Continuous control in a simple bacterial

1
Continuous control in a simple bacterial signaling
circuit
Eric Batchelor and Mark Goulian University of
Pennsylvania
2
Cell Signaling
Detection, Processing
Signals ions, small molecules, peptides,
carbohydrates, proteins, light, temperature,
electrical potential, mechanical stress, ...
Responses metabolism, cell cycle, development,
cell death, adhesion, taxis, chaperones,
virulence, ...
D. Hanahan and R.A. Weinberg, Cell 2000 10057
3
Porins in E. coli
OmpC OmpF
periplasm
outer membrane
inner membrane
cytoplasm
Permeability OmpF gt OmpC differential
permeability for antibiotics, bile salts,
4
Porin expression is regulated by extracellular
osmolarity
Low osmolarity
High osmolarity
OmpF
OmpC
OmpC
OmpF
5
Extracellular Osmolarity
EnvZ
EnvZ
EnvZ
P
ATP
ADP
control of OmpC/OmpF expression
6
Extracellular Osmolarity
EnvZ
EnvZ
EnvZ
P
ATP
ADP
control of OmpC/OmpF expression
7
EnvZP OmpR
(EnvZP)OmpR
ADP
ATP
EnvZ OmpR
Why not do this?
8
k1
EnvZP OmpR
(EnvZP)OmpR
kt
k-1
ADP
k-k
kk
k-2
k2
ATP
kp
EnvZ OmpR
EnvZ(OmpRP)
EnvZ 100 proteins/cell OmpR 3500 proteins/cell
Cai and Inouye, 2002
9
k1
EnvZP OmpR
(EnvZP)OmpR
kt
k-1
ADP
k-k
kk
k-2
k2
ATP
kp
EnvZ OmpR
EnvZ(OmpRP)
Steady State
Cp kk(kpk-2)/k2 Ct k-k(ktk-1)/k1
OmpRtotal gtgt EnvZtotal
OmpRtotal gtgt EnvZtotal , Cp, Ct OmpRP
f(Cp,Ct, OmpRtotal) OmpRP Cp
Output is insensitive to variations in
EnvZtotal , OmpRtotal , ... (i.e. output
is robust)
10
Chromosomal Insertions (operon fusions)
11
515 nm
YFP
525 nm
12
YFP (OmpF)
CFP (OmpC)
0 sucrose
15 sucrose
13
low osm.
(CFP/YFP)/(0 WT CFP/YFP)
high osm.
Log10( EnvZ/(WT EnvZ) )
EnvZ
Circuit Output as a function of EnvZ
14
(No Transcript)
15
CFP/YFP (OmpC/OmpF)
sucrose
16
low signal
medium signal
high signal
discrete (all or none)
continuous (graded)
17
low signal

• Systems Exhibiting
• all-or-none behavior
• Lactose operon in E. coli (Novick and Weiner
  1957)
• IL-2 activation in T-cells (Fiering et al 1990,
  Karttunen and Shastri 1991)
• Glucocorticoid inducible promoters (M.S.H. Ko et
  al 1990)
• Metal responsive promoters (Walters et al 1996)
• Arabinose inducible genes in E. coli (Siegele and
  Hu 1997)
• Xenopus oocyte maturation (Ferrell and Machelder
  1998)
• Jnk signaling in (Bagowski and Ferrell 2001,
  2003)

medium signal
high signal
of cells
response
Are there examples of continuous control of
transcription?
18
(No Transcript)
19
What sets the output level of the EnvZ/OmpR
circuit?
rheostat vs. on-off switch
20
An ompC deletion (in frame) does not affect
ompC/ompF transcription or OmpF expression.
21
OmpC over-expression has little effect on
ompC/ompF transcription.
22
The expression level of OmpC does not feed back
into the osmo-regulatory circuit. ?
open-loop continuous control
Extracellular Osmolarity
EnvZ
ompF
ompC
23
Why would cells possess circuits with continuous
control that lack feedback from the output back
into the circuit?
Hypothesis The biologically relevant
stimulus for a signaling circuit with graded
control is modulated by the circuit output.
24
What is the stimulus for EnvZ?
?
?
EnvZ
ompF
ompC
?
25
OmpR
osmolarity
EnvZ
26
OmpR
NarL
RcsB
osmolarity
EnvZ
NarX
NO3-, NO2-
YojN
PhoB
NarP
NarQ
PO42-
PhoR
YedW
YedV
RstA
DcuR
C4 dicarboxylate
RstB
DcuS
YgiX
YgiY
BaeR
BaeS
ArcA
O2 (quinones)
YfhA
YfhK
CreB
CreC
KdpE
osmolarity, turgor?
KdpD
YehT
YehU
HydG
HydH
PhoP
Mg2,Ca2
PhoQ
YpdA
YpdB
UvrY
unfolded periplasmic proteins
CpxR
???
RssB
CpxA
EvgA
CusR
FimZ
???
CusS
Cu2
DpiA
DpiB
UhpC
glucose6P
UhpB
GlnG
AtoC
NH4
GlnL
AtoS
TorR
(CH3)3NO
BasR
BasS
CheB
31 Homologous circuits in E. coli K-12
D,S,O2,...
CheA
CheY
27
RcsB
OmpR
NarL
osmolarity
EnvZ
NarX
NO3-, NO2-
YojN
PhoB
NarP
NarQ
PhoR
PO42-
YedW
YedV
RstA
RstB
DcuR
C4 dicarboxylate
DcuS
YgiX
YgiY
BaeR
BaeS
ArcA
O2 (quinones)
YfhA
YfhK
CreB
CreC
KdpE
YehT
osmolarity, turgor?
KdpD
YehU
HydG
HydH
PhoP
YpdA
YpdB
PhoQ
Mg2,Ca2
UvrY
???
RssB
unfolded periplasmic proteins
CpxR
CpxA
EvgA
FimZ
???
CusR
CusS
Cu2
DpiA
DpiB
UhpC
UhpB
GlnG
NH4
glucose6P
AtoC
GlnL
AtoS
TorR
(CH3)3NO
BasR
BasS
31 Homologous circuits in E. coli K-12
CheB
D,S,O2,...
CheA
CheY

• Current and Future Directions
• EnvZ stimulus, localization
• Positive Feedback and Phosphorelays (signaling
  cascades)
• Insulation and connectivity (interference vs.
  cross regulation)
• Plasticity (reengineering circuits)

28

Lab Members Eric Batchelor Paige Derr Justin
Kanoff Kizhevur Kandaswamy Sangmin Lee Tim
Miyashiro
Princeton Tom Silhavy Rockefeller Sandy Simon
(and the Simon lab) Penn Andy Binns (and the
Binns lab ) Dieter Schifferli Marjan van der
Woude Penn Biocomp Group Physics Department
Institute for Medicine and Engineering