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Two - Component Signal Transduction
modular 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
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E. coli:
Synechocystis sp:
Mycoplasma sp:
Bacillus subtilis:
Haemophilus influenza:
Helicobacter pylori:
30 HKs (5 hybrids) and 32 RRs
80
0
70
9
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
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Histidine Kinase
P
His
N-G1-F-G2
Kinase Catalytic Core:
~ 350 amino acids in length
dimerization domain
ATP/ADP-binding and phosphotransfer domain
phosphatase activity found in some
Histidine Kinase
P
His
Histidine-containing phosphotransfer domain
~ 120 amino acids in length
Histidine residue
No kinase or phosphatase activity
Histidine Kinase

Sensing domain:
N-terminal domain that senses external stimuli
Usually periplasmic receptor - not always
In many cases the ligand or stimulas is unknown
Little or no sequence similarity.

Transmembrane and Linker Domains:
Poorly understood
Critical for propagation of signal from
periplasmic binding domain to kinase core
Response Regulator
P

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
E. coli
osmoregulation
Modular Organization of tcs
P
P
His
Asp
N-G1-F-G2
OmpR
E. coli
Anoxic Redox
Regulation
EnvZ
P
His
N-G1-F-G2
P
P
P
Asp
His
Asp
ArcB
ArcA
P
E. coli
chemotaxis
His
N-G1-F-G2
KinA
P
His
N-G1-F-G2
P
P
P
Asp
His
Asp
SpoOF
SpoOB
SpoOA
KinB
B. subtilis
sporulation
P
Asp
P
His
N-G1-F-G2
CheA
CheY
P
Asp
CheB
Modular Organization of tcs

Phosphotransfer Systems: His --> Asp

Phosphorelay Systems:
His --> Asp --> His --> Asp
Added complexity provides for multiple regulatory
checkpoints and points of integration between
signaling pathways
Regulatory Mechanisms
The 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 HK
Dephosphorylation by specific phosphatases
Stimulation of intrinsic autophosphatase activity.

Inhibition of phosphotransfer
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Regulation of the expression of the two-component proteins.
Integration of Signals i
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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
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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
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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.)
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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…….
Cyclic-di-gmp-mediated regulation in
bacteria
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
diguanylate
cyclase
diguanylate
phosphodiesterase
2 GMP
c-di-GMP
2 GTP
2 PPi
GGDEF
EAL
Cyclic di-GMP as a Bacterial 2nd Messenger
diguanylate
cyclase
diguanylate
phosphodiesterase
2 GMP
c-di-GMP
2 GTP
2 PPi
GGDEF
EAL
Activity of Effector Protein
BrkA
Fimbrea
FHA
Tracheal
Colonization
Factor
Pertactin
Ptl
Adenylate
Cyclase
Toxin
Pertussis
Toxin
Tracheal
Cytotoxic
Toxin
Vrg18
Vrg6
Bvg+
BvgAS
Bvg-
25oC
Nicotinic Acid
MgSO4
DbvgAS
Vrg73
OM
D P
B
B
P
D
CM
K
I
N
BvgS
H
D
H
BvgA
D
HTH
37oC
OM
CM
ATP
ADP
BvgS
BvgA
K
K
I P~H
H H~ P I
N
N
DD
D
H
H
D
D
HTH
HTH
ATP
ADP
37oC
OM
CM
ATP
ADP
BvgS
K
I
N
K
I
N
H H
H
DD~ P
P~ D
H
H
BvgA
D
D
HTH
HTH
ATP
ADP
37oC
OM
CM
ATP
ADP
BvgS
K
I
N
K
I
N
H H
H
DD
D
H~P
BvgA
P~ H
D
D
HTH
HTH
ATP
ADP
37oC
OM
CM
ADP
BvgS
K
I
N
H H
H
D
H
H
~
P
BvgA
K
I
N
DD
D
ATP
ADP
P
~
ATP
D
HTH HTH
Virulence genes
25°C or 37°C + SO or Niacin
OM
CM
K
I
N
BvgS
BvgA
K
I
N
H H
H
DD
D
H
H
D
D
HTH
HTH
Virulence genes
bvgA
AUG
BvgA
Pvrg6
Pvrg18
Pvrg24
Pvrg53
Pvrg73
bvgS
AUG
P
bvgR
GUA
P
BvgS
BvgR
MgSO4
Nicotinic Acid
Temperature
?
RisS
BvgS
RisA
BvgA
+
BvgR
+
vrg
Cyclic di-GMP as a Bacterial 2nd Messenger
diguanylate
cyclase
diguanylate
phosphodiesterase
2 GMP
c-di-GMP
2 GTP
2 PPi
EAL
GGDEF
Activity of Effector Protein
BvgR
EAL
GGDEF/EAL proteins in Bacillus anthracis
BA 0548
EAL
BA 0628
EAL
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BA 2533
BA 3879
EAL
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EAL
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decompressor
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BA 4203
EAL
QuickTime™ and a
decompressor
are needed to see this picture.
BA 4263
BA 5543
BA 5593
BA 5664
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decompressor
areEAL
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decompressor
are needed
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Virulence of GGDEF/EAL Mutants
gevA = GGDEF/EAL virulence regulator A
Growth of gevA Mutant
GevA
TM
PAS
GGDEF
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.
EAL
GevA
TM
PAS
AAAAA
AAL
GGDEF
EAL