1. Using systems biology to learn how halo respond to their environment

2. bR converts light energy into chemical energy for making ATP Halobacterium cell Note: other proteins are required for this process, but we will simplify and focus on bR Bacteriorhodopsin (bR) LIGHT ATP

3. Bacteriorhodopsin (bR) Cell membRane Bacteriorhodopsin (bR) is made from a protein called bop and the molecule retinal Bop protein Bop + retinal = bacteriorhodopsin (bR) Retinal

4. Halobacterium cell LIGHT PRESENT LIGHT ABSENT (DARK) Halobacterium cell LIGHT When there is more light, halo respond by making more bR LIGHT When there is less light, halo make less bR Halo change the expression of bR in response to the amount of light in their environment

5. LightbR + The amount of bR increases when there is more light. Note that the size of nodes in this diagram relates to the amount of light and bR, not the actual size. In other words, the size of bR does not change; a larger node for bR indicates a larger amount of bR. Halo change the expression of bR in response to the amount of light in their environment

6. OUR QUESTION: How do Halobacterium cells control the amount of bR expressed in response to light? What is the gene and protein network that regulates the expression of bR?

7. Metabolic data group

8. lycopenebeta-caroteneretinal bacteriorhodopsin (bR) GG-PP phytoene bop

9. lycopenebeta-caroteneretinal bacteriorhodopsin (bR) GG-PP phytoene bop Question: How would increasing the amount of GG-PP affect the amount of bacteriorhodopsin (bR)?

10. lycopenebeta-caroteneretinal bacteriorhodopsin (bR) GG-PP phytoene bop Question: How would increasing the amount of GG-PP affect the amount of bacteriorhodopsin (bR)?

11. lycopenebeta-caroteneretinal bacteriorhodopsin (bR) GG-PP phytoene bop Question: If the enzyme that converts phyotene to lycopene were missing, how would the amount of bacteriorhodopsin (bR) be affected?

12. lycopenebeta-caroteneretinal bacteriorhodopsin (bR) GG-PP phytoene bop Question: If the enzyme that converts phyotene to lycopene were missing, how would the amount of bacteriorhodopsin (bR) be affected?

13. Homology

14. lycopenebeta-caroteneretinal bacteriorhodopsin (bR) GG-PP phytoene bop CrtYCrtB1brp Question: Which enzymes are part of the bacteriorhodopsin network? What other genes are involved in the system? bat

15. lycopenebeta-caroteneretinal bacteriorhodopsin (bR) GG-PP phytoene bop CrtYCrtB1brp Question: Which protein changes in response to light and affects the expression of other genes? bat LIGHT

16. Microarray gene expression

17. lycopenebeta-caroteneretinal bacteriorhodopsin (bR) GG-PP phytoene bop CrtYCrtB1brp bat To simplify, focus on the genes and gene products (proteins). Then we’ll see how these affect the metabolites and bacteriorhodopsin (bR).

18. bop CrtYCrtB1brp bat To simplify, focus on the genes and gene products (proteins). Then we’ll see how these affect the metabolites and bacteriorhodopsin (bR).

19. bop CrtYCrtB1brp bat Question: Which genes does bat affect?

20. bop CrtYCrtB1brp bat Question: What happens to the expression of the genes when bat is overexpressed?

21. bop CrtYCrtB1brp bat Question: What happens to the expression of the genes when bat is overexpressed?

22. bop CrtYCrtB1brp bat Question: What happens to the expression of the genes when bat is knocked out?

23. bop CrtYCrtB1brp bat Question: What happens to the expression of the genes when bat is knocked out?

24. bop CrtY CrtB1brp bat Question: What happens to the expression of the genes when bat is knocked out?

25. bop CrtY CrtB1brp bat Knocked out bat

26. bop CrtY CrtB1brp bat Overexpressed bat

27. bop CrtYCrtB1brp bat lycopenebeta-caroteneretinal bacteriorhodopsin (bR) GG-PP phytoene Question: Bat affects the amount of proteins in the bR network. How does this affect the amount of bacteriorhodopsin produced?

28. bop CrtY CrtB1brp bat Knocked out bat lycopenebeta-caroteneretinal bacteriorhodopsin (bR) GG-PP phytoene

29. bop CrtY CrtB1brp bat Overexpressed bat lycopenebeta-caroteneretinal bacteriorhodopsin (bR) GG-PP phytoene

30. bop CrtY CrtB1brp bat lycopenebeta-caroteneretinal bacteriorhodopsin (bR) GG-PP phytoene bop CrtY CrtB1brp bat lycopenebeta-caroteneretinal bacteriorhodopsin (bR) GG-PP phytoene Knocked out bat Overexpressed bat

31. OUR QUESTION: How do Halobacterium cells control the amount of bR expressed in response to light? What is the gene and protein network that regulates the expression of bR?

32. ADP + P ATP LIGHT bop CrtYCrtB1brp bat lycopenebeta-caroteneretinal bacteriorhodopsin (bR) GG-PP phytoene Our hypothesis – Light Present only when light is present

33. ADP + P ATP LIGHT bop CrtYCrtB1brp bat lycopenebeta-caroteneretinal bacteriorhodopsin (bR) GG-PP phytoene Our hypothesis – Light Absent only when light is present

34. Known Network – bR Production ADP + P ATP LIGHT bop CrtYCrtB1brp bat lycopenebeta-caroteneretinal bacteriorhodopsin (bR) GG-PP phytoene only when light is present Where do the other energy pathways fit (e.g. fermentation)? + + +

35. bop CrtYCrtB1brp bat lycopenebeta-caroteneretinal bacteriorhodopsin (bR) GG-PP phytoene L-arginosuccinate ArgG ArgH arginine citrulline ornithine carbamoyl-PO4 carbamate CO 2 NH3 ADP + P ArcA ArcB ArcC ATP Arginine (inside cell) Arginine (outside of cell) YhdG ADP + P ATP LIGHT only when light is present

36. bop CrtYCrtB1brp bat ArgG ArgH ArcA ArcB ArcC YhdG [+] [-]

37. bop CrtYCrtB1brp bat lycopenebeta-caroteneretinal bacteriorhodopsin (bR) GG-PP phytoene L-arginosuccinate ArgG ArgH arginine citrulline ornithine carbamoyl-PO4 carbamate CO 2 NH3 ADP + P ArcA ArcB ArcC ATP Arginine (inside cell) Arginine (outside of cell) YhdG ADP + P ATP LIGHT

38. L-arginosuccinate arginine citrulline ornithine carbamoyl-PO4 carbamate CO 2 NH3 ADP + P ATP Arginine (inside cell) Arginine (outside of cell) ArgG ArgH ArcA ArcB ArcC YhdG bop CrtY CrtB1brp lycopenebeta-caroteneretinal bacteriorhodopsin (bR) GG-PP phytoene bat LIGHT ADP + P ATP

39. L-arginosuccinate ArgG ArgH arginine citrulline ornithine carbamoyl-PO4 carbamate CO 2 NH3 ADP + P ArcA ArcB ArcC ATP Arginine (inside cell) Arginine (outside of cell) YhdG bop CrtY CrtB1brp lycopenebeta-caroteneretinal bacteriorhodopsin (bR) GG-PP phytoene bat ADP + P ATP LIGHT

40. L-arginosuccinate arginine citrulline ornithine carbamoyl-PO4 carbamate CO 2 NH3 ADP + P ATP Arginine (inside cell) Arginine (outside of cell) ArgG ArgH ArcA ArcB ArcC YhdG bop CrtY CrtB1brp lycopenebeta-caroteneretinal bacteriorhodopsin (bR) GG-PP phytoene bat LIGHT ADP + P ATP

41. L-arginosuccinate ArgG ArgH arginine citrulline ornithine carbamoyl-PO4 carbamate CO 2 NH3 ADP + P ArcA ArcB ArcC ATP Arginine (inside cell) Arginine (outside of cell) YhdG bop CrtY CrtB1brp lycopenebeta-caroteneretinal bacteriorhodopsin (bR) GG-PP phytoene bat ADP + P ATP LIGHT

42. L-arginosuccinate arginine citrulline ornithine carbamoyl-PO4 carbamate CO 2 NH3 ADP + P ATP Arginine (inside cell) Arginine (outside of cell) ArgG ArgH ArcA ArcB ArcC YhdG bop CrtY CrtB1brp lycopenebeta-caroteneretinal bacteriorhodopsin (bR) GG-PP phytoene bat LIGHT ADP + P ATP

43. L-arginosuccinate ArgG ArgH arginine citrulline ornithine carbamoyl-PO4 carbamate CO 2 NH3 ADP + P ArcA ArcB ArcC ATP Arginine (inside cell) Arginine (outside of cell) YhdG bop CrtY CrtB1brp lycopenebeta-caroteneretinal bacteriorhodopsin (bR) GG-PP phytoene bat ADP + P ATP LIGHT

44. Why is it so complicated? Why does the cell use this network of genes, enzymes, other proteins, and metabolites to make bR? Why does the cell go through the trouble of regulating the network for bR?

45. What could we do to test our network model? How could we validate our network? What other types of experiments could we do? What other types of information could we use? Would we have been able to define this network without multiple data types? Why or why not?