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MICR335 > Htin's lectures > Flashcards

Htin's lectures Flashcards

1
Q

outline hierarchical use of electron acceptors in e. coli?

A

e. coli have different electron acceptors for different oxygen concentrations

oxygen is the preferred one cause produces the most energy

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2
Q

how do bacteria and humans differ metabolically?

A

higher eukaryotes much more metabolically inflexible because we employ:

  • heterotrophic electron donors (sugars, aa’s, fats)
  • oxygen as sole terminal electron acceptor
  • fermentation transiently and products must be recycles

so e. coli more metabolically flexible cause can switch electron acceptor

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3
Q

discuss the metabolic flexibility of bacteria in the context of e. coli?

A

e. coli employs:

  • diverse organic and inorganic electron donors
  • oxygen and anaerobic electron acceptors
  • fermentation as terminal, sustained mode of energy gen

this flexibility enables e. coli to adapt to a much wider range of environments e.g. aerobic, microaerobic, anaerobic

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4
Q

what are the four types of metabolism in e. coli?

A

aerobic respiration

microaerobic respiration

anaerobic respiration

fermentation

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5
Q

what are the key enzymes and electron acceptor during aerobic respiration in e. coli?

A

electron acceptor; O2 –> H2O

key enzyme: cytochrome bo oxidase

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6
Q

what are the key enzymes and electron acceptor during microaerobic respiration in e. coli?

A

electron acceptor: O2 –> H2O

key enzyme: cytochrome bd oxidase

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7
Q

what are the key enzymes and electron acceptor during anaerobic respiration in e. coli?

A

electron acceptor/s:
NO3- –> NO2-
NO2- –> NH4+
fumarate –> succinate
DMSO –> DMS
TMAO –> TMA

key enzymes:
nitrate reductase
nitrite reductase
fumarate reductase
DMSO reductase
TMAO reductase

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8
Q

what are the key enzymes and electron acceptor during fermentation in e. coli?

A

electron acceptor/s:
pyruvate –> lactate
formate –> H2 + CO2

key enzymes:
lactate dehydrogenase
formate hydrogenlyase

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9
Q

what is the max ATP yield during aerobic respiration?

A

with oxygen as terminal e- acceptor (during high pO2) you get 38 ATP per mol glucose

this comes first in order of preferred e- acceptor

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10
Q

what is the max ATP yield during microaerobic respiration?

A

with oxygen as terminal e- acceptor (at low pO2) you get 15 ATP per mol glucose

this comes second in order of preference

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11
Q

what is the max ATP yield during anaerobic respiration?

A

with nitrate as terminal e- acceptor you get 15 ATP per mol glucose

with fumarate as terminal e- acceptor you get 12 ATP per mol glucose

so these come 3rd and 4th in order of preferred e- acceptors

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12
Q

what is the max ATP yield during fermentation?

A

with acetyl- as terminal e- acceptor you get 3 ATP per mol glucose

so this comes 5th in order of preference

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13
Q

why is ATP yield much less during fermentation than any form of respiration?

A

during respiration ATP yield greatly enhanced through oxidative phosphorylation (respiratory e- yields coupled to pmf generation)

during fermentation ATP can only be produced through substrate-level phosphorylation (direct transfer of phospho groups during glycolysis)

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14
Q

discuss how utilisation of electron acceptors is hierarchical?

A

most electropositive e acceptor yields greatest amount of energy

sequence used by e coli:
- aerobic resp
- microaerobic resp
- nitrate resp
- fumarate resp
- fermentation

hierarchical control depends on regulation of transcription of metabolic enzymes and terminal reductases in response to signals

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15
Q

discuss the signals and regulators responsible for controlling hierarchical electron acceptor utilisation?

A

low quinone signals low redox state; ArcBA is regulator which represses aerobic resp and activates microaerobic resp

no O2 signals anoxic conditions; FNR represses microaerobic resp and activates anaerobic resp

high nitrate signals presence of e acceptors; NarLP and NarPQ repress alternate modes of anaerobic respiration and activate nitrate respiration

high formate signals absence of e acceptors; FhlA activates fermentation

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16
Q

regulatory systems are carefully integrated to ensure what?

A

coordinated control and impose metabolic priorities

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17
Q

what is ArcBA and what does it regulate?

A

non-classical two-component system comprising a histidine kinase (ArcB) and response regulator (ArcA)

ArcBA the central regulator during initial response to hypoxia - regulates shift to microaerobic respiration

controls 9% of genome directly or indirectly; represses aerobic respiration enzymes, upregulates microaerobic respiration enzymes

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18
Q

how was the Arc system discovered?

A

by genetic screen

under anaerobic conditions succinate dehydrogenase is on but switched off under microaerobic cond

mutants that failed to anaerobically repress succinate dehydrogenase were isolated and ArcA identified as anaerobic repressor

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19
Q

discuss the structure of ArcB in the context of its domains and phosphotransfer?

A

ArcB is a membrane bound histidine kinase; phosphorelay increases affinity for target

transmembrane domain: two helices enable membrane-association

leucine zipper domain: dimerisation through alpha-helix interactions

PAS domain: enables redox-sensing via cysteine residues

1st transmitter domain: autophos his292 res through ATP binding

1st receiver domain: phosphotransfer from his292 to asp576

2nd transmitter domain: phosphotransfer from asp576 to his717

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20
Q

discuss the structure of ArcA in the context of its domains and phosphotransfer?

A

ArcA is response regulator activated by phos at asp54

2nd receiver domain: phosphotransfer from his717 of ArcB

helix-turn-helix domain: binds specific promoter sequences to control transcription

phosphorylated ArcA can specifically bind to promoter sequences as a multimer of dimers

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21
Q

discuss the phosphorelay system contained in the ArcBA system?

A

unlike classical TCS, phosphoryl groups transferred through his292-asp576-his717-asp54 relay for ArcA to activate

the reverse phosphotase relay also occurs during signal decay

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22
Q

how is ArcA both a repressor and an activator?

A

is a global regulator of carbon oxidation

recognises unusual 15bp consensus including two repeats

binding site generally overlaps -10/-35 regions in repressed genes (blocking RNAP), but is upstream in activated genes (prob recruiting RNAP)

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23
Q

what does ArcA do when it is on?

A

modulates the shift from aerobic to microaerobic respiration

it does this by repressing cytochrome bo oxidase and activating cytochrome bd oxidase

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24
Q

what genes does ArcA repress?

A

cytochrome bo oxidase
succinate dehydrogenase
lactate dehydrogenase
amino acid dehydrogenases

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25
Q

what genes does ArcA activate?

A

cytochrome bd oxidase
hydrogenase-1
ArcA (positive feedback loop)

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26
Q

why is ArcA controlled switching of terminal oxidases from cytochrome bo to cytochrome bd central to its response?

A

it ensures continued respiratory flow when pO2 low

cytochrome bo has low oxygen affinity but is fine when high pO2; cytochrome bd has high oxygen affinity so better in low pO2; trade off is that cytochrome bd makes less energy due to being proton-nontranslocating

so ArcA is a repressor for cyt bo and an activator for cyt bd

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27
Q

what is the signal activating ArcB?

A

ArcB must respond to signal that decreases its autophos activity as otherwise target genes would always be repressed

responds to membrane-associated signal; evidence for this is that membrane-detached ArcB constitutively on

further studies showed that quinones are the ArcB signal

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28
Q

how was it found that quinones are the signal for ArcB?

A

ArcB was purified and this showed it was autophos

so they did autoradiograms of P-ATP so measure autophos of ArcB and showed activity decreased by oxidised ubiquinone but not by ubiquinol (reduced)

thus e coli quinones (ubiquinone, menaquinone) repress ArcB during aerobic growth and quinones in diff states are the signal for ArcB to switch on/off

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29
Q

what are quinones?

A

membrane-associated electron carriers which are reduced by primary dehydrogenases and oxidised by terminal reductases

they signals of the intracellular redox state as quinone in oxidised state and quinol in reduced state - have lateral mobility in membrane allowing interaction with ArcB

under hypoxic conditions (low O2) reduced quinols accumulate and thus ArcB autophosphorylation increases and ArcA gets activated

30
Q

how does sensing take place in ArcB?

A

via cysteine residues 180 and 241 in the PAS domain; these are essential for redox switch

proposed these res form intermolecular disulfides between ArcB monomers

much evidence supporting this conclusion e.g. mutating to alanine dysregulates the protein function

31
Q

outline the overall proposed mechanism of ArcB redox switch and its effects?

A

electron transfer from thiol residues of cysteine residues to quinone proposed to maintain ArcB in inactive disulfide-bonded state

hypoxia causes quinone levels to fall and quinol levels to increase and equilibrium shifts to active thiol state of ArcB i.e. conformational change to the correct conformation for phosphorelay to ArcA

phosphorylation of ArcA means it can repress aerobic genes e.g. cyt bo and activate microaerobic respiration genes e.g. cyt bd which scavenges O2 better at expense of energy-generation

32
Q

what does heirarchical control of electron acceptor utilisation depend on?

A

regulation of the transcription of metabolic enzymes and terminal reductase in response to signals

e. coli uses most electropositive e- acceptor available in order to maximise ATP yield and growth rate

33
Q

what does FNR regulate and what is its signal?

A

regulates the switch to anaerobic respiration

represses microaerobic respiration and activates genes involved in anaerobic respiration

34
Q

what genes are switched off and on during the switch from microaerobic to anaerobic respiration?

A

cytochrome bo and cytochrome bd switched off

then switch on genes for anaerobic e- acceptors (in this order):

nitrate/nitrite
DMSO/DMS
TMNO/TMA

35
Q

during anaerobic respiration, what can e. coli can supplement electrons from NADH with?

A

highly electronegative donors such as formate and H2; these get reduced by enzymes like hydrogenase and formate dehydrogenase

36
Q

what are the two electron carriers in e. coli, and which one is the anaerobic electron carrier and why?

A

ubiquinone

menaquinone

menaquinone is used during anaerobic resp cause it is highly electronegative so better for transferring electrons between diverse anaerobic donors and acceptors

37
Q

outline nitrate and nitrite respiration in e. coli?

A

e. coli switches from aerobic respiration to nitrate respiration during anoxic conditions when nitrate is available

nitrate is primarily respired through activities of nitrate reductase and nitrite reductase

38
Q

what is FNR and how does it work?

A

FNR is the central regulator in the microaerobic to anaerobic shift

is a one-component system that directly senses oxygen; signals from sensory domain transduced to DNA-binding domain

FNR directly binds O2; O2 binding inactivates FNR (so its inactive in the presence of O2)

the absence of O2 causes it to dimerise and regulate transcription

39
Q

how was FNR discovered?

A

by mutant phenotyping; selected mutants unable to grow using fumarate as sole e- acceptor

three main types of mutations were mapped:

  • mutation in fumarate reductase structural gene
  • mutations in menaquinone biosynthesis pathway
  • mutations in a fumarate and nitrate reduction regulator (FNR)
40
Q

what are the domains of FNR and how they were studied?

A

FNR difficult to work with due to O2 sensitivity; secondary structure could be predicted and domain organisation modelled

sensory domain; binds O2 using oxygen-labile 4Fe4S cluster; sense high/low O2 conc

dimerisation domain; two O2; unbound FNR molecules dimerise through helix-turn-helix interactions

DNA-binding domain; recognises and binds DNA via helix-turn-helix domain; allows binding of specific FNR boxes

41
Q

how was it shown that FNR is a global regulator?

A

microarray profiling showed FNR controls gene expression of 16% of the genome

many of these genes involved in switch to anaerobic but also genes for other things too

42
Q

what genes does FNR repress?

A

cytochrome bo oxidase
cytochrome bd oxidase
NADH dehydrogenase II
succinate dehydrogenase
FNR (negative feedback loop)

42
Q

what genes does FNR activate?

A

formate dehydrogenase
hydrogenase
nitrate reductase
fumarate reductase
DMSO reductase
TMAO reductase
pyruvate formate lyase
fumarase B

43
Q

what does FNR activation depend on?

A

activation depends on recruiting RNAP

44
Q

discuss the promoter region of FNR-activated genes?

A

genes activated have FNR box at -42 region (class II promoters) OR -62 region (class I promoters) containing inverted repeat

in anoxic conditions FNR homodimer binds inverted repeats; FNR bends DNA to activate transcription

FNR-activated genes have weak basal promoter -35 sequences. FNR-activating regions compensate to recruit RNAP

45
Q

outline how FNR recruits RNAP?

A

FNR structure predictions show three regions that interact with RNAP to activate transcription

AR1 - enhance RNAP-binding by interacting with C-terminal domain of alpha-subunit accelerating closed to open complex formation

AR2 - also accelerates open complex formation by interacting w alpha subunit N-terminal domain

AR3 - recruits sigma factor 70 to directly activate RNAP; strongest interaction

46
Q

how does FNR activation at class I promoters differ to activation at class II promoters?

A

class I - FNR binds at -62 region; FNR adjacent to RNAP; AR1 interactions only

class II - FNR binds -42 region; FNR in middle of RNAP; AR1, AR2 and AR3 interactions

47
Q

how does FNR repress transcription?

A

by occluding RNAP binding

strong repression; FNR binds between the strong -35 and -10 elements; RNAP can’t bind either

moderate repression; FNR binds between -10 element and TSS; RNAP can still bind -35

weak repression; FNR binds upstream of -35 element; RNAP can still bind -10

so by looking at where FNR binds we can see if genes are repressed or activated by FNR

48
Q

outline FNR repression of NADH dehydrogenase II in aerobic, microaerobic and anaerobic conditions?

A

aerobic conditions: FNR completely inactive. NDH2 optimally transcribed

microaerobic conditions: FNR partially active; binds high-affinity -50 FNR box; some transcriptional repression

anaerobic conditions: FNR fully active; binds high-affinity -50 FNR box and low-affinity -95 FNR box; no transcription

49
Q

why does FNR repression of NADH dehydrogenase II occur?

A

non-translocating NADH dehydrogenase II is repressed anaerobically in favour of translocating NADH dehydrogenase I

50
Q

how was FNR repression of terminal oxidases at different air saturations experimentally shown?

A

put lac promoter in front of key terminal oxidase genes and measured beta-galactosidase activity (promoter activity) in wt, arcA mutant and FNR mutant under diff air saturations

showed that cytochrome bd expression highest around 10% air, activated by ArcBA below 20% air, repressed by FNR below 5% air

showed cytochrome bo expression highest above 20% air, repressed by ArcBA below 20% air, repressed by FNR below 5% air

showed cytochrome bo negatively regulated by both ArcBA and FNR and cytochrome bd neg reg by FNR and pos reg by ArcBA

51
Q

how can FNR synergise or antagonise with ArcBA?

A

repression of cytochrome bo; FNR and ArcBA synergistic cause both negatively regulate

repression of cytochrome bd; FNR antagonistic w ArcBA (cause ArcBA pos reg while FNR neg reg)

52
Q

what about FNR structurally allows it to sense oxygen?

A

FNR N-terminus has cysteine rich extension containing conserved cysteine residues which are ligands for iron; FNR binds an iron-sulfur centre (4Fe4S) which is perfectly suited for O2 binding and redox chemistry

so FNR senses O2 through iron-sulfur cluster which is bound to sensory domain

53
Q

how was it found that FNR switches between inactive monomer and active dimer depending on state of 4Fe4S cluster?

A

FNR mutants that activated transcription even under aerobic conditions were isolated - in these mutants FNR constitutively formed dimer that could activate transcription

spectroscopic studies showed 4Fe4S cluster of FNR directly binds O2 and is oxygen-labile; converted to inactive 3Fe4S by oxygen then disintegrates to 2Fe2S form then unbound apo form

under aerobic cond charge repulsion by side chains of FNR subunits inhibit dimerisation; under anaerobic cond 4Fe4S-dependent conf change allows FNR monomers to dimerise

54
Q

what is structurally different between monomeric and dimeric FNR?

A

dimeric FNR has intact 4Fe4S cluster allowing dimerisation helices to interact and HTHs orientated to bind DNA

monomeric FNR has inactive 2Fe2S cluster so dimerisation helices not aligned and HTHs cannot bind DNA

55
Q

give an overview of the phases of TB infection?

A

exposure to M.tb results in infection of about 25% exposed individuals

<10% of these experience early progression to active TB, immune system control it in the other 90% –> latent infection

of those latently infection 10% reactivate at some point due to failed immune control leading to active TB

56
Q

outline hypoxia and M.tb?

A

gradual depletion of oxygen leads to a stationary phase, slowed growth and persistent phenotypes (not replicating but metabolically active)

M.tb frequently encounters hypoxia in host and intracellular environments

57
Q

how can M.tb still survive in such low oxygen levels such as those experienced within granuloma?

A

has evolved to scavenge and metabolise traces of oxygen e.g. by upregulating cytochrome bd oxidase

58
Q

how was Rv3133c discovered and identified as hypoxia response regulator?

A

using 2D gel electrophoresis and taking samples from diff time points at diff O2 concs

this allowed identification of proteins upregulated under hypoxic conditions including acr and Rv3133c

later studies labelled Rv3133c DosR (dormancy survival regulator) and used microarray to identify it as a two-component response regulator upregulated under hypoxic cond; western blots showed Rv3134c mutants had polar effects and reduced Rv3133c and acr expression

same study found u could complement Rv3134c mutants with Rv3133c but not Rv3134c; identified Rv3133c as hypoxia response regulator

59
Q

what did it suggest when an experiment showed Rv133c deletions reduced survival phenotype as O2 depleted, Rv3132c mutants only had minor reduction, while no phenotypic change was seen under aerobic conditions?

A

DosR must be involved in survival/dormancy

Rv3132c (DosS) prob the sensor kinase for Rv3133c (DosR) - minor phenotype prob means theres another sensor kinase

(there is a second sensor kinase in this system (DosRT)

60
Q

outline discovery of the DosR/Rv133c regulon?

A

microarray of Mtb Rv3133c mutants under hypoxia and normoxia identified 26 genes upregulated under hypoxia which are activated by DosR

61
Q

how was the DosR regulon used to identify the DosR binding motif?

A

promoter regions of regulon members used to identify DosR-binding motif

20 bp consensus sequence was identified

deletions in this sequence reduced expression

DosR DNA-binding C-terminal domain shown to be required for promoter binding; DosR-Asp54 phosphorylation residue essential for expression

expression was looking at acr expression (activated by DosR)

62
Q

how was DosT/Rv2027c identified?

A

they knew DosR is a canonical DNA-binding response regulator and that deletion of DosS/Rv3132c had little effect on hypoxia response

looked for DosS homologs in Mtb and found Rv2027c/DosT

deletion of DosT also had little effect on hypoxia response (DosR activation and expression of acr) but deletion of both DosT and DosS resulted in NO acr expression

63
Q

what can be determined from the mutagenesis study showing only deleting BOTH DosT and DosS was sufficient to stop acr expression (DosR activation)?

A

this two-component system (DosRT) requires BOTH sensor kinases to be fully functional

64
Q

what is the currently accepted model of DosS and DosT?

A

they are heme-based oxygen sensors which work by signal integration

DosS is redox sensor; active when Fe2+; inactive when Fe3+

DosT is hypoxia sensor; active when deoxy; inactive when oxy

so both bind oxygen which causes reversible switch

65
Q

how was the physiological response of the DosR system experimentally shown?

A

mutagenesis studies showed dosR mutants had impaired survival under hypoxia and impaired ability to exit dormancy; also fail to slow O2 consumption as cells enter dormancy

66
Q

what is the physiological response of the DosR system?

A

DosS/DosT-DosR required for initiating metabolic downshift of mycobacterial cells in hypoxic conditions

DosR required for reactivation of metabolism allowing cells to exit dormancy

many functions of regulon members remain uncharacterised

67
Q

is cytochrome bd part of the DosRT regulon and what does this mean?

A

Nuk g

means there must be another regulator regulating it

68
Q

outline the process of identifying a regulator for cytochrome bd?

A

through looking at consensus sequence of cyt bd promoter CRP binding sites were found

CRP = cyclic AMP receptor protein

mutated these sites and cyt bd expression impacted

69
Q

outline the complexity of metabolic regulation in Mtb?

A

complex regulatory systems where multiple things regulating the process e.g. cAMP, DosR

70
Q
A
71
Q
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MICR335 (4 decks)

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