Lecture 6 - Journal Club Flashcards
1) What are polyPs?
2) What does synthesis of polyP require?
3) When is low levels of polyP made?
4) When is high levels of polyP made?
1) Polyphosphates (polyP) are polymers of inorganic phosphate.
2) Synthesis of polyP requires the PPK kinase in E. coli.
3) Low levels of polyP is made during NORMAL (log) E. coli growth.
4) High levels of polyP is made during E.coli cell STRESS CONDITIONS (heat shock, nutrient starvation, oxidative stress, etc)
E. coli ppk mutants (Δppk) display _________ sensitivity to cell stress.
increased
*ppk makes polyP
*polyP produced during stress
*Δppk = no polyP = increase sensitivity
*authors did not know why and how so their main question was “what is the role of PPK and polyP during an E. coli stress response?”
PolyP accumulation in bacteria is physiologically important.
What are the four main processes polyP plays a role in in bacteria?
1) Biofilm formation
2) Motility
3) Antibiotic resistance
4) Virulence
*human cells also have polyP
What is the role of PPK and PPX?
PPK (kinase) synthesizes polyP in stressful conditions, while PPX (phosphatase) disassembles polyP chains to adapt to no stress conditions!
(T/F) Cells that have Δppk mutants are able to adapt to stress conditions similar to WT cells.
False!
Cells that have Δppk mutants are NOT able to adapt to stress conditions similar to WT cells.
What was the objective, rationale, methodology, and outcomes of the authors’ investigation into proteomic differences between wildtype controls and Δppk E. coli during nutrient starvation?
Objective: to compare proteomic differences between WT and Δppk upon nutrient starvation.
Rationale: there may be some proteins that are expressed in WT but not in Δppk cells or vice versa.
Method: grow both cell types in stress condition (nutrient starvation); lyse cells and digest all the proteins in the cells with TRYPSIN (breaks protein into small peptides), use MASS SPECTROMETRY (MS) to identify/quantify peptides.
Outcomes: 78 proteins were identified that were significantly different in WT vs Δppk cells! Further, “All or none” proteins were detected - meaning these proteins were detected ONLY in WT or Δppk cells.
1) How did the authors confirm their mass spectrometry results?
2) What is 3Flag?
3) What does anti-Flag antibody allow for?
4) What is Ponceau S? Why is it used?
1) The results from the mass spectrometry were confirmed on WESTERN BLOTS.
2) If there is no commercial antibody for the protein of interest, you can add specific tags to POI via molecular biology techniques such as 3Flag.
3) Anti-Flag antibody recognizes the POI via the 3Flag tag and allows us to see the band in the blot.
4) Ponceau S (usually done with WBs) stains all cellular proteins non-specifically, serving as a LOADING CONTROL to ensure that we have loaded samples in the lanes and equal amounts loaded in each lane.
The researchers wanted to check if PPK regulated expression of proteins required for lipid A modification.
What was the rationale and methodology behind this experiement?
Rationale: based on the bubble plot and western blot results, the researcher found that some proteins involved in lipidA modification (ArnB, ArnC and EptA) were only present in WT strains and not Δppk strains.
Methodolgy: They did a western blot for Arn proteins and EptA from WT and mutant strains to see if they’re only present in WT.
Arn proteins and EptA modify lipid A (of LPS) to, in part, promote ______ ________ ___________.
cationic antibiotic resistance
What is pPPK and vec?
What were the observed results on the western blot for ArnC and ArnB proteins under the following conditions:
1) WT + vec
2) WT + pPPK
3) Δppk + vec
4) Δppk + pPPK
Why did the researchers perfom this experiment?
pPPK: plasmid that encodes for WT PPK protein
vec: empty plasmids
For both
WT + vec: band present
WT + pPPK: thicker band present
Δppk + vec: no band
Δppk + pPPK: band
This experiment helped solidify results with controls! If you add back PPK to mutant cells, you must see expression of Arn proteins if PPK regulated expression of these proteins! That’s exactly what had happened. Wt PPK regulates expression of proteins required for lipid A modification!
Which statement is true?
1) The proteomic profile of WT and Δppk cells are the same during stress.
2) WT cells and Δppk cells express Arn proteins and EptA during stress.
3) During stress conditions, PPK promotes expression of Arn proteins and EptA (lipid A modifiers).
3!
1) The proteomic profile of WT and Δppk cells are DIFFERENT during stress!
2) WT cells, BUT NOT Δppk cells, express Arn proteins and EptA during stress.
1) What led researchers to suspect that BasS/BasR might be influenced by PKR?
2) How did they test their hypothesis?
3) What were the results?
1) The genes that encode for Arn proteins and EptA are regulated by the BasS/BasR Two-Component System (based on literature). Thus, they wanted to test if PPK/polyP was also required for BasS/BasR expression during stress.
2) They performed a WB on Wt and Δppk cells for BasR and BasS.
3) BasR and basS are not present in Δppk mutants but are present in the WT strains! Thus, PPK is required for the expression of BasR/BasS which regulates the gene that encode lipid A modification proteins (Arn proteins and EptA)!
1) What are pEtN and L-Ara4N?
2) What hypothesis was tested using pEtN and L-Ara4N?
3) How was the hypothesis tested?
1) pEtN and L-Ara4N are lipid A modifications under stress!
2) If PPK is required for lipidA modification during stress, then lipid A modifications SHOULD NOT OCCUR in Δppk mutants (these should be downregulated).
3) They used CHROMATOGRAPHY FOR LIPIDS (separation of different types of lipids within a sample). They compared levels of pEtN and L-Ara4N in WT and mutant strains.
In the lipid chromatography, was pEtN and L-Ara4N seen in:
1) WT + vec
2) Δppk + vec
3) Δppk + pPPK
What does this tell us?
1) WT + vec: yes!
2) Δppk + vec: no!
3) Δppk + pPPK: yes!
Lipid A modifications are downregulated in Δppk mutants, further confirming that PPK is required for lipid A modification during stress.
1) What led researchers to suspect that PPK might be required for polymyxin (antibiotic) resistance?
2) How did they test their hypothesis?
1) Lipid A modifications during stress are required for polymyxin resistance. Since PPK is required for lipid A modifications during stress, then perhaps it is also required for polymyxin resistance.
2) They did a SPOT TEST to see effect on cell growth and viability. They took serially diluted cells in two conditions MOPS (nutrient starvation) only and MOPS + polymyxin.
