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Bacterial Physiology (BIOL 471) > Ch 19 > Flashcards

Ch 19 Flashcards

1
Q

What are the components of two-component systems?

A
  1. Histidine kinase (HK): receives a signal
  2. Response regulator (RR): receives signal from HK and transmits it to target
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2
Q

Two-components systems contain proteins of 3 types. What are the 3 types?

A
  1. Histidine kinase (HK)
  2. Response regulator (RR)
  3. Phosphatase: inactivates the RR
    - May be the HK, RR, or separate protein
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3
Q

What are the 2 domains of histidine kinases?

A
  1. Input domain
  2. Transmitter domain
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4
Q

What are the 2 domains of response regulators?

A
  1. Receiver domain
  2. Output domain
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5
Q

What does the phosphatase in a two-component system do?

A

Dephosphorylates the RR, returning it to the nonstimulated state, where it once again can respond to the signal

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6
Q

What are the steps of signal transduction in two-component systems?

A
  1. A transmembrane histidine kinase (HK) is activated by a signal at its N-terminal domain
  2. The activated protein autophosphorylates in the C-terminal domain
  3. The response regulator (RR) binds to the C-terminal end of the histidine kinase and the phosphoryl group is transferred from the HK to the RR, thus activating the latter
  4. The activated RR leaves the HK and stimulates its target
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7
Q

What defines histidine kinases and response regulators?

A
  • HK defined by conserved sequence of about 200 amino acids at the C-terminal end
  • RR defined by conserved amino-terminal domain of about 100 amino acids
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8
Q

What two-component systems regulate the genes that respond to anaerobiosis?

A
  • Arc system
  • FNR system
  • Nar system
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9
Q

Use the figure to describe the metabolic changes E. coli undergoes during anaerobiosis.

A

A. Aerobic conditions
- Oxidative TCA cycle feed electron into SDH and NADH dehydrogenase
B. Anaerobic conditions
- TCA pathway is noncyclic and reductive because α-ketoglutarate dehydrogenase activity is absent/diminished

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10
Q

What are the histidine kinase and response regulator proteins in the Arc system?

A
  1. HK: ArcB
  2. RR: ArcA
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11
Q

What is the Arc system responsible for?

A
  1. Anaerobic repression of genes for:
    - TCA cycle enzymes
    - Glyoxylate cycle enzymes
    - Dehydrogenases for aerobic growth
    - Fatty acid oxidation enzymes
    - Cytochrome o oxidase
  2. Anaerobic induction of the gene for pyruvate formate-lyase
  3. Induction in low oxygen of the genes for cytochome d oxidase and cobalamin synthesis
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12
Q

All of the following regulate gene expression by oxygen or
nitrate. Which one is not a two-component system?
- Arc system
- FNR system
- Nar system

A

FNR system

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13
Q

What types of genes does the FNR system activate? Which genes does it repress?

A
  1. Positive regulator of transcription for many genes expressed only during anaerobic growth
    - Fumarate reductase
    - Nitrate reductase
    - Formate dehydrogenase-N
    - Aspartase
    - Anaerobic fumarase B
    - Glycerol-3-phosphate dehydrogenase
  2. Repressor for certain genes expressed only during aerobic growth
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14
Q

What do mutations in the fnr gene result in?

A

Result in an inability to grow anaerobically on fumarte or nitrate as electron acceptors

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15
Q

How does E. coli regulate the activity of FNR?

A

Iron-sulfur cluster in FNR
- Active FNR: homodimer of an iron-sulfur protein with an oxygen labile [4Fe-4S] cluster in each monomer
- Upon exposure to oxygen: [4Fe-4S] cluster is oxidized and can even be lost from the protein
- Some of the FNR loses its iron clusters and becomes an apoprotein
- Both protein with oxidized cluster and apoprotein are inactive
- Bind with low affinity to DNA and don’t stimulate transcription

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16
Q

What are characteristics of FNR binding sites?

A
  1. Have only a partial match to the consensus sequence
  2. Located at variable positions within promoter regions
    - Directing whether FNR has either a positive or negative effect on
    transcription
17
Q

Where are FNR binding sites located at repressed vs. activated promoters?

A
  • Repressed: locations range from upstream of the 235 hexamer (which binds region 4.2 of RNA polymerase s70), to overlapping the transcription start site (TSS; +1)
  • Activated: center of the binding site is 41.5 nt upstream of the TSS
18
Q

Under anaerobic conditions: When E. coli is given a choice of electron acceptors such as nitrate, nitrite, or
fumarate, what will it choose? Why?

A

Nitrate
- Has a more positive redox potential (closest to oxygen out of all of those)
- Induces transcription of genes resulting in synthesis of a membrane-bound nitrate reductase
- Represses transcription of genes encoding other reductases

19
Q

Briefly summarize the path of nitrate reduction.

A
  • Nitrate (NO3-) first reduced to nitrite (NO2-) which is then reduced to ammonia (NH4+)
  • 8 electrons are required: 2 to reduced nitrate, 6 to reduce ammonia
  • Part of the ammonia is assimilated in to cellular compounds, part is excreted into the medium
20
Q

What enzymes are involved in nitrate reduction? What genes encode them?

A
  1. Nitrate reductase (membrane)
    - narG operon (narGHJI)
  2. Nitrite reductase (cytoplasmic)
    - nirB operon (nirBDC)
21
Q

Nar system: What is the purpose of the cytoplasmic nitrite reductase?

A

Prevent the accumulation of toxic nitrite in the cytoplasm

22
Q

Give an overview of gene regulation of the Nar system.

A
  • In the presence of excess nitrate: preferential synthesis of
    enzymes that use nitrate as an electron acceptor (i.e., nitrate reductase)
  • In the presence of excess nitrite: preferential synthesis of enzymes that use nitrite as an electron acceptor (i.e. respiratory nitrite reductase, cytochrome c522)
23
Q

What are the histidine kinase and response regulator proteins in the Nar system?

A
  1. HK: NarQ and NarX
  2. RR: NarL and NarP
24
Q

Explain the roles of NarX, NarQ, NarL, and NarP in the Nar system?

A
  • NarX and NarQ can autophosphorylate (using ATP as the phosphoryl donor) and transfer the phosphoryl group to NarL
  • NarX and NarQ can also negatively regulate the NarL protein by dephosphorylating NarL-P
25
Q

Research has also shown that NarX and NarQ have two transmembrane domains near the N-terminal end and a region that is exposed to the periplasm. What do mutations in the periplasmic region result in?

A
  • Interfered with the cell’s ability to respond to nitrate and nitrite
  • Cell either lost the ability to respond to the presence of nitrate and nitrite or started acting like nitrate and nitrite were present even if they were not
26
Q

How does a shift to a higher osmolarity affect the OmpC:OmpF ratio?

A

Ratio of OmpC:OmpF increases (OmpC inreases more than OmpF)

27
Q

Why are the changes to porin composition of the outer membrane as a response to higher osmolarity not homoestatic?

A

Because the changes in porin composition of the outer membrane don’t change the intracellular osmotic pressure

28
Q

What are the histidine kinase and response regulator proteins in the EnvZ/OmpR system?

A
  1. HK: EnvZ
  2. RR: OmpR
29
Q

Explain how a shift to high-osmolarity media represses ompF and stimulates ompC.

A
  • High osmotic pressures activate the kinase activity of EnvZ
  • Results in high levels of OmpR-P –> repress transcription of ompF and stimulate transcription of ompC
  • micF is transcribed from the ompC promoter but in the opposite direction (also activated by OmpR-P)
  • micF codes for an RNA transcript that is complementary to the 5’ end of the ompF mRNA and blocks translation
  • When external osmotic pressure is lower: concentrations of OmpR-P are too low to bind to DNA sites for repression of ompF and stimulation of ompC
  • Low levels of OmpR-P can still stimulate the transcription of ompF due to high-affinity binding sites for OmpR-P upstream of the ompF promoter
30
Q

Define catabolite repression. What does it result in?

A

The preferential use of one carbon source for growth over another when bacteria are grown in the presence of both carbon sources
- Results in diauxic growth

31
Q

What mechanisms are responsible for catabolic repression?

A
  • cAMP-CRP
  • Cra
  • CcpA system
32
Q

Summarize the cAMP-CRP system.

A
  • Controls several operons involved in catabolism
  • When CRP binds to cAMP, the complex can then bind to sites in the DNA and trigger transcription
  • Must also be bound to RNA polymerase to stimulate transcription
  • Low glucose levels activate adenylate cyclase –> synthesizes cAMP
  • Glucose uptake decreases cAMP levels –> decreases the transcription of cAMP-dependent genes
33
Q

What types of genes does Cra induce and repress?

A
  • Represses genes that code for enzymes that are necessary for growth on sugars –> inhibits the glycolytic and ED pathways
  • Induces genes that code for enzymes that are necessary for growth on organic acids and amino acids –> promotes the TCA, glyoxylate, and gluconeogenic pathways
34
Q

Explain the model for how Cra inactivates certain genes and represses others.

A
  • Cra activates certain genes and inhibits others depending on where it binds to the DNA in relationship to the RNA polymerase binding site
  • Upstream: activator
  • Downstream: inhbitor
35
Q

Compare the Cra and cAMP-CRP systems.

A
  • Both systems: sugar prevents catabolite-repressed genes from being activated
  • In the cAMP system: glucose lowers levels of cAMP–CRP by reducing IIAGlc–P levels via phosphorylation of incoming glucose
  • In the Cra system: Cra acts as the activator of gluconeogenic genes and is removed from DNA by F1P or FBP –> to catabolite repression of these genes
  • Cra also functions as a negative transcription regulator of genes essential for sugar catabolism
  • Both cAMP and Cra systems regulate hundreds of genes in enteric bacteria

Bacterial Physiology (BIOL 471) (15 decks)

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