Analysis of the Competence Signaling Pathway in Bacillus

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Analysis of the Competence Signaling Pathway in Bacillus Subtilis Gavin Price Note: this is

Analysis of the Competence Signaling Pathway in Bacillus Subtilis Gavin Price Note: this is my first presentation to the Arkin lab. Please be gentle as I am very delicate and sensitive. Gavin Price Arkin Lab 7/28/04

Background • Bacteria are called competent if they possess the ability to take up

Background • Bacteria are called competent if they possess the ability to take up extra-cellular DNA and combine it with their own • Uptake and recombination can be promiscuous or based on similarity • Some known competent species: B. subtilis, Streptococcus pneumoniae, Haemophilius influenzae • Several hypotheses regarding the reason for competence • DNA as food • Lateral gene transfer & diversification of the gene pool • DNA use as a template for repair • In B. subtilis, one of several responses to stress including degradative enzyme synthesis, chemotaxis, and sporulation. • Competence triggered by high population density and low resource levels • Small fraction of the population becomes competent – ~10 -20% in lab strains • Questions to be answered: • Why is competence development stochastic • How does the network integrate environmental and internal signals to turn competence on and off • How do the various stress response networks interact • We can suggest answers to some of these questions via mathematical modeling of the system Gavin Price Arkin Lab 7/28/04

phr. G/rap. G com. A switch phr. C/rap. C Overview of the competence pathway

phr. G/rap. G com. A switch phr. C/rap. C Overview of the competence pathway Pheromone I/O and sensing – quorum sensing or timing of competence Gavin Price Arkin Lab 7/28/04

Overview of the competence pathway Deg. U switch – switches between competence & degradative

Overview of the competence pathway Deg. U switch – switches between competence & degradative enzyme pathways deg. U/deg. S Gavin Price Arkin Lab 7/28/04

Overview of the competence pathway Com. K is the competence transcription factor and commits

Overview of the competence pathway Com. K is the competence transcription factor and commits cell to competence. Mutual inhibition with rok com. K/rok Gavin Price Arkin Lab 7/28/04

Overview of the competence pathway Com. K degradation reduced when quorum present via Com.

Overview of the competence pathway Com. K degradation reduced when quorum present via Com. S mec. A/com. S/clp. CP Gavin Price Arkin Lab 7/28/04

Overview of the competence pathway Abr. B/Cod. Y Gavin Price Arkin Lab Abr. B

Overview of the competence pathway Abr. B/Cod. Y Gavin Price Arkin Lab Abr. B provides link to sporulation pathway, competence shut off by Cod. Y when resources are high 7/28/04

Modeling the com. K/rok competitive inhibition subsystem Motivation for selection • All roads lead

Modeling the com. K/rok competitive inhibition subsystem Motivation for selection • All roads lead to com. K – easy to expand model from the core • I. e. , reductionist approach to modeling the entire competence signaling pathway • Rok binding behavior discovered in 2002, function in context of pathway is unclear • Thus, function of the subsystem is unclear: • Why does Abr. B 4 repress both rok and com. K? • Why is rok needed? System Details • Abr. B binds as a tetramer to Pcom. K and Prok and represses both. • Com. K binds as a tetramer to Pcom. K and Prok. It activates com. K and represses rok. • Rok represses both com. K and rok. Quaternary structure is unknown. • Deg. U binds between Com. K dimers to Pcom. K and acts as a ‘priming protein. ’ • Abr. B, Com. K, Rok, and Deg. U can all bind to com. K at the same time. Gavin Price Arkin Lab 7/28/04

Model Total: 19 parameters Gavin Price Arkin Lab 7/28/04

Model Total: 19 parameters Gavin Price Arkin Lab 7/28/04

Literature Search • Little physical data on this system Gavin Price Arkin Lab 7/28/04

Literature Search • Little physical data on this system Gavin Price Arkin Lab 7/28/04

Model Physical Constants • All but two chosen as a ‘reasonable’ guess, where ‘reasonable’

Model Physical Constants • All but two chosen as a ‘reasonable’ guess, where ‘reasonable’ means within typical ranges for similar constants Gavin Price Arkin Lab 7/28/04

Stress Response System Interactions • When the sporulation pathway is activated, Spo 0 A

Stress Response System Interactions • When the sporulation pathway is activated, Spo 0 A levels increase and repress abr. B 1. • Abr. B acts as a repressor of com. K at high concentrations, and an activator at low concentrations 2. • It is likely that this effect is due to de-repression of rok when Abr. B concentration is extremely low 3. • Assuming competence is active, Rok is the only other available repressor of com. K: Cod. Y must be shut off for the cell to be competent. • Questions: • Is the model sufficient to describe Abr. B’s effect on Com. K concentrations? • What is the system response to Spo 0 A repression of abr. B? 1. 2. 3. Gavin Price Arkin Lab Hahn, J. , Roggiani, M. , Dubnau, D. : The major role of Spo 0 A in genetic competence is to downregulate abr. B, an essential competence gene. J Bacteriol 1995, 177(12): 3601 -5. Hamoen, L. W. , Venema, G. , Kuipers, O. P. : Controlling competence in Bacillus subtilis: shared use of regulators. Microbiology 2003, 149: 9 -17. Hoa, T. T. , Tortosa, P. , Albano, M. , Dubnau, D. : Rok (Yku. W) regulates genetic competence in Bacillus subtilis by directly repressing com. K. Molecular Microbiology 2002, 43(1): 15 -26. 7/28/04

Pseudo-steady state analysis predicts correct Com. K behavior as fn([Abr. B]) • The current

Pseudo-steady state analysis predicts correct Com. K behavior as fn([Abr. B]) • The current model is sufficient to account for Abr. B’s positive and negative regulation of com. K • Rok represses com. K at low Abr. B concentrations Gavin Price Arkin Lab 7/28/04

A drop in Abr. B concentration causes a Com. K pulse • Abr. B

A drop in Abr. B concentration causes a Com. K pulse • Abr. B keeps com. K repressed at high concentrations, and Rok re-represses it at low Abr. B concentrations • The result is a pulse, activating competence while Abr. B is within a specific range • The subsystem, and rok in particular, seem to exist to regulate the switch from exponential growth to competence and finally to sporulation • Abr. B concentration changes set the timing for these transitions • How sensitive is this behavior to the system constants? Gavin Price Arkin Lab 7/28/04

Parameter List Gavin Price Arkin Lab 7/28/04

Parameter List Gavin Price Arkin Lab 7/28/04

 • The com. K basal transcription rate has virtually no effect Gavin Price

• The com. K basal transcription rate has virtually no effect Gavin Price Arkin Lab 7/28/04

 • Abr. B’s binding constant to rok sets the shape of the curve,

• Abr. B’s binding constant to rok sets the shape of the curve, with stronger binding resulting in a sharper peak • If the binding constant is below ~ 0. 05/n. M, the pulse amplitude is very small • Blue line is the curve resulting from the original parameter set Gavin Price Arkin Lab 7/28/04

 • Com. K’s binding constant to rok is strongly sensitive, but is known

• Com. K’s binding constant to rok is strongly sensitive, but is known to be ~ 0. 01/n. M. • If the binding is too strong, [Rok] cannot increase enough to repress com. K. Gavin Price Arkin Lab 7/28/04

 • The effect of Rok self repression is minimal, with less self repression

• The effect of Rok self repression is minimal, with less self repression resulting in a faster drop of [Com. K]. For those of you that are endlessly fascinated, there are 15 more plots at biospice. lbl. gov/~gavin Gavin Price Arkin Lab 7/28/04

 Gavin Price Arkin Lab 7/28/04

Gavin Price Arkin Lab 7/28/04

Critical and Robust Constants Highly Sensitive Constants rok basal transcription rate com. K transcription

Critical and Robust Constants Highly Sensitive Constants rok basal transcription rate com. K transcription rate in the Com. K 4 -Deg. U configuration Com. K 4 -Deg. U->com. K binding constant Com. K 4 -Deg. U-Rok->com. K binding constant Robust Constants com. K basal transcription rate com. K transcription rate in the Com. K 4 configuration Com. K 4 tetramer dissociation rate Com. K 4 ->com. K binding constant Rok->com. K binding constant Gavin Price Arkin Lab 7/28/04

 • Tried some 2 parameter experiments, but little illumination shed as a result.

• Tried some 2 parameter experiments, but little illumination shed as a result. 9 total experiments available at http: //biospice. lbl. gov/~gavin/ Gavin Price Arkin Lab 7/28/04

Summary • The Com. K/Rok/Abr. B subsystem may exist to regulate the switch from

Summary • The Com. K/Rok/Abr. B subsystem may exist to regulate the switch from exponential growth to competence and finally to sporulation • Note that competence and sporulation are usually considered to be mutually exclusive, although at least one experiment has induced both on the same media • With ‘reasonable guesses’ for constants, the model displays the qualitatively correct behavior. Matches the Com. K peak at ~ 2 hrs post stationary phase onset. • Highly sensitive parameters identified and behaviors characterized. Next • • • Response surface analysis and sensitivity analysis Develop experimental plan to test system behavior on plasmid, then in vivo Continue refining model and expanding analysis to the rest of the competence pathway Stochastic simulations to elucidate why only 20% of cells become competent System identification for parameter and model architecture estimation Comparative dynamics and genomics Acknowledgements Denise Wolf Gavin Price Adam Arkin Lab 7/28/04

Appendix Gavin Price Arkin Lab 7/28/04

Appendix Gavin Price Arkin Lab 7/28/04

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Gavin Price Arkin Lab 7/28/04