A systems biology approach for
studying complex chemotaxis pathways
Dr. Steven Porter
2
Systems biology of chemotaxis pathways
Systems Biology: The global investigation of how complex behaviour emerges
from the sum of the interactions of the components of a biological system
The aim of applying Systems Biology analysis to chemotaxis pathways is to
generate mathematical models of the signalling pathway that can accurately
predict the response of the system to stimuli.
These models require detailed knowledge of the operation of the system and its
governing parameters. Need to know:
• What are the inputs and the outputs of the system?
• Which proteins are involved in the signalling pathway and how do they
interact with one another?
• Reaction kinetics of the signalling reactions
• Cellular localization and protein copy number
Basic chemotaxis signalling pathway
•One of the best understood
signalling pathways.
•Detects changes in attractant/
repellent concentration.
•Controls swimming direction.
•CheA is the HPK; CheY and
CheB are the RRs.
Rhodobacter sphaeroides chemotaxis pathway
R. sphaeroides had 4 CheA homologues and 6 CheY homologues but no
homologues of known CheY-P phosphatases.
Like R. sphaeroides, over 45% of sequenced motile bacteria have multiple CheA
homologues.
Rhodobacter sphaeroides chemotaxis signalling
CheA1
CheY1
CheY2
CheA2
CheY3
Genome sequence
indicates 3 HPKs (CheAs)
and 6 RRs (CheYs).
CheA3A4
CheY4
CheY5
CheY6
Porter et al., 2002 (Mol. Microbiol.) & 2004 (JBC)
HPK-P
RR-P
CheA2
CheA3
C Y1 Y2 Y3 Y4 Y5 Y6
C Y1 Y2 Y3 Y4 Y5 Y6
Phosphotransfer profiling
showed that while CheA2
can phosphorylate all of the
RRs, CheA3 can only
phosphorylate CheY1 and
CheY6.
Transcriptomics indicated
that CheA1, CheY1, CheY2
and CheY5 are not
expressed. Deletion studies
confirmed that these
proteins are not required
for chemotaxis.
The CheAs localize to distinct signalling clusters
CheA2
CheA3
Porter and Armitage, 2002 (JMB) & 2006(JBC)
Signals from both clusters are required for chemotaxis.
All CheYs can bind the motor, but only CheY6-P is
capable of causing a change in swimming direction.
Wadhams et al., 2003
Modelling approach
We constructed a 2D spatiotemporal mathematical model of the cell and its
distinct chemosensory clusters using partial differential equations (PDEs).
In line with experimental data, the CheAs in the model are fixed to their
respective clusters, while the RRs and RR-Ps are free to diffuse throughout the
cell.
The PDEs representing the
activity of the cytoplasmic
cluster (W4)
A3
 k2 A3  k8 A3PY6  k8 A3Y6 P
t
A3 P
 k2 A3  k8 A3 PY6  k8 A3Y6 P
t
Y6
 D2Y6  k8 A3 PY6  k8 A3Y6 P  k12Y6 P
t
Y6 P
 DP 2Y6 P  k8 A3PY6  k 8 A3Y6 P  k12Y6 P
t
Porter and Tindall, in preparation
Model predicts the need for a phosphatase
• Model predicts levels of RR-P throughout
simulated chemotactic responses.
• Model predicts that the signal termination
process should take over 4 seconds.
• However, experimental data indicate that
cells can complete their entire response to
a short stimulus in less than 1 second.
• Need faster CheY-P dephosphorylation,
but there are no known CheY-P
phosphatases in R. sphaeroides.
Could CheA3 be the missing phosphatase?
Can detect more CheY6-P when CheA3P1-P is used as the phosphodonor
CheA3 is the missing phosphatase
CheY6-P half-life:
4.3 s
1.4 s
Porter et al., 2008 (PNAS)
The chemotaxis network of R. sphaeroides
Porter et al., 2008 Trends in Microbiology
Conclusions
• Spatiotemporal modelling of the R. sphaeroides chemotaxis
pathway predicted the need for a phosphatase.
• Experimentally, the CheA3 protein was shown to possess a novel
phosphatase activity.
• This phosphatase activity allows signal termination to occur
within the known signal response time of ~ 1 second.
• The discovery of this novel phosphatase is one of the first
examples of where the results of modelling work have fed back
into experimental design and successfully predicted the
existence of a biochemical reaction.
Acknowledgements
Oxford Centre for Integrative Systems Biology, University of Oxford
Judy Armitage, George Wadhams, Elaine Byles, Mark Roberts, Sonja
Pawelczyk, Gareth Davies, Jennifer De Beyer, Mostyn Brown, Nicolas
Delalez, David Wilkinson, Yo-Cheng Chang, Murray Tipping and Mila
Kojadinovic
STRUBI (Division of Structural Biology), University of Oxford
Christian Bell and David Stuart
Centre for Mathematical Biology, University of Oxford
Philip Maini
Department of Engineering Science, University of Oxford
Antonis Papachristodoulou
Institute for Cardiovascular and Metabolic Research, University of Reading
Marcus Tindall
The biochemical activities of CheA3
Reciprocal regulation of the kinase activity of CheA4 and the phosphatase
activity of CheA3 is likely to be a key point of control in the pathway.
Rhodobacter sphaeroides chemotaxis pathway
CheA1
CheY1
CheY2
CheA2
CheY3
Genome sequence
indicates 3 HPKs (CheAs)
and 6 RRs (CheYs).
CheA3A4
CheY4
CheY5
CheY6
Porter et al., 2002 (Mol. Microbiol.) & 2004 (JBC)
HPK-P
RR-P
CheA2
CheA3
C Y1 Y2 Y3 Y4 Y5 Y6
C Y1 Y2 Y3 Y4 Y5 Y6
Phosphotransfer profiling
showed that while CheA2
can phosphorylate all of the
RRs, CheA3 can only
phosphorylate CheY1 and
CheY6.
Transcriptomics indicated
that CheA1, CheY1, CheY2
and CheY5 are not
expressed. Deletion studies
confirmed that these
proteins are not required
for chemotaxis.
Rate of CheY6-P hydrolysis depends on [CheA3]
The three-fold stimulation of CheY6-P by CheA3 is concentration dependent.
The local concentration of CheA3 within the cytoplasmic chemosensory
cluster is ~ 1800 mM.