1. Microbial metabolism 4
Autotrophy

2. A tale of two microbes that oxidize sulfur
Sulfolobus
Purple sulfur bacteria
Photo: Chromatium okenii by Raymond Cox, University of Southern Denmark
"RT8-4" by Xiangyux - English Wikipedia. Licensed under Public Domain via Commons -

3. Sulfolobus Thermophile, thrives at 80-90 degrees
Acidophile, thrives at pH between 2-3
Lithoautotrophy – Oxidizes sulfur
Chemoheterotrophy –Uses sulfur to oxidize organic compounds

4. Purple sulfur bacteria
Found in anoxic zones of lakes
Sometimes hot springs
Anoxygenic phototrophs
Purple colored because of their photosynthetic pigments

5. Both species use sulfur in metabolism
Today we will learn how and why

6. Learning outcomes
Be able to explain why autotrophs need a source of electrons to reduce NADH or NADPH, in addition to electrons to generate PMF.
Explain how and why electrons for carbon fixation come from reverse electron transport
Be able to describe 2 mechanisms for CO2 fixation, and determine which genes you would look for to identify putative autotrophs
Explain lithotrophy and why the process differs depending on the electron donor
Explain the roles of pigments and oxidative phosphorylation in phototrophy
Compare and contrast lithotrophy and phototrophy
Compare and contrast different mechanisms of phototrophy

7. Lecture outline
Autotrophs need electrons (NADPH) to reduce CO2 and make more cellular material
Lithotrophs use inorganic electron donors to generate proton-motive force
Ex. Sulfur, nitrogen, iron
Phototrophs use light energy to generate proton-motive force
Bacteriorhodopsin
Pigments help them capture the light
Phototrophy can be oxygenic or anoxygenic
Anoxygenic phototrophy: Photosystem I
Anoxygenic phototrophy: Photosystem II
Oxygenic phototrophy uses both photosystems
These processes explain why and how Sulfolobus and purple bacteria use sulfur

8. Concepts you will be using today
Catabolism vs Anabolism
ETS can go backwards
Glucose
Glycolysis
Pyruvate
Citric acid
Cycle
NADH
ATP
NAD+
H2O
ETS
ATP synthase
Electrons down the tower (- to +)
Electrons up the tower (+ to -)

9. Two minute paper
How do you think a species can obtain energy from an inorganic electron donor?
(you might find it useful to draw a diagram of central metabolism for E. coli and then show what would change)

10. Carbon fixation requires +electrons to CO2
6 CO H2O → C6H12O6 + 6H2O
Carbon reduced from +4 to 0
Where do electrons come from?
Where does energy come from?

11. Carbon fixation requires NADH and ATP
Thus,
Complete overall reaction for carbon fixation:
6CO2 +12 NADH + 18ATP +18 H2O → C6H12O6 + 6H2O + 12 NAD+ +18 ADP(+Pi)
12 NADH and 18 ATP to make one glucose from CO2!!!

12. There are ≥ 4 mechanisms of CO2 fixation
Calvin cycle
Reverse TCA
Hydroxypropionate pathway
Reductive acetyl-CoA pathway

13. Mechanisms of carbon fixation
In which steps is carbon reduced?
Which enzymes and cofactors are responsible?
Where does the energy come from?

14. Calvin cycle: Rubisco adds CO2 to a 5C mol.

15. Calvin Cycle2: 3PG → G3P → Bkwd Glycolysis
Compare this to glycolysis
Cycles 6 times
Per glucose

16. Reverse TCA cycle

17. Reductive acetyl-CoA uses H2 and cofacters
How is CO2 reduced here? What do you think is next?

18. Take-home points: CO2 fixation
Carbon is reduced by endergonic reaction, Thus:
NADH and other electron carriers
ATP
Rubisco indicates carbon fixation pathway – genomics
Not used in every mechanism
Catabolic pathways re-used
*****Key to understanding autotrophs: Where do electrons for NADH come from??

19. Lithotrophy
Why sulfolobus oxidizes sulfur

20. Lithotrophy (how microbes eat rocks)
Glucose
Glycolysis
Pyruvate
Citric acid
Cycle
ATP
NAD+
H2O
NADH
ETS
ATP synthase

21. Lithotrophy H+ H+ ATP synthase H+ Electron donor = Inorganic molecule
Cell membrane e-
Terminal electron acceptor
Usually O2
ADP → ATP

22. Lithotrophs often survive with low energy

23. Diverse lithotrophs use O2 as e- acceptor
Why?

24. Lithotrophs often use G to reduce NAD
What do they need NADH for?

25. These lithotrophs use reverse ETS to get NADH

26. How to analyze lithotrophies
What is the electron donor?
What is the electron acceptor?
Where do electrons for CO2 fixation (NADH) come from?
How do they gain access to electron donor?
How much energy do they get per mole e-donor?

27. Iron oxidizers http://www.jcm.riken.jp/JCM/staff/skato/researchen.html

28. Energy and electrons scarce for Fe-oxidizers
Iron-oxidizers live in: soil/weathering, lakes that vary seasonally in oxygen, acid mine drainage.
At pH 2
Electron donor reaction
2Fe2+ → 2Fe3+ + 2e- (pH2) Eo’= +770mV Go’=+149 kJ/mol
Electron acceptor reaction
½ O2 + 2H+ + 2e- → H2O (pH2) Eo’= +1100mV Go’=-212 kJ/mol
Thus, ∆E is +330mV and ∆G is -63 kJ/mol
*ADP to ATP is 32kJ/mol
Conclusion:
E for NAD/NADH =-320mV

29. Process of lithotrophy in iron oxidizers
Metals must stay outside cell (rocks)

30. Sulfur can be oxidized in steps
H2S – HS- – S0 – S2O32- – H2SO4
Electron acceptors: oxygen (most), nitrate (some), iron (some)
reduced
oxidized
-220 mV
*Remember NAD/NADH is -320mV.

31. Sulfur oxidation
Different sulfur compounds come into the ETS at different points

32. Nitrogen oxidation: low energy, high E
NH4+ - NH2OH – HNO2 – HNO3
Electron donor reactions:
Ammonia oxidizing bacteria – ammonia to nitrite +440 mV
Nitrifying bacteria – nitrite to nitrate, +420 mV
Electron acceptors: Oxygen +820mV
Annamox uses Nitrite –N2: mV
How do they get their NADH?

33. Example ammonia oxidation
Oxygen used 2x
AMO = ammonia monooxygenase
HAO = hydroxylamine oxidoreductase

34. Do you think obligate chemolithoautotrophs have enzymes for glycolysis
Do you think obligate chemolithoautotrophs have enzymes for glycolysis? TCA cycle?
What if they are a chemolithoheterotroph?
What are the similarities between iron oxidation, sulfur oxidation, and ammonia oxidation?

35. Phototrophy Light and pigments generate pmf
PMF generates ATP through photophosphorylation

36. Simplist phototrophy: Bacteriorhodopsin

37. Bacteriorhodopsin in a membrane

38. Overview of photophosphorylation
Lower E H+ H+
ATP synthase e- H+ H+ H+ H+ e-
Cell membrane e- e-
Cyclic electron flow
Or to NAD
ADP → ATP
Reaction center/ Pigments
Sometimes comes from splitting water

39. Diverse chlorophyll capture light energy
Bacteriochlorophyll
Carotenoids

40. How pigments are arranged to capture energy

41. Pigments are arranged in membranes
Why?
Chlorosomes
Green sulfur bacteria
Purple bacteria

42. A few terms in phototrophy
Photophosphorylation: the process of making ATP from light
Photolysis: ‘lysing’ H2O or H2S - splitting a molecule with light
Photoexcitation: light excites an electron to higher energy
Photosynthesis = photophosphorylation + carbon fixation

43. Phototrophs are oxygenic or anoxygenic
Purple and green bacteria
Cyanobacteria, algae, green plants
Anoxygenic
Oxygenic
Reducing power
Carbon
Energy
Reducing power
Carbon
Energy
electrons
electrons
electrons
Light
Light

44. There are two photophosphorylation pathways
Anoxygenic uses:
PSI OR PSII
Oxygenic uses both (z pathway)

45. Light used to reduce NAD in Photosystem I
Ex. Chlorobia
Electron comes from photolysis of H2S

46. Photosystem I: in a cellular context
How is pmf generated?

47. Purple bacteria use photosystem II: cyclic
No photolysis
Electron recycled
For NADH, reverse flow
(electron from organic material or sulfur)

48. Photosystem II in a cellular context
Where do they get NADH?

49. Summary of photosystems I and II
Photosystem I
Green sulfur
Photosystem II
Purple sulfur
What does excited electron do?
Why is sulfur a byproduct?
Where does electron for ATP come from?
Where does electron for NADH come from?

50. Oxygenic phototrophs use both photosystems

51. Photophosphorylation in Cyanobacteria (Z)

52. Uses of sulfur in lithotrophy, phototrophy

53. Why and how do they oxidize sulfur?
Sulfolobus
Purple sulfur bacteria

54. Sulfolobus Thermophile, thrives at 80-90 degrees
Acidophile, thrives at pH between 2-3
Lithoautotrophy – Oxidizes sulfur
Chemoheterotrophy –Uses sulfur to oxidize organic compounds
What do you think this means?

55. How to guess the metabolism…..
Sulfur is oxidized
Sulfur is reduced