Plant-Growth-Promoting-Rhizobacteria (PGPR) are involved in plant growth
promotion through the production of phytohormones, solubilization of insoluble
phosphates and biocontrol of plant pathogens by the production of siderophores
(Mandal et al., 2007). Rhizobia are now considered as PGPR. Many
Rhizobium sp. are known for their production of siderophores, but
only few of them have been structurally characterized. These include anthranilate,
citrate, rhizobactin and other carboxylates, vicibactin as well as unidentified
catechols and hydroxamates (Carson et al., 2000). Many other strains
of rhizobia have not been examined for siderophore production. Basically,
siderophores are considered to be of two types, viz., catechol and hydroxamic
acids (Neilands, 1981). Further, hydroxamates are classified either into
mono, di and trihydroxamates based on maximum absorption in UV-spectrophotometer
scanning (Carson et al., 2000).
Sesbania sesban (L.) Merr. is an important green manure crop,
widely cultivated in South India. Very little is known about siderophore
synthesizing capacity of Rhizobium strains from this host. Hence,
the present work was taken up to study the siderophore synthesizing
capacity of 26 Rhizobium strains isolated from root nodules of
S. sesban and also attempted to optimize the cultural and nutritional
conditions of the Rhizobium strain, which produced maximum amount
MATERIALS AND METHODS
Microorganism and Growth Conditions
Twenty six Rhizobium strains were isolated from root nodules
of S. sesban, collected from different regions of Andhra Pradesh,
India. The identity of the strains as Rhizobium was confirmed by
Bergey`s Manual of Systematic Bacteriology (Jordan, 1984) as well as plant-infection
test (Vincent, 1970). The study was conducted in December 2006, in the
Department of Microbiology, Acharya Nagarjuna University.
For siderophore production, Fiss-glucose mineral medium (K2HPO4,
5.0 g; L-asparagine, 5.0 g; glucose, 5.0 g; ZnCl2, 0.05 g;
MnSO4, 0.01 g; MgSO4. 7 H2O, 4.0 g L-1)
was used (Vellore, 2001).
Hydroxamate-type of siderophores was detected and estimated in the
culture supernatant by ferric-perchlorate assay (Atkin et al.,
Catechol-type of siderophores was detected and estimated in culture
supernatant by Arnow`s assay (Arnow, 1937).
Optimization of Cultural Conditions
Siderophore Production as a Function of Time
The culture was grown in Fiss-glucose mineral medium with constant
shaking (120 rpm) at 30±2°C for 36 h. Samples were withdrawn
every 4 h intervals and measured for growth (Optical Density at 600 nm)
and siderophore production (OD at 480 nm).
Influence of Media Components
Fiss-glucose mineral medium was modified to determine the siderophore
production. Each of four media components was tested for its effect of
varying their concentration in the minimal media. Also the minimal medium
was supplemented with 1% mannitol or 1% sucrose as an alternate carbon
sources. NH4Cl or (NH4)2SO4
(0.1%) was also added to the media as an extra nitrogen sources and the
effects were evaluated. After 24 h incubation, growth and siderophore
production were measured in each media type.
Influence of Iron
The effect of different concentrations (0.5-100 μM) of iron (FeCl3.
6H2O) was added to Fiss-glucose mineral medium to determine
growth and siderophore production.
Extraction and Purification of Siderophores
The culture was grown in large volumes for 24 h at 28°C on a rotary
shaker. After incubation, the culture supernatant was collected by centrifuging
at 7000 rpm for 30 min. The supernatant was then acidified to pH 2.0 with
6M HCl in order to make the siderophores less soluble in water.
The acidified supernatant was passed through XAD-2 column (30x5 cm) and
eluted with methanol. Fractions were collected and tested on Thin Layer
Chromatography (TLC) plates using solvent system n-butanol: acetic acid:
distilled H2O (12: 3: 5). The plates were developed with 0.1
M FeCl3 in 0.1 N HCl.
Spectral Scan Analysis
A spectral scan (300-700 nm) was done on the purified siderophore
to determine whether this hydroxamate-type siderophore was a dihydroxamate
(maximum absorption range-500-520) or trihydroxamate (maximum absorption
range, 420-440 nm) as suggested by Jalal and Vander Helm (1991).
Amino Acid Analysis
The pure siderophore was hydrolyzed with acid (6N HCl) and alkali
(6N NaOH). Amino acid standards were prepared using 20 different amino
acids. Identification of the amino acids in the sample was done with TLC
using solvent system (methanol:ammonium acetate, 60:40) and sprayed with
ninhydrin (0.25% w/v) in acetone.
Analysis of Outer Membrane Receptor Proteins (OMRPs)
The culture was grown in presence and in absence of iron and whole
cell proteins and membrane proteins were prepared (Filip et al.,
1973). SDS-PAGE was carried out by the method described by Laemmli (1970).
RESULTS AND DISCUSSION
During the screening of 26 Rhizobium strains from S. sesban
for siderophore production, it was observed that nine strains produced
catechol-type of siderophores and 20 strains produced hydroxamate-type
of siderophores. Because, maximum number of strains produced hydroxamate-type
of siderophores, further studies were carried out for hydroxamate-type
of siderophores. However quantitative studies showed that, only the Rhizobium
strain 22 produced maximum amount of hydroxamates. The strain started
siderophore production at 8-9 h post inoculation, with maximum production
at 24 h (Fig. 1).
Fiss-glucose mineral media was used in the preliminary characterization
of siderophores produced by Rhizobium isolates. However, this media
needed to be optimized to achieve maximum siderophore production for purification.
Each component added to the media stock was varied separately to determine
its effect on siderophore production. No change in any of these components
was kept the same in minimal media. A variety of media combinations were
tried to optimize siderophore production. This include Fiss-glucose medium
supplemented with 1% mannitol, Fiss-glucose supplemented with 1% sucrose,
Fiss-glucose supplemented with 0.1% (NH4)2SO4,
0.1% NH4Cl, Fiss-glucose supplemented with 1% mannitol and
0.1% (NH4)2SO4, Fiss glucose medium supplemented
with 1% mannitol and 0.1% NH4Cl, Fiss-glucose supplemented
with 1% sucrose and 0.1% (NH4)2SO4 and
Fiss-glucose medium supplemented with 1% sucrose and 0.1 NH4Cl.
Addition of 1% sucrose and 0.1% (NH4)2SO4
to the original Fiss-glucose medium greatly increased the amount of siderophore
produced. Cultures grown in this media produced almost 4-fold more siderophore
than that produced in Fiss-glucose minimal media (Fig. 2).
||Effect of Incubation period on growth and siderophore production
by Rhizobium strain 22
||Hydroxamate production measured with various media
||Effect of iron concentration on growth and hydroxamate
production by Rhizobium strain 22
The addition of limited amount of iron in the media can increase growth
of the culture, which can lead to an increased production of siderophores.
However, higher concentrations of iron in the media can completely suppress
production of siderophores. In a medium where iron has been completely removed
2-dipyridyl, cell growth can not been sustained and the
culture can not produced any siderophores. Siderophore production is highest
when a ferric iron concentration of 0.5 μM is added to the modified
minimal medium (Fig. 3
). At a concentration higher than this, siderophore
production decreases. This is consistent with the optimum iron concentration
for siderophore production of other rhizobia, in which studies have been
found to be less than 1 μM (Carson et al
||UV-spectra of the purified compound isolated from Rhizobium
||Identification of amino acids in purified siderophore
Once the growth conditions had been optimized, it was possible to produced
large amount of siderophores by growing the culture in batch culture. Around
4-5 L of cultures were grown under optimized conditions and the acidified
supernatant was partially purified through XAD-2 column chromatography.
Different fractions were collected and the fraction that gave positive result
with ferric-perchlorate assay were collected and subjected to TLC. A wine
coloured spot was developed indicating hydroxamate-type of siderophores.
The spots were scraped out and the compounds eluted from that were analyzed
by UV-spectrophotometric scanning. Spectral scans (300-700 nm) of the purified
siderophores showed the absorption maxima at 500 nm indicating dihydroxamate-type
of siderophores (Fig. 4
). The dihydroxamate-type of siderophores was also
produced by Sinorhizobium meliloti
(Carson et al
When acid or alkali hydrolyzed siderophores were analyzed by TLC, it
was found to contain tryptophan and tyrosine (Table 1). Siderophores are
often conjugate of amino acids. Dihydroxamate-type of siderophores were
first identified in Enterobacter aerogenes, is a conjugate of L-lysine
(Gibson and McGrath, 1969).
SDS-PAGE analysis was performed on whole cell pellet and membrane pellet
of culture grown in no added iron and high iron in medium to detect a
possible Outer Membrane Receptor (OMR) proteins involved in siderophore
transport. This protein should be expressed in the no added iron pellets
and repressed in high iron conditions. The molecular weights for most
described OMRPs in the range of 70-90 kDa, which is the region of focus
in the gel (Fig. 5). The SDS-PAGE showed the presence of a band in this
molecular weight range that is only present in the no added iron cultures,
indicating that it is regulated by the amount of iron in the medium. This
protein is likely involved in siderophore transport. This described for
other iron regulated OMRPs in rhizobia (Reigh and O`connell, 1993). But,
OMRPs of cultures grown in high iron media revealed the presence of single
protein band of molecular mass of 45 kDa was observed. From this study,
it may conclude that hydroxamate-type of siderophore production is most
common among Rhizobium strains and strains vary in their production
of siderophores. Moreover, the ecological advantage in the synthesis of
siderophores may enable Rhizobium strains to compete with other
species for iron uptake.
||SDS-PAGE analysis of whole and membrane pellets: (A) Molecular weight
standard, (B) Rhizobium strain 22 grown in no added iron whole
cell pellet, (C) Rhizobium strain 22 grown in high iron whole
cell pellet, (D) Rhizobium strain 22 grown in no iron added
membrane pellet and (E) Rhizobium strain 22 grown in high iron
We thank Andhra Pradesh Council of Science and Technology (APCOST), Hyderabad,
India for financial assistance in the form of Young Scientist Fellowship
(YSF) to M.S.