Dissertation Report
1.1 SIDEROPHORES
Iron is the most vital element essential for all living organisms to perform cellular processes
such as electron transport chain and as a cofactor for many enzymes (Litwin and Calderwood,
1993). Surface iron get oxidized to insoluble oxyhydroxide polymer and reduced the level of
free iron due to aerobic atmosphere.Ferric iron (Fe3+) solubility under these conditions is 10-
17
M, whereas cytoplasmic iron concentrations are approximately 10-7 M in metabolically
active microbes (Ishimaru 1993). Thus uptake by diffusion is not an option for
microbes,therefore microorganism adopted a way for iron acquisition by producing iron
chelating molecule i.e. siderophore which is a low molecular weight molecules (< 10 KD)
that bind ferric iron with an extremely high affinity (Lankford 1973) and is specifically
recognized by a corresponding outer membrane receptor protein, which in turn actively
transports the complex into the periplasm of the cell.Many bacteria (Pseudomonas,
Azotobacter, Bacillus, Enterobacter, Serratia,Azospirillumand Rhizobium) and fungi are
capable of producing more than one type of siderophore or have more than one iron-uptake
system to take up multiple siderophores (Neilands 1981).
1.2CLASSIFICATION OF SIDEROPHORES
Catecholate (Phenolates) siderophore
Catecholate siderophores bind Fe3+ using adjacent hydroxyl groups of catechol rings.
Enterobactin, also known as enterochelin, the cyclic trimester of 2, 3-
dihydroxybenzoylserine, is produced by a various bacteria including E. coli, Klebsiella
pneumoniae and K.terrigena(Höfte 1993)and is the classic example of a catechol-type
siderophore. (O.Brien&Gibson 1970, Pollack et al. 1970). It possesses the highest known
affinity for Fe3+ with a stability constant (Kf) of 1052 (Höfte 1993).).Each catecholate group
provides two oxygen atoms for chelationwith iron so that a hexadentate octahedralcomplex is
formed as in the case of thehydroxamatesiderophores. Linearcatecholatesiderophore are also
producedin certain species. Erwiniacarotovoraproduces catecholates while Pseudomonas
produces mixed catecholate-hydroxamate siderophore (Leong and Neilands, 1982).
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Hydroxamate type
Hydroxamate siderophore are produced by bacteria and fungi. Most hydroxamate groups, C
(=O)N-(OH) R, where R is an amino acid or a derivative. Each hydroxamate group provides
two oxygen molecules, which form a bidentate ligand with iron. Therefore, each siderophore
forms a hexadentate octahedral complex with Fe 3+.Ferribactin, Gonobactin and nocobactin
produced by Pseudomonasfluorescens, Neisseria gonorrhoeae and N. meningitids
respectivelyare known to be a hydroxamate.
Carboxylate (complexones) siderophore
The detection of siderophore that are neither catecholates nor hydroxamates fascilitated by
the universal assay for siderophore detection (Schwyn and Neilands, 1987). Rhizobactin is
produced by Rhizobium melilotistrain DM4 with ethylenediaminedicarboxyl and
hydroxycarboxyl moieties as ironchelating groups. StaphyloferrinA,produced by
Staphylococcus hyicusDSM20459, is another member of this class of complex siderophores.
CATECHOLATE TYPE HYDROXAMATE TYPE
A)Enterobactin B) Vibriobactin A) Alcaligin B) Desferrioxamin B
CARBOXYLATE TYPE
A)Staphyloferrin A B)Achromobactin
Fig 1: Representative Siderophore Structures
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1.3 SIDEROPHORE TRANSPORT IN BACTERIA
It is both a receptor and an energy-dependent process (Sigel and Sigel, 1998). Such systems
have been well studied in Escherichia coli (Wandersman and Delepelaire, 2004).
Siderophores are part of a multi-component system for transporting ferric iron into a cell.
Other components include a specific outer membrane receptor protein Fec A, Fep A and
TonB-ExbB-ExbD protein complex in the inner membrane, a periplasmic binding protein,
and an inner membrane ATP dependent Fec CDE. Under iron deficiency bacteria synthesize
siderophore and increase number of receptor molecules once the siderophore excreted outside
of cell thorough membrane receptor it bind with iron complex and transport the iron in to the
cell via Fec A and Fep A outer membrane receptor(OM), after it transported to Fec C,D,E
and Fep C,D,E so called ABC-Transporter systems (from ATP-binding cassette) (Davidson
and Nikaido, 1991, Boos et al., 2001) assembled of two proteins, one to span the membrane
acting as a permease and a second one which can hydrolyse ATP to provide the energy for
transport. Later siderophore iron complex release in cytoplasm with the help of membrane
protein Ton B (Fig. 2). In the cell cytoplasm, the iron released from the complex either by
3+
hydrolytic destruction of the siderophore molecule or the reduction of Fe by a NAD (P) H
linked siderophore reductase or Ent A,B,C,D protein. The resulting Fe 2+ does not have a high
affinity for siderophore and therefore dissociated from the complex.
Fig. 2 Mechanism of siderophore mediates iron transport in bacteria
(Adapted from S.Sajid Ali, ijcmas 2013)
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1.4 APPLICATIONS OF SIDEROPHORE
Agricultural applications
In agriculture, inoculation of soil with Pseudomonas putida, producing pseudobactin,
increases growth and yield of various plants (Kloepperet al.,1980).Their plant growth
promoting activities include production of HCN, siderophores, protease, anti-microbials,
phosphate solubilizing enzymes (Chaiharnet al., 2008). Hydroxamate type siderophore
present insoil play important role to immobilize excessive accumulation of the heavymetals.
Environmental applications
Siderophores and other naturally occurring ligands may affect actinide mobility in waste
repositories and in the environment and may also used to treat radioactive wasteprior to
storage or to decontaminate soilsand water (Ruggiero et al., 2000; Von Gunten and Benes,
1995 ).
Medicinal application
a. Iron overload diseases, ß-thalassemia
As continued transfusion therapy leads to a steady build up of iron, these iron excesses, as
well as the primary iron overload diseases such as hemochromatosis and hemosiderosis, and
accidental iron poisoning, require the removal of iron from the body, especially from the
liver. Such disease can be efficiently treated with siderophore based drug and siderophore act
as principal model (Pietrangelo, 2002). Desferrioxamine B has also found therapeutic
application for various pathological conditions due to aluminum overload (Ackrillet al.,
1980)
b. Anti-Malarial
Siderophore produced by Klebsiella pneumoniae act as antimalarial agent (Gysin et al.,
1991).Desferrioxamine B produced by Streptomyces pilosus(Now produced by chemical
synthesis also) is active against P. falciparum in vitro as well as in vivo as well as in
Trypanosoma brucei. Siderophore enters inside P. falciparum cell and causes intracellular
iron depletion.
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1.5 OBJECTIVES
The main objectives were as follows:
Standardization ofironconcentration required for the production of catecholate.
Evaluation of optimum pH and temperature for catecholate production.
Evaluation of the effects of arsenic on growth, protein content and catecholate
production by E.coli.
Visualisation of the effect of catecholate siderophore on the growth of Aspergillus
nidulans.
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