livia's undergraduate research symposium poster 2015

1
RESEARCH POSTER PRESENTATION DESIGN © 2011 www.PosterPresentations.com 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 TimpanaroPerroJa, Mauro Salinas, David ZamoranoSánchez, 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 Measurements (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): 128602870. Print. 2. Sondermann, Holger; Shikuma, Nicholas J.; Yildiz, Fitnat H. , “You’ve come a long way: cdiGMP signaling.” Current Opinion in Microbiology (2012): 14146. 3. Ko, Junsang, KyoungSeok Ryu, Henna Kim, JaeSun Shin, JieOh Lee, Chaejoon Cheong, and ByongSeok Choi. "Structure of PP4397 Reveals the Molecular Basis for Different CdiGMP Binding Modes by Pilz Domain Proteins." Journal of Molecular Biology: 97110. 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.0020910 Wt ΔplzC Wt ΔplzC

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Page 1: Livia's Undergraduate Research Symposium Poster 2015

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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