Chapter 1 Introduction

INTRODUCTIONSiderophores

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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Chapter 1 Introduction

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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Chapter 1 Introduction

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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Chapter 1 Introduction

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

A variety of fungi are known to overproduce and excrete siderophores under iron

limitation, ferrichromes, being the most common group of fungal siderophores.

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Chapter 1 Introduction

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

their ironcharged forms and the prefix deferri- or desferri- is used to denote the iron-

free (deferrated) form of the ligand.

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Chapter 1 Introduction

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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Chapter 1 Introduction

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

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Chapter 1 Introduction

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

by excreted iron-complexing agents.

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Chapter 1 Introduction

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

Gliocladium virens

Fusarinines

Dihydroxamates Rhodutorula

Microbotryum

Fe-Rhodotorulic acid

Fusarium dimerum

Verticillium dahliae

Diemrum acid

Trihydroxamtes (Linear) Neurospora crassa

Penicillium spp

Coprogens

Fusarium dimerum

Verticillium dahliae

Coprogen B

Curvularia lunata

Epicoccum

purpurascens

Neocoprogen I

Neocoprogen II

Alternaria alternata

Alternaria lomgipes

Dimethylcoprogen

Hydroxycoprogen

Trihydroxamates (Cyclic) Ustilago spp

Penicillium

Ferrichrome

Neovossia indica Tetraglycylferrichrome

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Chapter 1 Introduction

Siderophore-Type Species Examples

Trihydroxamates (Cyclic) Aspergillus fumigates

Neurospora crassa

Ferricrocin

Aspergillus orchraceus

Aspergillus melleus

Asperchromes

Ferrichrysin

Aspergillus ochraceus

Penicillium variabile

Ferrirubin

Botrytis cineria Ferrirhodin

Fusarium cubense Fusarinin C

Mycelia sterilia

Aspergillus spp

Triacetylfusarinine C

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.

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Chapter 1 Introduction

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,

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

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Chapter 1 Introduction

difference between fungi and bacteria are mentioned.Properties Fungi Bacteria

Nucleus Eukaryotic; nuclear

membrane; more than

one

chromosome; mitosis

Prokaryotic;

no membrane;

nucleoid; only

one “chromosome”

Cytoplasm Mitochondria;

endoplasmic

reticulum; 80S ribosomes

No mitochondria;

no endoplasmic reticulum;

70S ribosomes

Cytoplasmic

Membrane

Sterols (ergosterol) No sterols

Cell wall Glucans, mannans, chitin,

chitosan

Murein, teichoic acids

(Gram-positive), proteins

Size, mean diameter Yeast cells: 3–5–10 lm.

Molds: indefinable

1–5 lm

Metabolism Heterotrophic;

mostly aerobes;

no photosynthesis

Heterotrophic; obligate

aerobes and anaerobes,

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

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Chapter 1 Introduction

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

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.

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Chapter 1 Introduction

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.

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.

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Chapter 1 Introduction

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.

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.

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Chapter 1 Introduction

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

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

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Chapter 1 Introduction

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.

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

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Chapter 1 Introduction

obscured in old cultures. Potato flake or PDA often promotes conidiation or

sporulation better than does (SDA).

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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