 mL of HCl 0.5 mol L−1. Subsequently, 1.0 mL of nitrite-molybdate reagent was added and then 1.0 mL of NaOH 1.0 mol L−1. The assay was incubated at room temperature, for approximately 5 min, to allow full colour development. As blank, 1.0 mL of deionised water was used. Nitrite-molybdate reagent was prepared by dissolving 10 g of sodium nitrite and 10 g of sodium molybdate in 100 mL of deionized water. If catecholate-type siderophore is present, the solution presents an orange-red colour. The colour intensity is dependent of the amount of catechol present (Arnow 1937).
The Csaky’s test detects hydroxamate-type siderophores and depends on oxidation to nitrite and formation of a coloured complex via diazonium coupling (Csáky 1948). Firstly, 1.0 mL of culture filtrate was hydrolysed with 1.0 mL of 6 N H2SO4, at 130 °C, for 30 min. The solution was then buffered with the aid of 3.0 mL of sodium acetate (350 g L−1) and 1.0 mL of sulfanilic acid [10 g L−1, 30% acetic acid (v/v)] was added, followed by 0.5 mL of iodine solution (13 g L−1, in glacial acetic acid). The solution was allowed to settle for 3–5 min, after which the excess of iodine was neutralized by the addition of 1.0 mL of 20 g L−1 sodium arsenite. Finally, 1.0 mL of α-naphthylamine solution [3 g L−1, in 30% (v/v) acetic acid] was added and the colour was allowed to develop for 20–30 min. The presence of hydroxamates in solution was confirmed by the presence of a deep pink colour.
Synthetic N,N-dihydroxy-N,N′-diisopropylhexanediamide (DPH), a hydroxamate, and Azotochelin, a catecholate, were used as controls (Martins et al. 2018). As blank, 1.0 mL of deionised water was used in both assays.
Complexation capacity assays
The complex capability of each culture medium containing siderophore was tested using a procedure adapted from Villen et al. (2007). Briefly, to a fixed volume of culture filtrate, FeCl3 was added, the solution pH was set to 9.0 ± 0.1 and let to rest for 30 min. Then, pH was corrected again and let to settle for 3 h. The solution was subsequently centrifuged (3000×g, 10 min) and filtered by a 0.45 µm pore size membrane. The amount of Fe in solution was then determined (see “Iron determination” bellow). A graphical representation of the ratio [Fe]complex/[L] versus [Fe]added/[L] was plotted in order to represent the iron complexation capability of the siderophore in solution; where [Fe]complex is the concentration of Fe found in the filtrate, [Fe]added is the total Fe concentration added and [L] is the concentration of siderophore determined by CAS method.
Bacteria staining
Bacterial cultures were collected and cells were fixed with 3.5% (w/v) formaldehyde for 2 h. Cells were then harvested by centrifugation and re-suspended in 10 mmol L−1 phosphate-buffered saline (PBS) solution (pH 7.0) with 3.5% (w/v) formaldehyde and stored at 4 °C, until to be observed.
Previously stored cells were pelleted by centrifugation and washed twice with PBS buffer (pH 7.0). Cells were re-suspended in PBS buffer with 3 µmol L−1 4,6-diamidino-2-phenylindole (DAPI) and incubated for 15 min in the dark at room temperature. DAPI is a cell membrane permeant stain, which exhibits a strong blue fluorescence upon bonded to adenine–thymine rich regions in DNA (Haugland 2005).
Microscopy
Bacteria were observed by phase-contrast or epifluorescence using a microscope with a HBO 100 mercury lamp and the A filter (excitation filter BP 340–380, dichromatic mirror 400, and suppression filter LP 425) from Leica. The images were captured with a Leica DC 300F camera (Leica Microsystems, Heerbrugg, Switzerland) using a 100 × oil immersion N plan objective and processed using Leica IM 50-Image manager software.
Iron determination
Iron determinations in cell cultures and in complexation capacity assays were carried out by atomic absorption spectroscopy with flame atomization (AAS-FA) using a Perkin Elmer AAnalyst 400 spectrometer (Norwalk, CT, USA).
Reproducibility of the results
All experiments were repeated, independently, at least, two times. Data is presented as mean values. Growth-curves were performed in duplicate; for each time, growth was monitored in triplicate. Data reported for siderophore concentrations and iron complexation experiments are the mean ± standard deviations of at least six determinations.