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For our secondary screen we tested selected mutants’ motility on soft agar motility plates.
Characterization of the Role of plzC in Motility Regulation of Vibrio cholerae
PlzC is a c-di-GMP receptor that is important for controlling motility, biofilm, and virulence in Vibrio cholerae. These type of receptors convey information through protein-protein interactions. In this study we designed a forward genetics approach to identify repressors of motility that might interact with PlzC.
Abstract
Introduc6on
The objective of this study was to determine proteins that may be interacting with PlzC and affecting motility.
Objec6ve
We used transposon
mutagenesis to introduce random
null mutations into the ΔplzC
genetic background.
Results Perspec6ve
References
C-di-GMP is an important secondary intracellular signaling molecule for Vibrio cholerae, and many other bacteria. It is synthesized by digaunylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs). A high internal concentration of c-di-GMP promotes the formation of biofilms. A low internal concentration of c-di-GMP promotes motility.
University of California – Santa Cruz Livia Timpanaro-‐PerroJa, Mauro Salinas, David Zamorano-‐Sánchez, Fitnat Yildiz
Methods
Primary Investigator: Fitnat Yildiz, Ph.D. Post-doctoral Researchers: David Zamorano-Sánchez, Ph.D., Namrata Rao, Ph.D. Research Technicians: Mauro Salinas
We then performed a series of motility screens to determine secondary mutations that restored motility to a wild-type (WT) phenotype. We used Arbitrary PCR and DNA Sequencing to determine where the transposon had inserted.
ΔRR Screened: 4,076 Selected: 235 Sequenced: 33
Selected for Clean Deletions: 9
• Do Clean Deletions to recapitulate the motility phenotype • Do Epistatic Analysis • Do Bacterial Two Hybrid Assay to show protein-protein
interactions • Evaluate the role of c-di-GMP in the interactions
Acknowledgements
1.73
1.6 1.63 1.63
1.43
2.17 2.13
2.3
2.1
1.63
2.25 2.25
2.07
1.68 1.6 1.6
2.35
2.47 2.37
2.16 2.17
2.33
2.53 2.43
1.78
2.23
1.53
2.18
1.6
2.52
2.35
2.63
2.3
1.57
1.759
0
0.5
1
1.5
2
2.5
3
Colony M
easuremen
ts (cm)
Mutant
ΔplzC Mo6lity Screen
PlzC
?
An in-frame deletion of the c-di-GMP receptor plzC causes a decrease in motility of Vibrio cholerae in soft agar plates. This would suggest that PlzC is inhibiting a repressor of motility. Since c-di-GMP has been shown to inhibit motility we speculate that in the presence of this second messenger PlzC wont be able to repress the hypothetical motility repressor.
Our primary screen was done in a 96-well format stamping cells grown on selective liquid media over soft agar plates.
Examples of a non-selected and selected mutants
Controls: ΔplzC SWT
We did arbitrary PCR to determine the gene responsible for the suppressor mutation.
1.PraJ, J. T., R. Tamayo, A. D. Tischler, and A. Camilli. "PilZ Domain Proteins Bind Cyclic Diguanylate and Regulate Diverse Processes in Vibrio Cholerae." Journal of Biological Chemistry 282.17 (2007): 12860-‐2870. Print. 2. Sondermann, Holger; Shikuma, Nicholas J.; Yildiz, Fitnat H. , “You’ve come a long way: c-‐di-‐GMP signaling.” Current Opinion in Microbiology (2012): 14-‐146. 3. Ko, Junsang, Kyoung-‐Seok Ryu, Henna Kim, Jae-‐Sun Shin, Jie-‐Oh Lee, Chaejoon Cheong, and Byong-‐Seok Choi. "Structure of PP4397 Reveals the Molecular Basis for Different C-‐di-‐GMP Binding Modes by Pilz Domain Proteins." Journal of Molecular Biology: 97-‐110. Print. 4. Liu, X., Beyhan, S., Lim, B., Linington, R. G., & Yildiz, F. H. (2010). Iden.fica.on and Characteriza.on of a Phosphodiesterase That Inversely Regulates Mo.lity and Biofilm Forma.on in Vibrio cholerae . Journal of Bacteriology, 192(18), 4541–4552. doi:10.1128/JB.00209-‐10
Wt ΔplzC
Wt ΔplzC