Microbiology Exam 2 Practice: Growth, Metabolism, and Information Flow

Comprehensive practice flashcards covering Weeks 5 to 7 lecture topics: bacterial growth kinetics, calculation protocols, the protein budget, metabolic electron pathways and redox calculations, the carbon ledger, transcription, translation, and operon regulatory gates.

What is the measured reality of bacterial cells during the stationary phase of a batch growth curve, contrary to the textbook claim that 'births equal deaths'?

Measurements in E. coli show that fewer than $7\,\%$ of cells are lysed and fewer than $5\,\%$ are dividing; the cells remain intact and continue synthesizing protein at a lower rate.

How is final cell number calculated in a forward generation time problem?

Find the number of generations using $n = \text{minutes elapsed} \div g$, then calculate final cell count using $N = N_0 \times 2^n$.

If a culture starts at $400\,\text{cells/mL}$ with a generation time of $25\,\text{minutes}$, how many cells per mL are present after $2\,\text{hours}$ and $5\,\text{minutes}$?

Time elapsed is $125\,\text{minutes}$. Number of generations $n = 125 \div 25 = 5$. Final count $N = 400 \times 2^5 = 400 \times 32 = 12{,}800\,\text{cells/mL}$.

How is generation time ($g$) calculated backward from starting and ending cell numbers?

Determine $N / N_0$ and express it as a power of two ($2^n$) to find the number of doublings $n$. Convert total elapsed time from hours to minutes, then divide by $n$: $g = \text{minutes elapsed} \div n$.

A culture grows exponentially from $3{,}000$ to $192{,}000\,\text{cells/mL}$ in $3\,\text{hours}$. What are the values of $n$ and $g$?

$N / N_0 = 192{,}000 / 3{,}000 = 64 = 2^6$, so $n = 6$. Total time is $3\,\text{hours} = 180\,\text{minutes}$. Generation time $g = 180 \div 6 = 30\,\text{minutes}$.

What cellular mechanism occurs when an organism's generation time ($g$) is shorter than the $\sim 40\,\text{minutes}$ ($C$ period) required to replicate its chromosome?

The cell starts a new round of replication before the previous round has finished, meaning a newborn cell already carries more than one genome's worth of DNA (overlapping rounds of replication).

Why does oxygen kill obligate anaerobes that possess superoxide dismutase (SOD) and catalase?

$\text{O}_2$ and its reactive radicals destroy essential enzymes that are built around iron-sulfur centers.

In hydrogen peroxide detoxification enzymes, what are the correct redox directions for the substrate and electron donor?

Hydrogen peroxide ($\text{H}_2\text{O}_2$) is reduced to water, while the electron donor is oxidized.

What is the true function and atmospheric composition of a candle jar?

It raises $\text{CO}_2$ levels to approximately $3\,\%$; it leaves oxygen levels near $17\,\%\,\text{O}_2$ rather than consuming most of the oxygen.

What are the pH classification boundaries based on growth optimum?

Acidophile: optimum $< 5.5$; Neutrophile: optimum $5.5\text{--}8$; Alkaliphile: optimum $> 8$.

How does GC content correlate with growth temperature in thermophiles?

Total genome GC content does not correlate with growth temperature; however, GC content in rRNA and tRNA stems does track growth temperature.

What structural adaptation characterizes proteins of cold-adapted psychrophiles?

They possess a less hydrophobic, more flexible core rather than a rigid hydrophobic core.

Why is 'piezophile' preferred over 'barophile', and what type of pressure does it require?

Piezophiles require high hydrostatic pressure, not high atmospheric pressure.

How is growth rate established in a chemostat continuous culture?

The environment is nutrient-limited, and the flow rate of medium sets the growth rate.

How does shifting a bacterial culture from poor medium to rich medium affect its protein budget and RNA/protein ratio?

Ribosomes and metabolic proteins share a fixed proteome budget. Faster growth in richer medium requires a higher ribosome share, which increases the RNA/protein ratio.

Why does slowing translation with chloramphenicol increase the ribosome share of a bacterial proteome?

Slowing translation lowers the output per ribosome, forcing the cell to allocate more of its protein budget to synthesizing additional ribosomes to maintain protein synthesis.

What are the five steps of the bacterial stringent response?

1. An amino acid runs short. 2. An uncharged tRNA enters the ribosomal A site. 3. RelA synthesizes (p)ppGpp. 4. (p)ppGpp with DksA acts directly on RNA polymerase. 5. rRNA promoters are downregulated while amino acid biosynthesis operons are upregulated.

What are three things that ppGpp is not?

It is not an off switch, not a DNA-binding protein, and not solely an emergency signal.

What are the three independent nutritional axes used to classify microbial metabolism?

1. Energy source: phototroph (light) vs chemotroph (chemical redox). 2. Electron donor: organotroph (organic) vs lithotroph (inorganic). 3. Carbon source: autotroph ($\text{CO}_2$) vs heterotroph (organic carbon).

Where does a chemotroph's energy actually come from, contrary to the idea of 'breaking bonds'?

Energy comes from redox reactions where electrons transfer from a donor to an acceptor. Breaking chemical bonds always requires energy input; energy release occurs because reaction products are more stable.

What direct cellular work is powered by the proton motive force (PMF) rather than ATP?

Bacterial flagellar rotation and many membrane transport systems run directly on the proton motive force.

What is the course definition of cellular respiration?

The passage of electrons through a membrane electron transport chain to a terminal electron acceptor (which can be inorganic or organic, and does not require oxygen).

What is the course definition of fermentation?

The transfer of electrons directly onto an organic molecule produced internally by the catabolic pathway itself, operating without a membrane electron transport chain.

What is the single distinguishing feature that separates respiration from fermentation?

The presence of a membrane electron transport chain (respiration uses a chain; fermentation does not; oxygen is not the dividing line).

What formula calculates free energy change from the redox tower, and what do its variables represent?

$\Delta G^{\circ\prime} = -n F \Delta E_0'$, where $n$ is the number of electrons transferred, $F = 96.5\,\text{kJ/(V}\cdot\text{mol)}$, and $\Delta E_0' = E_0'(\text{acceptor}) - E_0'(\text{donor})$ in volts.

How much free energy is released when two electrons transfer from NADH ($-0.32\,\text{V}$) to fumarate ($+0.03\,\text{V}$), and how does this compare to transfer to $\text{O}_2$ ($+0.82\,\text{V}$)?

$\Delta E_0' = 0.03 - (-0.32) = 0.35\,\text{V}$. $\Delta G^{\circ\prime} = -2 \times 96.5 \times 0.35 = -68\,\text{kJ}$. This yields about one-third of the energy of transferring to oxygen ($\Delta E_0' = 1.14\,\text{V}; -220\,\text{kJ}$), meaning anaerobic respiration on fumarate yields less ATP per electron pair.

What is reverse electron flow, and how is it energetically financed?

The thermodynamically uphill movement of electrons from a weak donor (such as $\text{Fe}^{2+}$ at $+0.76\,\text{V}$) to $\text{NAD}^+$ ($-0.32\,\text{V}$), which generates positive $\Delta G^{\circ\prime}$ and is driven by consuming the proton motive force.

What is the complete carbon ledger per molecule of glucose through glycolysis (EMP), transition reaction, and the Krebs cycle?

Glycolysis: $2\,\text{ATP}$, $2\,\text{NADH}$, $0\,\text{CO}_2$. Transition reaction: $0\,\text{ATP}$, $2\,\text{NADH}$, $2\,\text{CO}_2$. Krebs cycle (2 turns): $2\,\text{ATP}$, $6\,\text{NADH}$, $2\,\text{FADH}_2$, $4\,\text{CO}_2$. Total: $4\,\text{ATP}$, $10\,\text{NADH}$, $2\,\text{FADH}_2$, $6\,\text{CO}_2$.

Why does a fermenting cell transfer electrons from NADH to pyruvate even though this step generates no ATP?

It oxidizes NADH back to $\text{NAD}^+$ so glycolysis can continue generating ATP via substrate-level phosphorylation, leaving approximately $25\,\text{kJ}$ of redox energy uncollected.

Why is '38 ATP per glucose' incorrect for cellular respiration?

Mitochondrial conversion yields $\sim 2.5\,\text{ATP/NADH}$ and $\sim 1.5\,\text{ATP/FADH}_2$, giving $\sim 30\text{--}32\,\text{ATP}$. In bacteria, the yield varies depending on how many protons their specific electron transport chain pumps per electron pair and how many protons their ATP synthase requires per ATP.

What primarily determines whether a facultative cell respires or ferments?

Genetic and metabolic regulation determines the pathway, not simple energetic yield (for example, Saccharomyces yeast ferments in the presence of air when glucose is abundant via the Crabtree effect).

What is the difference between noncompetitive and allosteric enzyme inhibition?

'Noncompetitive' describes a kinetic pattern (lowers maximum velocity $V_{\max}$, maintains substrate affinity $K_m$), whereas 'allosteric' refers to the physical location of the binding site away from the active site.

What are the subunits of bacterial RNA polymerase core enzyme and holoenzyme?

Core enzyme consists of 5 subunits: two $\alpha$, $\beta$, $\beta'$ (the claw), and $\omega$. Holoenzyme consists of the core enzyme plus the $\sigma$ factor (6 subunits total).

What are the specific functional roles of the bacterial RNA polymerase core enzyme and the sigma ($\sigma$) factor?

The $\sigma$ factor recognizes the $-35$ and $-10$ promoter sequences (the Pribnow box, not a TATA box) and helps open the DNA duplex; the core enzyme synthesizes the RNA strand.

What are the four discrete steps of bacterial transcription initiation?

1. Bind (formation of the closed complex). 2. Open (DNA bubble formation; $\sigma_2$ stabilizes the non-template strand). 3. Stutter (synthesis of short abortive transcripts). 4. Commit (promoter clearance, during which $\sigma$ often dissociates).

Given a DNA template strand reading $3'\text{-TAC GGA CCT ATC-}5'$, what are the non-template strand, the mRNA, and the resulting peptide?

Non-template strand: $5'\text{-ATG CCT GGA TAG-}3'$. mRNA: $5'\text{-AUG CCU GGA UAG-}3'$. Peptide: Met-Pro-Gly (UAG is stop). The mRNA sequence matches the non-template strand with U replacing T.

During translation elongation, what happens to the growing polypeptide chain when a peptide bond forms?

The peptide chain moves from the tRNA in the P site onto the amino acid on the tRNA in the A site; this reaction is catalysed by rRNA.

How do Bacteria, Archaea, and Eukaryotes compare regarding RNA polymerases, promoter recognition, and initiating amino acid?

Bacteria: 1 RNAP, $\sigma$ factor recognizes Pribnow box, starts with fMet. Archaea: 1 RNAP (homologous to Pol II), TBP and TFB recognize TATA box, starts with plain Met. Eukaryotes: multiple RNAPs, TBP/transcription factors recognize TATA box, starts with plain Met.

In the trp operon, what molecule acts as the corepressor, and what phenotype results from a repressor that cannot bind it?

Tryptophan is the corepressor (it binds and activates the repressor). A mutant repressor unable to bind tryptophan cannot bind the operator, leaving trp biosynthesis genes constitutively ON.

What is the molecular mechanism of Gate 1 in the lac operon?

Basal expression leaks lactose into the cell, where $\beta$-galactosidase converts it to allolactose (the inducer); allolactose binds the lac repressor, causing it to dissociate from the operator.

What is the molecular mechanism of Gate 2 in the lac operon, and how does glucose maintain it shut?

Strong transcription requires CAP-cAMP bound at $-61$. Glucose keeps expression low in two ways: 1) Glucose uptake lowers cAMP, causing CAP to dissociate. 2) Inducer exclusion: glucose transport leaves EIIA unphosphorylated, which blocks lactose permease.

What are the states of the repressor, CAP-cAMP, and transcription in Flask 1 (glucose present, lactose absent)?

Repressor is ON the operator; CAP-cAMP is ABSENT from the promoter; transcription is OFF (very low basal leak).

What are the states of the repressor, CAP-cAMP, and transcription in Flask 2 (glucose present, lactose present)?

Repressor is MOSTLY OFF the operator (inducer exclusion limits lactose entry); CAP-cAMP is MOSTLY ABSENT; transcription is LOW.

What are the states of the repressor, CAP-cAMP, and transcription in Flask 3 (glucose absent, lactose present)?

Repressor is OFF the operator; CAP-cAMP is ON the promoter; transcription is HIGH (both gates open).

What are the states of the repressor, CAP-cAMP, and transcription in Flask 4 (glucose absent, lactose absent)?

Repressor is ON the operator; CAP-cAMP is ON the promoter (with nothing to do); transcription is OFF (very low basal leak).

What occurs in a lac operon mutant where the repressor protein cannot bind allolactose?

The repressor remains permanently bound to the operator regardless of lactose presence, keeping the operon continuously OFF.