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
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
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):
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