Two - Component Signal Transductionmodular stimulas-response systems

Response Regulator: conserved receiver domain + specific effecter domain

Histidine Kinase Sensor: conserved kinase core (transmitter domain) + specific sensory domain

Phospo - transfer Reactions

T

The -phosphoryl group is transferred to the conserved histidine side chain of the HK. The RR catalyzes the transfer of the phosphoryl group from the phospho-His

residue to the conserved aspartic acid side chain of the RR. Finally the phosphoryl group is transferred from

the phospho-Asp residue to water in a hydrolysis reaction

genomic distribution

E. coli: 30 HKs (5 hybrids) and 32 RRs

Synechocystis sp: 80 Mycoplasma sp: 0 Bacillus subtilis: 70 Haemophilus influenza: 9 Helicobacter pylori: 11

Histidine Kinase

Most are periplasmic membrane receptors.

Function as homodimers: autophosphorylation is a bimolecular event.

Periplasmic, N-terminal binding domain.

Transmembrane domain. Linker domain. Histidine-containing phosphotransfer domain

C-terminal kinase core.

CheA & NtrB are soluble, cytoplasmic HKs

Histidine Kinase

Kinase Catalytic Core:~ 350 amino acids in length

dimerization domainATP/ADP-binding and

phosphotransfer domainphosphatase activity found in some

His

P

N-G1-F-G2

Histidine Kinase

Histidine-containing phosphotransfer domain

~ 120 amino acids in lengthHistidine residue

No kinase or phosphatase activity

His

P

Histidine Kinase

Sensing domain:N-terminal domain that senses external stimuli Usually periplasmic receptor - not alwaysIn many cases the ligand or stimulas is unknownLittle or no sequence similarity.

Transmembrane and Linker Domains:Poorly understoodCritical for propagation of signal from periplasmic binding domain to kinase core

Response Regulator

N-terminal Receiver or Regulatory domain

C-terminal Effector domain: DNA-binding transcriptional regulator

enzymatic activity (CheB or RegA)

protein-protein interactions

Catalyze the transfer of phosphryl group from phospho-HK to conserved aspartic acid: phosphorylation results in conformational change of response regulator.

Many also catalyze auto-dephosphorylation.

Asp

P

Modular Organization of tcsE. coli

osmoregulation

E. coli

Anoxic Redox

Regulation

E. coli

chemotaxis

B. subtilis

sporulation

His

P

Asp

P

N-G1-F-G2

His

P

N-G1-F-G2

His

P

N-G1-F-G2

Asp

P

Asp

P

His

P

His

P Asp

P

N-G1-F-G2

Asp

P

EnvZ OmpR

His

P

Asp

P

His

P

N-G1-F-G2 Asp

P

ArcB ArcA

SpoOASpoOB

KinA

KinB

SpoOF

CheA

CheY

CheB

Phosphotransfer Systems: His --> Asp

Phosphorelay Systems: His --> Asp --> His --> Asp

Added complexity provides for multiple regulatory checkpoints and

points of integration between signaling pathways

Modular Organization of tcs

Regulatory MechanismsThe whole point of signal transduction is regulation. The signaling pathway provides steps at which the flow

of information can be modulated.

Regulation of the Histidine Kinase:Autokinase activity either stimulated or repressed by specific stimulas. RR phosphatase activity of the histidine kinase can be modulated.

Regulation of the Response Regulator:Phosphorylation by cognate HKDephosphorylation by specific phosphatasesStimulation of intrinsic autophosphatase activity.

Inhibition of phosphotransfer

Regulation of the expression of the two-component proteins.

Integration of Signals i

Five related HKs are capable of phosphorylating the RR SpoOF (KinA, KinB, KinC, KinD and KinE).

KinA, KinB, KinC, KinD and KinE share sequence similarities surrounding the phosphorylatable histidine residue but differ in their sensing domains.

RapE is expressed during vegetative growth.

RapA and RapB are induced by the ComA/ComP TCS

Therefore sporulation is prevented during vegetative growth and competence development

Integration of Signals II

ResD/ResE regulates expression of genes required for anaerobic respiration.

PhoP/PhoR regulates expression of genes required for phosphate uptake.

When phosphate is low, phosphorylated PhoP induces expression of res operon while repressing the PhoP-independent promoter.

Phosphorylated ResD activates phoP-phoR expression (positive feedback loop)

Integration of Signals III

The product of the udg gene is required for both the Pmr-regulated modification of LPS and the Rcs-dependent production of capsule.

Both PmrA and RcsB can bind and activate transcription from the ugd promoter.

PmrD activates PmrA post-transcriptionally independently of PmrB in response to Mg++.

The ugd gene is expressed in response to Mg++, Fe+++ OR cell envelope stress.

1) High osmotic pressure changes the conformation of the outer segment of EnvZ sensor protein.

2) The change is transmitted inwards and EnvZ phosphorylates itself using ATP. It then transfers the phosphate group to OmpR. The OmpR-P form binds DNA.

When OP is low, there is only a trace of OmpR-P, but this is sufficient to bind to the high affinity site in front of the ompF gene and activate transcription.

At high OP, the concentration of OmpR-P rises and it can now occupy the low affinity sites. This stops transcription of the ompF gene and activates transcription of the ompC gene.

In addition the micF gene is transcribed to give MicF RNA. This binds to the front of the ompF message and prevents translation. Thus whenever expression of ompC is increased, expression of ompF is decreased. (Actually micF is more probably important for temperature control than for osmoregulation.)

CheA is HK that phosphorylates RRs CheY and CheB.

Phosphorylation of CheA stimulated by unoccupied receptors (requires CheW).

Phosphorylated CheY binds the flagellar motor and stimulates CW rotation of the motor which results in enhanced tumbling.

CheZ is a phosphatase that dephosphorylates CheY

Upon phosphorylation by CheA, CheB removes methyl groups from MCP resulting sensory adaptation.

Ligand bound MCP undergoes conformational change that inhibits autophosphorylation of CheA…….

The discovery of c-diGMP dates back to work published by Moshe Benziman on the regulation of cellulose

biosynthesis in Gluconacetobacter xylinum (formerly called Acetobacter xylinum) and Agrobacterium

tumefaciens.

In two landmark papers, published in 1987 and 1998, Benziman and colleagues first described the

identification of c-diGMP as an allosteric regulator of cellulose synthase (CS)

CS activity is almost completely dependent on the presence of c-diGMP

Cyclic-di-gmp-mediated regulation in bacteria

2 GTP c-di-GMP 2 GMP

2 PPi

diguanylate cyclase

diguanylatephosphodiestera

se

GGDEF EAL

Cyclic di-GMP as a Bacterial 2nd Messenger

2 GTP c-di-GMP 2 GMP

2 PPi

diguanylate cyclase

diguanylatephosphodiestera

se

GGDEF EAL

Activity of Effector Protein

EAL

Adenylate

Cyclase Toxin

Pertussis

Toxin

Pertactin

Fimbrea

FHA

BrkA

Tracheal Colonization

Factor

Ptl

TrachealCytotoxic

Toxin

Vrg6 Vrg73

Vrg18

BvgASBvg+ Bvg-

25oC

Nicotinic Acid

MgSO4

bvgAS

D

D

CM

OM

BvgS

BvgA

KIN

H

HTH

H

DBP

PBD

D

H H

CM

OM

ATP

ADP

ATP

ADP

D

KIN

H

H

KIN

D

H

H~P

BvgS

~ P

37oC

DBvgA

HTH HTH

D

D

H H

CM

OM

ATP

ADP

ATP

ADP

D

KIN

H

H

KIN

D

H

H

BvgS ~ P ~P

37oC

DBvgA

HTH HTH

D

D

H H

CM

OM

ATP

ADP

ATP

ADP

D

KIN

H

H

KIN

D

H

H

DBvgA

HTH HTH

D

BvgS

~ P ~P

37oC

D

H H

CM

OM

ATP

ADP

ATP

ADP

D

KIN

H

H

KIN

D

H

H

BvgA

BvgS

D

HTH HTH

D

~

P

~

P

Virulence genes

37oC

D

H H

CM

OM

D

KIN

H

H

KIN

D

H

H

BvgA

BvgS

D

HTH HTH

D

Virulence genes

25°C or 37°C + SO or Niacin

BvgRPvrg6

vrg18

vrg24

vrg53

vrg73

P

P

P

P

bvgA bvgS bvgRAUG AUG GUA

BvgA BvgSPP

vrg

BvgS

BvgA

RisS

RisA

BvgR

MgSO 4

Nicotinic Acid Temperature -?

+

+

Cyclic di-GMP as a Bacterial 2nd Messenger

Activity of Effector Protein

BvgR

EAL

2 GTP c-di-GMP 2 GMP

2 PPi

diguanylate cyclase

diguanylatephosphodiestera

se

GGDEF EALEAL

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BA 5543QuickTime™ and a

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BA 2533

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BA 4263

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BA 0548

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BA 5593

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BA 5664

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BA 3879

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BA 4203

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EAL

EAL

EAL

EAL

EAL

EAL

EAL

GGDEF/EAL proteins in Bacillus anthracis

Virulence of GGDEF/EAL Mutants

gevA = GGDEF/EAL virulence regulator A

Growth of gevA Mutant

GGDEF EALPASTM

GevA

PAS domains act as sensory modules for oxygen tension, redox potential or light intensities.

The domain functions through protein-protein interactions or through binding cofactors within

their hydrophobic cores to regulate protein-protein

interactions in response to stimuli.

GGDEF EALPASTM

GevA

AAAAA AAL