unit 2 lec 14

Why do bacteria regulate proteins?

To adapt to environmental changes and conserve cellular energy.

What are the two main ways bacteria regulate proteins?

By controlling protein amount or protein activity.

Allosteric regulation

Binding of another protein changes a target protein's shape, activating or inactivating it.

Reversible modification

Adding or removing chemical groups such as P, CH3, or AMP changes protein activity.

Regulation by sRNA

Small regulatory RNA binds and alters protein function.

Operon

A transcriptional unit containing a promoter, two or more protein-coding genes, and a terminator.

Promoter

DNA region where RNA polymerase binds to initiate transcription.

Operator

DNA control region where a repressor binds to block transcription.

What is the function of the lac operon?

To regulate production of proteins needed to metabolize lactose.

lacZ

Encodes beta-galactosidase, which breaks down lactose.

lacY

Encodes permease, which transports lactose into the cell.

lacA

Encodes a transacetylase with an uncertain function.

lacI

Regulatory gene encoding the LacI repressor protein.

What happens to the lac operon without lactose?

LacI binds the operator and blocks most transcription.

What happens to the lac operon when lactose is present?

Allolactose binds and inactivates LacI, allowing transcription.

Allolactose

Inducer formed from lactose that binds and inactivates the LacI repressor.

Leaky expression

Low basal transcription that produces small amounts of LacZ and LacY even without lactose.

Why is leaky expression important?

It allows lactose to enter the cell and be converted into allolactose.

How is the lac operon used as a reporter?

LacZ breaks down X-gal to produce a blue pigment indicating gene expression.

Catabolite repression

Regulation that prioritizes glucose use over other carbon sources.

What happens to cAMP when glucose is present?

cAMP decreases because adenylate cyclase is inhibited.

What happens when glucose is low?

cAMP increases, binds CRP, and activates transcription of other sugar metabolism genes.

What two conditions favor strong lac operon expression?

Lactose must be present and glucose must be low.

Microbial stress

Unfavorable environmental conditions that threaten normal bacterial functions or survival.

Why do bacteria have stress responses?

To adapt to unfavorable conditions and increase their chances of survival.

Nutritional stress

Lack of essential nutrients such as carbon, amino acids, or phosphate.

Osmotic stress

Changes in salt concentration that affect cellular water balance.

Temperature stress

Exposure to temperatures that are too high or too low.

Toxic stress

Exposure to harmful substances such as antibiotics or oxidative chemicals.

What are the four steps of a microbial stress response?

Sensing, signal transduction, response, and feedback control.

Sensing

A protein or nucleic acid sensor detects an environmental stress signal.

Signal transduction

The sensor transmits the stress signal to a regulator.

Response

Regulators control target gene expression to produce proteins needed for adaptation.

Feedback control

Turns off the stress response once the stressful condition is resolved.

Two-component system

A bacterial signaling system consisting of a sensor histidine kinase and a response regulator.

Sensor protein (histidine kinase)

Detects environmental signals and initiates signal transduction by autophosphorylation.

Response regulator

Receives phosphate from the histidine kinase and controls target gene expression.

Sensing domain

Region of the sensor protein that detects environmental signals.

Histidine kinase domain

Region that autophosphorylates its histidine residue using ATP.

Autophosphorylation

A protein adds a phosphate group to itself.

Dimerization

Two protein molecules come together following a signal-induced conformational change.

What happens after the histidine kinase detects stress?

It changes shape, dimerizes, and autophosphorylates its His residue.

Where does phosphate transfer occur in a two-component system?

From the histidine (His) of the sensor kinase to the aspartate (Asp) of the response regulator.

What happens when the response regulator is phosphorylated?

Its DNA-binding activity is activated, allowing it to control target promoters.

What does the response regulator's DNA-binding domain do?

Binds target DNA regions to activate or repress gene transcription.

Phosphatase

An enzyme that removes phosphate groups from proteins.

How is a two-component system turned off?

A phosphatase dephosphorylates the response regulator after the stress passes.

CheA–CheY system

Two-component system that controls flagellar rotation during chemotaxis.

MCP (Methyl-accepting Chemotaxis Protein)

Senses environmental signals and influences CheA activity.

CheA

Histidine kinase that autophosphorylates and transfers phosphate to CheY.

CheY

Response regulator that controls flagellar motor rotation when phosphorylated.

CheZ

Phosphatase that removes phosphate from CheY.

What happens when CheY phosphorylation is high?

Clockwise (CW) flagellar rotation is favored, causing tumbling.

What happens when CheY phosphorylation is low?

Counterclockwise (CCW) flagellar rotation is favored, causing forward running.

How does CheA–CheY signal transduction work?

MCP detects a signal, CheA autophosphorylates, and phosphate transfers from CheA to CheY.

How does the CheA–CheY system turn off its phosphorylation signal?

CheZ dephosphorylates CheY.

Pho system

Two-component system that responds to inorganic phosphate limitation.

PhoR

Histidine kinase that activates PhoB when phosphate is limited.

PhoB

Response regulator that activates phosphate-acquisition genes when phosphorylated.

Pst

Phosphate transport system that helps bacteria obtain inorganic phosphate.

PhoU

Protein linking phosphate transport through Pst to regulation of PhoR.

Does PhoR directly bind phosphate to detect phosphate limitation?

No, PhoR senses phosphate availability indirectly through the Pst–PhoU system.

What happens when phosphate is sufficient?

PhoR does not autophosphorylate, PhoB remains inactive, and the Pho regulon is OFF.

What happens when phosphate becomes limited?

Phosphate transport slows, PhoR autophosphorylates, and phosphate transfers to PhoB.

What happens when PhoB becomes phosphorylated?

PhoB binds target promoters and activates the phosphate response regulon.

What is the purpose of the phosphate response regulon?

To activate genes that help bacteria obtain more phosphate.

What happens to the PhoU–PhoR connection when phosphate becomes limited?

A conformational change disrupts the connection, allowing PhoR activation.

How does the Pho system turn off when phosphate returns?

PhoR promotes PhoB dephosphorylation, shutting off the phosphate response regulon.

Main difference between CheA–CheY and PhoR–PhoB

CheA–CheY controls flagellar movement; PhoR–PhoB controls phosphate-acquisition genes.

How do antibiotics cause bacterial stress?

They disrupt essential cellular functions, potentially causing cell death or activating stress responses.

What happens if an antibiotic stress response succeeds?

Bacteria may survive, persist, or evolve antibiotic resistance.

What do quinolone antibiotics target?

DNA gyrase and topoisomerase IV, disrupting DNA replication.

What can sublethal quinolone exposure cause?

Increased mutation rates, recombination rates, and persister formation.

What are three mechanisms of quinolone resistance?

Efflux pumps, mutations in DNA gyrase or topoisomerase IV, and plasmid-encoded resistance genes.

Efflux pumps

Transport antibiotics out of bacterial cells, reducing intracellular antibiotic concentration.

Target mutations

Changes in DNA gyrase or topoisomerase IV that reduce quinolone effectiveness.

Plasmid-encoded resistance genes

Resistance genes carried on plasmids that can be transferred between bacteria.

Persister cells

Dormant or slow-growing bacteria that temporarily tolerate antibiotics without necessarily being genetically resistant.

Persister cells vs. resistant bacteria

Persisters temporarily tolerate antibiotics; resistant bacteria have resistance mechanisms that can be inherited.

How can antibiotic resistance be tested?

Expose bacteria to antibiotics and determine whether bacterial growth is inhibited.

Disk diffusion vs. MIC

Disk diffusion measures zones of growth inhibition; MIC is the lowest antibiotic concentration preventing visible growth.

Error-prone DNA repair

DNA repair mechanisms that introduce mutations while repairing or bypassing damaged DNA.

Translesion synthesis (TLS)

Specialized DNA polymerases copy past damaged DNA, allowing replication to continue but potentially introducing mutations.

Alternative end joining (alt-EJ)

Repairs DNA double-strand breaks by joining broken ends, sometimes introducing mutations.

How can sublethal ampicillin exposure increase mutations?

Ampicillin damages peptidoglycan, activating stress responses involving RpoS that increase DNA errors and reduce mismatch repair.

RpoS

General stress-response regulator that increases error-prone DNA polymerase IV and indirectly decreases MutS.

DNA polymerase IV (Pol IV)

Low-fidelity DNA polymerase that introduces more DNA copying errors, including base substitutions.

High-fidelity vs. low-fidelity DNA polymerases

High-fidelity polymerases make few mistakes; low-fidelity polymerases make more mistakes.

MutS

Mismatch repair protein that recognizes DNA copying errors so they can be repaired.

SdsR

Small regulatory RNA activated by RpoS that reduces MutS expression.

How does RpoS reduce mismatch repair?

RpoS increases SdsR, which downregulates MutS, allowing more DNA mistakes to remain uncorrected.

How does antibiotic stress increase the mutation rate?

Increased Pol IV creates more DNA errors, while decreased MutS allows fewer errors to be repaired.

How can error-prone DNA repair lead to antibiotic resistance?

Increased mutations create genetic variation, and some mutations may provide antibiotic resistance.