Oxygen Sensing and Regulation

Thursday, May 23, 2024 1:44 AM

Oxygen sensing imp as it allows for repression of unnecessary pathways (aerobic/anaerobic) as well as
induction of necessary ones to prevent wastage of protein expression.

Sinorhizobium, Bradyrhizobium and Rhodobacter both employ two-component oxygen-sensing systems.

These are FixLJ and RegAB respectively. Oxygen inhibits nitrogen fixation and photosynthetic electron
transport respectively in these bacteria. Response: Up regulation of certain genes

Methods of sensing oxygen:

Direct: Via haem domains which bind oxygen directly and alter conformation of protein and change
signalling activity.
Indirect: Changes in oxygen level -> Changes in electron transport rate. This causes build up of 1) a
metabolic intermediate which can bind to a sensor (quinone, pyruvate, lactate for ArcAB) or 2) change in
redox state of a metal containing protein (Fe loss from Csy residues of FNR)
Oxidation: removal of electron
Reduction: addition of electron

Succinate or NADH is made from Krebs Cycle when O2 is high, dehydrogenases remove H and donate
them to quinone, reducing them to QH2. They give them to cytochrome bc1 as 2H and 2e- and goes
back to oxidised form Q. These electrons travel through the electron transfer chain and combine with
oxygen and H+ to make water. At high oxygen ETC goes very fast, quinone pool is in Q state with QH2
only briefly made. Q used as metabolic intermediate for sensing purposes of high oxygen conditions, Q is
chemically different to QH2 and Q can bind to or interact differently with sensory proteins which QH2
cannot.

TCA cycle operates during aerobic conditions, pyruvate buildup from glycolysis results in anaerobic
enyzmes forming products such as lactate, formate, ethanol and acetate. The TCA cycle normally uses
pyruvate quickly aerobically but fermentation pathways making lactate work more slowly. Thus lactate
and pyruvate and acetate build up is another sign to E. coli that it is now experiencing anaerobic
conditions. E.coli sense this to regulate the gene expression of TCA cycle enzymes (such as succinate
dehydrogenase sdhABCD.)

ArcAB: two component system, allows E coli to live in upper gut (lower oxygen conditions)
ArcB: sensor kinase, ArcA: response regulator. ArcB has three autophosphorylation sites.
In aerobic conditions: H1 gets -P, which is then sent to site 2 (D1), from there can be lost
In anaerobic conditions, -P gets transferred to final site H2 which phosphorylates the D on ArcA (D2) as
expected of system.
At high oxygen, oxidised Q interacts and oxidises sensory domain, turning off kinase property of H1; the
quinones in the ETC are free to oxidise the nearby SH groups on the Cys residues in the PAS domains of
the ArcB, causing them to form S-S bonds. In aerobic conditions, ArcB even dephosphorylates ArcA-P.

QH2 does not interact with sensor. At low oxygen, lactate and other products accumulate and interact
with H1, activating ArcB as a kinase. Allows for -P transfer from D1 to H2 and lactate binding at D1
allows H2 to transfer -P to D2
ArcB is a dimer but only one monomer autophosphorylates when active.

ArcA-P dimerises on promoter, but only one monomer is phosphorylates .ArcA dimerises head to head.
Represses TCA Cycle genes, induces fermentative process genes (products make ethanol) and cydAB (a
gene encoding a high oxygen affinity cytochrome D to respond to low oxygen conditions and keep
electron transport flowing).

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electron transport flowing).

Up to three ArcA-P ArcA dimers can bind at single promoters at low but not no oxygen conditions in the
cydAB promoter. CytD is expressed, allowing for higher affinity for oxygen at end of ETC. At no oxygen
conditions, FNR represses expression of CytD.

FixLJ regulates Sinorhizobium genes required for nitrogen fixation so that they are only expressed under
anaerobic conditions (oxygen scavenged by plant to make leghaemoglobin in root nodules). Nitrogenase
inhibited by oxygen.
FixL is a 505 aa membrane protein dimer, senses oxygen by haem domain partially bound to His 194. O2
binding causes inactive conformation of FixL. Autophosphorylates itself on His 285 and phosphorylates
FixJ in absence of oxygen.
FixJ forms a dimer and recruits RNA polymerase, binds to nitrogen fixation genes and activates
transcription. FixJ-P also transcribes a secondary regulator known as FixK2 which gets a second stimulus
in low oxygen. FixK1 is used to regulate other genes for free living.

Rhodobacter use either photosynthetic or respiratory ETC to maintain proton motive force to create
ATP, photosynthesis only carried out in low oxygen conditions.
Gene products needed for photosynthesis: Reaction centre, Light harvesting proteins, synthesis of
carotenoids and chlorophyll that protein binds.
The PufLM and PuhA proteins make the photosynthetic reaction centre (RC) and the two PufBA gene
pairs encode light harvesting (LH) proteins. PufLM and the LH 1 PucBA & LH2 PufBA proteins each bind
chlorophyll and carotenoid pigments for photosynthesis to work. The chlorophylls and carotenoids are
the products of long pathways of enzymes encoded by many genes (& also heavily regulated).

RegB: Membrane bound HK, how it senses oxygen is a mystery, oxidised Q binds directly to it (found in
2006), inhibiting kinase activity. QH2 not able to bind to it.
RegA: Phosphorylated by RegB, dimerises on promoters and activates transcription of regulated genes
such as puf and puc. RegA without -P does not dimerise

FNR is activated anaerobically by gaining 4 Fe in its iron sulphur cluster structure (Cys residues). This lets
it dimerise. When it is affected by oxygen in vivo, it switches to an inactive monomeric form by losing

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it dimerise. When it is affected by oxygen in vivo, it switches to an inactive monomeric form by losing
first 2Fe and then all Fe from the clusters. This makes it monomerise. Further the no-Fe form can also
get proteolytically degraded.
FNR as a regulator turns off the CytD gene promoter in conditions of no oxygen (total anaerobic). Not a
sensor kinase system, but a one component system. (FNR: Regulator of fumarate and nitrate reduction).
Can activate or repress gene funtion depending on position on promoter in terms of binding site. Has
helix turn helix at C terminus which bind to consensus sites at promoter and can recruit DNA
polymerase.
Binding sites for FNR at centered at either -41.5 or around -61.5 (to -71.5). At these positions,
dimerisation of FNR exposes one of the faces of FNR for the interaction with alpha C-terminal domain of
RNA polymerase. This recruits the polymerase to the promoter and activates transcription. If the FNR
binding site is at -41.5 the polymerase surrounds the FNR dimer and interacts at the 5’ face of the left
hand FNR subunit. If the site is at the -61.5/-71.5 position the polymerase binds at one side with the 3’
face of the right hand FNR subunit.

If two FNR binding sites are at -50.5 and -94.5 then the promoters are repressed by high levels of FNR
binding. These genes encode proteins required for aerobic or low oxygen metabolism but NOT required
during anaerobic conditions.
In this case both the FNR binding sites overlap two sequences where the RNA polymerase alpha C-
terminal domain normally naturally can bind activating transcription. FNR here works against this
natural activation. If only one of these (the -50.5) is occupied by an FNR dimer (as at low oxygen
conditions some FNR but not all may be active) then the RNA polymerase alpha C-terminal domain still
works by “reaching” and binding activating DNA upstream of it. If both FNR sites are occupied by dimers
(as at anaerobic conditions) then RNA polymerase cannot get DNA contact for its alpha C-terminal
domain & is not active and the promoter is repressed giving no transcription

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