Adaptive and environmental challenges Flashcards
What are the 3 components of a 2C regulatory system?
Histadine kinase, response regulator, and a regulating phosphatase
Where are the HK and RR phosphorylated?
hk: autophosphorylates at a histadine
RR: phosphorylated by HK as an aspartate residue
What domain of the HK senses the signal? what part transmits the signal to the RR?
the N terminal part senses the signal and the conserved C-terminal domain transmits the signal
What is the composition of the conserved C terminal domain?
conserved sequence of ~200 aa that contains the autophosphorylation (histadine) site
Most HKs are transmembrane, what is one exception that is cytoplasmic?
CheA in the chemotaxis system
What is the structure of RR like?
Cytoplasmic proteins
Have a conserved N-terminal domain of about 100 aa, which interacts with the C-terminus of HK.
- aspartate residue in this domain is what gets phosphorylated
What can RR-P do?
either activate or repress transcription of target genes directly or indirectly.
What is the C- terminus of the RR regulator thought to do?
Interact with target
What is catabolite repression?
the preferential use of one carbon source for growth over another when both carbon sources are present
What 3 systems mediate catabolite repression? Which are cAMP dependent/independent?
- PTS (cAMP dependent)
- Cra (cAMP independent)
- CcpA (cAMP independent)
What does the Cra system stand for?
catabolite repressor/activator system
What is Cra?
A transcriptional regulator
What does Cra do?
represses genes coding for enzymes of the glycolytic pathway and induces genes encoding enzymes required for growth on organic acids and amino acids
What does the pykF gene code for? when is the product used?
Pyruvate kinase F which converts PEP to pyruvate during glycolysis
What does pckA code for? when is the product used?
PEP carboxykinase which converts oxaloacetate to PEP during gluconeogenesis
Under glucose rich conditions, what happens to Cra?
in the presence of a PTS sugar, the Cra complexes with the catabolite.
It can’t repress pykF (pyruvate kinase) or enhance pckA (PEP carboxykinase A)
Under glucose limiting conditions, what does Cra do?
Binds upstream of both the pykF and pckA genes
- inhibits transcription of pykF –> prevents pyruvate kinase F being made for glycolysis
- enhances transcription of pckA –> more PEP carboxykinase A made for gluconeogenesis
What kind of bacteria posses a CcpA system?
low %G+C Gram-positive bacteria
What does CcpA stand for?
Catabolite control protein A
What is CcpA?
transcriptional inhibitor of genes that are catabolite repressed by glucose
What is another role of CcpA in Streptoccoci?
plays a role in controlling virulence of pathogenic streptococci
- acts as a transcriptional activator in this case
Explain how glucose can confer catabolite repression via CcpA
- When glucose is present, it is metabolized. One of the intermediates F-1,6-BP activates an ATP dependent Hpr kinase
- Hpr is phosphorylated
- Hpr-P binds to CcpA and F-1,6-BP to form a ternary complex
- Ternary complex then binds to CRE: Catabolite-responsive element, a conserved DNA sequence
- prevents gene expression of catabolite repressible genes
What are the two kinda of nitrogen sources bacteria can use?
inorganic (NH4+, NO3-, N2) and organic (amino acids, urea, peptides)
What are all nitrogen sources ultimately converted to?
Ammonia
What is ammonia then assimilated into?
Glutamate or glutamine
What does Glutamate dehydrogenase catalyze?
assimilation of ammonia into glutamate
- used NADPH and alpha ketoglutarate as the starting materials
Explain the Glutamine synthetase/GOGAT system. What does each enzyme do?
- Glutamine synthetase converts glutamate to glutamine by adding an NH2 group from ammonia
- That glutamine is then converted back glutamate as the amino group is transferred to alpha ketoglutarate
- End result is 2 molecules of glutamate
What does GOGAT stand for?
glutamine oxoglutarate aminotransferase
When does glutamine synthetase become more active?
Under nitrogen limiting conditions
What regulon contains the genes regulated by ammonia supply?
Ntr regulon
Which operon in the Ntr regulon encodes glutamine synthetase, NRII (histidine kinase), and NRI (response regulator).
glnALG
How many promoters are there in the glnALG operon? What are they called?
3.
Ap1, Ap2, and Lp
What sigma factors bind to each promoter in the glnALG operon? which are high affinity and which are lower
sigma 70 ( low affinity housekeeping sigma) binds to Ap1 and Lp
Sigma 54 is the high affinity factor and binds to Ap2
Describe what happens under ammonia limiting conditions in terms of NRI and NRII
When ammonia is limiting, NRII acts as a kinase
- stimulated by PII-UMP?
it phosphorylates NRI (RR)
NRI-P levels increase which stimulates higher transcription rate of Ap2 (with help from sigma 54)
Describe what happens under excess ammonia conditions in terms of NRI and NRII
When ammonia is in excess, NRII acts as a phosphatase
-stimulated by PII
NRII dephosphorylates NRI-P and transcription rates from Ap2 decrease
What is the product of transcription from the Ap2 promoter
glutamine synthetase
What increases the levels of PII vs PII-UMP?
as glutamine levels increase in the cell, PII levels increase
as levels of alpha ketoglutarate incease in the cell, PII-UMP levels increase
What enzyme controls the switch between PII vs PII-UMP?
uridylyl transferase/uridydyl removal (UT/UR)
When is inhibition of the Ntr regulon relieved?
when ammonia conc falls below 1 mM.
What factor is required for activation of some NRI dependent promoters in the Ntr regulon?
Integration host factor (IHF)
- Job is to bind the DNA together and bring the promoter close
What example was given in class for the action of IHF?
NifA : positive regulator of the nif (nitrogen-fixation) genes
IHF binds a region of DNA between NiFA binding site and promoter. The binding bends the DNA allowing NiFA to interact with RNA polymerase
What is the effect of the PII/PII-UMP ratio on the enzyme activity of glutamine synthetase? what other enzyme is involved in this?
Under limiting-ammonia conditions, PII-UMP stimulates the adenylyl removal activity of the enzyme adenylyl removal/adenylyl transferase (AR/AT), resulting in GS in the active form.
Under excess ammonia conditions, PII activates the adenylyl transferase activity of AR/AT, resulting the GS as inactive GS-AMP.
How is Pi transported into the cell under excess conditions? What is the transporter and what is it driven by?
it is transported by a low-affinity transporter called Pit. Pit consists of a single transmembrane protein and the transport is driven by the ∆P
What happens when Pi becomes limiting?
least 38 genes involved in Pi assimilation are transcribed (in E. coli).
What is formed by the transcription of the Pi limiting genes?
the PHO regulon
What is PhoA
alkaline phosphatase
What is PhoBR?
2 component system
PhoR is the histadine kinase
PhoB is the response regulator
What is PhoE?
outer membrane protein
What is Pst
high affinity Pi uptake system
How is Pi transported in a saturated state?
By an osmotic shock sensitive transport system
- uses an ABC transporter
- sensing by PhoR
How does signalling occur when Pi is limiting?
PhoR autophosphorylates and dimerizes
This causes PhoB to dimerize
PhoB dimer can then translocate to the nucleus and activate the PHO regulon
What are the 4 key changes that occur when a facultative anaerobe goes from aerobic to anaerobic conditions?
- final electron acceptor is not O2 but others (e.g. nitrate, fumarate or DMSO).
- replacement of fumarase A and succinate dehydrogenase (SDH) by fumarase B and fumarate reductase (FRD).
- repression of alpa-ketoglutarate dehydrogenase (ODHG) synthesis.
- acetyl-CoA is made by pyruvate-formate lyase (PFL) instead of pyruvate dehydrogenase (PDHG).
What happens to the TCA cycle under anaerobic conditions? Why?
becomes non-cyclic and reductive
- b/c alpha ketoglutarate dehydrogenase is absent
What are the 4 systems (briefly) in E-coli that bring about the aerobic–>anaerobic switches
- Arc system
- FNR system
- Nar system
- FhlA system/formate regulon
What are the 2 components of the Arc system? what are their roles?
ArcB is the sensor, ArcA is the response regulator
What is different about ArcB
an “unorthodox” HK
- has 3 histadine residues that are used to pass the phosphate through
What kinds/how many genes does the Arc system regulate
up to 30
Mainly repression of: Cytochrome o and d oxidases TCA cycle enzymes Glyoxylate cycle enzymes Several dehydrogenases for aerobic growth (e.g. pyruvate dehydrogenase) Fatty acid oxidation enzymes
What is the end result of the Arc system?
Induction of pyruvate formate lyase (PFL)
What does the Fnr system stand for?
Fumarate, nitrate, reductase regulation
how many genes does the Fnr system contol?
over 100
What does Fnr repress?
cytochrome o, cytochrome d, and nar operon
What does Fnr activate?
formate dehydrogenase, fumarate reductase, DMSO reductase, and nitrate reductase
How does FNR sense O2 or no O2?
via an oxygen-sensitive (4Fe-4S) center
What happens to FNR is there is oxygen?
The 4Fe-4S centre becomes oxidized and disassembles into 2x 2Fe-S centres
- without the dimer, FNR can no loner bind to to DNA and therefore no activation or repression occurs
What happens to FNR if there is no oxygen?
The 4Fe-4S centre remains reduced and the dimer binds to the DNA to control anaerobic gene expression
What is the acid tolerance response? why does it occur?
exposure of bacteria to non-lethal acidic pH results in the induction of sets of genes whose products can protect the cell when exposed to lethal pH conditions
what kind of proteins are produced by the ATR? what response do they have some crossover with?
Acid shock proteins, some of which are also heat shock induced
What are the 3 mechanisms of the ATR (briefly)
- Production of glutamate decarboxylase + specific antiporter
- Production of arginine deaminase
- Production of urease
How does glutamate decarboxylase/antiporter help with the ATR? How does it work?
It is unclear how it helps.
specific antiporter exports g-amino butyric acid (the decarboxylation product of glutamate) and imports more glutamate
How does arginine deaminase help with the ATR?
- converts arginine to ammonia and citrulline.
- Citrulline is further converted to ammonia and CO2.
- The ammonia generated can alkalinize the environment
How does urease help with the ATR?
converts urea to ammonia and CO2
- ammonia alkalizes
What increases the rate of heat shock protein synthesis?
increase stability and translation of sigma factor 32 (also called RpoH)
How does increased temperature affect stability and translation of sigma 32?
- melts secondary structures in the 5’ region of rpoH mRNA resulting in increased translation
- at lower temps, sigma 32 is bound by a bunch of chaperones and easily degraded by proteases. When temperatures increase, the chaperones leave sigma 2 to help other proteins fold and leave it free
What causes envelope stress?
accumulation of misfolded proteins in the periplasm
- result of altered pH, osmolarity, increases in temperature, over-production of recombinant proteins, or even carbon/energy starvation
What are the two response pathways to deal with envelope stress?
- extracytoplasmic function (ECF) sigma factor
2. CpxRA two-component regulatory system
What are the two proteins involved in the ECF system?
RseA and RseB
How does the ECF system work when there is no envelope stress ?
- RseA is bound by RseB from the periplasm.
- The C-terminus of RseA binds sigma factor E and sequesters it from RNA pol.
- Thus, RseA is acting as an anti-sigma factor
How does the ECF system work when envelope stress occurs?
- RseB leaves RseA to bind to misfolded proteins in the periplasm
- RseA is now vulnerable to degradation by proteases
- sigma factor E is then released from RseA and can bind with RNA polymerase
How does the CpxRA system work? what does it activate that links it to the ECF system?
- CpxA is the sensor, autophosphorylates when it senses envelope stress
- transfers P to CpxR
- CprR-P activates expression of dsbA (disulfide oxidoreductase) and ppi (peptidyl-prolyl-isomerase) - enzymes needed to refold misfolded proteins
- also activates periplasmic protease DegP which requires sigma E
- links it to ECF
What are 3 oxygen reduction products that cause oxidative stress?
Superoxide radical (O2-), hydrogen peroxide (H2O2), and hydroxyl radical (OH-)
What does superoxide dismutase do?
superoxide radical to O2 and H2O2
What does catalase do?
H2O2 to O2 and H2O
What do organic reductants (like glutathione and ascorbic acid), and peroxidases do?
H2O2 to H2O
superoxide dismutase, catalase, and organic reductants/peroxidases are used by..?
aerobic bacteria
What 2 methods do anaerobic bacteria use to protect themselves from oxygen radicals
NADH oxidase and superoxide reductase system
What does NADH oxidase do?
the direct reduction of O2 to H2O
What does the superoxide reductase system accomplish?
converting superoxide to H2O2 then to H2O
What are the two oxidative stress regulons in E. coli?
OxyR and SoxRS
How many proteins are produced by the OxyR regulon? what induces it?
9 proteins, induced by H2O2
is OxyR a positive or negative regulator
Positive
How does OxyR exert its effect (what does it bind to? )
binds to target DNA and interacts with the C-terminal domain of RNA polymerase to activate gene expression
What activates OxyR?
oxidation, which results in the formation of a disulfide bond between two cysteine residues in OxyR
- only this form can bind DNA
How many proteins are comprised in the SoxRS regulon and what is it induced by?
9 proteins induced by superoxide (O2-)
how does the SoxRS system work?
- SoxR senses oxidative stress from O2-
- and triggers expression of soxS
- SoxS then activates the expression of genes in the regulon
How does SoxR sense oxidative stress?
uses two (2Fe-2S) centers to sense oxidative stress. Under normal physiological conditions, the Fe-S centers remain reduced and become oxidized when superoxide is present
The oxidized active conformation is what allows it to interact with RNA polymerase
