Chapter 1

Introduction

INTRODUCTION
Siderophores
Iron is the fourth most abundant element of the earth’s crust and amongst metals; it is
second only to aluminum. While iron is widespread in the environment, it is often
considered biologically unavailable as it is often only found in the form of highly
insoluble Fe (III) (oxyhydr) oxides. Under anaerobic conditions, Fe (II) is soluble,
readily available and may be taken up by anaerobic bacteria without the help of iron
chelators. Under aerobic conditions, Fe (II) is readily soluble but it is quickly
oxidized to Fe (III) and forms a complex of precipitated Fe (III) minerals, such as
amorphous ferrihydrite, goethite and hematite. Iron is a versatile and necessarily
nutrient. Iron is a component of electron transport proteins such as cytochromes,
ferredoxines and iron-sulfur proteins. Most microbial life requires between 10-8 to
10-6 M for optimal growth, such that, without chelators, most microbes inhabiting
aerobic, neutral or alkaline environments would live in a state of permanent iron
deficiency.1
Lankford coined the term siderophore in 1973. Siderophores are low
molecular weight organic molecules, which can compete for ferric iron in ferric
hydroxide complexes2. Siderophore was derived from a Greek term meaning – iron
carrier. This is an appropriate term because the siderophore binds iron with an
extremely high affinity and is specifically recognized by a corresponding outer
membrane receptor protein, which in turn actively transports the complex into the
periplasm of the cell. The molecular weights of siderophores range from
approximately 600 to 1500 Daltons, and because passive diffusion does not occur for
molecules greater than 600 Daltons, siderophores must be actively transported3.
Siderophores are commonly produced by aerobic and facultative anaerobic bacteria
and by fungi under iron limiting condition. They are apparently absent in animal
tissue, but plants secrete analogous compounds dubbed phytosiderophores, most of
which contains the azetidine nucleus4. Hence siderophores can be defined as low
molecular weight extra cellular organic compounds secreted by microorganisms
under iron-starved conditions, used by them to chelate and solubilize iron.4

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Classification of siderophores
Siderophores are novel structures, many containing modified amino acids not
found in nature. Great variation is seen in siderophore structure from one species to
other. There are three main kinds of iron containing functional groups known as
Hydroxamate, Catecholate and Carboxylate siderophores.5
Over 500 siderophores have been described5. Siderophores are classified on
the basis of the chemical functional groups they use to chelate iron.
• Catecholate-type (phenolate) siderophores bind Fe3+ using adjacent hydroxyl
groups of catechol rings. Enterobactin, also known as enterochelin, is
produced by a number of bacteria including E. coli and is the classic example
of a catechol-type siderophore (Figure 1A) 6. It possesses the highest known
affinity for Fe3+ with a stability constant (Kf) of 1052. Enterobactin production
has been demonstrated in some nitrogen-fixing bacteria, including Klebsiella
pneumoniae and K. terrigena7.
• Fe3+ is chelated using nitrogen atoms of thiazoline and oxazoline rings in
hydroxamate-type siderophores8. Ferrichrome is the classic hydroxamate-type
siderophore (Figure 1B). It is produced by a number of fungi including
Ustilago sphaerogena. Although produced by fungi, ferrichrome is used by a
number of bacterial species with the appropriate receptor protein7.

• A third class of siderophores utilizes N-hydroxy amino side chains with an

oxygen atom as one of the ligands for Fe3+, Anguibactin, produced by Vibrio
anguillarum incorporates this functional group, but it is also a combination of
all three siderophore types in that it is made up of all three functional groups,
with three different methods of binding Fe3+.

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A B

C D
Figure No.1:- Representative Siderophore Structures. A) Enterobactin
(catechol-type) B) Ferrichrome (hydroxamate-type) C) Aerobactin (Citric
acid based type) D)Anguibactin(mixed)8

Applications of Siderophores
The importance of siderophores extends beyond their role in microbial
physiology and their applications in biotechnology. Addition to transporting iron,
siderophores have other functions and effects, acting as intracellular iron storage
compounds and suppressing growth of other microorganisms. Siderophores and their
derivatives can complex other metal apart from iron which shows a lot of
applications.5

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Other applications are development of treatment options for diseases associated

with iron overload. An example, haemochromatosis is a disorder wherein there is
progressive increase in body iron content causing iron deposits in liver which is
associated with any form anemia. Desferroxime in the form of Desferal, which is a
synthesized siderophore of the ferrichrome family used in treatment of
haemochromatosis. It has also been used to treat patients with β-thalassemia who
produce defective hemoglobin and must undergo periodic blood transfusions, which
leads to iron accumulation in the body. Desferal is used to chelate excess iron and has
also been used in treating iron poisoning9.
One of the important applications of siderophores is its conjugation with some
iron-chelating antibiotics which behave like siderophores called sideromycins against
whom the microorganisms have become resistant. Dr. Miller's group has developed
conjugate that contain a siderophore component bound to carbacephalosporins, other
conjugate incorporates an erythromycin analog, whereas, last conjugate incorporates
a siderophore and a novel antifungal agent related to the neoenactins10.
In agriculture, inoculation of soil with Pseudomonas putida, which produces
pseudobactin, increases growth and yield of various plants. Hydroxamte
siderophores are present in soil at high concentration which is enough to be taken up
by plant roots.
Siderophores can be successfully used in removing many toxic metals off the
soil which poses a serious health threat. Siderophore can complex with heavy metals
like Cadmium, Lead, Nickel, Arsenic (III, V), Aluminium, Magnesium Zinc,
Copper, Cobalt, and Strontium other than Iron11.

Other potential biotechnological applications for siderohores in medicine as

nontoxic, organ selective magnetic resonance imaging (MRI) contrast agents,
reprocessing of nuclear fuel, biomineralization, bioremediation, industrial waste
treatment and in cosmetics as deodorants.

Advantages of Fungal Siderophores over Bacterial Siderophores

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A variety of fungi are known to overproduce and excrete siderophores under iron
limitation, ferrichromes, being the most common group of fungal siderophores.
Solubilization, transport and storage are three important functions assigned to
siderophores. Measurement of transport activities is best performed with fungi
because of easier handling during transport assays. Fungal siderophores are known
for their use in wood biodegradation and deinking process. Monohydroxymates such
as fusarinines, Dihydroxymates like dimerum acid, Trihydroxymates like Coprogen,
Neocoprogen and Ferrichrysin are few examples of fungal siderophores.12

Iron and Fungal Cells

Iron is required by most living systems. The metal has two readily available ionization
states, Fe (II) and Fe (III) which are often used as a cofactor for oxidation-reduction
enzymes. Iron in nature is usually present in insoluble form in aerobic environment
Fungi overcomes this problem in various ways.
The uptake of iron across the plasma membrane is considered the primary
regulatory point for controlling iron homeostasis. Two major systems for acquisition
of iron by fungi exists viz, reductive iron assimilation and siderophore-assisted iron
uptake. Recent reports have reported that some fungal siderophores act as virulence
determinants useful in maintaining plant-fungi symbiotic interactions. Multiple iron
acquisition strategies separated into high affinity and low-affinity iron uptake systems
have evolved in fungi. High affinity systems function during iron-limiting conditions,
whereas low-affinity systems are important during periods of relative iron abundance.
In fungi, Siderophore-mediated iron uptake requires synthesis and excretion of
an iron-free siderophore (desferrisiderophore), chelation of iron, uptake of the
siderophore–iron complex and intracellular release of iron.
Siderophore Biosynthesis
Siderophores are produced during extreme iron-depleted conditions for the
solubilisation of extracellular ferric iron by most bacteria and fungi. The majority of
fungal siderophores are hydroxamates, apart from the carboxylate-type siderophore
rhizoferrin produced by zygomycetes. Siderophores are generally named based on

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their ironcharged forms and the prefix deferri- or desferri- is used to denote the iron-
free (deferrated) form of the ligand.

The general fungal siderophore biosynthetic pathway can be shown as:

Fig No.2: Schematic representation of Fungal Siderophore Biosynthetic Pathway

All fungal siderophores are derived from L-ornithine, and all share N5-acyl-N5-
hydroxyornithine as the common basic unit. All reactions are catalysed by enzymes.
The various enzymes involved in the fungal siderophore biosynthetic pathway are L-
ornithine N5-oxygenase, N5-transacylases and N2-transacetylase.13
Some of the examples of fungi and their mechanism of iron-acquisition type
are given in the tabular format as: 14

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Table No 1: - Mechanism of iron-acquisition by pathogenic fungi

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Siderophore-mediated Iron Transport

Initially, the siderophore binds to ferric iron in the external environment. The
iron-siderophore complex is then recognized by the corresponding outer membrane
receptor protein. Binding of the ferric-siderophore complex induces considerable
conformational changes, perhaps signaling to initiate TonB interaction. Using energy
presumably provided by the TonB complex (proton motive force), the ferric-
siderophore complex is actively transported into the periplasm. Once in the
periplasm, the ironsiderophore complex is bound to a periplasmic binding protein that
transports the complex to the ABC-type transporter in the cytoplasmic membrane,
which transports the complex into the cytoplasm utilizing energy from the hydrolysis
of ATP (Figure 2). Iron is released from the siderophore by either reduction via ferric
reductases, or by chemical modification or breakdown of ferric siderophore
complexes by acetylation and esterases, respectively15.

Fig No.2:- Schematic representation of General Siderophore-Mediated Iron Transport

Transport methodology of fungal siderophores

Fungi are ubiquitous saprophytes which propogate by sexual as well as asexual
spores. Numerous filamentous and yeast like fungi have been shown to sequester iron

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by excreted iron-complexing agents.

Three functions have been assigned to them: Solubilization, transport and storage of
iron. The measurement of transport activities is best performed with yeast like fungi,
because easier handling during transport assays. Trasnport assays are carried out
using membrane filters, but usually in few cases where in osmotically fragile cells to
be treated.
The available data on fungal siderophore indicate that most of fungal
siderophores are transported as a whole across the plasma membrane, delivering iron
to acceptors inside the cell and in certain genera of fungal siderophores do not
penetrate membrane barrier but deliver iron to membrane bound iron acceptors. There
are some examples of siderophores produced by various fungi
Siderophore-Type Species Examples
Monohydroxamates Fusarium Fusarinines
Gliocladium virens
Dihydroxamates Rhodutorula Fe-Rhodotorulic acid
Microbotryum
Fusarium dimerum Diemrum acid
Verticillium dahliae
Trihydroxamtes (Linear) Neurospora crassa Coprogens
Penicillium spp
Fusarium dimerum Coprogen B
Verticillium dahliae
Curvularia lunata Neocoprogen I
Epicoccum Neocoprogen II
purpurascens
Alternaria alternata Dimethylcoprogen
Alternaria lomgipes Hydroxycoprogen
Trihydroxamates (Cyclic) Ustilago spp Ferrichrome
Penicillium
Neovossia indica Tetraglycylferrichrome

Siderophore-Type Species Examples

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Trihydroxamates (Cyclic) Aspergillus fumigates Ferricrocin

Neurospora crassa
Aspergillus orchraceus Asperchromes
Aspergillus melleus Ferrichrysin
Aspergillus ochraceus Ferrirubin
Penicillium variabile
Botrytis cineria Ferrirhodin

Fusarium cubense Fusarinin C

Mycelia sterilia Triacetylfusarinine C

Aspergillus spp

Table No 2: - Examples of fungi and corresponding siderophores produced

General siderophore structural traits
The selectivity of siderophores for iron depends upon the optimal selection of number
and type of metal binding groups in addition to the stereochemical arrangement. To
the date, Hydroxamates, Catecholate and o-hydroxycarboxilic acid binding subunits
arranged in various configurational patterns like linear, tripodal, endocyclic and
exocyclic and have been found to be most promising iron-binding ligands in the
nature. The number of iron binding functional groups, or denticity, is an important
component of the siderophore architecture.
A main structural element of fungal all fungal siderophores is the amino acid
ornithine which after δN-hydroxylation and δN-acylation gives Fe (III) complexing
hydroxamic acid bidentate.

Fig No.4: - Schematic representation of structure of fungal siderophore

The hydroxamic acid residues (R-CO-) may originate from acetic acid, a
hydromevalonic or glutaconic acid but others may also occur. According to structure,

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Fungi shows three group of siderophore families viz, Ferrichromes, Cyclic

triacetylfusarinines, Coprogens.

Ferrichromes represent a group of cyclic peptide siderophores with structural

alterations in the peptide backbone or in the hydroxamic acid moieties. The typical
ferrichromes may differ in the peptide ring and posses three acetyl residues as
hydroxamic acid moieties. Other ferrichrome type siderophores posses a ferrichrysin
peptide backbone but may have different hydroxamic acids linked to the N-hydroxy-
ornithyl residues.
Cyclic triacetylfusarinines represent a group of siderophores which contains
cyclic trimesters of fusarinine residues possessing three ester bonds.
Coprogens represent a family of siderophores which contains linear
trihydroxamic siderophores composed of trans-fusarinine residues linked to trans-
anhydromevalonyl groups. The coprogens possess both ester and peptide bonds.12

Fungi
Fungi are eukaryotic organisms that do not contain chlorophyll, but have cell
walls, filamentous structures, and produce spores. The study of fungi is known as
MYCOLOGY. These organisms grow as saprophytes and decompose dead organic
matter. There are between 100,000 to 200,000 species depending on how they are
classified. About 300 species are presently known to be pathogenic for man. Fungi
are microorganisms in the domain eucarya. They show less differentiation than
plants, but a higher degree of organization than the prokaryotic bacteria. Only about a
dozen of these “pathogenic” species cause 90% of all human mycoses. Many mycotic
infections are relatively harmless, for instance the dermatomycoses. In recent years,
however, the increasing numbers of patients with various kinds of immune defects
have resulted in more life-threatening mycoses.
Although Fungi differ from bacteria in various ways, some of the difference between
fungi and bacteria are mentioned.
Properties Fungi Bacteria
Nucleus Eukaryotic; nuclear Prokaryotic;

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membrane; more than no membrane;

one nucleoid; only
chromosome; mitosis one “chromosome”
Cytoplasm Mitochondria; No mitochondria;
endoplasmic no endoplasmic reticulum;
reticulum; 80S ribosomes 70S ribosomes
Cytoplasmic Sterols (ergosterol) No sterols
Membrane
Cell wall Glucans, mannans, chitin, Murein, teichoic acids
chitosan (Gram-positive), proteins
Size, mean diameter Yeast cells: 3–5–10 lm. 1–5 lm
Molds: indefinable
Metabolism Heterotrophic; Heterotrophic; obligate
mostly aerobes; aerobes and anaerobes,
no photosynthesis facultative anaerobes

Table No. 3:- Differences between properties of fungi and bacteria

Classification and Taxonomy
The taxonomy of the fungi is essentially based on their morphology. In medical
mycology, fungi are classified according to practical aspects as dermatophytes,
yeasts, molds, and dimorphic fungi. Molds grow in filamentous structures, yeasts as
single cells and dermatophytes cause infections of the keratinized tissues (skin, hair,
nails, etc.). Dimorphic fungi can appear in both of the two forms, as yeast cells or as
mycelia.

Fungi are carbon heterotrophs. The saprobic or saprophytic fungi take carbon
compounds from dead organic material whereas biotrophic fungi (parasites or
symbionts) require living host organisms. Some fungi can exist in bothsaprophytic
and biotrophic form

Morphology

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Morphologically fungi exist in two forms. The basic elements of fungi are shown
below.

Fig No.5: - a) Hypha, septate, or nonseptate b) Mycelium: web of branched

hyphae.
c) Yeast form, budding (diameter of individual cell 3–5 lm).
d) Pseudomycelium.

 Hyphae: This is the basic element of filamentous fungi with a branched,

tubular structure, 2–10µm in width.
 Mycelium: This is the web or mat like structure of hyphae. Substrate mycelia
penetrate into the nutrient substrate, whereas aerial mycelia develop above the
nutrient medium.

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 Fungal thallus: This is the entirety of the mycelia and is also called the
fungal body or colony.
 Yeast: The basic element of the unicellular fungi. It is round to oval and 3–10
µm in diameter. Several elongated yeast cells chained together and resembling
true hyphae are called pseudohyphae.
 Dimorphism: Some fungal species can develop either the yeast or the
mycelium form depending on the environmental conditions, a property called
dimorphism. Dimorphic pathogenic fungi take the form of yeast cells in the
parasitic stage and appear as mycelia in the saprophytic stage.

Metabolism
All fungi are carbon heterotrophs, which mean they are dependent on
exogenous nutrient substrates as sources of organic carbon, and with a few
exceptions, fungi are obligate aerobes. Many species are capable of maintaining
metabolic activity in the most basic of nutrient mediums. The known metabolic types
of fungi include thermophilic, psychrophilic, acidophilic, and halophilic species. The
metabolic capabilities of fungi are exploited in the food industry (e.g., in the
production of bread, wine, and in the pharmaceutical industry (e.g., in the production
of antibiotic substances, enzymes The metabolic activity of fungi can also be a
damaging factor. Fungal infestation can destroy foods, wooden structures, textiles,
etc. Fungi also cause numerous plant diseases, in particular diseases of crops.

Reproduction in Fungi
• Asexual reproduction. This category includes the vegetative propagation of
hyphae and yeasts as well as vegetative fructification, i.e., formation of
asexual spores.
• Hyphae elongate in a zone just short of the tip in which the cell wall is
particularly elastic.
• Yeasts reproduce by budding. This process begins with an outgrowth on the
mother cellwall that develops into a daughter cell or blastoconidium.

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• Vegetative fructification: A type of propagative form, the asexual spores, is

formed in this process. These structures show considerable resistance to
exogenous noxae and help fungi spread in the natural environment. These
forms rarely develop during the parasitic stages in hosts, but they are observed
in cultures.

• Sexual fructification. Sexual reproduction in fungi perfecti (eumycetes)

follows essentially the same patterns as in the higher eukaryotes. Sexual
reproduction structures are either unknown or not present in many species of
pathogenic fungi, known as fungi imperfecti.17

IDENTIFICATION OF FUNGI
There are four methods presently in use for detecting and identifying fungi
histological observation, most recently with molecular methods
• surface sterilization of the host tissue and isolation of them emerging fungi on
appropriate growth media,
• detection by specific chemistry, e.g. immunological methods or
• By direct amplification of fungal DNA from colonized plant tissues.
Fungi frequently colonize the internal and external plant environment as
pathogens, mutualists or organisms without apparent effect on the plant. Fungal
structures, including hyphae, fruiting bodies and spores can be visualized by light-
and electron-microscopy.
Light microscopes belong to the standard equipment of every microbiology
laboratory and are relatively inexpensive. Several reporter genes with enzymatic
functions such as β-glucuronidase (GUS) encoded by gusA and β-galactosidase
encoded by lacZ are used to visualise cells and gene expression.
Scanning electron microscopy (SEM) has the advantage of high resolution and is,
therefore, a powerful tool with which to follow the process of seed and root
colonization by microorganisms. Single bacterial cells and bacterial

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microcolonies can be visualised. SEM is also very suitable for showing the
morphological differences within endogenous communities of microorganisms.
SEM is ideally suited to visualising the total microflora since it does not require
the tagging of microorganisms with reporters.
Fluorescence microscopy is based on the presence of fluorescent compounds,
including proteins, which, after excitation with light of a certain wavelength, will
emit light of a longer wavelength due to energy loss during the process of
absorption and excitation. Confocal laser scanning microscopy (CLSM) is a
highly sophisticated form of fluorescence microscope. The use of CLSM for
visualising fluorescent molecules results in higher resolution and lower auto
fluorescence background compared to traditional fluorescence microscopy In
addition, the resolution and sharpness of the digital images produced by CLSM
can be improved by the use of deconvolution software that corrects for small
defects in the optical lenses. In many applications fluorescent tags are coupled to
compounds specifically binding to certain molecules, such as DNA or RNA, in
the living cell. Green fluorescent protein (GFP) has become the most frequently
used reporter in the biological sciences since its application as a marker.
General characteristics of fungi

Fungi are one of the five kingdoms of organisms. Like higher plants (of the
kingdom Plantae), most fungi are attached to the substrate they grow on. Unlike
plants, fungi do not have chlorophyll and are not photosynthetic. Another key
difference from plants is that fungi have cell walls composed of chitin, a nitrogen
containing carbohydrate. All fungi have nuclei and the nuclei of most species are
haploid at most times. Many species have two or more haploid nuclei per cell during
most of the life cycle. All fungi reproduce asexually by spore production. Most
species reproduce sexually as well. The different taxonomic groups of fungi have
different levels of cellular organization. Some groups, such as the yeasts, consist of
single-celled organisms, which have a single nucleus per cell. Some groups, such as
the conjugating fungi, consist of single-celled organisms in which each cell has
hundreds or thousands of nuclei.

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Groups such as the mushrooms, consist of multicellular, filamentous

Fungi which form mycelia are called moulds or filamentous fungi, hyphae may be
Septate or nonseptate. And septa have holes through which free flow of cytoplasmic
material can take place, and mycelium can be divided into the vegetative mycelium
which grows into the medium and the aerial mycelium which projects from the
surface.

Macroscopic examination of cultures

After initial inoculation and incubation, cultures should be examined for
growth every 2-3 days during the first week and at least weekly thereafter. Rapid
growers will appear by the first or second time the culture tubes are checked, whereas
slow growing fungi may not be evident for 2-3 weeks or longer. It is imperative that
any yeast, mould, or actinomycetes that grow on a primary medium be subcultured
immediately to ensure the viability and isolation of the organism. When mature
growth has developed on Saburaud dextrose agar (SDA), the texture and surface
colour of colony should be carefully noted. The colour of reverse (underside) of the
colony must also be recorded, along with any pigment that diffuses into the medium.
To ensure that cultivation of the fungi in a specimen (especially the slower-
growing pathogen), it is advisable in many cases to hold the culture for at least a
month, eventhough some fungi may have been isolated. When more than one fungus
is seen on the slant, a carefully streaked plate is usually necessary for isolation. The
lead may be taped close in a several places for safety and prevention of the
dehydration, but care must be taken not to create anaerobic conditions.
Microscopic Examination of Growth
It is best to examine a fungus microscopically when the culture first begins to
grow and form conidia or spores and again a few days later. In many instances the
manner of conidiation or sporulation, which is so important to identification, is
obscured in old cultures. Potato flake or PDA often promotes conidiation or
sporulation better than does (SDA).

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Tease mount: Place a drop of lactophenol cotton blue(LPCB) on a clean glass slide.
With a bent dissecting needle, remove a small portion of the colony from the agar
surface and place it on LPCB. Wit two dissecting needle gently tease apart the
mycelia mass of the colony on the slide, cover with a coverslip, and observe under
microscope with low power or and high-dry (430X) magnifications. Unfortunately,
this method does always preserve the original position and structure of the conidia,
spores and other characterizing elements, but it is very rapid method and always
worth a try.

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