2. Molecular Microbiology Bioenergetics Bacteriology for 2001

3. Molecular Microbiology2 Outline Bacterial bioenergetics –What does this mean? –Mitchell’s hypotheses –Why  G and mV do not appear in these lectures

4. Molecular Microbiology3 Bacterial bioenergetics What I expect you to understand –electron transport chain and ATP synthesis –How bacteria are adapted to pursue life according to Mitchell’s hypotheses What is possible irrelevant –measuring PMF –the concept of Gibbs free energy

5. Molecular Microbiology4 How does bacterial metabolism work Central pathways of metabolism Electron transport chain ATP synthase Are these separate entities or do they work together?

6. Molecular Microbiology5 What is the point of the ETC? To reduce oxygen to water? To transport electrons? To move protons across a membrane? All of the above with a side order of chips?

7. Molecular Microbiology6 Mitchell’s postulates Four postulates to explain –respiratory phosphorylation –transport of solutes –all membrane-based enzymology

8. Molecular Microbiology7 Periplasmic space Building a chemiosmotic organism Cell membrane ATP ADP + P i H+

9. Molecular Microbiology8 Building a chemiosmotic organism ATP ADP + P i H+ ATP ADP + P i H+

10. Molecular Microbiology9 Building a chemiosmotic organism H+ ATP ADP + P i H+ Solute

11. Molecular Microbiology10 Mitchell’s postulates Four postulates to explain –respiratory phosphorylation –transport of solutes –all membrane-based enzymology

12. Molecular Microbiology11 Postulate 1 The respiratory or photosynthetic electron transport chains should translocate protons –i.e. the electron transport chain and other reactions should function as proton pumps

13. Molecular Microbiology12 Building a chemiosmotic organism ATP ADP + P i H+ ATP ADP + P i H+

14. Molecular Microbiology13 Postulate 2 The ATP synthase should function as a reversible proton-translocating ATPase –f1f0 ATPase –activity in the absence of a PMF –reverse flow in presence of PMF

15. Molecular Microbiology14 Building a chemiosmotic organism ATP ADP + P i H+ ATP ADP + P i H+

16. Molecular Microbiology15 Postulate 3 Energy-translocating membranes should have a low effective proton conductance –For a chemiosmotic organism to survive,protons must only be able to move through proteins, and not through membranes

17. Molecular Microbiology16 Building a chemiosmotic organism ATP ADP + P i H+ ATP ADP + P i H+

18. Molecular Microbiology17 Postulate 4 Energy-transducing membranes should possess specific exchange carriers to permit metabolites to permeate, and high osmotic stability to be maintained, in the presence of a high membrane potential

19. Molecular Microbiology18 Ports, symports, antiports Solute Basic port

20. Molecular Microbiology19 Ports, symports, antiports Solute Symport H+

21. Molecular Microbiology20 Ports, symports, antiports Solute Antiport SoluteH+ or H+ or

22. Molecular Microbiology21 So what drives ATP synthesis? What is the primary pump? The electron transport chain pumps 10 protons per NADH + H + The NADH + H + produced during glycolysis etc. is used to create ATP chemiosmotically

23. Molecular Microbiology22 The ETC is the primary pump in aerobic respiratory organisms

24. Molecular Microbiology23 Bacteria are chemiosmotic organisms Paracoccus denitrificans is an obligately respiratory organism. Has an f 1 f 0 ATPase which is coupled to a conventional electron transport chain. E. coli is similar but has a strange ETC and can ferment

25. Molecular Microbiology24 The bacterial f 1 f 0 ATPase

26. Molecular Microbiology25

27. Molecular Microbiology26

28. Molecular Microbiology27 What can  pH be used for? Solute transport –substrates –drug efflux –maintenance of ionic balance Bacterial flagellar motion

29. Molecular Microbiology28 What is the point of the ETC? To reduce oxygen to water? To transport electrons? To move protons across a membrane?   Sometimes

30. How does it all link in?

31. Molecular Microbiology30 H+ ATP ADP + P i H+ NADH + H+ reduced FMN NADH + H+ reduced FMN Reduced electron acceptor Carbon Source Glycolytic Pathway TCA

32. Molecular Microbiology31

33. Molecular Microbiology32 Short questions for group work What other means are there of generating PMF? What other uses can a proton gradient be put to in bacterial cells?

34. Molecular Microbiology33

35. Molecular Microbiology34

36. Molecular Microbiology35

37. Section II Chemiosmotic solutions to environmental problems

38. Molecular Microbiology37 Aerobic neutrophiles H+ ATP ADP + P i H+ Solute External pH 6-8 Internal pH 6-8 - - - - - - - -- - -

39. Molecular Microbiology38 Aerobic acidophiles H+ ATP ADP + P i H+ Solute External pH 1-4 Internal pH 5.5 + + + + + + + ++ + +

40. Molecular Microbiology39 Aerobic alkaliphiles ATP ADP + P i H+ Na+ Solute External pH 10-11 Internal pH 8.2 - - - - - - - -- - - Na+ H+ + + + + + + ++ + + +

41. Molecular Microbiology40 Halophiles ATP ADP + P i H+ Na+ Solute - - - - - - - -- - - Na+ H+ + + + + + + ++ + + + Light External pH 6-10 Internal pH 6-8

42. Molecular Microbiology41 Marine/halotolerant ATP ADP + P i Na + or H+ Na+ Solute - - - - - - - -- - - Na+ H+ + + + + + + ++ + + + Na+ or H+ External pH 6-10 Internal pH 6-8

43. Section III An example of a proton pump Bateriorhodopsin

44. Molecular Microbiology43 Halobacterium salinarum A member of the Archaea Also known as Halobacterium halobium When grown aerobically utilises a normal respiratory chain In the light under very low O 2, purple patches appear on their membranes

45. Molecular Microbiology44 Halobacterium salinarum Cells lack chlorophyll Still can generate ATP from light Protons are pumped by a simple (ish) to understand mechanism centered on bacteriorhodopsin

46. Molecular Microbiology45 Bacteriorhodopsin, crystal structure

47. Molecular Microbiology46 Bacteriorhodopsin, crystal structure, showing retinal

48. Molecular Microbiology47 Bacteriorhodopsin, crystal structure, showing retinal, top view

49. Molecular Microbiology48 Clever bacterium Halobacterium salinarum possesses four rhodopsins –bacteriorhodopsin (bR) –halorhodopsin (HR) –sensory rhodopsins I and II

50. Molecular Microbiology49 See overhead Mechanism p184 Nicholls & Ferguson

51. Molecular Microbiology50

52. Molecular Microbiology51 References Nichols and Ferguson - Bacterial Bioenergetics (Academic Press) Lehninger, Stryer or another good biochemistry text book ASM News