Module 6 - Mycology

Question 1

What is the serial endosymbiosis theory, and how does it relate to the evolutionary relationship between fungi, animals, and plants?

Answer 1

The serial endosymbiosis theory suggests that eukaryotic cells evolved through a series of symbiotic mergers between different prokaryotic cells. Opisthokonts, a group within eukaryotes that includes animals and fungi, are characterized by cells that propel themselves with a single posterior flagellum. This theory, as clarified by Cavalier-Smith in 1987, proposed that fungi are more closely related to animals than to plants, changing the previous assumption that fungi were closely related to plants.

Question 2

How do fungal cell walls differ from plant and bacterial cell walls?

Answer 2

Fungal cell walls contain mannans, glucans, and chitin. Plant cell walls contain xyloglucan, cellulose, and pectins. Bacterial cell walls contain peptidoglycan.

Question 3

What are the common growth forms of fungi?

Answer 3

Yeast: Round to ovoid cells that separate readily from each other.
Pseudohyphae: Elongated, ellipsoid yeast cells that remain attached at a constricted septation site, often growing in a branching pattern.
True Hyphae: Characteristic of moulds, they are long, highly polarized cells with parallel sides and no constrictions between cells.

Question 4

What is lignin and its role in plants?

Answer 4

Lignin is a heterogeneous polymer, second only to cellulose as the most abundant organic compound in the biosphere.
It provides structural rigidity to plants.
Lignin is highly resistant to degradation, making it a key component of plant durability.

Question 5

What is Phanerochaete chrysosporium and its role in lignin degradation?

Answer 5

Phanerochaete chrysosporium is the best-studied white rot fungus.
It produces enzymes that generate reactive free radicals, catalyzing lignin depolymerization.
Key components of its lignin degradation system are manganese-dependent peroxidase and lignin peroxidase.
These enzymes are encoded by large gene families in the P. chrysosporium genome.

Question 6

What does the comparison of 31 fungal genomes reveal about lignin-degrading peroxidases?

Answer 6

Lignin-degrading peroxidases expanded in a common ancestor of fungi.
This gene family expansion is estimated, using a molecular clock, to have occurred at the end of the Carboniferous Period.

Question 7

What are the two main types of fungal/plant symbioses and how do they function?

Answer 7

Mycorrhiza: Fungal growth enlarges the root surface area, increasing the plant’s absorption of water and minerals. In return, the plant provides the fungus with sugars.
Lichens: The fungus helps absorb moisture from the air, while the algae partner performs photosynthesis to produce sugars for both organisms.

Question 8

What are the characteristics and effects of Russula emetica, commonly known as “the sickener”?

Answer 8

It is a mycorrhizal species that associates with conifers.
Has a peppery taste.
Causes diarrhea and vomiting when consumed.

Question 9

How does Russula persanguinea relate to Russula emetica?

Answer 9

Russula persanguinea is a closely related species found in Australia.
It associates with eucalyptus trees.

Question 10

What is Amanita phalloides, and why is it known as the “death cap mushroom”?

Answer 10

Amanita phalloides belongs to a genus with 900–1,000 species, some of which are edible, but others, like A. phalloides, produce potent cyclic peptide toxins.
These toxins survive digestion and target liver cells by inhibiting RNA polymerase II.
Human LD₅₀ for α-amanitin is ~0.1 mg/kg, with 10–12 mg present in one mushroom.
Symptoms include gastrointestinal distress within 6–12 hours, temporary remission for 12–24 hours, followed by progressive liver failure, leading to death within 3–5 days.

Question 11

What is Ustilago maydis and what unique characteristic does it have?

Answer 11

Ustilago maydis is a basidiomycete pathogen that infects maize.
During its sexual cycle, it forms tumors on the plant that are filled with black spores.
Interestingly, it is also considered a delicacy and “tastes awesome.”

Question 12

What is the significance of the Fertile Crescent in early civilization?

Answer 12

Known as “The cradle of civilization,” it was a moist and fertile region compared to surrounding areas.
It was home to the eight Neolithic founder crops: Emmer, Einkorn, Barley, Flax, Chickpea, Pea, Lentil, and Bitter vetch.
Evidence shows that bread and beer production dates back approximately 8,000 years in this region.

Question 13

What is the origin of the name Saccharomyces cerevisiae?

Answer 13

Derived from sakkharon, the Greek word for “sugar.”
Mykēs is Greek for “fungus.”
Cerevisia is Latin for “beer.”

Question 14

What is yeast?

Answer 14

Yeast:
A frothy substance produced by fermentation of malt, etc. such as in the production of beer, often used for leavening in baking
A unicellular fungal growth form where the organism reproduces either by budding or fission
Saccharomyces cerevisiae, also known as budding yeast, baker’s yeast and brewer’s yeast
Two species of yeast used:
- Saccharomyces cerevisiae
- Saccharomyces carlsbergensis (syn. pastorianus)
Yeast Extract:
A processed yeast product commonly used in microbiological media
Vegemite

Question 15

Why are fungi easy to work with?

Answer 15

• Easy to grow, short life cycles
• Small genome relative to other eukaryotes (only ~10-40 Mbp)
• Many fungal genes are homologous to those in other eukaryotes
• An extremely wide array of molecular techniques
• Most have haploid genomes amenable to mutation
• Sexual stage for analysis of segregation and recombination of genes; all products of meiosis can be retrieved in haploid spores
• Asexual (clonal) reproduction

Question 16

What is Saccharomyces cerevisiae?

Answer 16

• Most well-known member of the phylum Ascomycota
• Cells are spherical to oval, cell division through budding
• 5-10 µM diameter
• Flourish in habitats where sugars are present
• Sexual reproduction process called mating

Question 17

What is the lifecycle of S.cerevisiae?

Answer 17

• Stable as a haploid (n=16), stable as a diploid (2n=32)
• Exists primarily as a diploid, particularly in industry
• Sexual cycle begins readily, but completion requires special sporulation conditions

Question 18

What are the key characteristics of the genome of Saccharomyces cerevisiae, including its chromosome count, orientation of genes, and notable features of Chromosome 1?

Answer 18

Saccharomyces cerevisiae has 16 chromosomes totaling 12 Mb. The orientation of genes is generally random, with ORFs transcribed in one direction typically not overlapping with those in the reverse direction. Chromosome 1 is one of the smallest at 230 kb (0.23 Mb).

Question 19

What are the key points of Das Reinheitsgebot?

Answer 19

The German Beer Purity Law, established in 1516, allowed only water, barley, and hops as ingredients in beer. If other ingredients were used, the beer would be confiscated.
To avoid competition with bakers for wheat and rye, brewers could only use barley.
Hops were required as a preservative to prevent the use of other harmful ingredients.
The Vorläufiges Biergesetz (Provisional German Beer Law, 1993) now allows yeast, wheat malt, and cane sugar, but not unmalted barley.

Question 20

What enzymes are involved in starch breakdown in barley and how do they function?

Answer 20

The major energy store in barley endosperm is starch, composed of branched amylopectin and linear amylose.
α-amylase acts at random locations along the starch chain, producing maltotriose and maltose.
β-amylase operates from the non-reducing end of the starch molecule, hydrolyzing the second α-1,4 glycosidic bond to cleave off maltose units one at a time.

Question 21

What is wort and how is it prepared in the brewing process?

Answer 21

Wort is a nutrient-rich liquid produced during the brewing process.
The preparation begins by milling malted grain, which is then combined with water and heated in a process called mashing.
During mashing, enzymes such as amylases act to convert starches into sugars.
After mashing, the wort is separated from the spent grain through a process known as lautering.
Flower clusters of Humulus lupulus (hops) are added to the wort for preservation and to impart bitterness.
The wort solution is then boiled for up to two hours and subsequently filtered.

Question 22

What is sourdough and what role do yeast and bacteria play in its fermentation process?

Answer 22

Sourdough is a symbiotic and stable culture of wild yeasts and bacteria created from a mixture of flour and water, often passed down for years.
Lactic Acid Bacteria (LAB), such as Lactobacillus sanfranciscensis, L. pontis, L. fermentum, and others, hydrolyze starch and convert sugars into lactic and acetic acids, creating a sour taste and acidifying the dough to a pH of approximately 3.8, which inhibits the growth of harmful microbes.
Acid-tolerant yeasts, like Candida humilis and C. krusei, help leaven the bread by metabolizing the byproducts of lactic acid fermentation into carbon dioxide and ethanol.
Saccharomyces cerevisiae (baker’s yeast) has less tolerance to acetic acid compared to other sourdough yeasts.

Question 23

What is the Pasteur effect in yeast metabolism?

Answer 23

The Pasteur effect refers to the phenomenon where energy production from glucose is maximized in the presence of oxygen through the TCA cycle.
In contrast, less energy is produced through anaerobic fermentation, specifically the conversion of glucose to ethanol and carbon dioxide:
C6H12O6 -> 2CO2 + 2CH3 + 2CH3CH2OH
In most fungi, essential genes exist whose products require oxygen to function.
Saccharomyces spp. have adapted to acquire non-oxygen requiring variants via horizontal gene transfer from anaerobic bacteria.

Question 24

How does horizontal gene transfer relate to pyrimidine biosynthesis in Saccharomyces species?

Answer 24

Pyrimidine biosynthesis requires dihydroorotate dehydrogenase, a protein located in the mitochondria of eukaryotes that depends on oxygen.
Anaerobic bacterial species possess a cytoplasmic version of this enzyme that does not require oxygen.
Saccharomyces species have acquired the anaerobic bacterial version through horizontal gene transfer, allowing them to synthesize pyrimidines in oxygen-limited environments.

Question 25

What is the Crabtree effect in Saccharomyces species?

Answer 25

The Crabtree effect refers to the phenomenon where Saccharomyces spp. produce ethanol even in the presence of very high concentrations of glucose and sufficient oxygen. This results in aerobic fermentation, where the yeast favors the production of ethanol over respiration.

Question 26

How does beer production contribute to the safety of potable water?

Answer 26

During beer production, the water is boiled, which kills microbes present in the water.
The growth of Saccharomyces yeast produces ethanol, which robs competing microbes of a carbon source and kills them.
Hops, a key ingredient in beer, also inhibits microbial growth, further ensuring the safety of the beverage.

Question 27

What are the two main types of brewery yeast strains and their characteristics?

Answer 27

Top Fermenting Yeast (Ancient):
Remain uniformly distributed in the fermenting wort.
Carried to the top by the CO₂ produced during fermentation.
Used in the production of ales (e.g., porters and stouts) at temperatures of 14-23°C.
Example: Saccharomyces cerevisiae.

Bottom Fermenting Yeast (Modern):
Settle to the bottom of the fermenting wort.
Used in the production of lagers at cooler temperatures (5-14°C).
Example: Saccharomyces pastorianus (syn. carlsbergensis).

Question 28

What are the key differences between primary and secondary metabolites?

Answer 28

Primary Metabolites:
Produced during exponential growth.
Essential for cell growth and metabolism.
The best known example is alcohol.

Secondary Metabolites:
Produced during the stationary phase of growth.
Not essential for the growth of the organism.
Formation depends on specific growth conditions.
Typically produced as a group of related compounds.
Often significantly overproduced.
Frequently produced by spore-forming microbes during sporulation.

Question 29

What are the steps involved in purifying the metabolite of interest during whiskey production?

Answer 29

1. Fermentation: A fermentation process is performed using malted barley.
2. Distillation: This step increases the alcohol content and removes undesired impurities, resulting in whiskey.
3. Aging: Aging the whiskey in wood casks imparts additional flavors to the final product.
4. Boilermaker: A Boilermaker, which is a pint of beer with a shot of whiskey added, essentially combines aged distilled beer back into beer.

Question 30

What advancements have been made in the transformation of fungi, particularly in S. cerevisiae?

Answer 30

Whole genome deletion sets have been established in S. cerevisiae (both haploid and diploid), and most fungi can now be transformed using simple techniques, extending beyond just the “model” species.

Question 31

What is the methodology for transforming S. cerevisiae and how are transformants selected?

Answer 31

To transform S. cerevisiae, cells are exposed to lithium acetate to induce competence. They then take up DNA after the addition of PEG, heterologous carrier DNA, and heat shock. Transformants are selected using vectors that carry the DNA of interest and a selectable marker; if the cell takes up the marker, it indicates that it has also incorporated the gene of interest.

Question 32

What are some key characteristics of the methylotrophic yeast Pichia?

Answer 32

Pichia is a methylotrophic yeast from the phylum Ascomycota that grows to extremely high cell density. It efficiently secretes heterologous proteins into defined media and has tightly controlled methanol-inducible transgene expression.

Question 33

What is electroporation and how is it used in the transformation of Pichia pastoris?

Answer 33

Electroporation: application of an electrical field creates transient pores in the cell membrane, enabling polar molecules such as DNA to enter. In P. pastoris the process of electroporation yields transformants containing multiple copies of the inserted DNA

Question 34

What criteria must be met to successfully express foreign genes in fungi?

Answer 34

1. No introns: Use cDNA created with reverse transcriptase or buy a synthetic construct.
2. Native promoter: A tightly regulated native promoter should be used.
3. Typical codon usage: The codon usage should align with that of the fungus being utilized, as each organism has a preference for certain codons for specific amino acids.

Question 35

What are the key aspects of advanced genetic engineering in producing therapeutic proteins like erythropoietin in Pichia pastoris?

Answer 35

Post-Translational Modifications: Many proteins require modifications for stability, regulation, or function.
Glycosylation Requirement: Erythropoietin must be glycosylated to effectively stimulate red blood cell production.
Production: Erythropoietin, a powerful therapeutic agent, is now produced in the methylotrophic yeast Pichia pastoris.

Question 36

What is Coccidioidomycosis and what are its causative species?

Answer 36

Coccidioidomycosis, also known as Valley Fever, desert bumps, or desert rheumatism, is caused by the fungi Coccidioides immitis and Coccidioides posadasii. It is a haploid dimorphic fungus that adopts a yeast form in tissues to form large spherules. This infection is primarily found in the south-west USA, Central America, and South America.

Question 37

What are the types of diseases and the route of infection associated with Coccidioidomycosis?

Answer 37

Coccidioidomycosis is the most virulent known fungus, leading to several types of disease:
- Mild (flu-like) respiratory infection
- Serious pulmonary infection
- Dissemination to skin and CNS, more common in pregnant women, dark-skinned individuals, and the immunocompromised.

Question 38

What is Penicilliosis and what are its key characteristics?

Answer 38

Penicilliosis is caused by the opportunistic intracellular pathogen Talaromyces marneffei. It is a haploid dimorphic fungus that can adopt a yeast form in human hosts and is primarily found in Southeast Asia. The disease can present as superficial infections or systemic infections that affect multiple organs, including the skin, kidneys, and lungs. Untreated infections can be fatal, particularly in immunocompromised individuals, making it the third most common life-threatening fungal infection in Southeast Asia and an AIDS-defining pathogen. The route of infection is primarily through the inhalation of spores.

Question 39

What are the key features of Cryptococcus neoformans?

Answer 39

• Haploid basidiomycete yeast
• Worldwide distribution
• Most common life-threatening fungal infection in AIDS patients
• Causes systemic mycoses, commonly meningoecephalitis
• Responsible for up to a quarter of a million deaths per year
• Fatal if untreated
• Relapse is common

Question 40

What are the key details of the case study in Cryptococcosis?

Answer 40

Scenario:
- 60-year-old male with kidney transplant 3 years prior
- Immunosuppressed with prednisone and cyclosporine A
- Minor scratch on hand by cat

Symptoms:
- Local inflammation, treated with amoxicillin and clavulanic acid
- Inflammation spread rapidly up the arm
- Continued worsening of inflammation

Treatment:
- Incision on palm and removal of dead tissue
- Administered clindamycin, ciprofloxacin, and fluconazole
- Severe soft tissue necrosis, edema, and fluid collections
- Imipenem and liposomal amphotericin B
- Amputation at wrist
- Amphotericin B for 3 weeks intravenously; fluconazole orally for 15 weeks

Diagnosis:
- Encapsulated yeast identified as C. neoformans
- After 9 months, no symptoms of skin or systemic infection
- Wound almost completely healed