2. What is PCM?
• L-isoaspartyl protein carboxyl methyltransferase
• Repairs protein damage
Visick, J. E. 2014. Clarke, S., et al., Ageing Res. Rev. 2:263-285.
3. Importance of PCM
• Aging, Alzheimer’s and some autoimmune diseases
• Nearly universal enzyme
Lee, C., et al., J. Biochem. Mol. Biol. 38:275-280. Clarke, S., et al., Ageing Res. Rev. 2:263-285.
Kim, E., et al., Proc. Natl. Acad. Sci. 94:6132-6137, Dreamatico.com, Daviddarling.info
4. E. coli and PCM
Visick, J. E., et al., J. Bacteriol. 180: 2623-2629 (1998).
• Long-term stationary phase
• Secondary stresses
Untreated
WT
WT
ΔPCM
ΔPCM
5. Filamentation
• Elongation of a cell
• Response to stress or survival mechanism
• PCM-deficient mutants (Δpcm) will filament under low
salt conditions?
Justice, S. S., et al. 2008. Nat. Rev. Microbiol. 6:162-168.
6. Research Questions
• Is there a significant difference in filamented cell length
between the Δpcm and WT?
• Are filamented cells multinucleate or anucleate?
• Is the sepatation inhibitor, SulA, the mechanism causing
filamentation?
Cliparts.co
9. 0
10
20
30
40
50
60
70
80
90
WT 0% ΔPCM 0% WT 0.05% ΔPCM
0.05%
WT 1% ΔPCM 1%
%Filamented
Using a one-way ANOVA, there was no significant difference
between WT and Δpcm at 0% at Day 4 (F =1.808, p =0.183).
10. 0
10
20
30
40
50
60
70
80
90
WT 0% ΔPCM 0% WT 0.05% ΔPCM
0.05%
WT 1% ΔPCM 1%
%Filamented
Using a one-way ANOVA, there was no significant difference between
WT and Δpcm at 0.05% at Day 4 (F =0.171, p =0.681).
14. Measuring Genetic
Material
• Genetic material proportional to area
• Consistent Salmonella filaments under high salt
Pratt, Z. L., et al. Appl. Environ. Microbiol. 78:6704-6713.
15. SOS Response
• Extensive DNA damage
• Promotes DNA repair
• SulA - septation inhibition
• Mechanism for
filamentation?
McCool, J. D., et al. Mol. Microbiol. 53:1343-1357.
Gottesman, S. J., et al. Bacteriol. 148:265-273.
Justice, S. S., et al. 2008. Nat. Rev. Microbiol. 6:162-168.
16. SulA
• Visualize the activation of sulA
• SulA-GFP reporter
• Quantitation of filamented cells activating sulA
20. SulA Activity
• Small percentage of cells expressed GFP
• SulA is not the only mechanism of filamentation
21. Future Direction
• Better understanding of genetic material in cells
• Investigate other possible mechanisms for filamentation
• Nutrient restoration
Spontaneous isomerization of aspartate/asparagine resulting in a kink in the backbone could effect the protein activity i.e. folding or positioning of aa.
PCM repairs by methylation, which stabilizes the backbone.
Make sure you know the mechanism if asked about it!!!
Intramolecular nucleophilic attack of the peptide-bond N on the side chain carbonyl carbon
Succinimide intermediate
Intermediate can either undergo spontaneous hydrolysis to produce either: isoapartate or aspartate
PCM will methylate the hydroxyl group
Protein damage is an implication of aging
Studied in bacteria, plants, mice, fruit flies and nematodes and required for aging and/or long term survival but found nearly everywhere
A study found that PCM deficient mice die at an earlier age due to an on set of a epilepsy-like disorder.
In order to study PCM, we used E. coli that have been starved. This causes them to essential “age” and is called stationary phase.
Stationary phase is entered when bacteria are placed a nutrient poor environment/growth limiting factors.
Secondary stresses: heat, methanol, low salt and oxidative
Graph is in the presence of paraquat stress
MC1000 – WT
JV1068 – dPCM
JV1083 – complement
Stresses: protein damage or possible mechanism to survive better/ survival response
Resistance of phagocytosis and rapid invasion epithelial:
UTI, epithelial cells of the bladder
Uropathogenic E. coli (UPEC) respond to host’s immune system by filamentation
Epithelial cells die and allow filaments exposure to the surface
Filaments resist neutrophil phagocytosis
Katie’s data
Get better picture with cell that is filamented
Introduce more…
Percentages are referring to salt conc.
There is a clear difference between the standard conditions and the low salt
At 0% added salt, there is no significant difference between the mutant and the WT; mutant is not significantly filamented more than WT
Need to look up how much salt is in media without added salt
N= 50
Again, at the 0.05% there is still not significant difference between the mutant and WT
Connect filamentation and PCM more, what do these finding suggest?
Difference from low salt to normal conditions
Contradicts previous preliminary data, but it is possible that PCM reduces filamentation under specific conditions, i.e. Christina’s data with oxidative stress
PCM is only needed under very specific conditions
DAPI is a nuclear stain, it allows us to quantify the presences of genetic material
Fix this graph…
Get rid of outliers and make plots bigger
Fix labels
Possible reason for multinucleoid? With a restoration of nutrients, filamented cells can quickly divide and multiply?
DNA replication as cells elongate?
Salmonella filaments were found to be multinucleoid
Insert some data here
UV exposure/oxidative radicals cause extensive DNA damage and the SOS response is activated.
RecA binds to ssDNA and stimulates the autoproteolysis of the SOS transcriptional repressor LexA.
LexA disassociation allows a cell-division inhibitor sulA to stop cell division by binding to the FtsZ monomer.
sulA stops the polymerization of the division protein FtsZ
Without FtZ, this blocks the FtZ ring formation and leads to a formation of non-septate bacterial filaments.
In order to explore this question, we need to find a way to know when SulA is being activated.
We can do this by creating a sulA-GFP reporter strain, GFP = Green flour. Protein.
Bigger pictures.
Vivid expression on day 0 and by day 4 there is fluorescence only visible at the poles.
This could indicate that the GFP tag was degraded and was sequestered to the poles to be disposed of
(We know that damaged proteins are sequestered to the poles of the cells)
GFP isn’t actually expressed at the poles, but is sequestered to the poles we think.
GFP between WT and dPCM @ 0% and 0.05% conditions
Christina’s research also suggests there is both a SulA dependent and independent mechanism for filamentation.
No stress, Day 5 – while there is a reduction in filamentation for the SulA mutant, there is still filamentation. This suggests other mechanisms behind filamentation
Delete the Day 0 and Day 5 stressed data… we don’t care at this point. This presentation is about filamentation only
Christina’s research suggests that deletion of SulA led to a reduction in filamentation but not total elimination
Other possible mechanisms? We really don’t know of other mechanisms at this time…
Add some pictures
