Chapter 3

Review of Literature

REVIEW OF LITERATURE

Siderophores play an extremely important role in microbial pathogenicity.

Microbial uptake of siderophore-iron complexes through active transport systems
allow microbes to survive and proliferate even under iron deficient environments
during invasion of a host. Due to their structural complexity, unique iron (III)
chelation, acquisition properties, and their therapeutic potential, siderophores have
attracted much attention in a broad range of disciplines. Tremendous progress has
been made in siderophore syntheses, in determination of the structures and functions
of outer membrane receptors (e.g. FhuA and FepA), and in the mechanistic insight
into siderophore-iron-mediated active transport processes20.Siderophores are
commonly produced by aerobic and facultative anaerobic bacteria and by fungi under
iron limiting condition. They are apparently absent from animal tissue, but
phytosiderophores (mugineic acid) from plant have been reported. Lots of work has
been done on their production, purification, identification and study of their
antimicrobial activity.

Fungal siderophores

Fungi have also been studied for their siderophores and their role in iron
transport has also been studied. Some research work on fungal siderophore has been
given below.

Winkelmann G gave the evidence that a variety of fungi are known to

produce and excrete desferi-siderophore complexes under iron limitation.
Siderophore-specific transport systems take such complexes and represent energy
consuming systems as they are inferred from their sensitivity to respiratory inhibitors,
uncouplers and changes of the membrane potential and recognized the structure and
stereochemical configuration of siderophore molecule.21

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Santos MA, Gapsar M, Simoes Gonclaves M.L.S et al studied new

hydroxymate type siderophore was synthesized and characterized in terms of its acid-
base behaviour. They form a stable ferric complex and were investigated by
spectrophotometric methods. Molecular mechanical methods revealed most probable
structure of ferric complex.22
Schobert R, Stanl A, Hennemann K et al Biscatechol- hydroxymate chelators
of similar structure were synthesized set of acylation reactions and were tested for
siderophoric activity in various receptor-deficient mutants of E.coli 23
Johnson L studied that most of the fungi express specific mechanisms for iron
acquisition from the hosts they infect for own survival.24 New strategies for the
control of multidrug resistant Mycobacterium strains have become a necessity for
proper management of tuberculosis. One of the strategies is the use of the iron-
sequestering agents like siderophore as active therapeutic agent in treatment of
tuberculosis. This report describes for the first time the inhibition of growth of
Mycobacterium tuberculosis H37Ra in vitro by a phytosiderophore isolated from the
root washings of Tephorsia purpurea.25
Illmer P and Buttinger R studied the influence of iron, aluminium and of the
combined application of both metals on microbial biomass and production of
siderophores by three fungi (Aspergillus nidulans, Neurospora crassa and
Hymenoscyphus ericae) were investigated. All three species showed a strong iron
regulation and Al-sensitivity of siderophore biosynthesis although several differences
remained species dependent.

Inhibitory effects of Fe and Al on siderophore-production were additive and the

higher binding capacity of siderophores towards iron could be compensated by a
higher Al-availability. Although pH itself was also important for regulation of
siderophore biosynthesis, an indirect effect of Al on siderophore production via an
Al-induced pH decrease could be outlined. The toxic effects of Al resulting in a

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reduced biomass production were compensated by high Fe-availability, whereas the

addition of DFAM, a bacterial siderophore, enhanced Al-toxicity.

Bartholdy BA, Berreck M and Haselwandter K produced siderophore from

various isolates of Phialocephala fortinii and they assessed quantitatively as well as
qualitatively in batch assays under pure culture conditions at different pH values and
iron (III) concentrations. They found a distinct effect of both of these parameters on
siderophore synthesis and as well as on fungal growth. In comparative analyses of
two of the isolates, maximum siderophore production was found at a pH in the range
of 4.0 to 4.5 while, under the experimental conditions employed, the optimal
concentration of ferric iron was determined to be between 20–40 µg iron (III)/L
(0.36–0.72 µM, respectively). HPLC analysis of the culture filtrate of most of the
isolates of P. fortinii revealed the excretion of ferricrocin as main hydroxamate
siderophore, followed by ferrirubin and ferrichrome C. The pattern of release of these
three substances proved to be dependent on pH and iron (III) concentration of the
culture medium, and to be specific for each isolate under investigation26.

Antelo L, Hof C, Welzel K, Eisfeld K, Sterner O and Anke H studied the

siderophores production by Magnaporthe grisea in the presence and absence of iron.
An analysis of siderophores produced by Magnaporthe grisea revealed the presence
of one intracellular storage siderophore, ferricrocin, and four coprogen derivatives
secreted into the medium under iron depletion. Structural analysis showed that the
compounds are coprogen, coprogen B, 2-N-methylcoprogen and 2-N-methylcoprogen
B. Siderophore production under low and high iron conditions was quantified27.

Dexter HH, Rafie R, Tiwari A and Faull KF reported hydroxamate

siderophores of Histoplasma capsulatum. The fungus was grown in a synthetic
medium deferrated with the cationic exchange resin Chelex 100. Siderophores were
detected after 4 days of incubation at 37°C in media containing 0.3 to 1.0 µM iron.
The secretion was suppressed by 10 µM iron. The hydroxamates were purified by

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reverse-phase and size-exclusion chromatography. On the basis of ions observed

during electrospray mass spectroscopy, five hydroxamate siderophores were
tentatively identified: dimerum acid, acetyl dimerum acid, coprogen B, methyl
coprogen B, and fusarinine (monomeric). A polyclonal antibody to dimerum acid was
generated. This reagent was cross-reacted with coprogen B and fusarinine. Thus, the
antibody detects hydroxamates in all three families of siderophores excreted by H.
capsulatum28.

Haselwandter K, Passler V, Reiter S, Schmid DG, Nicholson G, Hentschel

P, Albert K and Winkelmann G isolated a novel trishydroxamate siderophore,
named basidiochrome, as the principal siderophore from low-iron culture filtrates of
Ceratobasidium and Rhizoctonia species which are known as mycorrhizal fungi
associated with orchid roots. Ion-exchange chromatography and preparative HPLC
yielded a pure compound which contained two components according to GC–MS
analysis: L-N5-hydroxy-ornithine and 3-methyl-2-cis-pentenedioic acid (3-methyl-cis-
glutaconic acid). FTICR-ESI-MS of both the iron-free and ferric form indicated an
elemental composition of C33H47N6O16Fe (MW=839) for the ferric form of
basidiochrome. The connectivity was further elucidated by 2D-NMR techniques
(HSQC, HMBC, COSY, NOESY) indicating that basidiochrome is a novel linear
tripeptide consisting of three L-N5-hydroxyornithines each linked to 3-methyl-2-cis-
pentenedioic acid residues29.

Wiebe MG reported the siderophore production by Fusarium venenatum

A3/5. This fungus was grown in iron-restricted batch cultures and iron-limited
chemostat cultures to determine how environmental conditions affected siderophore
production. The specific growth rate in iron-restricted batch cultures was 0.22 h-1,
which was reduced to 0.12 h-1 when no iron was added to the culture. Siderophore
production was correlated with specific growth rate, with the highest siderophore
production occurring at D = 0.08 h-1 and the lowest at D = 0.03 h-1. Siderophore
production was greatest at pH 4.7 and was significantly reduced at pH above 6.0.
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Siderophore production could be enhanced by providing insoluble iron instead of

soluble iron in continuous flow cultures.30

Plant Siderophores

Siderophores have also been reported from the plants. Plants siderophores are
also called as phytosiderophores.

Boron (B) phytotoxicity affects cereal-growing regions worldwide. Although

B-tolerant barley (Hordeum vulgare) germplasm is available, molecules responsible
for this tolerance mechanism have not been defined. Patterson J, Ford K, Cassin A,
Natera S and Bacic A described a new comparative proteomic technique, iTRAQ
peptide tagging (iTRAQ), to compare the abundances of proteins from B-tolerant and
B–intolerant barley plants from a ‘Clipper’ X ‘Sahara’ doubled-haploid population
selected on the basis of a presence or absence of two B-tolerance quantitative trait
loci. iTRAQ was used to identify three enzymes involved in siderophore production
(Iron Deficiency Sensitive2 [IDS2], IDS3, and a methylthio-ribose kinase) as being
elevated in abundance in the B-tolerant plants. Following from this result, they report
a potential link between iron, B, and the siderophore hydroxymugineic acid31.

A sensitive method for the separation of different phytosiderophores of the

mugineic acid family, and the candidate ligand for intracellular metal transport in
plants nicotianamine, and respective metal complexes in plants by zwitterionic
hydrophilic interaction liquid chromatography (ZIC-HILIC) coupled to electrospray
ionization mass spectrometry was described by Xuana Y, Scheuermann EB, Meda
AR, Hayena H, Nicolaus von Wiren, and Weber G. Separation of mugineic acid,
2’-deoxymugineic acid (DMA), 3-epi-hydroxymugineic acid (epi-HMA),
nicotianamine, FeIII-DMA, FeIII-NA,MII-DMA, and MII-NA complexes (MII = ZnII,
CuII, NiII, and FeII), was achieved within 22 min on the ZIC-HILIC column by using a

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gradient elution with a mobile phase consisting of ammonium acetate and acetonitrile
at pH 7.3, at a flow rate of 0.15 mL/min. The on-line coupling to ESI-MS in the
negative ionization mode enables the detection of these compounds in the µmol/L
range, which is the relevant concentration range in real plant samples.32

Ueno D, Rombola AD, Iwashita T, Nomoto K and Ma JF identified and

characterized the phytosiderophores secreted by two perennial grasses, Lolium
perenne cv. Tove and Poa pratensis cv. Baron. Root exudates were collected from the
roots of Fe-deficient grasses and then purified with various chromatographies. The
structure of the purified compounds was determined using both nuclear magnetic
resonance and fast atom bombardment mass spectrometry. Both species secreted
phytosiderophores in response to Fe deficiency, and the amount of phytosiderophores
secreted increased with the development of Fe deficiency. The type of
phytosiderophores secreted differed with plant species; L. perenne cv. Tove secreted
3-epihydroxy-2′-deoxymugineic acid (epiHDMA), 2’-deoxymugineic acid (DMA)
and an unknown compound, whereas P. pratensis cv.Baron secreted DMA, avenic
acid A (AVA) and an unknown compound.33

Ma JF, Ueno H, Ueno D, Rombola AD and Iwashita T investigated the

response to iron (Fe) deficiency in two cultivars of Festuca rubra L. (Rubina and
Barnica) used in correction of chlorosis of fruit trees cultivated on calcareous soils.
They found that a Fe-chelating compound, identified as 2’-deoxymugineic acid
(DMA), was secreted from the roots in response to Fe-deficiency in both cultivars.
The amount of DMA secreted into solution increased with the development of Fe-
deficiency. The secretion showed a distinct diurnal rhythm characterized by a
secretion peak at between 2 and 5 hours after sunrise at 20 oC. However, this secretion
peak was delayed by 3 hour at low temperature (<10oC) and occurred 3 h earlier at
high temperature (30oC). When water used for the collection of root exudates was
pre-warmed (25oC) or pre-cooled (10oC), this led to an earlier or a delayed secretion
compared to control (15oC) under the same air temperature, respectively. These
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results demonstrate that the secretion time of DMA from the roots is, at least partly
controlled by the temperature in the root environment.34

Detection and Separation of Siderophores

Bernhard Schwyn, Neilands J.B. developed a universal method to detect and

determine siderophores using high affinity for iron (III). The ternary complex chrome
azurol S/iron(lII)/hexadecyltrimethylammonium bromide, with an extinction
coefficient of approximately 100,000 M-’ cm-’ at 630 nm, serves as an indicator.
When a strong chelator removes the iron from the dye, its color turns from blue to
orange. Because of the high sensitivity, determination of siderophores in solution and
their characterization by paper electrophoresis chromatography can be performed
directly on supematants of culture fluids. The method is also applicable to agar plates.
Grange halos around the colonies on blue agar are indicative of siderophore
excretion.35
Miranda Perez S., Cabirol N. et.al, developed redesigned and optimized to
produce a new, fast, non-toxic and easy method to determine a wide variety of micro-
organisms capable of siderophore production on a solid medium. Detection was
optimized through adjustments to the medium composition and a quantifying
strategy. A total of 48 micro-organisms were isolated from three different types of
samples (fresh water, salt water and alkaline soil), of which 36 were determined as
siderophore producers. The compounds identified through this method belonged to
both hydroxamate and catechol types previously reported to cause a colour change
that differ from previous descriptions.36
Adriane M.F. Milagres, Angela Machuca and Diovana Napoleao
developed a modified method of CAS agar plate assay which was made by
incorporating the CAS blue dye in a medium with no contact with the micro-
organisms tested. Half of each plate used in our experiments was filled with the most

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appropriate culture medium for each type of microorganism and the other half with
CAS-blue agar. This modification allowed us to study several strains of fungi
(basidiomycetes, deuteromycetes, ascomycetes and zygomycetes) and bacteria (Gram
positive and negative), some of them appearing for the first time in the literature. All
the microorganisms grew properly and reacted in different manners to the CAS assay.
This modified method could facilitate optimization of culture conditions, since both
CAS-blue agar and growth medium were prepared and added in the Petri plate
separately.
Arefa Baakza, A.K. Vala, B.P. Dave H.C. Dube done a comparative study of
siderophore production by fungi from marine and terrestrial habitats. Siderophore
producing potential of 20 fungal isolates (same 10 species from each marine and
terrestrial habitat) were examined and compared. Examination of the chemical nature
of siderophores revealed that mucoraceous fungi produced carboxylate, while others
produced hydroxamate siderophores. Thus, the nature of siderophore was found to be
independent of habitat. Comparison of quantification of siderophore production
between marine and terrestrial revealed that four terrestrial isolates (Aspergillus
niger, Aspergillus ochraceous, Penicillium chrysogenum, Penicillium citrinum) were
ahead in siderophore production, while, the other four marine isolates (Aspergillus
versicolor, C. elegans, Rhizopus sp., Syncephalastrum racemosum) were found to be
more potent siderophore producers, indicating that they were equally competent.37
Sung Heui Shin, Yong Lim et al., developed a simple and universal method,
by modifying the universal CAS _Chrome azurol S. assay, measuring siderophores in
various biological fluids.They named the assay as CAS agar diffusion CASAD. assay.
CAS plate devoid of nutrients was prepared by using Bacto-agar _1.5%, wrv. as a
matrix. Holes with 5-mm-diameter were punched on the CAS agar plate. Each hole
was added by 35 ml of the test fluids containing Desferal that was twofold serially
diluted. After incubating at 378C or room temperature for 4–8 h, the size of orange
haloes formed around the holes was measured. The size of orange haloes correlated
well with the concentration of Desferal in all the biological fluids tested in this study.
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CASAD assay showed consistent results in wide pH range from 5 to 9. Addition of

iron to the test fluids containing Desferal decreased the size of orange haloes in a
dose-dependent manner, which suggests that the CASAD assay detects only iron non-
bound siderophore. These results suggest that CASAD assay would serve as a simple,
stable, and highly reproducible test for screening and quantitative siderophore
analysis in biological fluids.
Frank A. Fekete, Jack T.Spence and Thomas Emery, developed a rapid
and sensitive assay for detection of of microbial siderophores (iron-binding
compounds) is described. Nine representative fungal and bacterial cultures including
Ustilugo sphaerogena, Penicillium sp., Fusarium roseum, Rhodotorula pilimanae,
Bacillus subtilis W 23, Bacillus subtilis W 168, Bacillus megaterium, Azotobacter
vinelandii OP, and Escherichia coli B, were nutritionally stressed for iron by
sequential transfers on iron-deficient solid-plating media. In response to Fe-stress
conditions, the microorganisms excreted siderophore compounds into the
extracellular solid culture medium. The solid agar matrix effectively concentrated and
restricted the migration of the siderophore compounds to the region immediately
adjacent to colonial growth. Agar-block samples from this region were removed and
placed at the origin of an electrophoresis paper strip. The resultant absorbed material
from the agar-block sample was subjected to high-voltage paper electrophoresis
which separated the siderophore compounds by size and molecular net charge.
Phenolic acid (“catechol”)-type siderophores were detected by fluorescence under uv
light. Hydroxamic acid-type siderophores were visualized by spraying the
electrophoretogram with ferric iron solution.38
A highly sensitive spectrophotometric method for the selective detection of
catechol compounds such as catechol siderophores (e.g., enterobactin) was described
by Rioux C, Jordan DC and Rattray JBM. The basis of the method involves the
ability of the vicinal aromatic hydroxyl groups under acidic conditions to bring about
a reduction of Fe3+ (from ferric ammonium citrate) to Fe2+. Detection of Fe2+ in the
presence of Fe3+ was made with 1, 10-phenanthroline under previously established
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conditions. The assay mixture was heated at 60°C for 1 h to accelerate the
development of color which is subsequently measured at 510 nm. Compared to the
Arrow nitration method, the assay was more responsive, was approximately seven
times more sensitive. The method has been adapted to determine catechol compounds
in the culture medium of bacterial cells grown at different iron concentrations39.

Mucha P, Rekowski P, Kosakowska A and Kupryszewski G carried out the

separation of siderophores by capillary electrophoresis. Capillary electrophoresis
(CE) was applied as a fast method of siderophore separation. Siderophores are iron
binding and regulating cell products, which facilitate iron transport into cells. A fast
and efficient method of siderophore analysis is important for better understanding of
the iron pathways in a sea environment or marine organisms. The best results of CE
analysis were obtained using free zone CE in 25 mM phosphate buffer at basic pH
using a constant voltage of 20 kV. Under these conditions it was possible to detect the
presence of siderophores in seawater40.

Trskova R, Rychlovsky P, Nemcova I and Jegorov A developed a method

for the spectrophotometric determination of siderophores using flow-injection
analysis (FIA) based on the reaction of siderophores with the ternary complex
Eriochrome Cyanine R-Fe (III)-cetyltrimethylammonium bromide.
2,3-Dihydroxybenzoic acid, 2,3-dihydroxynaphthalene, and tolypocine were used as
the model iron-binding ligands. The calibration curve for one of the siderophores
(tolypoone) was found to be linear in the concentration range 2.6 x 10-6-1.5 x 10-4M.
The determination limit (10σ) for tolypocine was 2.6 x 10-6M. The applicability of
the method was demonstrated on the determination of the complexation ability of
siderophores produced by some entomopathogenic fungi41.

Fuhrmann JJ reported a technique for recovering siderophores from culture

supernatants of fluorescent Pseudomonus spp. Siderophores produced in a modified
succinate medium were purified by ion-exchange chromatography and if needed, by

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subsequent acetone precipitation. Relative to alternative procedures commonly

employed, this method was simple, rapid and minimizes the use of potentially
hazardous reagents. Although possibly not as universal in its application as previously
described techniques, it may possess significant advantages for a number of
experimental systems42.

A column chromatographic method using a polyamide support has been

developed by Robinson AV, which allows rapid isolation of catechol-type
siderophores directly from culture supernatants. The method was superior to presently
used methods with regard to speed, convenience, and lack of degradation products.
Siderophores from Micrococcus denitrifcans (ATCC 19367), Klebsiella nerogenes
(ATCC 13882), and Bacillus subtilis (ATCC 6051) have been purified and the
method has been tested with two synthetic catechol-type chelators, N1,N2-bis(2, 3-
dihydroxybenzoyl)ethylenediamine & N1,N4-bis(2,3-dihydroxybenzoyl)putrescine43.

Albert A. Minnick, Laura E. Eizember,et al., developed convenient plate

assay which is sensitive to medium pH has been developed to evaluate potential
siderophores of Candida albicans. Adding a siderophore to a filter paper disk on
chemically defined Lee’s agar (final pH 7.2) seeded with the test strain reversed the
growth inhibitory effects of the supplemented (25-100 pg/ml) iron chelator
ethylenediaminedi(o-hydroxyphenylacetic acid), to provide a zone of growth
stimulation. This bioassay has been used to demonstrate the structureactivity
relationships of ferrichrome and several water-soluble hydroxamate peptide building
blocks of this natural siderophore.
B.P.Dave, Kena Anshuman & Puja Hajela, studied nine halophilic archaea
bacteria (Halobacterium salinarium, Halococcus saccharolyticus, Halorubrum
saccharovorum, Haloterrigena turkmenica, Halogeometricum spp and Natrialba spp)
isolated from marine salterns around Bhavnagar coast and screened for siderophore
production. Out of nine, five isolates were found to produce siderophores giving
positive FeCl3 test CAS assay and CAS agar plate assay. Determination of chemical
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nature of siderophores by chemical and bioassays identified them as carboxylates.

Quantification of siderophores indicated Halorubrum saccharovorum to be the
maximum siderophore producer. The present study is the report on siderophore
production in Indian haloarchaeal strains.
M. A. F. JALAL, RAMAN MOCHARLA and DICK VAN DER HELM
studied Iron(II1) chelates of nineteen trihydroxamate siderophores of fungal origin,
including ferrichromes, coprogen and triacetylfusarinine C, were separated on a
preparative scale with a reversed-phase column using the octadecyl silica gels LRP-I
or LRP-2 as the stationary phase and a water-methanol gradient as the mobile phase.
Using this system in combination with silica gel column chromatography, most
siderophores can be obtained in pure form. Factors affecting the mobility of these
compounds in the reversed-phase system are discussed.

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