Htin's lectures Flashcards

Question

what genes does ArcA activate?

Answer 1

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

Answer 2

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

Answer 3

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

Answer 4

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

Answer 5

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

Answer 6

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

Answer 7

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

Answer 8

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

Answer 9

regulates the switch to anaerobic respiration represses microaerobic respiration and activates genes involved in anaerobic respiration

Answer 10

cytochrome bo and cytochrome bd switched off then switch on genes for anaerobic e- acceptors (in this order): nitrate/nitrite DMSO/DMS TMNO/TMA

Answer 11

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

Answer 12

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

Answer 13

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

Answer 14

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

Answer 15

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)

Answer 16

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

Answer 17

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

Answer 18

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

Answer 19

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

Answer 20

activation depends on recruiting RNAP

Answer 21

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

Answer 22

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

Answer 23

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

Answer 24

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

Answer 25

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

Answer 26

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

Answer 27

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

Answer 28

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)

Answer 29

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

Answer 30

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

Answer 31

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

Answer 32

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

Answer 33

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

Answer 34

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

Answer 35

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

Answer 36

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)

Answer 37

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

Answer 38

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)

Answer 39

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

Answer 40

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

Answer 41

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

Answer 42

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

Answer 43

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

Answer 44

Nuk g means there must be another regulator regulating it

Answer 45

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

Answer 46

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