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Question 2

What are the components of a two-component regulatory system?

Answer 2

A sensor protein (typically in the plasma membrane or cell wall), a response regulator protein (usually in the cytoplasm), and a signal. The sensor detects environmental changes and transmits the signal via conformational changes.

Question 3

Describe how a typical two-component system functions when a stimulus is present.

Answer 3

The sensor protein detects a stimulus, leading to its autophosphorylation. The phosphate group is then transferred to the response regulator, which can then bind to DNA and alter gene expression.

Question 4

What is quorum sensing?

Answer 4

Quorum sensing is a communication mechanism used by bacteria to coordinate group behaviours based on population density. It involves the production and detection of signalling molecules called autoinducers.

Question 5

What is the main difference in autoinducer molecules between Gram-negative and Gram-positive bacteria?

Answer 5

Gram-negative bacteria typically use acylated homoserine lactones (AHLs) as autoinducers, which are membrane permeable. Gram-positive bacteria use secreted peptides as autoinducers, requiring ABC transporters for secretion.

Question 6

What are small non-coding RNAs (sRNAs)?

Answer 6

sRNAs are short RNA molecules that do not code for proteins but regulate gene expression. They can act by binding to mRNA, affecting translation or stability.

Question 7

How do sRNAs like RyhB function in iron regulation?

Answer 7

RyhB is negatively regulated by the ferric uptake regulator (Fur). In iron-limited conditions, Fur repression of RyhB is alleviated. RyhB then suppresses genes involved in iron storage and iron-using proteins, making more iron available.

Question 8

What is the general stress response in yeast?

Answer 8

It is a conserved, initial response to various stresses, aiming to protect critical cell functions until a specific response can be activated.

Question 9

What are protein chaperones, and what are the main classes?

Answer 9

Chaperones are proteins that bind to other proteins to protect them from denaturation. The main classes include: Small HSPs (HSP12-42), Intermediate HSPs (HSP60-90), Large HSPs (HSP100).

Question 10

Describe the role of Msn4 in the general stress response in yeast.

Answer 10

Msn4 is a transcription factor that is induced to activate the general stress response. It binds to specific sequences in the promoter region of genes like SOD1.

Question 11

What is the function of SOD1 in the general stress response?

Answer 11

SOD1 is a superoxide dismutase that converts superoxide radicals into hydrogen peroxide, which is then broken down by catalases. It is part of the cell’s defense against oxidative stress.

Question 12

What are alternative sigma factors, and how do they function?

Answer 12

Alternative sigma (σ) factors are proteins that direct RNA polymerase to specific sets of promoters. They enable the polymerase to recognize promoter regions, allowing for the transcription of specific genes in response to various conditions.

Question 13

Describe the heat shock response and the role of sigma factors.

Answer 13

The heat shock response is triggered by a sudden increase in temperature, which can lead to protein denaturation. It involves the increased expression of chaperones and proteases. Sigma factors such as σ32 (encoded by rpoH) are key regulators of this response.

Question 14

How is the sigma factor σE regulated in E. coli?

Answer 14

σE is complexed with an anti-sigma factor, RseA, which sequesters σE in the cytoplasm. When outer membrane stress is detected, RseA is degraded, releasing σE, which can then activate its regulon.

Question 15

What is a biofilm and what are the main steps in its formation?

Answer 15

A biofilm is a community of microorganisms attached to a surface, encased in a self-produced matrix of extracellular polymeric substances (EPS). Steps include: 1. Attachment to a surface 2. Accumulation of signalling molecules that activate biofilm formation genes 3. Development of structures and channels 4. Detachment of cells to colonize new locations.

Question 16

What are some advantages and disadvantages for bacteria adopting a biofilm lifestyle?

Answer 16

Advantages: Increased resistance to antibiotics and host defenses, increased access to nutrients, and enhanced cell-to-cell communication. Disadvantages: Can lead to chronic infections, biofouling of medical devices, and increased difficulty in eradication.

Question 17

How do bacteria use quorum sensing to regulate biofilm formation?

Answer 17

Bacteria produce signalling molecules that accumulate as the cell population increases. When a threshold concentration is reached, these molecules activate genes involved in biofilm formation.

Question 18

What is quorum quenching?

Answer 18

Quorum quenching is the process of disrupting or inhibiting quorum sensing. This can be achieved by degrading the autoinducers, blocking their receptors, or inhibiting their synthesis.

Question 19

How are bacterial cell walls different between Gram-positive and Gram-negative bacteria?

Answer 19

Gram-positive bacteria have a thick peptidoglycan layer and lack an outer membrane. Gram-negative bacteria have a thin peptidoglycan layer and an outer membrane containing lipopolysaccharides (LPS).

Question 20

What are some major components of the lipopolysaccharide (LPS)?

Answer 20

LPS is composed of three parts: Lipid A: Anchors LPS to the outer membrane. Core: A repeating sugar unit attached to Lipid A. O-antigen: A repeating sugar structure linked to the core, unique to each bacterial strain.

Question 21

What are bacterial capsules and slime layers?

Answer 21

Both are types of glycocalyx composed of exopolysaccharides (EPS). Capsules are tightly attached, organized matrices that exclude small particles, while slime layers are loosely attached, easily deformed, diffuse layers.

Question 22

What is the role of FtsZ in bacterial cell division?

Answer 22

FtsZ is a protein essential for cell division. It assembles at the division site between segregated chromosomes, forming a ring that constricts the cell membrane.

Question 23

What are the main components of a bacterial flagellum?

Answer 23

A flagellum consists of: Filament: composed of flagellin, Hook: a rigid structure connecting the filament to the motor, Basal body: the motor of the flagella, inserted into the plasma membrane.

Question 24

What are the key steps in nitrogen-fixing nodule formation in plant-bacteria symbiosis?

Answer 24

1. Rhizobia stimulate root hair growth, and are captured in curled root hairs. 2. Infection threads form, allowing bacteria to enter cortical cell layers. 3. Nodule primordium forms and bacteria are released into the plant cytoplasm. 4. Bacteria differentiate into bacteroids which fix nitrogen.

Question 25

What are Nod factors and how do they function in symbiotic associations?

Answer 25

Nod factors are lipochitooligosaccharides secreted by bacteria which are specific to the plant they are infecting. They are recognized by receptors on plant cells, triggering a response that activates nod genes in the bacteria.

Question 26

How does a cell use multiple drug resistance (MDR) transporters to combat drugs?

Answer 26

MDR transporters are efflux pumps that actively transport drugs out of the cell, reducing their intracellular concentration. This is a common resistance mechanism against many structurally and chemically different compounds.

Question 27

What are the key differences between MFS and ABC transporters?

Answer 27

ABC Transporters: Utilize ATP hydrolysis to drive transport (active transport). MFS Transporters: Utilize the electrochemical gradient of protons (H+) across the membrane (secondary active transport).

Question 28

What are some of the mechanisms used to achieve drug resistance in fungi?

Answer 28

Mechanisms include: Mutations in drug target proteins, Overexpression of target proteins, Efflux of the drug via MDR transporters, Metabolic tolerance to the drug.

Question 29

How does the drug 5-FC work?

Answer 29

5-FC is a prodrug that is converted into 5-FU inside the pathogen. 5-FU disrupts DNA and protein synthesis.

Question 30

What is the target of Azole drugs?

Answer 30

Azoles target Erg11p, an enzyme required for ergosterol biosynthesis in fungal cell membranes.

Question 31

What is the function of lipid rafts in cell membranes?

Answer 31

Lipid rafts are tightly packed platforms formed by sphingolipids and sterols. They serve as sorting and segregation devices for proteins, particularly in the plasma membrane.

Question 32

What are some markers of apoptosis in yeast cells?

Answer 32

Markers include chromosome condensation, DNA fragmentation, ROS generation, exposure of phosphatidylserine, and loss of membrane integrity.

Question 33

What is the purpose of programmed cell death in unicellular eukaryotes such as yeast?

Answer 33

Apoptosis in unicellular organisms allows the population to function as a multicellular-like system, with damaged or aged cells sacrificing themselves for the benefit of the collective.

Question 34

What is the function of ubiquitination in the general stress response?

Answer 34

Ubiquitination is involved in tagging proteins for degradation. In the general stress response, it helps remove damaged or misfolded proteins.

Question 35

What methods can be used to study TF-DNA interactions?

Answer 35

Indirect Methods: Expression studies to identify genes that are affected by the presence or absence of the TF. Direct Methods: More rigorous methods to determine direct binding including: Chromatin Immunoprecipitation (ChIP): Determines TF binding sites in vivo. Electrophoretic Mobility Shift Assay (EMSA): Identifies protein-DNA interactions in vitro. DNase Footprinting Assay: Identifies specific DNA regions bound by proteins in vitro.

Question 36

What are transcription factories?

Answer 36

Transcription factories are discrete sites in the nucleus where multiple active RNA polymerases are concentrated. Actively transcribed genes can be located at the same location, allowing for efficient transcription.

Question 37

Describe how the transcription factor Yap1 is activated by oxidative stress.

Answer 37

Oxidative stress induces the formation of disulphide bonds in cysteine residues of Yap1, causing conformational changes that prevent its export from the nucleus and leading to nuclear accumulation and activation of MDR genes.

Question 38

Name three examples of genes controlled by the PDR network in yeast.

Answer 38

PDR5, SNQ2 and YOR1 which encode for efflux pumps.

Question 39

What are some of the key virulence factors associated with Burkholderia cepacia complex (Bcc)?

Answer 39

Virulence factors include pili (CblA), flagella, siderophores, lipases, haemolysin, melanin, zinc metalloproteases, a type III secretion system, quorum sensing systems, lipopolysaccharide (LPS), and exopolysaccharide (EPS) such as cepacian.

Question 40

What is the role of the exopolysaccharide (EPS), cepacian, in Bcc?

Answer 40

Cepacian is a virulence factor produced by Bcc bacteria that forms a protective layer and contributes to biofilm formation. EPS-negative mutants are less virulent.

Question 41

What are bce gene clusters in Bcc bacteria?

Answer 41

The bce gene clusters are responsible for producing exopolysaccharide (EPS), including cepacian. These clusters include genes involved in nucleotide sugar biosynthesis, polymerization, and export of EPS.

Question 42

What is the LD50?

Answer 42

The LD50 (lethal dose 50) is the amount of a pathogen required to kill 50% of the hosts in a test group. A lower LD50 indicates higher virulence.

Question 43

What are the main steps in the process of protein ubiquitination?

Answer 43

This process involves the attachment of ubiquitin to target proteins in a multi-step process involving: Ubiquitin-activating enzyme (E1), Ubiquitin-conjugating enzyme (E2), Ubiquitin ligase (E3).

Question 44

What is the role of the protein FNR?

Answer 44

FNR system cooperates with the NarQ/NarP and NarX/NarL systems to activate the fumarate reductase and nitrate reductase as part of a newly formed respiratory chain.

Question 45

What are the three domains of the general structure of a transcription factor?

Answer 45

DNA binding domain, transactivation domain and a dimerization domain.

Question 46

Describe the general mode of action of a two-component regulatory system (TCRS), using the example of the Acinetobacter baumannii BfmRS system.

Answer 46

A TCRS consists of a sensor histidine kinase (HK), such as BfmS, and a response regulator (RR), such as BfmR. The BfmS sensor protein detects a signal and autophosphorylates. The phosphate group is then transferred to the BfmR regulator, which can then bind to DNA and alter gene expression. The system is activated by a structural change that aligns a conserved histidine residue with the ATP binding site of the kinase.

Question 47

How would you experimentally demonstrate that BfmR is regulated by BfmS?

Answer 47

An experiment could involve creating a mutant strain of A. baumannii with a non-functional bfmS gene and comparing the phosphorylation state of BfmR in this mutant versus a wild-type strain. If BfmR phosphorylation is reduced or absent in the bfmS mutant, it would indicate that BfmS is required for BfmR activation. Another approach would be to use purified BfmS and BfmR proteins and assess the transfer of a phosphate from BfmS to BfmR in vitro, confirming a direct interaction.

Question 48

Define quorum sensing (QS) and describe the regulatory circuitry of the Acinetobacter baumannii AbaIR QS system.

Answer 48

Quorum sensing is a regulatory mechanism where bacteria communicate via autoinducers to coordinate gene expression based on population density. The AbaI protein synthesizes the autoinducer N-hydroxy-dodecanoyl-L-homoserine.

Question 49

Describe how a two-component regulatory system (TCRS) responds to a stimulus, including the specific mechanisms of activation of the histidine kinase (HK).

Answer 49

A TCRS responds to a stimulus through a sensor histidine kinase (HK) and a response regulator (RR). Upon stimulus detection, the HK autophosphorylates, transferring the phosphate to the RR, which then alters gene expression. HK activation can occur via: ◦ Conformational Changes: Large structural shifts in the protein expose a conserved histidine residue to the ATP binding site, enabling phosphate transfer. ◦ Helix Sliding: A helix containing the histidine slides within the dimerization domain to facilitate the phosphate transfer.

Question 50

Design an experiment using purified proteins to demonstrate that BfmS directly phosphorylates BfmR.

Answer 50

This experiment would use purified BfmS and BfmR proteins, and would include the following: ◦ Incubation: Mix purified BfmS with ATP and purified BfmR in a reaction buffer and incubate. ◦ Controls: ▪ BfmS with ATP, but without BfmR. ▪ BfmR with ATP, but without BfmS. ▪ BfmS and BfmR without ATP. ◦ Detection: Use phospho-specific antibodies against phosphorylated BfmR to detect the transfer of a phosphate from BfmS to BfmR.

Question 51

Compare and contrast the regulatory mechanisms of the LasI/LasR and RhII/RhIR quorum sensing systems in Pseudomonas aeruginosa.

Answer 51

Both are LuxI/LuxR homologs. ◦ LasI/LasR: LasI synthesizes an autoinducer that, upon binding to LasR, activates the expression of several virulence genes, including lasB, lasA, toxA, and aprA, as well as lasI itself (positive feedback). ◦ RhII/RhIR: The LasR-autoinducer complex induces the expression of rhIR. RhIR, when bound to its autoinducer, activates some of the same target genes as the Las system, such as lasB and aprA, but also unique targets like rpoS and rhlAB.

Question 52

How can quorum quenching be used to combat bacterial infections? Describe three main strategies.

Answer 52

Quorum quenching disrupts bacterial communication, which can reduce virulence and biofilm formation. ◦ Autoinducer Degradation: Enzymes that degrade AHLs (autoinducers in Gram-negative bacteria) can be used. For example, AHL lactonases can break down AHLs. ◦ Receptor Blocking: Molecules that bind to and block the autoinducer receptor but do not activate it, preventing the signal from being transduced. ◦ Inhibition of Synthesis: Drugs that inhibit autoinducer synthases such as AbaI and LasI can reduce the accumulation of the signalling molecules.