7. Signal transduction can be recognised as three steps: reception,
transduction, induction.
Reception – the binding of the signal molecule to its specific receptor.
Transduction – the second messenger is formed in or released into the
cytosol.
Induction – activation of the cellular process.
8.
9. The complexity of signalling networks in model organisms, such as
Escherichia coli and Bacillus subtilis, has long hindered their
systematic analysis.
The first description of a two-component system was quickly followed
by the discovery of crosstalk between nitrogen assimilation and
chemotaxis, suggesting complex interactions between different
regulatory systems and signal integration.
The availability of complete genomic sequences allowed the
researchers for the first time to evaluate the total number and
composition of the signal transduction proteins.
The rapid rate of genome sequencing is contributing to the progress
in comparative genome analysis.
10. It has been found, for example, that parasitic bacteria usually
encode fewer signalling proteins than free-living bacteria, even if one
takes into account their smaller genome sizes. Gram-positive
bacteria and archaea turned out to have fewer signal transduction
proteins than proteobacteria or cyanobacteria of the same genome
size.
Complete genome sequences allows increasingly accurate
reconstructions of the metabolic pathways in poorly studied
organisms and even prediction of their nutritional requirements.
In a similar fashion, one could hope that some day it would be
possible to reconstruct microbial signalling pathways and predict
responses of a given microorganism to various environmental
factors, based on its genome content.
11. Comparative studies revealed certain general trends in
the organization of diverse signalling systems:
i. Modular structure of signalling proteins.
ii. Common organization of signalling components with the flow of
information from N-terminal sensory domains to the C-terminal
transmitter or signal output domains (N-to-C flow).
iii. Ability of some organisms to respond to one environmental signal by
activating several regulatory circuits.
iv. Abundance of intracellular signalling proteins, typically consisting of
a PAS or GAF sensor domains and various output domains.
v. Importance of secondary messengers, cAMP and cyclic diguanylate.
vi. Crosstalk between components of different signalling pathways.
13. Recently Described Signaling domains
Components of the signal transduction system can be
subdivided into the following domains:
I. Sensory (ligand-binding) domains
II. Signal transduction (phosphorylation, methylation,
homodimerization) domains
III. Signal output (DNA binding) domains
14. I. Sensory Domains
Different transmembrane receptors: histidine kinases, methyl-accepting
proteins, adenylate or diguanylate cyclases and phosphodiesterases
have different sensory domains, which include:
Periplasmic or extracytoplasmic domains,
Cytoplasmically located domains,
Integral membrane domains.
These domains have well defined and narrow substrate specificity e.g
nitrate-binding NIT domain and citrate-binding CitAP domain.
15. Periplasmic Domain
Periplasmic solute-binding proteins have long been
known to function as ligand-binding domains of
sensor histidine kinases, for example, EvgS protein
from Escherichia coli.
16. Cytoplasmically located Domains
1) Pseudomonas aeruginosa AmiC:
AmiC serves as the receptor and negative regulator
for amide-inducible aliphatic amidase operon
amiEBCRS.
Together with the RNA-binding response regulator
AmiR, AmiC regulates expression of the AmiE
amidase, as well as expression of its own gene,
amiC, in response to amides.
17. 2) KdpD from E. coli:
E.coli respond to K limitation and high osmoloraty
by induction of KdpFABC operon.
This operon encode high affinity k translocating
channel.
The expression of operon is controlled by two
component system.
Contain cytoplasmically located N-terminal turgor-
sensing i.e KdpD and response regulator KdpE.
18. 3) PAS and GAF domains:
The most common cytoplasmic signalling domains
are PAS and GAF domains.
The PAS and GAF domains were shown to have
similar structures, characterized by a presence of a
ligand-binding pocket that can accommodate a
variety of small-molecule ligands, from haeme to
flavin to adenine and guanine.
19. PAS Domain
PAS domain sences signals i.e oxygen,light and
redox potential.
Responsible for transmigration of bacteria into the
microenvironment.
Presence of oxygen was shown to affect the position
of the PAS-bound haeme molecule, causing a
change in the general conformation of the PAS
domain and thereby allowing the sensing of oxygen
to effect signal transmission to the C-terminally
located signal transduction domains
20. Integral Membrane domain
1) Ethylene-binding domain(ETR1):
The first membrane domain with proven sensory
function was the ethylene-binding
domain of the Arabidopsis thaliana.
This domain was found later in cyanobacteria and
several proteobacteria.
21. 2) Proline Permease-like N-terminal domains:
Histidine kinases from P. aeruginosa (PA3271), Vibrio
cholerae (VC0303), and several other bacteria
contain proline permease-like N-terminal domains,
which led to a suggestion that these proteins might
serve as sensors of sodium-motive force.
23. II. Signal Transduction Domains
Environmental changes, sensed by the sensory
domains of transmembrane receptors then transfer
to the cytoplasmically located domains of these
receptors to trigger appropriate cellular responses.
These responses include increased gene
expression, changes in motility (chemotaxis),
changes in secretion and many other processes.
A dimerization domain, HAMP is involved in
transmission of signals from sensory to cytoplasmic
domains
24. Alone histidine kinases and methyl-accepting proteins
are not capable of sensing extracellular signals.
Fusions of periplasmic sensory domains to adenylate
cyclase, serine/threonine protein kinase, diguanylate
cyclase and phosphodiesterase domains have been
described.
1).
The fusion of adenylate cyclase domain to a periplasmic
sensor domain was originally recognized in
cyanobacteria Spirulina platense and Anabaena sp.
Experimentally verified to have adenylate cyclase
activity
25. 2) Cytoplasmic GGDEF and EAL
domains:
Bacterial membrane receptors also combine periplasmic sensory
domains with the cytoplasmic GGDEF and EAL domains.
The GGDEF domain is often paired with the EAL domain, forming a
diguanylate cyclase/phosphodiesterase combination that catalyses
synthesis and hydrolysis of cyclic diguanylate (c-diGMP).
C-diGMP regulates formation of extracellular cellulose and
extracellular polysaccharide.
Eal stimulate degredation of c-diGMP and regulating cell surface
adhesiveness in bacteria.
26. III. Output Domain
Response regulators are simpler in structure than
histidine kinases.
Two domains: a receiver domain that accepts a
phosphoryl group from a cognate HK and an
effector domain that generates the outputs of the
signaling events.
DNA-binding output domain that activates or
represses transcription of specific target genes
27. Cont..
The receiver domain is also known as the
phosphorylation domain or regulatory domain.
There is an invariable aspartate residue in the
receiver domain that accepts a phosphoryl group
from a cognate HK.
Phosphorylation results in conformational changes
in the domain, which is transmitted to the effector
domain to regulate its activities.
28. Structures of the receiver domain and
effector of response regulators
30. Intracellular signalling network
Several well-studied histidine kinases have no
transmembrane segments
For example, chemotaxis histidine kinase CheA and nitrogen
regulation protein NtrB from E. coli, sporulation kinase KinA
from B. subtilis, or rhizobial oxygen sensor FixL etc.
Free-living bacteria typically encode a significant number of
intracellular histidine kinases, adenylate cyclases,
diguanylate cyclases and phosphodiesterases.
31. Intracellular signalling network
The genome of M. loti, for example, encodes 13 copies of the
adenylate cyclase domain. Of these, only one appears to be
fused to a periplasmic sensor domain, and another one is
fused to an integral membrane sensor domain. All the rest are
found in cytoplasmic proteins.
32. Quorum sensing
The ability of bacteria to sense
and respond to environmental
stimuli such as pH,
temperature, the presence of
nutrients, etc has been long
recognized as essential for their
continued survival
It is now apparent that many
bacteria can also sense and
respond to signals expressed
by other bacteria
33. Cont…
Quorum sensing is the
regulation of gene
expression in response to
cell density and is used by
Gram positive and Gram
negative bacteria to
regulate a variety of
physiological functions
It involves the production
and detection of
extracellular signaling
molecules called
autoinducers.
34. Cont…
Bacteria utilize numerous
mechanisms to monitor and
adapt to their external
environment.
One such mechanism
involves the ability to ‘count’
their local population
numbers.
Once a specific number of
cells, or quorum, is reached,
bacteria are able to modify
their group behaviour through
a mechanism known as
35. Quorum Signalling in Streptococcus
pneumoniae
Competency in S. pneumoniae is regulated competence
stimulating peptide (CSP) quorum signal encoded by the gene
comC.
CSP is transported by ATP-dependent ComAB transporter. The
CSP then accumulates on the outside of the cell and is
detected by other cells and itself (autoinduction), via the
membrane bound histidine kinase receptor, ComD.
ComD autophosphorylates and subsequently transfers its
phosphate group to the response regulator ComE.
Phosphorylated ComE activates transcription of the alternative
sigma factor ComX, which goes on to induce expression of
multiple genes involved in DNA uptake and recombination
37. Quorum Signalling in Bacillus
subtilis
This species codes for two types of quorum-sensing
system—ComQXP and Rap-Phr
The ComQXP system is encoded by the comQXP
operon.
The ComX polypeptide is cleaved, modified, and
exported out of the cell by the ComQ protein.
Extracellular ComX is sensed by the ComP sensor
kinase.
ComP phosphorylates the response regulator ComA,
which induces the comS gene. ComS blocks proteolysis
of the transcriptional activator ComK, which upregulates
the production of many genes that stimulate competency
39. Quorum Signalling in V. fischeri
supernatants from stationary phase cultures could be
added to cells at low density and trigger light
production.
In 1981, the V. fischeri autoinducer was purified and
determined to be the AHL 3-oxo-hexanoyl-HSL
(3OC6-HSL).
Either Free-living or in a symbiotic relationship with
squid Euprymna scolopes.
40. Quorum Signalling in V. fischeri
The V. fischeri LuxI protein synthesizes 3-oxo-C6-HSL
from S-adenosylmethionine (SAM) and other carrier
protein.
3OC6-HSL freely diffuses across the membranes and
out of the cell.
A critical concentration of AI-1 stimulates AI-1 to
interact with and activate the response regulator
LuxR.
Activated LuxR promotes transcription of the luxR
gene as well as the luxICDABEG operon
42. Bacterial signal transduction in Eukaryotic
cells and vice versa
Durring Early days it was concluded that
2 Component system is restricted to Bacteria.
Serine, Threonine and Tyrosine-dependent kinase
restricted to Eukaryotes.
43. However, recent advancement in the field of microbiology
show some of the previously
Unseen signaling domain in Bacteria, plants and animals.
• The discovery of GGDEF, FAL and HisK signaling
domains mosquito “Anopheles gambiae” shows that
some of the signaling domains have their roots in
bacteria now transfered due to bacterial contamination.
44. Concluded Remarks
Besides so much advancement we are still far from
understanding many key aspects of bacterial signal
transduction Such as,
The effect of particular parameter on sensor domain such
as,
Osmolarity on intracellular K+ concentration
Ionic strength in the cytoplasm,
Effects of changes in the external pH values or
temperature.
45. Cont….
A list of signalling domains is probably far from
complete and new domains of poorly defined function are
still being described such as,
The hydrolase of HD superfamily (COG1639) works as
the output domain of the P. aeruginosa response regulator
PA0267 and several related proteins but for this protein
the substrate is unknown.
46. Cont…
There are many domains that are likely involved in signalling but
whose functions are still unknown the best example is,
The tryptophan-rich sensory protein TspO/CrtK/MBR, an integral
membrane protein found in representatives of all domains of life,
from archaea to human (PF03073, COG3476).
The possible function of this is to regulate photosynthesis gene
expression in
Rhodobacter sphaeroides, nutrient stress in Sinorhizobium
meliloti however, the presence of this domains in cells of B.
subtilis or Archaeoglobus fulgidus, which do not carry out
photosynthesis are remain a question
47. Future prospects
What are the exact biochemical activities of the GGDEF
and EAL domains?
What are the principal targets and mechanisms of the c-
diGMP action?
What is the function of the C-terminal CheY domains in
hybrid histidine kinases?
48. Cont..
Are the effects of tandem sensory domains (e.g.
PAS, GAF) in a single protein additive or
hierarchical (or both)?
What is the extent of crosstalk between different
histidine kinases and their response regulators in
vivo?
Is there an order or hierarchy in signal
transduction from different membrane receptors
sharing the same sensory domain?
