The Effect of Cyclic Peptides on Sigma E Regulation in Escherichia

coli

Catherine Shea and Shruti PanchavatiDr. Sarah Ades Lab

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

Cyclic Peptide Inhibitors• Created a SICLOPPS library through intein-catalyzed cyclic peptide production

Naumann 2008

σE Pathway

• σE transcribes the sRNA rybB• rybB works with the Hfq protein• Targets the mRNA of OmpC for degradation

σE rybB + HfqOmpCmRNA

Let’s Find Inhibitors!

• Set up an artificial system with two plasmids

rpoErybB

Plasmid 1 Plasmid 2

OmpC yfp

Plasmid 1 Action

rpoErybB

σE

rybB

Plasmid 2 Action

rybB

Hfq

OmpC yfp

OmpC/yfp mRNA

RNase RNase

Possible Outcomes

• If rybB is present in the cell, OmpC/yfp will be degraded

• If pathway is blocked, OmpC/yfp will not be degraded

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

Selecting for the SICLOPPS plasmidBright cell growing on Kan/Amp/Chlor plate = contains all three plasmids

Miniprep Transform into DH5α

Chlor plate

Chlor plate

Kan/Amp plate

MiniprepTest in screening strain

Previous FACS Results•The image shows fluorescence of control (OFF) strain

•6802 (screening strain without ydcQ deletion)

•ompC-yfp repressed by rybB

D13 + arabinose E8 + arabinose

E15 + arabinose F3 + arabinose

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

Amp

rpoE rybB

SigmaE sRNA

Kan

Ompc-yfp

Chl

SICLOPPS

Plasmid 1 Reporter

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

Observation under Microscope

- .0002% arabinose + .0002% arabinose(Longer and Fatter)

Problem with growth at 37 degrees Celsius. Toxic intermediates are produced during formation. Fixed by growth at 30 degrees Celsius

Preparation of New Strains

• Screening Strain

16

17

18

19

• Chromosome of Screening Strain• Deletion of digestive enzymes• Deletion of ydcQ

SGWMH(Q)VS

SGWSW(Q)EP

SGWSER(Q)T

SGWAD(Q)CK

Observation under Florescence Microscope

“OFF” screening strainrpoE-rybB plasmid + ompC-yfp reporter

“ON” screening strainVector + ompC-yfp reporter

“ON” screening strain 17 + .0002% arabinose“OFF” screening strain

F3 + arabinose“OFF” strain

E15 - arabinose

E15 + arabinose

832 - arabinose

832 + arabinose

Conclusion

• 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

ompc-ypfrybBsigmaE

??