Bacterial Chemotaxis

1. Bacterial Chemotaxis • Bacteria swim toward attractants and away from repellents. • Their motion is a biased random walk due to control of tumbling frequency. • They measure gradients in time, not space. They adapt over time. • Chemotaxis probably relies on clustering and cooperation among receptors. Jason Kahn, UMCP

2. Fundamental mechanics of chemotaxis • Textbooks and Review Sources: • Macnab, R. M. (1987). Motility and chemotaxis. In Escherichia coli and Salmonella typhimurium: Cellular and molecular biology. 1st ed. (Neidhardt, F. C., ed.), pp. 732-759. Am. Soc. Microbiol., Washington, D.C. • Stock, J. B. & Surette, M. G. (1996). Chemotaxis. In Escherichia coli and Salmonella typhimurium: Cellular and molecular biology. 2nd ed. (Neidhardt, F. C., ed.), pp. 1103-1129. Am. Soc. Microbiol., Washington, D.C. • White, D. (2000). The physiology and biochemistry of prokaryotes. 2nd ed., Oxford University Press, Oxford. • Parkinson, J. S. (2004). Signal amplification in bacterial chemotaxis through receptor teamwork. ASM News 70, 575-582. Jason Kahn, UMCP

3. Swimming driven by a flagellar motor • The flagellum is an organelle consisting of (1) a basal body that is a rotary motor, (2) a connecting hook, and (3) a long filament. • Counter-clockwise rotation leads to all of the filaments of the cell twining together about a common axis, and swimming in a straight line. • Clockwise rotation leads to separation of separate filaments and tumbling, leading to random reorientation of the cell. • Typically the cell swims for 1 sec at 25 um/sec and then tumbles for 0.1 sec. A random walk. Chemoattractants increase the time spent swimming when the bacterium is going up the gradient. A biased random walk. Jason Kahn, UMCP

4. Bacterial Outboard Motor • The flagellum is a complex molecular motor driven by the transmembrane proton potential (pH) generated from oxidative phosphorylation or a proton-pumping ATPase. • Regulation and mechanism of assembly of flagellar subunits is a subject of current research. Jason Kahn, UMCP

5. Tumbling and the Biased Random Walk • From Macnab, 1987, E. coli and S. typhimurium • Left hand side shows CCW flagellar rotation. The corkscrewing action of the left-handed helical filament drives the bacterium in a straight line. Right hand side showsCW flagellar rotation, with the different filaments separating and driving in different directions, tumbling the cell. • Increasing the time spent swimming between tumbles leads to netmotion toward attractant. Decreasing the time spent swimming leadsto motion away from a repellent. Jason Kahn, UMCP

6. Assays for chemotaxis • Swarm plates: on semisolid agar, observe motion away from thecenter of a self-generated nutrient gradient. • Microscopic observation of free-swimming cells. • Tethering to an immobile surface allows observation of the rotation of the body of the cell. • Temporal stimulation (rapid changes in chemoeffector concentration). From Berg, Physics Today, 1999 Jason Kahn, UMCP

7. Dynamics of Chemotaxis • Bacteria respond to temporal, not spatial signals. Given their size, the spatial gradient is undetectable unless they move! • Thus they have a short term “memory” of the chemoeffector concentration over the previous few seconds. • A change in concentration leads (after a brief latency phase) to excitation (a change in tumbling frequency) followed by adaptation: after a few seconds at a new effector concentration the cell resumes random tumbling. This allows the cell to continue following a gradient up or down. Jason Kahn, UMCP

8. MCP Receptors and Stimuli • MCP = methyl-accepting chemotaxis protein. These are specifically engaged in chemotaxis, whereas periplasmic binding proteins for nutrients are primarily for transport. • Aer: Energy-linked attractants (O2). Monitors redox state of the cell. • Tar: Aspartate attraction, maltose attraction via binding MBP, the periplasmic binding protein (PBP) for maltose. Also Co2+ and Ni2+ repulsion • Tsr: Serine attraction, external pH • Trg: Sugar attraction via PBP’s • Tap: dipeptide attraction via PBP’s • Organic repellents act at much higher concentrations: may screw up membranes • Figure from Parkinson foreshadows higher-level organization Jason Kahn, UMCP

9. Che Proteins: CheA • Che = Chemotaxis genes. (Mot = motility, and many other flagellum mutants are defective in chemotaxis as well.) • Chemotaxis is a classic bacterial two-component signalling system. • Is general, these systems have a histidine kinase sensor protein and a response regulator that is controlled by phosphorylation via phosphotransfer from the kinase • The chemotaxis genes are CheA, CheW, CheY, CheZ, CheR, and CheB. • Extensively studied! • CheA histidine kinase is a dimer in solution. It autophosphorylates using phosphate from ATP: CheA + ATP  CheA~P + ADP (on His 48) • CheA auto-phosphorylation is enhanced by CheW Jason Kahn, UMCP

10. Tar Structure and Interactions • Extracellular ligand-binding domain binds chemoeffector. • Signal is somehow transduced inside (through helix translation or rotation?) • Cytoplasmic helical domains interact with CheA, CheW • Ligand binding domain from 2LIG (Pymol), Stock 1997 Jason Kahn, UMCP

11. Tar-CheA-CheW ? • Cryo-EM image reconstruction / fitting X-ray structure model for the cytoplasmic domain of Tar interacting with CheA and CheW. (Francis et al., PNAS 2004). Suggests 6 receptor dimers per CheA dimer, plus 3 CheW monomers • Each red or yellow pillar is a trimer of dimers of TAR, but not the trimer of dimers seen in a crystal structure. • Controversial, mechanism of signalling to CheA unresolved. Jason Kahn, UMCP

12. Signal Transduction: CheY~P • Phosphorylated CheA transfers its phosphate to CheY, the response regulator: CheA~P + CheY  CheA + CheY~P (on Asp 57) • CheY~P interacts with FliM and other flagellar proteins to switch the motor to CW rotation: tumbling. • Thus inactivation of CheA upon attractant binding to MCP’s leads to more swimming and less tumbling. Jason Kahn, UMCP

13. Stimulation: Control of CheY • CheY has an autophosphatase activity stimulated by CheZ: CheY~P + H2O  CheY + Pi • So the steady-state level of CheY~P leads to the swimming / tumbling random walk, and the steady-state level changes in the presence of chemoeffectors. • CheZ may be unregulated, may interact with CheA. • This is a rapid response (< 1 sec) CheZ Jason Kahn, UMCP

14. Adaptation: Methylation • Adaptation means that bacteria stop responding to chemoeffectors when their concentrations do not change. They have a memory of the concentration over the previous few seconds. Hence adaptation is slower than excitation, allowing for a brief episode of biased random walking after a change in chemoeffector level. • Adaptation is mediated by the CheR methyltransferase and the CheB methylesterase. • CheR methylates the MCP’s using (Tar, Tsr, et al.). MCP-Glu + S-Adenosyl-Methionine (SAM)  MCP-Glu-CH3 + Adenosyl-homocysteine • Methylated MCP’s are less responsive to attractant: CheA is not inactivated, CheY continues to be phosphorylated, bacteria continue to tumble. • There are 4 or 5 methylation sites per MCP, and if they occur in clusters this means there are many possible methylation states for a cluster of receptors: wide dynamic range. • CheR is slow, leading to the delay time in adaptation. Jason Kahn, UMCP

15. Adaptation II • CheB methylesterase removes methyl groups from MCP’s: MCP-Glu-CH3 + H2O  MCP-Glu + CH3OH • Allows system to reset to be responsive to low concentrations of attractants again. • CheB is activated by phosphorylation by CheA. • Thus, CheA causes immediate excitation via phosphorylation of CheY, but also initiates a slower negative feedback loop that reduces the sensitivity of the MCP’s that regulate it! • MCP’s have allosteric responses to attractants that make them more susceptible to methylation: damping. • CheB auto-dephosphorylates slowly, perhaps unregulated. Jason Kahn, UMCP

16. Overall Summary of Circuitry Jason Kahn, UMCP