Fungal growth

Lecture 2

How many Fungal species

5 million species (although probably more)

• > 10 million

Fungal role

• Crucial role in environmental mineral and nutrient recycling

• ideal model orgnaisms for research

  • food

  • beverage

  • pharmaceutical and biotechnology

• Devastate crops

  • phytopathogens

• Cause human disease

  • mycoses

What is a true fungus?

• Should be closer realted to us than to brown seaweed etc

Fungal like:

• oomycetes e.g Phytophthora infestans- potato late blight

• Plasmodiophorids

• similar patterns of growth and nutrition to fungi but less related to us

True fungus:

• Penicillium notatum

  • distantly related to the p.infestans

What are fungi? (structurally)

• Eukaryote (can be multi-nucleate)

• cell wall: chitin and glucan

• multi or unicellular (sometimes)

• Vacuoles

  • like plant vaculose: hydrolytic enzymes, storage

• Chloroplast-free zone

  • most important determining factor

  • heterotrophs

Roles of Fungi

1. recycle N and C

   • cellulose and lignin degradation

   • hard to digest

   • saprophytes

2. assist or destroy crops

   • arbuscles good

   • Haustoria bad/pathogenic

3. save or kill humans

   • antibiotics

   • mycosis from bad fungus- aspergillus

4. can be utilised

   • Yeast for research

1% are unicellular

• Spheroids

  • small SA per unit volume

  • wall manufacture is economical

• Habitat

  • plant/animal surfaces

  • plant exudates

  • animal mucous membranes

• Two main types

  • budding yeast (Saccharomyces cerevisiae)

  • fission yeast (Schizosaccharomyces pombe)

Multicellular fungi (filamentous)

• ‘tube in search of food’- Hyphae

  • vegetative growth form

  • emerges from a spore

• Habitat

  • everywhere

What are spores

• dispersive propagule (ananolgous to a plant seed)

• carries genetic code

Features of spores

Made in vast quantities from differentiated hyphae

1. Asexual or sexual

2. Low metabolic activity

3. Germination trigger

   • environmental triggers

   • e.g temperature

   • Response= imbibing water→ germinating

Asexual spores

Sexual Spores

What happens during germination of spores

Sphere→ Hypha

• to anisotropic (polar) growth

• why?

  • germ tube can locate nutrients

  • then first cross wall formed

  • race against time

  • must locate new food supply

    • before endogenous reserve are exhausted

How do the new hyphae find food?

• Tropisms

  • e.g towards amino acids

  • enable targeted growth towards food supply

  • found = germ tube matures→ hypha

Structure of hypha

• Series of zones of specific…

  • structure and role

• Divided into

  • discrete, regular compartments by cross walls

How have protoplasmic continuity?

Pores (septa)

Types:

1. Woronin bodies: in simple pores

   • Block the pores

   • Porteinaceous

2. Parenthosome: in Dolipores

Apical growth zone: where grow from

• restricted to the apex

• addition of new materials helps it grow

• fungal polar growth

How found out the model of the fungal polar growth

• pharaseutical and genetic

  • manipulation of putative compondents

Generalised model for polar growth: Step 1

1. Wall component and enzymes to catalyse wall formation

   • delivered continuously to the tip

   • via Vesciles

     • e.g Neurospora crass: 38,000 delivered per minute

Generalised model for polar growth: Step 2

2. Vesicles are formed by

   • sub-apical ER/Goli

   • directed to the apex by

     • actin cytoskeleton

Generalised model for polar growth: Step 3

3. Vesicles fuse to the apical plasma membrane

   • exocytosis

   • deliver the cargo to the area to

     • where the nascent wall is thin and deformable

Generalised model of polar growth: Step 4

4. Cell wall synthesising enzymes

   • secreted as zymogens

   • Hydrostatic pressure “pushes” out the tip

Generalised model of polar growth: Step 5

5. The wall matures to form a rigid, layered lateral wall

Why is control of this system needed

1. achieved regular shape

2. regular growth rate 1mm/hour

3. directional changes much centre on targeted exocytosis

Where are vesicles found

Densely packed in the tip

• forms Spitzenkorper

  • vescile organisation centre

• If disrupted

  • fungal growth ceases

Involvement of Ca²⁺ ions with actin and exocytosis

1. Apical plasma membrane Ca2+ permeable ion channels permit localised Ca2+ influx

   • helps create tip with high [Ca²⁺]_cyt (microM) gradient

2. High [Ca²⁺]_cyt stimulates exocytosis

   • maintains actin in F-actin (filamentous) form

3. F-actin gives the cytosol mechanical strength

   • without high viscosity

How are vesicles directed?

• SNARE proteins help move them to specific fusion sites

  • Cognate pairs of SNAREs exist in vesicles and target membrane

  • have been found in animal and yeast cells

    • Ustilago maydis

What is the tip also used for?

Exoenzyme secretions

• Used to degrade insoluble external substrate

  • cellulose, lignin, chitin

• So it can be absorbed

Where secreted?

• Some small molecules

  • from the lengths (depends on wall porosity)

• Larger

  • secreted at the nascent tip

When do hyphae secrete exoenzymes

• When need to grow

  • adaptive and economical

Catabolite repression

• Can grow Trichoderma with cellulose and only C source

  • secretes enzymes

  • breaks it into glucose

But

• Grow it on glucose

  • production of enzymes inhibited

    • Catabolite repression

      • In all fungi

Why have catabolite repression

• Use the nutrition source that is in greatest abundance

• Switch off metabolism that becomes unnecessary

  • less energy wasted

E.g of catabolite repression: Neurospora

• Grow on no arginine

  • Makes own: anabolic reaction

    • made from ornithine

• Supply with arginine

  • arginine inhibits enzymes that make ornithine

  • so no more arginine is made

  • arginase activity in cell increases

  • Catabolic production

    • makes arginine→ ornithine

Taking up materials (once digested etc)

Needed for new materials for growth

• Fungal growth could be infinite

Many transport proteins

• e.e yeast has 19 hexose transporters

Many of the transport proteins

• Fungu run on proton economy

• Many pumps are P-type A+-ATPase

  • In plasma membrane

  • encoded by PMA1 and PMA2

What H+ pumps do

• uses 50% of fungal ATP to set up

  • proton electrochemical potential graidnet

  • Drives H+ coupled nutrient uptake

    • symport

    • or

    • Explusion of potentially toxic ions

      • (e.g SOD2 Na+/H+ antiporter in pombe)

What do experiments show sabout H+ arounf the hyphae

Use vibrating microeletrode experiments

• Show extracellular H+ circuit around growing hyphae

• acts as a DC power supply

  • H+ coupled nutrient symporters at the apical membrane act as resistor in the circuit

• Points of re-entry

  • indicate nutrient absorption

    • at and just behind the apex

  • Disappear in absence of nutrients

Expression of nutrient uptake transporters

• Tightly regulated

  • as metabolic enzymes

Example of transport regulation: Saccharomyces cerevisiae

HXT expression- hexose transporter

1. At low external glucose

   • HXT2 and HXT4 are expressed

   • high affinity H+ coupled glucose sympoters

2. entry of glucose triggers phosphoylation (via cAMP?)

   • of Regulatory C-terminus of plasma membrane H+-ATPase

   • Activit increase to aid the H+ coupled glucose symporters

Other genes encode for low affinity non-coupled transporters

Storage zones

• In the older sub apical region

  • Excess C is stored as:

    • Glycogen

    • lipid

  • Vacuoles

    • excess N: in vacuolse

    • ions such as K+

When storaged stuff is needed

Mobilised for growth

What storage zones also do

• Generate osmotic gradient

• ensure water uptake to generate hydrostatic pressure

  • needed for growth

What this means

Nutrient uptake and extension:- INEXTRICABLE LINKED

Senescence zone

Older regions are not wasted!

• Vacuoles release hydrolytic enzymes

  • autolysis

• Adjacent younger regions absorb breakdown products

Colony strucuture

1. Newly emergent hyphae near maximum extension rate

2. Branch will form

3. Makes new hyphae

4. Lateral branches continue to form

   • at exponential rate

   • grow at the tip and absorb nutrients

Why are many fungal hyphae autotropic?

So that it can make sure it grows in new environment, even if it doesn’t have any food available yet??

How hyphae colonise uncolonised substatum

1. senses presence of other hyphae

2. makes sure to grow away from it

3. So grows into place where not colonised yet$

Mycelium

Hyphae network

Fungal conidiation (spore formation) regulated by…

Circadian clock

Mycelium advantages

• efficient at maximising nutrient uptake from substratum

Make sure to not grow in rubbish places

• sets up nutrient depletion zones beneath the colony

  • when depletion is acute:

    • growth in these regions is switched off

    • growth only in leading esge mycelium

    • It is out there foraging

Advantage of concentrating growth capacity at the leading edge

Not limited to rate of diffusion

• out there foraging

unlike:

• yeast and bacterial colonies

  • rely on diffusion

Metabolic flexibility: vast range of growth substrates

Metabolic flexibility: Glycoxylate cycle

1. Under low C conditions (e.g when fungus is phagocytoses by macrophages)

   • Glycolate cycle switched on

   • Phagosome is nutrient poor environment

2. Fungus upregs expression of enzymes of glyoxylate cycle

3. Diverts TCA

4. into gluconeogenesis for production of hexoses for growth

C.glabrata strain carrying mutation in isocitrate lyase genes

Are non-pathogeneic

• Therefore: glyoxylate cycle imporant for

  • Pathogenesis

Metabolic flexibility: Oxygen

Majority of fungi are aerobes

but

some are obligate anaerobes

• niche habitats

  • cow rumen

• no longer have mitochondria

Why useful to be facultative anearobe

Increases survival chances:

• TCA is switched off

• mitochondria production suppressed

• glycolysis relied upon for ATP

• ethanol end-product may be metabolised as a C source when an oxygen supply returns