BIOL 2051 - MOCK EXAM #2

What is the definition of a coliform?

Coliforms are aerobic or facultatively anaerobic, Gram-negative, non-spore-forming rods that produce gas from lactose fermentation within 48 hours at 35˚C. They are part of the normal intestinal microbiota of humans and large animals.

Which organism is used as the indicator of fecal contamination in the U.S.?

Escherichia coli is used as the primary indicator organism because it is found in large numbers in sewage and survives longer in water than most intestinal pathogens.

What does MPN stand for?

Most Probable Number

What are the three phases of MPN?

Presumptive Test, Confirmed Test, & Completed Test

What media is used for the MPN Presumptive test?

Lauryl Sulfate Lactose (LSL) broth (also known as lauryl tryptose broth). The lauryl sulfate selects for intestinal bacteria, while the lactose serves as the differential component for fermentation.

How is a positive result identified in LSL and BGLB broths?

A positive result is the presence of gas bubbles trapped in the inverted Durham tubes. In BGLB broth, this must occur within 48 ± 3 hours at 35 degrees Celsius

Which agar is used for the MPN Completed test?

Eosin Methylene Blue (EMB) agar.

How does E. coli appear on EMB agar?

E. coli produces very dark/black colonies that typically exhibit a characteristic green metallic sheen. This is due to the large amount of acid produced during lactose fermentation, which causes significant dye uptake.

How does K. aerogenes appear on EMB agar?

It produces larger, mucoid "fish-eye" colonies. These have a dark center (where more dye is taken up) and pale pink outer edges (where less acid is produced).

What does the acronym IMViC stand for?

Indole, Methyl Red, Voges-Proskauer, and Citrate (the lowercase "i" is added for pronunciation)

What enzyme is tested for in the Indole test, and what is its substrate?

The enzyme is tryptophanase, and its substrate is the amino acid tryptophan.

What reagent is added to detect Indole, and what is a positive result?

Kovac’s reagent is added. A positive result is the formation of a red transparent layer at the top of the broth.

At what pH does Methyl Red (MR) turn red?

Methyl Red is yellow at neutral pH and turns red when the pH drops below 4.2.

What does a positive Voges-Proskauer (VP) test indicate?

It indicates the production of acetyl methyl carbinol (acetoin), which is a neutral product of butanediol fermentation.

What pH indicator is in Simmons' Citrate Agar?

bromothymol blue

what is a positive color in Simmons’ Citrate Agar?

a change from green to deep blue, indicating the organism used citrate as its sole carbon source and produced alkaline products; Alkaline Shift (Positive Result): If an organism can transport and metabolize citrate, it grows and produces alkaline byproducts. This rise in pH causes the bromothymol blue to change from green to a deep blue.

what is a negative indicator in Simmons’ Citrate Agar?

Neutral/No Change (Negative Result): If the organism cannot use citrate, it will not grow, and the medium remains its original green color.

What is the symbiotic relationship between Rhizobium and clover?

It is a mutualistic relationship. Both benefit: the bacteria fix nitrogen for the plant, and the plant provides the bacteria with a stable environment and nutrients.

What protein regulates oxygen in root nodules and gives them a pink color?

Leghemoglobin. It binds oxygen to keep levels high enough for aerobic bacterial respiration but low enough to prevent the inactivation of the nitrogenase enzyme.

Name a free-living (nonsymbiotic) aerobic nitrogen-fixing soil microbe.

Azotobacter vinelandii.

What medium is used to selectively enrich Azotobacter?

N-free (nitrogen-free) broth containing mannitol as a carbon/energy source. The absence of a nitrogen source selects for organisms capable of fixing gaseous nitrogen N2 from the air.

Which enzyme degrades cellulose, and what type of bond does it break?

The enzyme is cellulase. It breaks the beta(1→4) glycosidic bonds between glucose residues in cellulose.

Identify two soil microbes that produce cellulase.

Verified examples include bacteria in the genus Cytophaga (e.g., C. hutchinsonii) and fungi in the genus Aspergillus. Bacteria in the genus Clostridium also produce it anaerobically.

Define the Stationary phase of bacterial growth

It is the phase where the number of cells undergoing division is equal to the number of cells dying, usually due to nutrient exhaustion

What is a major limitation of using a spectrophotometer (turbidity) to measure microbial growth?

This method cannot distinguish between live and dead cells, which can skew counts during the stationary and death phases

Explain the concept of Viable But Non-Culturable (VBNC) cells.

These are living cells that cannot be detected or grown on standard solid agar media, necessitating advanced detection methods like RT-PCR or fluorescence staining.

How do repressor proteins physically stop the process of transcription?

They bind to a specific DNA sequence called an operator near the promoter, physically blocking RNA polymerase from attaching.

By what mechanism does the antibiotic Rifampicin interfere with bacterial growth?

It binds to RNA polymerase and blocks it at various stages, such as preventing it from binding to or clearing the promoter region.

How does an anti-sigma factor regulate gene expression?

It binds to a sigma factor to prevent it from associating with the RNA polymerase holoenzyme, thereby blocking the recognition of specific promoters.

Under what condition does the 3’ →5’ exonuclease activity of DNA polymerase predominate over its polymerase activity?

It predominates when the last nucleotide inserted is not correctly base-paired to the template.

What is the primary difference between Base Excision Repair (BER) and Nucleotide Excision Repair (NER)?

BER removes a single damaged base using a DNA glycosylase, while NER recognizes bulky lesions (like pyrimidine dimers) and removes a multi-nucleotide portion of the damaged strand.

Why do RNA viruses typically have much higher mutation rates than prokaryotes?

Many viral RNA-dependent RNA polymerases lack the proofreading ability found in prokaryotic DNA polymerases.

Explain why the AUG (methionine) and UGG (tryptophan) codons are at a "higher risk" regarding mutations.

They are non-degenerate; any single nucleotide mutation in these codons will lead to a change in the amino acid or start signal, whereas degenerate amino acids are protected by silent mutations.

How do Vancomycin-resistant staphylococci modify their cell wall to survive treatment?

It acts as an efflux pump that actively expels a wide range of antibiotics (like tetracyclines) from the cell's interior to the extracellular environment.

How do the membrane lipids of thermophilic archaea differ from those of bacteria to withstand high heat?

They often use more stable ether linkages (rather than ester linkages) and may form lipid monolayers using tetraether lipids for added stability.

Why do psychrophilic enzymes require lower activation energy Ea to function?

They have highly flexible structures that allow for catalysis even with the reduced kinetic energy available in cold environments.

How does Helicobacter pylori neutralize its immediate environment in the acidic stomach?

It uses the enzyme urease to hydrolyze urea into ammonia and CO2; the ammonia then combines with protons to buffer the periplasm and cytoplasm.

In alkaliphiles, why is the Proton Motive Force (PMF) converted into a Sodium Motive Force (SMF)?

Because the PMF is not strong enough to drive all cellular processes at high external pH, the energy is used to power Na+/H+ antiporters to create an SMF.

What is the mechanism of "photoreactivation" in repairing UV damage?

Energy from visible light is utilized by enzymes to directly break the cyclobutane ring of a pyrimidine dimer, restoring the bases to their normal state.

How does the bacterium Deinococcus radiodurans survive high doses of gamma radiation?

It is exceptionally skilled at repairing the double-strand and single-strand DNA breaks caused by ionizing radiation.

What are "compatible solutes" (organic osmolytes) and why are they used during osmotic stress?

They are small organic molecules (like proline or glycine betaine) that can accumulate to high concentrations without disturbing cellular metabolism, protecting the cell from dehydration.

Contrast the potassium K+ uptake mechanisms of the TrkG and TrkH transporters in E. coli.

TrkG is Na+-dependent and transport is partially repressed by H-NS, while TrkH is Na+-independent and its expression is not controlled by H-NS.

RNA viruses and viroids have the highest mutation rates because their polymerases often lack ______________

proofreading

Prokaryotes have _________________ genomes than eukaryotes but a higher mutation rate

smaller

Organisms with _________________ replication cycles, such as viruses and bacteria, tend to accumulate mutations more quickly.

faster

A _________________ mutation occurs in a gene's coding region but does not alter the final protein or its function.

silent

The _________________ of the genetic code provides a buffer against mutations, allowing multiple codons to code for the same amino acid

degeneracy

Unlike degenerate codons, the codons for _________________ (AUG) and _________________ (UGG) are at a higher risk because any single nucleotide change leads to an amino acid substitution

methionine; tryptophan

A _________________ mutation creates a premature stop codon, leading to a shortened and often non-functional protein

nonsense

DNA polymerase can correct its own errors during replication if it possesses ________________ exonuclease activity

3’ → 5’

UV radiation primarily kills bacteria by creating _________________, which distort the DNA chain and block transcription or replication

pyrimidine dimers (or thymine dimers)

________________ is a repair process where energy from visible light is used to break the cyclobutane ring of a pyrimidine dimer

Photoreactivation (aid of photolyase enzyme)

In _________________ excision repair, a multienzyme complex recognizes a bulky lesion and removes a portion of the damaged strand

nucleotide

Gamma radiation causes _________________ damage by creating free radicals and reactive oxygen species that attack DNA

indirect

The bacterial species _________________ is exceptionally skilled at surviving radiation by repairing double-strand and single-strand DNA breaks.

Deinococcus radiodurans

Some bacteria survive gamma rays by forming highly resistant _________________

spores (or endospores)

In Nucleotide Excision Repair, the resulting gap in the DNA is filled by _________________ and sealed by _________________

DNA polymerase; ligase

A _________________ is a type of microorganism that has an optimal growth temperature below 15°C

psychrophile

To maintain membrane fluidity in the cold, psychrophiles incorporate more _________________ and branched-chain fatty acids

unsaturated (polyunsaturated)

Thermophiles increase protein stability by using a higher number of _________________, which are electrostatic interactions between oppositely charged amino acid side chains

salt bridges

Enzymes in psychrophiles have a more flexible structure resulting in _________________ activation energy

lower

In response to high temperatures, bacteria produce _________________ proteins to refold denatured proteins and prevent aggregation

heat shock (or HSPs/chaperone)