Microbial metabolism 4 Autotrophy

A tale of two microbes that oxidize sulfur Sulfolobus Purple sulfur bacteria Photo: Chromatium okenii by Raymond Cox, University of Southern Denmark http://www.sulfosys.com/sulfolobus-solfataricus.html "RT8-4" by Xiangyux - English Wikipedia. Licensed under Public Domain via Commons - https://commons.wikimedia.org/wiki/File:RT8-4.jpg#/media/File:RT8-4.jpg http://courses.mbl.edu/microbialdiversity/

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

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

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

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

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

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 -)

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)

Carbon fixation requires +electrons to CO2 6 CO2 + 18 H2O → C6H12O6 + 6H2O Carbon reduced from +4 to 0 Where do electrons come from? Where does energy come from?

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!!!

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

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

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

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

Reverse TCA cycle

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

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??

Lithotrophy Why sulfolobus oxidizes sulfur

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

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

Lithotrophs often survive with low energy

Diverse lithotrophs use O2 as e- acceptor Why?

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

These lithotrophs use reverse ETS to get NADH

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?

Iron oxidizers http://www.jcm.riken.jp/JCM/staff/skato/researchen.html http://lonelyspore.com/category/microbes-by-habitat/deep-biosphere-microbes-by-habitat/ http://www.legend-group.com/node/170

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

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

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.

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

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: +740 mV How do they get their NADH?

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

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?

Phototrophy Light and pigments generate pmf PMF generates ATP through photophosphorylation

Simplist phototrophy: Bacteriorhodopsin

Bacteriorhodopsin in a membrane

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

Diverse chlorophyll capture light energy Bacteriochlorophyll Carotenoids

How pigments are arranged to capture energy

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

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

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

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

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

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

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

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

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?

Oxygenic phototrophs use both photosystems

Photophosphorylation in Cyanobacteria (Z)

Uses of sulfur in lithotrophy, phototrophy

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

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?

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