Using systems biology to learn how halo respond to their environment

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

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

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

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

5. Light bR Halo change the expression of bR in response to the amount of light in their environment 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.

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. GG-PP phytoene lycopene beta-carotene retinal bop bacteriorhodopsin (bR)

9. GG-PP phytoene lycopene beta-carotene retinal bop bacteriorhodopsin (bR) Question: How would increasing the amount of GG-PP affect the amount of bacteriorhodopsin (bR)?

10. GG-PP phytoene lycopene beta-carotene retinal bop bacteriorhodopsin (bR) Question: How would increasing the amount of GG-PP affect the amount of bacteriorhodopsin (bR)?

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

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

13. Homology

14. GG-PP phytoene lycopene beta-carotene retinal CrtB1 CrtY brp bop bacteriorhodopsin (bR) bat Question: Which enzymes are part of the bacteriorhodopsin network? What other genes are involved in the system?

15. GG-PP phytoene lycopene beta-carotene retinal CrtB1 CrtY brp bop bacteriorhodopsin (bR) bat LIGHT Question: Which protein changes in response to light and affects the expression of other genes?

16. Microarray gene expression

17. GG-PP phytoene lycopene beta-carotene retinal CrtB1 CrtY brp bop bacteriorhodopsin (bR) bat To simplify, focus on the genes and gene products (proteins). Then we’ll see how these affect the metabolites and bacteriorhodopsin (bR).

18. CrtB1 CrtY brp bop bat To simplify, focus on the genes and gene products (proteins). Then we’ll see how these affect the metabolites and bacteriorhodopsin (bR).

19. CrtB1 CrtY brp bop bat Question: Which genes does bat affect?

20. CrtB1 CrtY brp bop bat Question: What happens to the expression of the genes when bat is overexpressed?

21. CrtB1 CrtY brp bop bat Question: What happens to the expression of the genes when bat is overexpressed?

22. CrtB1 CrtY brp bop bat Question: What happens to the expression of the genes when bat is knocked out?

23. CrtB1 CrtY brp bop bat Question: What happens to the expression of the genes when bat is knocked out?

24. CrtY CrtB1 brp bop bat Question: What happens to the expression of the genes when bat is knocked out?

25. CrtY CrtB1 brp bop bat Knocked out bat

26. CrtB1 brp CrtY bop bat Overexpressed bat

27. GG-PP phytoene lycopene beta-carotene retinal CrtB1 CrtY brp bop bacteriorhodopsin (bR) bat Question: Bat affects the amount of proteins in the bR network. How does this affect the amount of bacteriorhodopsin produced?

28. GG-PP phytoene lycopene beta-carotene retinal CrtY CrtB1 brp bop bacteriorhodopsin (bR) bat Knocked out bat

29. GG-PP phytoene lycopene beta-carotene retinal CrtB1 brp CrtY bop bacteriorhodopsin (bR) bat Overexpressed bat

30. GG-PP phytoene lycopene beta-carotene retinal CrtY CrtB1 brp bop Knocked out bat bacteriorhodopsin (bR) bat GG-PP phytoene lycopene beta-carotene retinal CrtB1 brp CrtY Overexpressed bat bop bacteriorhodopsin (bR) 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. Our hypothesis – Light Present GG-PP phytoene lycopene beta-carotene retinal CrtB1 CrtY brp bop bacteriorhodopsin (bR) bat LIGHT ATP ADP + P only when light is present

33. Our hypothesis – Light Absent GG-PP phytoene lycopene beta-carotene retinal CrtB1 CrtY brp bop bacteriorhodopsin (bR) bat LIGHT ATP ADP + P only when light is present

34. Known Network – bR Production GG-PP phytoene lycopene beta-carotene retinal CrtB1 CrtY brp + bop + + bacteriorhodopsin (bR) bat LIGHT ATP ADP + P only when light is present Where do the other energy pathways fit (e.g. fermentation)?

35. GG-PP phytoene lycopene beta-carotene retinal CrtB1 CrtY brp YhdG bop Arginine (outside of cell) Arginine (inside cell) bat bacteriorhodopsin (bR) LIGHT ATP ADP + P only when light is present arginine ornithine ArgH carbamate CO2 L-arginosuccinate ArcA ArcB ATP NH3 ArcC ArgG citrulline carbamoyl-PO4 ADP + P

36. CrtB1 CrtY brp [+] [+] YhdG bop [-] [+] bat [-] [-] [-] [-] [-] ArgH ArcA ArcB ArcC ArgG

37. GG-PP phytoene lycopene beta-carotene retinal CrtB1 CrtY brp YhdG bop Arginine (outside of cell) Arginine (inside cell) bat bacteriorhodopsin (bR) LIGHT ATP ADP + P arginine ornithine ArgH carbamate CO2 L-arginosuccinate ArcA ArcB ATP NH3 ArcC ArgG citrulline carbamoyl-PO4 ADP + P

38. GG-PP phytoene lycopene beta-carotene retinal CrtB1 brp CrtY YhdG bop Arginine (outside of cell) Arginine (inside cell) bat bacteriorhodopsin (bR) LIGHT ATP ADP + P arginine ornithine ArgH carbamate CO2 L-arginosuccinate ArcA ArcB ATP NH3 ArcC ArgG citrulline carbamoyl-PO4 ADP + P

39. GG-PP phytoene lycopene beta-carotene retinal CrtY CrtB1 brp YhdG bop Arginine (outside of cell) Arginine (inside cell) bat bacteriorhodopsin (bR) LIGHT ATP ADP + P arginine ornithine ArgH carbamate CO2 L-arginosuccinate ArcA ArcB ATP NH3 ArcC ArgG citrulline carbamoyl-PO4 ADP + P

40. GG-PP phytoene lycopene beta-carotene retinal CrtB1 brp CrtY YhdG bop Arginine (outside of cell) Arginine (inside cell) bat bacteriorhodopsin (bR) LIGHT ATP ADP + P arginine ornithine ArgH carbamate CO2 L-arginosuccinate ArcA ArcB ATP NH3 ArcC ArgG citrulline carbamoyl-PO4 ADP + P

41. GG-PP phytoene lycopene beta-carotene retinal CrtY CrtB1 brp YhdG bop Arginine (outside of cell) Arginine (inside cell) bat bacteriorhodopsin (bR) LIGHT ATP ADP + P arginine ornithine ArgH carbamate CO2 L-arginosuccinate ArcA ArcB ATP NH3 ArcC ArgG citrulline carbamoyl-PO4 ADP + P

42. GG-PP phytoene lycopene beta-carotene retinal CrtB1 brp CrtY YhdG bop Arginine (outside of cell) Arginine (inside cell) bat bacteriorhodopsin (bR) LIGHT ATP ADP + P arginine ornithine ArgH carbamate CO2 L-arginosuccinate ArcA ArcB ATP NH3 ArcC ArgG citrulline carbamoyl-PO4 ADP + P

43. GG-PP phytoene lycopene beta-carotene retinal CrtY CrtB1 brp YhdG bop Arginine (outside of cell) Arginine (inside cell) bat bacteriorhodopsin (bR) LIGHT ATP ADP + P arginine ornithine ArgH carbamate CO2 L-arginosuccinate ArcA ArcB ATP NH3 ArcC ArgG citrulline carbamoyl-PO4 ADP + P

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?