Questions & explanations
1. How do fungi regulate the production of cellulases so they only make them when needed?
Fungi regulate cellulase production through a system that senses available sugars. When glucose is present, a repressor protein blocks cellulase genes. When glucose is low and cellulose or its breakdown products (like cellobiose) are present, a sensor triggers a signaling pathway that activates transcription factors. These factors turn on cellulase genes. This ensures the fungus does not waste energy making cellulases when easier sugars are available. Some fungi also use a positive feedback loop where small amounts of cellulase release inducers that boost more production. This regulation helps fungi adapt to changing food sources.
2. What is the difference between a bacterial biosensor and a bacterial bioreporter?
The terms are often used interchangeably, but a biosensor typically includes the whole system (bacteria plus detection device), while a bioreporter usually refers only to the engineered bacterium that produces the signal. A biosensor outputs a quantifiable signal that can be read by an instrument, whereas a bioreporter may simply indicate presence or absence. Bioreporters often use reporter genes like GFP (green fluorescent protein) that can be seen under a microscope. Biosensors are designed for field use and may include a handheld reader. Both rely on the same principle of genetic engineering to link detection to a signal.
3. What is the main difference between broth microdilution and disk diffusion methods for testing antifungal susceptibility?
Broth microdilution uses liquid medium with increasing drug concentrations in wells to find the minimum inhibitory concentration (MIC), which is the lowest drug amount that stops fungal growth. Disk diffusion places drug-soaked paper disks on an agar plate with fungus; a clear zone around the disk shows inhibition. Broth microdilution gives a precise MIC number, while disk diffusion gives a zone diameter that is compared to breakpoints. Broth microdilution is more quantitative and standard, whereas disk diffusion is simpler but less precise. Both methods help determine if a fungus is susceptible or resistant to a drug.
4. Compare the risk of parasitic infections in HIV and transplant patients.
Both HIV and transplant patients have weakened immune systems, but the causes differ. HIV destroys CD4 cells, while transplant drugs suppress overall immunity. HIV patients are more prone to reactivation of latent parasites like Toxoplasma, while transplant patients may acquire new infections from the donor organ. For example, Strongyloides hyperinfection is more common in transplant patients. Both groups need preventive measures, but HIV patients benefit from antiretroviral therapy to restore immunity, whereas transplant patients must stay on immunosuppressants. Monitoring and prophylaxis are tailored to each group.
5. What is a bacterial biosensor and how does it work?
A bacterial biosensor is a genetically engineered bacterium that detects a specific chemical or physical signal and produces a measurable output, like light or color. The bacterium contains a sensor part that recognizes the target molecule and a reporter gene that produces a signal when the sensor is activated. For example, a biosensor for arsenic uses a promoter that turns on in the presence of arsenic, causing the bacteria to produce green fluorescent protein. The amount of light or color indicates the concentration of the target. These biosensors are cheap, portable, and can detect pollutants or disease markers.
6. How do you analyze 16S amplicon sequencing data to compare two microbial communities?
First, sequence reads are quality-filtered and clustered into operational taxonomic units (OTUs) based on 97% similarity. Each OTU represents a group of closely related sequences. Then, OTU abundances are compared between samples using statistical tests. Alpha diversity measures within-sample richness, while beta diversity measures between-sample differences. Principal coordinate analysis (PCoA) can visualize community dissimilarity. Finally, taxonomic assignment is done by matching OTUs to reference databases. This workflow reveals which microbes differ between communities, such as healthy vs. diseased gut.
7. How does a polyketide synthase (PKS) differ from an NRPS in the type of compounds it makes?
A PKS builds polyketides by repeatedly adding small units like acetate (two carbons) to a growing chain, while an NRPS adds amino acids. Polyketides include compounds like statins (cholesterol-lowering) and aflatoxins (toxins), which have carbon chains with ketone groups. NRPS products are peptides with amide bonds. Both enzymes use a similar modular logic, but PKS uses acyl-CoA substrates and NRPS uses aminoacyl-tRNA-like substrates. Some hybrid PKS-NRPS enzymes make mixed compounds. The final structures differ: polyketides are often cyclic or aromatic, while NRPS products are linear or cyclic peptides.
8. Compare Aspergillus and Cryptococcus: which one is more likely to cause disease in a person with a healthy immune system?
Aspergillus species are common molds that people breathe in every day. They rarely cause disease in healthy individuals, but can cause allergic reactions or lung infections in people with lung disease or weak immunity. Cryptococcus neoformans is a yeast found in soil and bird droppings. It mainly causes meningitis in people with weak immune systems, such as those with HIV/AIDS. Both are opportunistic, but Cryptococcus more specifically targets immunocompromised hosts, while Aspergillus can also affect people with chronic lung conditions. Neither typically causes disease in fully healthy individuals.
9. Compare adaptive laboratory evolution with rational genetic engineering. What are the advantages and disadvantages of each?
Rational engineering requires knowing which genes to change, which is hard for complex traits like stress tolerance. Adaptive evolution does not need this knowledge; it lets nature find the beneficial mutations. However, evolution is slower and may take months, while rational engineering can be faster if the right targets are known. Also, evolved strains may have multiple mutations, some of which might be harmful. Rational engineering can make precise changes without unwanted side effects. Often, the best approach is to combine both: evolve first, then identify and transfer the key mutations.
10. Why are fungi better than bacteria for degrading some pollutants like lignin-derived compounds?
Fungi secrete powerful extracellular enzymes (lignin peroxidases, laccases) that can break down large, insoluble pollutants like lignin and PAHs outside their cells. Bacteria often need to take up pollutants inside, which is hard for large molecules. Fungal enzymes are non-specific and can attack a wide range of aromatic compounds. Fungi also grow as hyphae that can penetrate solid materials, reaching pollutants in soil or wood. This makes fungi effective for treating complex mixtures of pollutants. Additionally, fungi tolerate harsh conditions like low pH and high pollutant concentrations.
11. Compare the drug development process for malaria and leishmaniasis.
For malaria, drug development has more funding and many approved drugs, like artemisinin combinations. Targets include the parasite's digestive vacuole and mitochondria. For leishmaniasis, fewer drugs exist, and they often have severe side effects, like antimony compounds that damage the heart. Leishmania lives inside immune cells, making drug delivery harder. Both diseases require drugs that are effective against different life stages. Malaria drugs are tested in mice and then humans, while leishmaniasis drugs are tested in hamsters. Newer approaches use high-throughput screening for both.
12. Why do some fungi become resistant to multiple drug classes at the same time? Give one example of a mechanism that causes multidrug resistance.
Multidrug resistance often arises from overexpression of efflux pumps that can export several different drug types. For example, the ATP-binding cassette (ABC) transporters in Candida can pump out azoles, echinocandins, and other drugs. This reduces the intracellular concentration of each drug, allowing the fungus to survive. Another cause is mutations in regulatory genes that upregulate multiple resistance pathways. Biofilm formation also contributes by physically blocking drug penetration. Multidrug resistance makes infections hard to treat and requires combination therapy or new drugs.