3. Adaptation

6. Essential for sensory perception Nearly universal Salmonella How to move towards unseen food?

7. Bacterial view of the world

9. Strategy If things are getting better, keep on going

10. Directed movement through a random walk Temporally biased Adaptation critical

11. How does Salmonella achieve adaptation? Sensory input Behavioral output Signal transduction Figure from Bren and Eisenbach (2000). J Bacteriol 182:6865-6873 Methylation-accepting chemotaxis protein (MCP) CheA (histidine kinase) CheY (response regulator) Flagellar motor

12. How does Salmonella achieve adaptation? High sensory input Reduced sensitivity Low sensory input Methylation of MCP Loss of methylation

13. How does Salmonella achieve adaptation? High sensory input Reduced sensitivity Low sensory input Increased sensitivity Methylation of MCP Loss of methylation

14. How does Salmonella achieve adaptation? High sensory input How does methylation of MCP cause reduced sensitivity? Lower affinity Hypothesis #1 Reduced input Hypothesis #2 Reduced output High sensory input Less signal transduction

15. How does Salmonella achieve adaptation? How does methylation of MCP cause reduced sensitivity? Reduced affinity Reduced signal transduction vs Tuning the Responsiveness of a Sensory Receptor via Covalent Modification Pete Dunten and Daniel Koshland J Biol Chem (1991) 266:1491-1496

16. Does methylation of MCP reduce binding affinity? Strategy: Add excess aspartate (methylates MCPs) Measure affinity of MCPs for aspartate Problem: Measuring affinity varies aspartate Low aspartate reduces methylation Measure response of Salmonella to aspartate

17. Does methylation of MCP reduce binding affinity? Strategy: Lock in methylation state of MCPs Lock in demethylated state Mutate cheR gene

18. Does methylation of MCP reduce binding affinity? Strategy: Lock in methylation state of MCPs Lock in demethylated state Mutate cheR gene (loss of methylation)

19. Does methylation of MCP reduce binding affinity? Strategy: Lock in methylation state of MCPs C O -O - O glutamate methylation COCH 3 O methyl-glutamate site-specific mutation CNH 3 O glutamine Lock in fully methylated state Amidation equivalent to methylation?

20. Does methylation of MCP reduce binding affinity? Strategy: Lock in methylation state of MCPs Measure affinity of MCPs for aspartate * * * * * * * * * * High affinity binding Monitor binding of radioactive aspartate * * * * * * * * * * * * * * * Low affinity binding * * * * * * * Low K D High K D K D = Asp required for ½ sites bound

21. Does methylation of MCP reduce binding affinity? Strategy: Lock in methylation state of MCPs Measure affinity of MCPs for aspartate Measure response of Salmonella to aspartate Measure net distance traveled in fixed time (velocity)

22. Does methylation of MCP reduce binding affinity? Strategy: Lock in methylation state of MCPs Measure affinity of MCPs for aspartate Measure response of Salmonella to aspartate Measure net distance traveled in fixed time (velocity) Determine [Asp] half-maximal velocity V max Adapted low [Asp] 1/2 Unadapted high [Asp] 1/2

23. Does methylation of MCP reduce binding affinity? Expect: If affinity affected: If affinity unaffected - No methylation (receptor unadapted) sensitive (low) - Full methylation (receptor adapted) insensitive (high) - No methylation (receptor unadapted) - Full methylation (receptor adapted) sensitive (low) strong (low) weak (high) normal Response [Asp] 1/2 Affinity (K D ) insensitive (high)

24. Does methylation of MCP reduce binding affinity? Observed: 15 µM Asp Full methylation (receptor unadapted) 3000 µM Asp No methylation (receptor adapted) 30 45 Response [Asp] 1/2 Affinity (K D ) Glutamine can replacement methyl-glutamate Adaptation does not affect affinity 200-fold 1.5-fold Difference

25. How does Salmonella achieve adaptation? Sensory input Behavioral output Signal transduction Figure from Bren and Eisenbach (2000). J Bacteriol 182:6865-6873

26. Adaptation