BIOL 2051 Lecture 3 - Bacterial Endospores

Practice questions covering bacterial endospore formation, transcription mechanisms, gene regulation cascades, and clinical/industrial applications of spore-forming bacteria.

Why is a 48- to 72-hour culture of Bacillus used for endospore staining?

Endospore formation is a survival mechanism triggered by nutrient depletion and environmental stress, which occurs as the culture enters the stationary or death phase.

Why are 16- to 18-hour cultures typically used as examples of vegetative cells?

They are in the exponential (log) growth phase where nutrients are abundant and cells are actively dividing.

What are the two primary genera of Gram-positive bacteria that form endospores?

Bacillus and Clostridium.

What characterizes the B-DNA structure?

The standard right-handed helix with approximately $10.5$ base pairs (bp) per turn.

What structural change occurs to DNA during endospore core dehydration?

It shifts from B-DNA to the more robust A-DNA conformation, which is wider and more compressed with approximately $11$ bp per turn.

What is the function of Small Acid Soluble Proteins (SASPs)?

They saturate and bind tightly to DNA in the minor groove, causing it to condense into an A-like conformation and protecting it from UV radiation, chemicals, and heat.

What is the role of Calcium-Dipicolinic acid (Ca-DPA) in the endospore?

It creates a low water content (dehydration) and stabilizes DNA to help the core survive heat stress.

In bacteria, which subunits make up the RNA polymerase?

It contains four catalytic subunits and a single regulatory subunit known as the sigma ($\sigma$) factor.

What is the function of sigma ($\sigma$) factors?

They are discriminatory regulatory subunits that recognize and bind to distinct set of promoter sequences to initiate transcription.

How does eukaryotic transcription differ from prokaryotic transcription regarding polymerase types?

Eukaryotes lack sigma factors and use three types of RNA polymerase: RNA pol I for rRNAs, RNA pol II for mRNAs and small regulatory RNAs, and RNA pol III for tRNAs.

What are housekeeping genes (HKGs)?

Genes that are constitutively and stably expressed to provide essential functions like DNA replication or metabolism, such as $rrsA$ ($16S$ rRNA) and $gyrB$.

What is the primary purpose of using housekeeping genes in Semiquantitative RT-PCR?

Normalization: they serve as an internal control to correct for variations in starting material or technical errors.

What are the four main stages of bacterial transcription?

1. Promoter Engagement, 2. Initiation, 3. Elongation, and 4. Termination.

Which protein is responsible for polymerizing the Z-ring at the mid-cell?

FtsZ.

What protein initiates the relocation of the Z-ring from the mid-cell to the pole during sporulation?

SpoIIE.

Identify the cascade of sigma factors involved in Bacillus spore development.

$\sigma^F$ (prespore), $\sigma^E$ (mother cell), $\sigma^G$ (prespore), and $\sigma^K$ (mother cell).

What is the function of anti-sigma factors (ASMs)?

They are regulatory proteins that bind to and inhibit specific sigma factors, blocking RNA polymerase from initiating transcription.

How do Bacillus thuringiensis (Bt) toxins kill insect pests?

Larva ingest inactive crystals which are activated in the alkaline midgut; activated toxins then bind to receptors and form pores in the gut wall, leading to cell lysis and death.

What organism is used as a biological indicator for autoclave effectiveness?

Geobacillus stearothermophilus.

What are the standard parameters for autoclave steam sterilization?

$121^\circ C$ at $15\,psi$ for approximately $20\,\text{min}$.

In a biological indicator test, what does a color change from purple to yellow indicate?

Sterilization failure; the spores survived and germinated, resulting in acid production which lowered the pH of the medium.