Dr. Steven Porter

1. A systems biology approach for studying complex chemotaxis pathways Dr. Steven Porter 2

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

3. 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.

4. 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.

5. 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 CheA2 CheA3 HPK-P Y6 Y6 Y1 Y1 Y2 Y2 Y3 Y3 Y4 Y4 Y5 Y5 C C RR-P CheY1 CheY2 CheY3 CheY4 CheY5 CheY6 Porter et al., 2002 (Mol. Microbiol.) & 2004 (JBC)

6. 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

7. 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

8. 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.

9. Could CheA3 be the missing phosphatase? Can detect more CheY6-P when CheA3P1-P is used as the phosphodonor

10. CheA3 is the missing phosphatase CheY6-P half-life: 4.3 s 1.4 s Porter et al., 2008 (PNAS)

11. The chemotaxis network of R. sphaeroides Porter et al., 2008 Trends in Microbiology

12. 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.

13. 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

15. 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.

16. 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 CheA2 CheA3 HPK-P Y6 Y6 Y1 Y1 Y2 Y2 Y3 Y3 Y4 Y4 Y5 Y5 C C RR-P CheY1 CheY2 CheY3 CheY4 CheY5 CheY6 Porter et al., 2002 (Mol. Microbiol.) & 2004 (JBC)

17. 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.