Dr. Steven Porter

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A systems biology approach for studying complex
chemotaxis pathways

• Dr. Steven Porter

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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

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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.

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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.
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Rhodobacter sphaeroides chemotaxis signalling
Genome sequence indicates 3 HPKs (CheAs) and 6
RRs (CheYs). 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.
CheA1
CheA2
CheA3A4
CheY1
CheY2
CheY3
CheY4
CheY5
CheY6
Porter et al., 2002 (Mol. Microbiol.) 2004
(JBC)
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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
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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)
Porter and Tindall, in preparation
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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.

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Could CheA3 be the missing phosphatase?
Can detect more CheY6-P when CheA3P1-P is used as
the phosphodonor
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CheA3 is the missing phosphatase
Porter et al., 2008 (PNAS)
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The chemotaxis network of R. sphaeroides
Porter et al., 2008 Trends in Microbiology
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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.

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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 I
nstitute for Cardiovascular and Metabolic
Research, University of Reading Marcus Tindall
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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.
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Rhodobacter sphaeroides chemotaxis pathway
Genome sequence indicates 3 HPKs (CheAs) and 6
RRs (CheYs). 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.
CheA1
CheA2
CheA3A4
CheY1
CheY2
CheY3
CheY4
CheY5
CheY6
Porter et al., 2002 (Mol. Microbiol.) 2004
(JBC)
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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.