Advanced Aspects of Chemotactic Mechanisms: Chemotaxis II Advanced Aspects of Chemotactic Mechanisms: Clustering of receptors Dynamic range of the response to chemoattractors Allostery among receptors Robustness of the control network Sensitivity to chemoattractors Integration of signals Theory vs. experiment Connection to other processes Jason Kahn: Chemotaxis II

How does chemotaxis work so well? Chemotaxis operates rapidly, with terrific sensitivity to chemoattractors, over a wide range of attractor concentration, and in response to multiple signals simultaneously. It has been difficult to understand how the process can work so well based on fundamental physical principles. Clustering and communication among receptors provides some answers. Theoretical approaches expand the concept of allostery to large aggregates of receptors. Jason Kahn: Chemotaxis II

Jason Kahn: Chemotaxis II Review of the Basics Source: Sourjik (2004), Trends in Microbiology CheA is the central control nexus, forming a dimer and interacting with CheW, receptors, CheB, and CheY Jason Kahn: Chemotaxis II

Chemotaxis as Engineering Chemotaxis is often discussed using the languages of electrical engineering or computer science Defaults for simple binding interactions vs. actual values: Amplification (Gain): 1 vs. 100 Hill coefficient: 1 vs. 10 Dynamic range: ~ 10 fold vs 100,000-fold This puzzle has engaged many brilliant people Noise and cell-cell fluctuations have recently emerged as important as well. Sourjik, 2004 Jason Kahn: Chemotaxis II

Jason Kahn: Chemotaxis II The Issue of Gain Sourjik and Berg, PNAS 2002, with accompanying commentary by Bray, PNAS, 2002. Gain = (% change in Bias/% change in receptor occupancy) A change in occupancy of only ~0.1% can be detected, corresponding to 1-2 molecules bound per cell, over a wide concentration range. Simple kinetic models fail to account for this. Gain occurs at two stages: (1) The fractional change in the level of CheY~P changes ~35x more than the fractional change in receptor occupancy for small changes in occupnacy (see below). (2) The Hill coefficient for CheY~P control of the motor bias is about 10, as measured in single cells. Experiment: Fluorescence resonance energy transfer (FRET) was measured between CheY-YFP and CheZ-CFP. YFP and CFP are two variants of Green Fluorescent Protein. When they come into close proximity, excitation of CFP is transferred to YFP and detected as YFP fluorescence. Since the substrate of CheZ is CheY~P, FRET reports on the abundance of CheY~P, which in turn is a measure of the activity of CheA. The apparent Kd for Asp can be measured from the concentration-dependence of the response to methyl-Asp (not metabolized). This value is used to calculate change in receptor occupancy; there’s very little apparent cooperativity. Then the FRET response can be compared to the calculated change in receptor occupancy to give the gain (see next page). This doesn’t explain how gain comes about! Could be at the receptor level or something to do with methylation. Jason Kahn: Chemotaxis II

Jason Kahn: Chemotaxis II Evidence for gain Sourjik and Berg’s demonstration of gain at the receptor to CheA step Receptor occupancy is calculated from Kd’s derived from response curves for receptor variants in cheR-cheB mutant cells Slope = -36 Removal Addition Jason Kahn: Chemotaxis II

Gain and Integration via Clustering Cooperative interactions among clustered receptors (MCP’s) can result in both gain and also integration of different signals, because mixed receptor complexes can affect each others’ activity. This means that the effects of low-abundance Trg, Aer, and Tap receptors are amplified by the high-abundance Tsr and Tar receptors. Both MWC and more complex models (Shimizu et al., JMB 2003) for cooperativity have been developed/applied. Jason Kahn: Chemotaxis II

Methylation provides dynamic range Levit and Stock, JBC 2002 Receptor methylation has minor effects on receptor occupancy. The response (CheA inactivation) is markedly different. Jason Kahn: Chemotaxis II

Connection to Engineering “Integral control” is a robust control mechanism whereby the integral of the error in a response is fed back in to the system in order to control it. Thermostats work this way. Chemotaxis is an example of integral control, as long as CheB demethylates only active receptors (those that are stimulating CheA to cause tumbling) and a few other constraints on rate constants are met. Application of engineering principles makes testable predictions for how the system should behave in order to exhibit “exact adaptation,” which is the idea that as long as the chemoattractant concentration is constant, no matter what the actual value of the concentration is, the bug swims randomly. Yi et al., PNAS 2000 Jason Kahn: Chemotaxis II

Connection to Quorum Sensing So far we have thought only about single bacterial cells seeking out nutrients. Starving cells are attracted to each other, due to nutrient leakage or pheromones. Alteration of behavior in response to neighbors is called “quorum sensing.” It may be adaptive in allowing e.g. biofilm formation, virulence gene expression, or plasmid sharing. Thus chemotaxis is an important mechanism for quorum sensing, and tends to direct cells to migrate to small enclosed areas. Park et al. (Stock lab), Science 2003, and see PNAS 2003 Jason Kahn: Chemotaxis II