Abstract: The Rhizobium sp. isolated from stem nodules of Sesbania procumbens (Roxb.) W and A was studied for its ability to produce siderophores on Chrome-Azurol S agar medium. The symbiont was able to produce catechol-type of siderophores in culture after 4 h of incubation. Maximum siderophore production was observed after 24 h. Carbon and nitrogen sources greatly influence the siderophore production. Among the carbon and nitrogen sources, mannitol (2%) and glutamine (0.1%) were found to increase the siderophore production. Thin Layer Chromatography (TLC) of the siderophore extract showed the presence of 2, 3-Dihydroxy Benzoic Acid (DHBA) and 3, 5-DHBA. Arginine, glutamine and proline were identified as conjugated amino acids of siderophore extract.
INTRODUCTION
Leguminous plants undergo a symbiotic relationship with respective root and stem nodule bacteria. The relationship is iron dependent, since iron is required for nodule formation as well as synthesis of components, such as nitrogenase complex and leghaemoglobin, required for nitrogen fixation (Raychaudhuri et al., 2005). Siderophores are low molecular weight high-affinity ferric iron chelators, synthesized and secreted by many microorganisms in iron deprivation. The compounds solubilize and bind iron and transport back into the microbial cell, usually through specific membrane receptors (Payne, 1994). Besides microbial iron nutrition, many siderophores also play a very important role in microbial infection and the antagonism of Plant Growth Promoting Rhizobacteria (PGPR) against plant pathogens (Franza et al., 2005). Basically siderophores are considered to be two types, viz., secondary hydroxamic acids and catechol type (Neilands, 1981). Further, most of the catechols are derivatives of 2,3-Dihydroxy Benzoic Acid (DHBA) and consists of 2,3-DHBA and one or more amino acid residues (Xie et al., 2006).
Sesbania procumbens (Roxb.) W and A. is an important weed legume growing in waterlogged soils of India. It possesses stem nodules in addition to root nodules (Shenbagarathi and Shanmughasundaram, 1992). Very little is known about the Rhizobium sp. associated with stem nodules of this host. The aim of this study was therefore to investigate siderophore production in culture of Rhizobium sp. isolated from stem nodules of S. procumbens and regulation of various factors such as incubation time, pH, carbon and nitrogen sources along with characterization and identification of conjugated amino acids in the siderophore.
MATERIALS AND METHODS
Microorganism and Growth Conditions
The symbiont was isolated from the fresh healthy stem nodules of S. procumbens
plants grown in the Acharya Nagarjuna University Campus in December 2006, using
Yeast Extract Mannitol Agar (YEMA) medium. Identification of the isolate was
carried out on the basis of morphological, cultural and biochemical characteristics
on YEM broth by standard method (Holt et al., 1994) and by plant infection
test (Vincent, 1970).
Chrome-Azurol S (CAS) Agar Medium for Detection of Siderophores
For siderophore detection Chrome-Azurol S (CAS) agar medium (Schwyn and
Neilands, 1987) devoid of iron was used. The isolate was grown on synthetic
medium (with 10 μM iron and without iron) of known composition (Jadhav
and Desai, 1992) for 24 h on a rotary shaker at 30±2°C. After 24
h, the culture was centrifuged and the cell free supernatant was applied to
CAS plates containing wells made with cork borer.
Arnow’s Assay for Detection and Estimation of Catechol-type of Siderophores
Catechol-type of siderophores in the culture supernatant was detected and
estimated by Arnow’s assay (Arnow, 1937) and concentration of catechol
was measured in μg mL-1 by the standard curve prepared with
2,3-DHBA.
Atkin’s Assay for Detection of Hydroxamate-type of Siderophores
The culture supernatant was further tested using Atkin’s assay for
the detection of hydroxamate-type of siderophores (Atkin et al., 1970).
Siderophore Production as a Function of Time
The culture was grown in 50 mL of basal medium with constant shaking on
rotary shaker (120 rpm) at 30±2°C for 24 h. Samples were withdrawn
after every 4 h intervals and measured for growth (optical density at 600 nm)
and siderophore concentration.
Effect of pH on Siderophore Production
Basal medium with different pH in the range 4-10 were prepared separately
and inoculated with Rhizobium sp. to test the effect of various pH levels
on growth and siderophore production.
Effect of Carbon and Nitrogen Sources
In order to study the effect of carbon sources (1%) on growth and siderophore
production, mannitol in the original basal medium is replaced with different
carbon sources (glucose, mannose, galactose, sucrose, arabinose, citrate, fumarate,
acetate, lactate and malate) and the effect of different concentrations (0.5,
1.0, 1.5, 2.0, 2.5, 3.0%) of most effective carbon source on growth and siderophore
production were also studied.
The effect of nitrogen sources (0.1%) was also studied by replacing sodium glutamate in the basal medium with lysine, ornithine, arginine, urea, proline, glutamine, glycine and aspartic acid. Growth and siderophore production were measured after incubation.
Extraction and Identification of Siderophore
Extraction of catecholate-type of siderophores in culture supernatant was
done by the method described by Jadhav and Desai (1992). Identification of 2,3-DHBA
and 3,5-DHBA was done by Thin Layer Chromatography (TLC) with a solvent system
containing benzene: toluene: acetic acid (2:2:1).
Identification of Conjugated Amino Acids
The siderophore extract was taken and hydrolyzed with 6 N HCl. It was neutralized
with 1N NaOH and the identification of amino acids in the sample was done by
paper chromatography with a solvent system containing butanol: acetic acid:
water (12:3:5).
RESULTS AND DISCUSSION
Based on morphological, cultural and biochemical characteristics, the isolate was identified as Rhizobium sp. The identification of bacteria was done according to Bergey’s Manual (Jordan, 1984). When culture supernatant was applied to the wells of the CAS plates, orange to yellow colour halo was produced around the well, indicating the production of siderophores (Fig. 1). A halo was observed from the supernatant of cultures grown in iron restricted media and the cultures grown under high iron conditions create no colour change. The supernatant was further tested using Arnow’s and Atkin’s assay for the detection of catechol and hydroxamate type of siderophores respectively, because CAS assay does not indicate the type of siderophores being produced. When the culture supernatant was tested with Arnow’s reagent, pink to deep red colour was produced, indicating the presence of catechol type of siderophores. But, hydroxamate type of siderophores was not detected in the culture supernatant.
Siderophore production started after 4 h of incubation. Maximum concentration was observed after 24 h, when the organism just entered into stationary phase (Fig. 2). That the Rhizobium sp. isolated from cowpea also produces catechol-type of siderophores after 4 h and reached maximum after 22-24 h (Jadhav and Desai, 1992).
Effect of pH on growth and siderophore production by the Rhizobium sp. in iron containing medium revealed that the growth and siderophore production started at pH 4.5 and reached maximum at neutral pH (Fig. 3). At pH 10.0, there was no siderophore production. This may be due to the presence of insoluble form of iron at neutral pH and therefore not available to the bacteria. With increasing pH (towards alkalinity), siderophore production decreased. This may be due to the fact that alkaline pH helps in excess solubilization of iron, which increases the iron content of the medium and ultimately results in decrease in siderophore production as reported by Schwyn and Neilands (1987).
Among the 11 carbon sources tested, maximum siderophore concentration was observed in mannitol containing medium (Table 1). Mannitol at 2% concentration promoted both growth and siderophore production (Fig. 4). Siderophore production was not detected in mannose, galactose, arabinose, citrate, acetate, lactate and malate.
| Fig. 1: | Chrome-Azurol S Assay. A) high iron supernatant (10 μM). B) Uninoculated medium C) no iron supernatant D) low iron supernatant |
| Fig. 2: | Growth and siderophore production by Rhizobium sp. from stem nodules of S. procumbens. Data are the mean of triplicates and bar at the points indicate±SE |
| Fig. 3: | Effect of pH on growth and siderophore production by Rhizobium sp. from stem nodules of S. procumbens |
| Table 1: | Effect of carbon sources on siderophore production |
| *: Without carbon source | |
That the production of siderophores varied with type of carbon sources was reported earlier in Pseudomonas sp. (Sayyed et al., 2005). Among the eight nitrogen sources tested, maximum siderophore concentration was observed in glutamine followed by arginine, proline and ornithine (Table 2). Lysine has no effect on siderophore production. Deelip et al. (1998) found that arginine had stimulatory effect on siderophore production in Pseudomonas sp.
TLC of the extracted sample was carried out and then spots were developed with Hathway’s reagent (K-ferricyanide, 0.3 g; FeCl3, 0.3 g; distilled water, 100 mL). Two clear blue spots developed. The position of one spot matched that of authentic 2,3-DHBA and the position of other spot exactly matched that of authentic 3,5-DHBA (Table 3). That the two types of compounds (2, 3-DHBA and 3, 4-DHBA) were also observed in siderophore extract of cowpea Rhizobium (Jadhav and Desai, 1992).
| Fig. 4: | Growth and siderophore production by Rhizobium sp. from stem nodules of S. procumbens on different concentrations of mannitol |
| Table 2: | Effect of nitrogen sources on siderophore production |
| *: Without nitrogen source | |
| Table 3: | Identification of DHBA in the siderophore extract by TLC |
| Table 4: | Identification of amino acids in the siderophore extract by TLC |
When acid hydrolyzed siderophore was analyzed by paper chromatography, it was found to contain arginine, glutamine and proline (Table 4). Amino acids are known to conjugate with DHBA in the number of siderophores reported earlier. In cowpea Rhizobium lysine and alanine have been reported to be conjugated amino acids (Jadhav and Desai, 1992). The conjugation of amino acids with DHBAs in our siderophore might be responsible for the increased efficiency of the compound in iron transport, as it has been reported in the case of azotobactin and azotochelin, where unconjugated DHBA does not promote significant iron uptake, whereas the conjugated azotobactin and azotochelin could promote iron uptake in Azotobacter vinelandii (Knosp et al., 1984).
ACKNOWLEDGEMENTS
We thank Andhra Pradesh Council of Science and Technology (APCOST), Hyderabad, India for financial assistance in the form of Young Scientist Fellowship (YSF) to MS.