1. Catherine Shea and Shruti Panchavati Dr. Sarah Ades Lab
The Effect of Cyclic Peptides on Sigma E Regulation in Escherichia coli
Catherine Shea and Shruti Panchavati
Dr. Sarah Ades Lab

2. Project Summary
Part of Dr. Ades’ lab studying σE in E. coli
σE is an essential sigma factor necessary for cell envelope homeostasis
Complete inhibition of σE causes cell death
It would be beneficial to find σE inhibitors when trying to discover new antibiotics

3. Cyclic Peptide Inhibitors
Created a SICLOPPS library through intein-catalyzed cyclic peptide production
Naumann 2008

4. σE Pathway + Hfq rybB σE σE transcribes the sRNA rybB
rybB works with the Hfq protein
Targets the mRNA of OmpC for degradation
+ Hfq
OmpC
mRNA
rybB σE

5. Let’s Find Inhibitors! Set up an artificial system with two plasmids
rybB
OmpC yfp
rpoE
Plasmid 1
Plasmid 2

6. Plasmid 1 Action σE
rybB
rpoE
rybB

7. Plasmid 2 Action
rybB
Hfq
OmpC yfp
OmpC/yfp mRNA
RNase
RNase

8. Possible Outcomes
If rybB is present in the cell, OmpC/yfp will be degraded
If pathway is blocked, OmpC/yfp will not be degraded

9. Inhibitors Found:
Plasmid 1 (rpoE σE rybB) contains a gene for Ampicillin resistance
Plasmid 2 (OmpC/yfp) contains a gene for Kanamycin resistance
SICLOPPS library plasmid contains a gene for Chloramphenicol resistance
Bright cells growing on Kan/Amp/Chlor plates contained all three plasmids and inhibited the pathway and were selected for further study

10. Selecting for the SICLOPPS plasmid
Bright cell growing on Kan/Amp/Chlor plate = contains all three plasmids
Chlor plate
Miniprep
Transform into DH5α
Test in screening strain
Miniprep
Chlor plate
Kan/Amp plate

11. Previous FACS Results
The image shows fluorescence of control (OFF) strain
6802 (screening strain without ydcQ deletion)
ompC-yfp repressed by rybB

12. D13 + arabinose
E8 + arabinose
E15 + arabinose
F3 + arabinose

13. Recent Findings Specific genetic background for optimal success
Remove enzymes which digest arabinose
Bacteria normally digest arabinose and a large amount of arabinose is deadly to cells
Deletion of ydcQ gene
ΔydcQ allows cells to live without sigma E
Strain 6491: ΔydcQ
Strain 6716: ΔrybB

14. Amp
rpoE
rybB
SigmaE
sRNA
Kan
Ompc-yfp
Chl
SICLOPPS
Plasmid 1
Reporter

15. Preparation of strains
Transformed strains with cyclic peptides
E15 SGWEYVRP, D13 SGWSAYTL, F3 SGWLGPQR, E8 SGWRSVWA
Streaked on Kan, Amp, Chl plates to screen for sensitivity
Added lacZ gene and performed beta-glucosidase to test for high sigma E level
6491
E15
D13 F3 E8
6716
E15
D13 F3 E8

16. Observation under Microscope
% arabinose
% arabinose
(Longer and Fatter)
Problem with growth at 37 degrees Celsius. Toxic intermediates are produced during formation. Fixed by growth at 30 degrees Celsius

17. Preparation of New Strains16 17 18 19
SGWMH(Q)VS
SGWSW(Q)EP
Screening Strain
SGWSER(Q)T
SGWAD(Q)CK
Chromosome of Screening Strain
Deletion of digestive enzymes
Deletion of ydcQ

18. Observation under Florescence Microscope
“OFF” screening strain
rpoE-rybB plasmid + ompC-yfp reporter
“ON” screening strain
Vector + ompC-yfp reporter

19. “ON” screening strain
“OFF” screening strain
% arabinose

20. F3 + arabinose
“OFF” strain
E15 - arabinose
E15 + arabinose
832 - arabinose
832 + arabinose

21. Conclusion
ompc-ypf
rybB
sigmaE ?
While cyclic peptides here have shown to inhibit the system, the mechanism of action is still unclear
Next step is to test what site the cyclic peptides block