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Dynamic Range and Specificity in Bacterial Two-Component Signaling. Mark Goulian University of Pennsylvania. RssB. FimZ. RstB. KdpD. UhpB. CusS. PhoQ. TorS. DpiA. ArcB. NarQ. BasS. PhoR. DcuS. CpxA. YedV. EnvZ. QseC. BarA. YehU. YfhK. HydD. YpdA. NarX. RcsC. RcsD.

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Dynamic Range and Specificity in Bacterial Two-Component Signaling


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slide1

Dynamic Range and Specificity in Bacterial

Two-Component Signaling

Mark Goulian

University of Pennsylvania

slide2

RssB

FimZ

RstB

KdpD

UhpB

CusS

PhoQ

TorS

DpiA

ArcB

NarQ

BasS

PhoR

DcuS

CpxA

YedV

EnvZ

QseC

BarA

YehU

YfhK

HydD

YpdA

NarX

RcsC

RcsD

NtrB

TorR

BasR

RcsB

KdpE

DpiB

NarL

CheB

CheY

RstA

PhoB

PhoP

DcuR

CreB

CusR

UhpC

NarP

YedW

YfJR

UvrY

YehT

YpdB

QseB

OmpR

CpxR

HydG

ArcA

NtrC

CheA

CreC

BaeR

AtoC

BaeS

AtoS

envelope

stress

PO42-

osmolarity

Mg2+

Cu2+

TMAO

glucose 6P

NO3-,NO2-

quin-ones

K+

~30 two-component signaling systems

in E. coli K-12

EvgA

EvgS

Zn2+

Fe3+

slide3

RssB

FimZ

RstB

UhpB

KdpD

CusS

PhoQ

TorS

DpiA

NarQ

ArcB

DcuS

BasS

PhoR

CpxA

QseC

YedV

BarA

HydD

YehU

YfhK

YpdA

EnvZ

NarX

RcsC

RcsD

NtrB

TorR

BasR

RcsB

KdpE

CheY

CheB

DpiB

NarL

RstA

DcuR

PhoP

CreB

PhoB

CusR

UhpC

NarP

YehT

CpxR

OmpR

HydG

YfJR

UvrY

ArcA

YedW

QseB

YpdB

NtrC

P

P

CheA

CreC

BaeR

AtoC

BaeS

AtoS

envelope

stress

PO42-

osmolarity

Mg2+

Cu2+

TMAO

glucose 6P

NO3-,NO2-

quin-ones

K+

~30 two-component signaling systems

in E. coli K-12

HK

HK

HK

EvgA

RR

RR

EvgS

Zn2+

Fe3+

histidine kinase:

response regulator:

slide4

PhoQ

CpxA

EnvZ

PhoP

OmpR

CpxR

cell envelope

stress

osmolarity?,

procaine

low Mg2+, antimicrobial peptides

outer membrane

permeability (porins), ...

secreted chaperones,

proteases ..

magnesium transporter,

cell envelope modifications ...

slide5

PhoQ

CpxA

EnvZ

PhoP

OmpR

CpxR

P

P

cell envelope

stress

osmolarity?,

procaine

low Mg2+, antimicrobial peptides

outer membrane

permeability (porins), ...

secreted chaperones,

proteases ..

magnesium transporter,

cell envelope modifications ...

Many histidine kinases are bifunctional – they phosphorylate and dephosphorylate

their cognate response regulator

HK

HK

HK

RR

RR

  • The phosphorylation cycle leads to:
  • Insensitivity to changes in levels of the HK and RR
  • Dynamic range modulation through autoregulation
  • Cross-talk suppression
slide6

RssB

FimZ

RstB

KdpD

UhpB

CusS

TorS

PhoQ

DpiA

ArcB

NarQ

DcuS

BasS

PhoR

CpxA

QseC

YedV

EnvZ

YfhK

YehU

YpdA

HydD

BarA

NarX

RcsC

RcsD

NtrB

TorR

BasR

RcsB

KdpE

CheY

NarL

CheB

DpiB

RstA

DcuR

CreB

PhoP

PhoB

CusR

UhpC

NarP

YpdB

ArcA

YedW

CpxR

OmpR

QseB

YfJR

YehT

UvrY

HydG

NtrC

CheA

CreC

BaeR

AtoC

BaeS

AtoS

RR

HK

Some two-component systems

are auto-regulated

EvgA

EvgS

Why autoregulate?

~30 two-component systems in E. coli

Tim Miyashiro

slide7

PhoQ

PhoP

low Mg2+, antimicrobial peptides

phoP

phoQ

slide8

cfp

Reference promoter

PhoQ/PhoP regulated

promoter

yfp

P

PhoP

slide9

cfp

Reference promoter

PhoQ/PhoP regulated

promoter

yfp

Average YFP/CFP

Mg++ (mM)

P

PhoP

YFP:

The output is graded,

not switch-like

CFP:

30mM Mg++

100 μM Mg++

slide10

phoP

phoQ

slide11

PhoQ

PhoP

PhoP

PhoP

P

P

low Mg2+

PhoQ

PhoQ

phoP

phoQ

What happens to the level of PhoP-P

when you increase expression of PhoP and PhoQ?

slide12

HK

HK

HK

RR

P

P

kk

RR

KMp, kcat p

Output is insensitive (robust)

to variations in total [HK]

and total [RR}.

Observed experimentally for

EnvZ/OmpR and several

other systems

( kk /kcat p ) KM p

steady-state [RR-P]

total [RR]

slide13

phoP

phoQ

phoP

phoQT281R

PhoP-regulated transcription

YFP/CFP

YFP/CFP

PhoP / PhoPwild type

PhoP / PhoPwild type

inducible

promoter:

(phosphatase- mutant)

slide14

autoregulation

High signal:

 autoregulation boosts

the output

[RR-P]

For low to intermediate signal the system operates in saturation

 autoregulation has little effect on the output.

[RR]total

slide15

+ autoregulation:

phoP

phoQ

phoP

phoQ

- autoregulation:

PhoP-P regulated transcription (mgrB transcription)

+ autoregulation

- autoregulation

 Autoregulation has no effect on steady-state behavior down to 10 M Mg++

slide16

Very high stimulus (growth-limiting) Mg++

mgrB transcription

+ autoregulation

- autoregulation

Autoregulation increases the dynamic range

slide17

PhoQ

PhoP

PhoP

PhoP

P

P

low Mg2+

PhoQ

PhoQ

What happens when we get rid of the phosphatase activity?

phoP

phoQ

slide18

cfp

Reference promoter

PhoQ/PhoP regulated

promoter (mgrB, … )

yfp

Wild-type PhoQ

PhoQT281R (phosphatase-)

Mixed Population

slide19

PhoQT281R (phosphatase-)

Phase

CFP

YFP

“Phenotypic Switching”

slide20

PhoQ

PhoP

PhoP

PhoP

P

P

low Mg2+

PhoQ

PhoQ

phoP

phoQ

For a simple two-component systems containing a bifunctional histidine kinase,

positive autoregulation boosts the response range at high stimulus without

affecting the response to low and intermediate stimulus.

 provides a stable form of positive feedback that prevents the steady-state output from rolling off at high stimulus.

slide21

RssB

FimZ

RstB

UhpB

KdpD

CusS

TorS

PhoQ

DpiA

ArcB

NarQ

DcuS

BasS

PhoR

CpxA

BarA

QseC

YedV

YehU

YfhK

YpdA

EnvZ

HydD

NarX

RcsC

RcsD

NtrB

TorR

BasR

RcsB

KdpE

DpiB

NarL

CheY

CheB

RstA

PhoP

PhoB

CreB

DcuR

CusR

UhpC

NarP

CpxR

OmpR

QseB

HydG

YedW

UvrY

YfJR

YehT

YpdB

ArcA

NtrC

CheA

CreC

BaeR

AtoC

BaeS

AtoS

envelope

stress

PO42-

osmolarity

Mg2+

Cu2+

TMAO

glucose 6P

NO3-,NO2-

quin-ones

K+

EvgA

EvgS

Zn2+

Fe3+

slide22

RssB

FimZ

RstB

CusS

UhpB

KdpD

PhoQ

TorS

DpiA

NarQ

ArcB

PhoR

BasS

DcuS

CpxA

YehU

EnvZ

YfhK

YpdA

YedV

HydD

QseC

BarA

NarX

RcsC

RcsD

NtrB

TorR

RcsB

BasR

KdpE

NarL

CheB

CheY

DpiB

RstA

CreB

DcuR

PhoP

PhoB

CusR

UhpC

NarP

YpdB

YehT

YfJR

YedW

CpxR

UvrY

QseB

OmpR

ArcA

HydG

NtrC

CheA

CreC

BaeR

AtoC

BaeS

AtoS

envelope

stress

PO42-

osmolarity

Mg2+

Cu2+

TMAO

?

glucose 6P

NO3-,NO2-

quin-ones

K+

Can we evolve cross-regulation

between a histidine kinase

and a non-cognate

response regulator?

Albert Siryaporn

EvgA

EvgS

Zn2+

Fe3+

slide23

CpxA

EnvZ

CpxR

OmpR

P

P

degP

ompC

osmolarity,

procaine, ...

misfolded proteins

in the periplasm, ..

, ...

, ...

outer membrane

permeability (porins), ...

secreted chaperones,

proteases ..

HK

HK

HK

RR

RR

EnvZ and CpxA

are bifunctional

slide24

CpxA

EnvZ

CpxR

OmpR

P

P

ompC

, ...

outer membrane

permeability (porins), ...

CpxA

CpxA

CpxA

OmpR

Cross-talk can be detected in the absence of EnvZ and CpxR, but it is

blind to signal and shows no evidence of phosphatase activity.

OmpR

slide25

Helix 2

Helix 1

catalytic

Histidine Kinase HK853 fromThermotoga maritima

(Marina et al, 2005)

Catalytic

domain

histidine

Helix 2

Helix 1

DHp domain

CpxA SQQRLLSDISHELRTPLTRLQLGTALLRRRSGESKELERIETEAQRLDSMINDLLVM

EnvZ DRTLLMAGVSHDLRTPLTRIRLATEMMSEQDG--YLAESINKDIEECNAIIEQFIDY

slide26

Helix 2

Helix 1

catalytic

Histidine Kinase HK853 fromThermotoga maritima

(Marina et al, 2005)

Catalytic

domain

histidine

Helix 2

Helix 1

DHp domain

CpxA SQQRLLSDISHELRTPLTRLQLGTALLRRRSGESKELERIETEAQRLDSMINDLLVM

EnvZ DRTLLMAGVSHDLRTPLTRIRLATEMMSEQDG--YLAESINKDIEECNAIIEQFIDY

Residues important for specificity of interaction with response regulator

Skerker et al. Cell 2008. (Laub lab)

Change of specificity mutants are blind to signal and show no evidence of

phosphatase activity in vivo.

slide27

Acknowledgements

Lab members

  • Albert Siryaporn
  • David Chow
  • Melissa Lasaro
  • Elizabeth Libby
  • Andrew Lippa
  • Sri Ram
  • Manuela Roggiani
  • Manan Shah
  • Michele Kim

Michael Laub (MIT)

  • (former)
  • Eric Batchelor
  • Paige Derr
  • Tim Miyashiro
  • Justin Kanoff
  • Ben Kline
  • Sangmin Lee