Influence of Environmental Factors on siderophore production by Streptomyces fulvissimus ATCC 27431
M.S. Bendale, B. L. Chaudhari and S. B. Chincholkar*
School of Life Sciences, North Maharashtra University, P B 80, Jalgaon 425001,Maharashtra, India
*For Correspondence- firstname.lastname@example.org
Streptomyces fulvissimus ATCC 27431 was studied for siderophore production. Biosynthesis of siderophore was found to be influenced by environmental factors. Out of various media tried, chemically defined low iron medium gave maximum siderophore production (94 % siderophore units). A number of carbon and nitrogen compounds were tested for their effect on growth of Streptomyces fulvissimus and siderophore production. Glycerol, sucrose and glucose were good carbon sources while ammonium chloride, sodium nitrate and urea were good nitrogen sources. Moreover, 0.05 - 0.3 M of Carbon, 0.05 - 0.1 M of Nitrogen sources and 20 - 30 mM of phosphate was required for maximum siderophore production (92 % siderophore units). Increase in Fe (III) concentration > 5 µM had a negative effect on siderophore production. Optimum pH for siderophore production was 8 while temperatures below 20 ºC and above 40 ºC were not suitable for culture growth and siderophore production. To the best of our knowledge this is the first report on effects of environmental factors on siderophore production by S. fulvissimus.
Key Words: Siderophore, Streptomyces fulvissimus, Carbon source, pH
Most of the living organisms require iron for metabolic activities and growth. Aerobic metabolism of microbes requires iron for variety of functions including the electron transport chain, in deoxyribonucleotide synthesis, in the synthesis of heme and for incorporation in the proteins involved in nitrogen fixation (1, 2). Iron is the fourth most abundant element in the earth’s crust (3). Although iron is abundant in the environment, it is not readily available since it exists mostly in ferric Fe (III) state. Reduced form iron Fe (II) is soluble and biologically available that can be easily taken into the living cell using various mechanisms under aerobic conditions, ferrous is oxidized to ferric form existing as insoluble oxyhydroxide polymers (4) and hence such complexes are not easily assimilated by microorganisms. Acquisition of iron thus becomes a challenge. To solve this bioavailability problem, microorganisms synthesize and excrete selective and strong Fe (III) binding low molecular weight molecules known as siderophores (3).
Streptomyces, Gram-positive soil bacteria are well known for contributing majority of antibiotics. Apart from this typical character, the only commercially available siderophore, Desferrioxamine B, called Desferal is produced by Streptomyces pilosus which is used to treat iron overload in humans (5). Streptomyces pilosus produces a group of desferrioxamines (A1, A2, B, D1, D2, E, F, G, H) wherein B and E are predominantly present (6), S. coelicolor A 3(2) is known to produce coelichelin (7), coelibactin, DFO G1 and DFO E (8). S. antibioticus produces desferrithiocin (9) and S. griseoflavus produces ferrimycins (6). Shimi and Dewedar (10) reported production of gluconimycin by Streptomyces A S 9. Enterobactin is produced by S. tendae Tu 901/8c (11) and oxachelin by Streptomyces sp.GW9/1258 (12).
Streptomyces fulvissimus is known for production of an antibiotic, valinomycin (13), but siderophore production by Streptomyces fulvissimus has not been studied earlier. Attributes like production of spores, antibiotic and siderophore may be helpful in exploitation of this organism in plant nutrition and biocontrol of plant diseases. But as the biosynthesis and secretion of siderophores is strictly regulated by environmental factors (14), the aim of this study was to find out the suitable parameters for maximum siderophore production.
Materials and Methods
Microorganism and growth conditions
Streptomyces fulvissimus was used for this study which was maintained by frequent transfer on medium composed of (gl-1): Yeast extract 4, malt extract 4, and glucose 10, agar 20, and pH 7 + 0.2. For siderophore production, Chemically Defined Low Iron Medium (CDLIM) consisting of (gl-1): K2SO4 2, K2HPO4 3, NaCl 1, and NH4Cl 5 was used. To this solution, 2 mg thiamine and the following elements were added (mgl-1): CaCl2.2H2O 100, MgSO4.7H2O 80, ZnSO4.7H2O 2, and MnSO4 0.0035, CuSO4 0.005 and 2.5 % glycerol as an energy source (15). The culture was inoculated in CDLIM and incubated at 28 ºC for 120 h with shaking at 220 rpm.
Factors affecting siderophore production Growth medium
Various media were used to study the siderophore secretion viz Chemically Defined Low Iron Medium (CDLIM) (15). Glycerol Aspergin Medium [(gl-1): Glycerol 10 , K2HPO41, L-Aspergin 1, trace salt solution 1, pH 7.4 (Pridham and Gotttlieb trace salt solution (gl-1): CuSO4.5H2O 0.64, FeSO4.7H2O 0.1, MnCl2 0.79, and ZnSO4 0.15)], Synthetic Medium [(gl-1): K2HPO4 1, MgSO4.7H2O 0.5, KCl 0.5, NaNO3 2, Glycerol 30, pH 7.2], Casein Starch Medium [(gl-1): Casein 0.3, Starch 10, NaCl 2, KNO3 2, K2HPO4 2, MgSO4 0.09, CaCO3 0.02 , pH 7]. Siderophore production was checked after completion of 120 h of incubation at 28 °C, on a rotary shaker (Remi, Mumbai) at 220 rpm. Unless and otherwise specified in the text through out the experimentation cultivation conditions were incubation period of 120 h at 28 °C temperature and 220 rpm shaking speed.
The effect of different carbon sources such as glucose, sucrose, glycerol, arabinose, maltose, sodium acetate, lactose was studied on siderophore production by Streptomyces fulvissimus. All carbon sources were added at concentration 2.5 % w/v. Also experiments were carried out by varying the concentrations of sucrose, glucose and glycerol in the range of 0.05 – 0.4 M.
Effect of urea, sodium nitrate, ammonium chloride was studied at concentration 0.5 % w/v and varied in the concentration range of 0.04 – 0.5 M. CDLIM was fortified with different nitrogen sources.
In CDLIM, K2HPO4 is the source of phosphate. Different concentrations of K2HPO4 in the range of 0.005 – 0.05 M were used to study the effect of phosphate on siderophore production.
Iron content of CDLIM was varied by the addition of ferric chloride. Effect of iron was studied in the range of 0 - 60 µM concentration.
The effect of pH on siderophore production was studied by varying the pH of CDLIM in the range of 5 – 10.
To check the influence of different incubation temperatures on siderophore production, culture grown in CDLIM was incubated at different temperatures in the range of 20 - 44 ºC for 120 h at 220 rpm.
The culture Streptomyces fulvissimus was inoculated in CDLIM and one was incubated on rotary shaker at 220 rpm and 28 °C and another set was incubated at stationary conditions and at 28 ºC for 120 h.
Siderophore detection and partial characterization
Qualitatively siderophore production was detected by Universal chemical assay (16) and comparative account of siderophore production was done in terms of percent siderophore units (17). After incubation of 120 h at 28 ºC at 220 rpm, siderophore production by Streptomyces fulvissimus was determined. Centrifuged (10,000 rpm for 15 min) cell free supernatant was subjected to estimation of siderophore as per Chrome Azurol S (CAS) liquid assay of Payne (17). Briefly, 0.5 ml of culture supernatant was mixed with 0.5 ml of CAS assay solution. Un-inoculated medium was used as reference. The optical density (OD) at 630 nm was measured for loss of blue colour resulting from siderophore production. Siderophore produced was calculated by using following formula,
%Siderophore Units = [(Ar - As) / Ar] x 100
Ar - Absorbance of Reference
As - Absorbance of Sample at 630 nm
Hydroxamate type of siderophore was determined by Csaky’s Assay (18). After growth, cell mass was separated by centrifugation at 10,000 rpm at 4 ºC for 15 min. Siderophore was extracted by using amberlite XAD 4 column chromatography. Thin layer chromatography of the purified siderophore was carried out. Desferrioxamine mesylate (Desferal, Novartis) was used as standard.
HPLC of siderophores
Analytical HPLC of purified siderophores was carried out on chromeleon (c) Dionex version 6.60 SP8 build 1544 (Switzerland) using REFTEK, pinnacle II C18 reverse phase column (250 × 4.6 mm, 5µM integrated pre column) as stationary phase with detector (UV-Visible, PDA 100) and methanol: water (8:2 v/v) as mobile phase at flow rate of 1 ml min-1 at 25 ºC and at 220 nm.
Results and Discussion
Siderophore production by S. fulvissimus was studied by cultivating it in chemically defined low iron medium and detection by Universal Chemical Assay for siderophores developed by Schwyn and Neilands (1987). Further factors affecting siderophore production were also studied including carbon source, nitrogen source, iron, inorganic phosphate, pH, temperature and aeration.
Siderophore detection and partial characterization:
The Chrome Azurol S (CAS) assay is based on the principle of higher affinity of siderophores towards Fe (III) than CAS reagent. Siderophores acquire iron from its complex with weak chelator in the reagent due to which it undergoes decolorization. For this, the pH of the culture supernatant was adjusted to neutrality and equal amount of CAS reagent was added to it. Colour change from blue to orange was observed at λ max 630 nm which indicated siderophore production. The type of siderophore was detected by Csaky’s test where formation of red coloration indicated the presence of hydroxamate group in the siderophore.
Centrifuged cell free supernatant was concentrated under rotary vacuum evaporator (Buchi, Switzerland) and subsequently subjected to amberlite XAD 4 column chromatography. CAS test positive fractions were pooled together dried and weighed. A 90 – 100 mg / L of purified siderophore powder was obtainable and subjected to HPLC. Thin layer chromatography of the purified siderophore showed Rf value 0.65 in butanol: acetic acid: water (9:1:1) which matched with desferrioxamine mesylate (Desferal, Novartis).
HPLC analysis of purified siderophores:
The HPLC of desferrioxamine showed the peak at 6.60 min while the partially purified sample showed three peaks among which one peak had similar retention time to standard desferrioxamine (Fig. 1a, 1b), which clearly suggested the presence of desferrioxamine in the sample. Other two peaks are being studied for their characterization.
Influence of media on siderophore production
The influence of medium on siderophore production was studied in four different media such as CDLIM, GASPM, SM, and CSM. Optimum production of siderophores relies on performance of producer organism, the choice of medium, available iron content of the medium used and the iron requirement level of the organism (19). As depicted in Fig. 2, CDLIM was found to be the best medium among all others compared since it gave maximum yield of siderophores (94 % siderophore units); where as Glycerol - Asperagin medium gave 89.14 % siderophore units. In case of casein starch medium, siderophore production was lower with 83 % siderophore units, followed by synthetic medium for actinomycetes. Hence for further experiments CDLIM was used.
Influence of different carbon sources on siderophore production
Streptomyces species is reported for the utilization of simple sugars (20, 21), alcohol and some organic acids (22). Pridham and Gottlieb (23) characterized actinomycetes according to the utilization of different carbon sources. In antibiotic novobiocin production, the producer organism S. niveus favored citrate over glucose (24) while S. kanamyceticus M 27 can utilize dextrose excellently for Kanamycin production (25). Hence, Streptomyces fulvissimus was grown in CDLIM fortified with different carbon sources for 120 h at 28 ºC at 220 rpm. Sucrose followed by glucose and glycerol were proved to be the best carbon sources (Fig. 3) achieving maximum siderophore production i.e. 93.08, 92.05, 91.61 % siderophore units, respectively within 0.07 – 0.2 M range of carbon source concentration. While sodium acetate and lactose, did not support siderophore production.
Glucose, sucrose and glycerol gave higher siderophore production. Concentration of these three carbon sources was varied in a range of 0.05 – 0.4 M. Glucose did not support siderophore production above 0.2 M concentration (Fig. 4) with about 91 % siderophore units in range of 0.05 – 0.1 M concentration. In case of Sucrose (Fig. 4), the siderophore production was highest (93.25 % siderophore units) at 0.05 M concentration and thereafter it declined gradually. When glycerol (Fig. 4) was used as carbon source over a range of 0.05 to 0.4 M concentration, the siderophore production remained constant in between concentration range 0.1 – 0.3 M i.e. 91 %. The results indicated that glycerol can achieve the maximum siderophore production over a wide range of concentrations.
Influence of different nitrogen sources on siderophore production
The production of secondary metabolites is influenced by the availability of nutrients. The availability of nitrogen and its source can affect the production of secondary metabolites (26, 27). In this study, the regulation of siderophore production by nitrogen using different nitrogen sources and synthetic culture medium was investigated. Wherein urea, ammonium chloride, sodium nitrate were used. Each nitrogen source was studied at the concentration of 0.5 % w/v (Fig. 5). When sodium nitrate was used as nitrogen source, highest siderophore production i.e. 93.12 % siderophore units was obtained. Urea as nitrogen source was observed to give 83.3 % siderophore units and ammonium chloride gave 90.05 % siderophore units. Urea at concentration at 0.05 M showed 92 % siderophore units (Fig. 6) on 120 h of incubation at 28 ºC. Siderophore production gradually decreased to 23 % siderophore units as concentration was increased to 0.4 M. Ammonium chloride gave maximum % siderophore units (91 %) at concentration range of 0.05 - 0.1 M (Fig. 6) which gradually decreased to 70 % siderophore units at 0.5 M concentration. Ammonium salts are the principal nitrogen sources which have been reported to interfere with antibiotic production. Gonzalez et al., reported an increase in the growth and specific production of gentamicin, proportional to the amount of ammonium present in the culture medium for ammonium concentration ranging from 20 - 150 mM (28). In case of sodium nitrate, siderophore production was 93.19 % siderophore units at 0.05 M concentration and declined with the increase in concentration. At 0.5 M concentration it was 30 % siderophore units (Fig. 6).
Influence of inorganic phosphate on siderophore production
Phosphate concentration is one of the important factors that affect siderophore production (26). Effect of phosphate concentration in the range of 0.005 – 0.05 M on siderophore secretion by Streptomyces fulvissimus was studied. As depicted in Fig. 7, the range of 0.02 – 0.03 M phosphate concentration was found to be optimum for siderophore production, since within this range maximum (92 % siderophore units) siderophore production was recorded which declined at higher concentrations. Very high or low phosphate concentration did not support siderophore production. It needs to have specific concentration of phosphates which is in agreement with results obtained by Barbhaiya and Rao (22).
Influence of iron concentration on siderophore production
Among environmental factors, iron concentration is the most important that mainly regulates the biosynthesis and secretion of siderophore (29, 30). Taking into account this factor, the influence of exaneously added Fe (III) in increasing order to the CDLIM on the siderophore production was observed. Although cell growth reached maximum at value above 5 µM of added Fe (III), (Fig. 8) siderophore production was lowered at this concentration. In the absence of exogenous iron / less than 1 µM Streptomyces fulvissimus produced maximum siderophores while in iron containing media, siderophore production was decreased proportionally with the increase in iron concentration. Concentration > 5 µM of iron could not induce siderophore production. Thus indicating that 5 µM of iron showed the iron sufficiency which lead to negligible amount of siderophore production. Iron more than 5 µM enhanced the growth of S. fulvissimus but responsible for repression of siderophore production. Similar results have been reported by Yang et al. (31), for siderophore production by P. pseudomallei U7.
Influence of pH on siderophore production
The pH plays vital role in the solubility of iron in production medium and thereby siderophore production. Iron is insoluble at neutral to alkaline pH but solubility increases at acidic pH values (32). So at the acidic conditions iron is available to microorganisms which in turn reduce the siderophore production.
For Streptomyces fulvissimus, pH 8 was found to be optimum for siderophore secretion (Fig. 9). At pH 8, highest siderophore production (93 % siderophore units) was seen, while very sharp decrease at pH 10. At alkaline pH, solubility of iron is highly affected therefore theoretically siderophore yield should increase as the available iron is less. But poor growth of Streptomyces fulvissimus would be the main reason for sharp decrease in siderophore production at high alkaline pH (above 9). Very poor growth and the higher alkaline conditions did not support siderophore production. In contrast, at acidic pH, iron is in soluble form, can repress the siderophore production and so the yield was negligible.
Influence of temperature on siderophore production
To check the influence of temperature on siderophore production, S. fulvissimus was grown at various temperatures (Fig. 10). At 28 ºC, maximum siderophore production was observed i.e. 93.47 % siderophore units which was little bit lowered at 37 ºC but no siderophore production was induced at 20 ºC and at 44 ºC when grown in chemically defined low iron medium.
This may be because at these temperatures, S. fulvissimus was not able to grow. Similar results have been reported earlier. Worsham and Konisky (33) have reported decreased siderophore production by S. typhimurium at elevated temperatures, Garibaldi (34), and Kochan (35), have also reported that in E. coli and S. typhimurium, enterochehin biosynthesis was inhibited at temperatures greater than 40 ºC. The temperature range of 28 ºC to 37 ºC was suitable for optimum siderophore production by S. fulvissimus.
Influence of aeration on siderophore production
When incubation was carried out at stationary conditions, siderophore production (Fig. 11) either did not take place or was too negligible. Incubation with aeration at 220 rpm had achieved high production i.e. 90 % siderophore units. Pseudomonas stutzeri, strain CCUG 36651, a facultative anaerobe was shown to produce siderophore when grown under aerobic conditions. In contrast no siderophores were observed from anaerobically grown P. stutzeri (36).
Streptomyces fulvissimus was found to produce siderophores under iron depleted conditions. Environmental factors such as temperature, pH, iron concentration, growth medium, carbon, nitrogen and phosphate influence siderophore biosynthesis. As siderophores have wide application in biocontrol of plant diseases and plant nutrition, these attributes are very important.
Authors are thankful to UGC for financial help through SAP- DRS project.
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