LON-CAPA MCB 229 Spring 2000: Metabolism: autotrophs (chemolithotrophs & phototrophs)

		Quiz Me! (not yet available)	Metabolism: autotrophs (chemolithotrophs & phototrophs)
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Last revised: Thursday, February 24, 2000
Ch. 9 (p. 179-187); Ch. 10 (p. 193-195) in Prescott et al, Microbiology, 4th Ed.
Note: These notes are provided as a guide to topics the instructor hopes to cover during lecture. Actual coverage will always differ somewhat from what is printed here. These notes are not a substitute for the actual lecture!
Copyright 2000. Thomas M. Terry

Imagine being hungry, walking outside, taking off your shirt, lying in the sun for a few hours, becoming totally full (fat even!), and being done eating. No stores, no lines, no choices, just sunlight --- and the machinery of an autotroph --- and some CO₂ and a couple of other requirements (water --- and H₂S or Hydrogen gas, if you happen to be an anerobe)
Autotroph = gets all carbon from CO₂, organic C not required (for C-source). Use special metabolic cycle: Calvin-Benson cycle (see handout)
Refers to C-source only; some organisms still require organic C as energy source

Calvin-Benson cycle

Pathway details (see handout)

Each CO₂ is added to a 5-C acceptor molecule (ribulose 1,5 bis-phosphate)
Immediately split into two 3-C molecules (3-phosphoglyceric acid)
Must add phosphate group (from ATP) and hydrogen (from NADPH) to get reduced product, 3-phospho-glyceraldehyde (PGA)
Cannot take all (PGA) as product --- must regenerate some more acceptor to keep cycle going. How?
Take 5 PGA molecules (5 x 3C = 15 C atoms). Rearrange through series of reactions to make 3 5-C molecules (still 15 C atoms). Add ATP to each, make 3 acceptor molecules (ribulose 1,5 bis-phosphate)
Net result: To get 1 PGA (3-C) as reduced product, need 3 CO₂ molecules, added to 3 acceptor molecules ----> six 3-C molecules, use 6ATP and 6NADPH ----> 6 PGA molecules; five of these are used to regenerate acceptor molecules (+ 3ATP), one PGA can leave cycle and be used by cell.

Summary:

Actual cyce exports 3-C reduced molecules: look at balanced equation:

3 CO₂ + 9 ATP + 6 NADPH -----> 3-phospho-glyceraldehyde (PGA) + 9 ADP + 9 NADP⁺

Often want to look at balanced equation relative to 6C synthesis. Must multiply all terms in balanced equation above by two (since 2 PGA ~ 1 glucose)

6 CO₂ + 18 ATP + 12 NADPH -----> glucose + 18 ADP + 12 NADP⁺

Note for reaction: glucose + O₂ ----> 6CO₂ + 6 H₂O; delta G_o'= - 688kcal/mole
If each ATP contains ~7.3 kcal/mole (from delta G_o' for hydrolysis) and each NADPH contains ~54 kcal/mole (from delta G_o' for oxidation), then to make glucose costs 780 kcal/mole, more than the energy available by oxidizing glucose.
Conclusion: making sugar is expensive! Cell needs to supply large quantities of ATP and NADPH.

Types of Autotrophs - two basic types

Chemolithotrophs -- use inorganic compounds as Energy sources
Phototrophs -- use light as Energy source

Group Practice

Chemolithotrophs
(see handout on Chemolithotrophs)
Hydrogen Bacteria

Gain energy by oxidizing hydrogen gas:
H₂ + NAD⁺ -------(hydrogenase enzyme)------> NADH + H⁺
alternative: electrons can be donated directly to ETS chain, bypassing NAD
Note: only need one special enzyme to carry this step out: hydrogenase.
Many different genera of bacteria include members that can induce hydrogenase. When hydrogen disappears, back to heterotrophic life. Hydrogen bacteria are usually facultative chemolithotrophs.

Sulfur Bacteria

Called "colorless" in contrast to chlorophyll-containing sulfur bacteria, usually green or purple
Oxidize sulfur compounds: Example: Thiobacillus thiooxidans
thiosulfate: S₂O₃⁼ -----> SO₄⁼
free sulfur: 2 S^o + 2 H₂O + 3 O₂ -----> 2 H₂SO₄
Note product: sulfuric acid!! Cells can grow even in pH 0 (1M sulfuric acid). But cell internal pH is ~7, so difference across membrane can be 6 or 7 pH units.
Acid mine drainage: common in Western Penn., E. Ohio, W. Virginia. Rivers can run rust red. Mines have been major sources of pollution. Water seeps in, sulfur deposits exposed during coal mining allow microbial growth ------> megatons of H₂SO₄
Sulfuric acid leaches out, dissolves iron, precipitates in river with bicarbonate to form rusty deposits.
Quantities involved: Ohio River carries 100 million tons of 98% conc. H₂SO₄ per year.
To cure problem, must seal up old mines, prevent oxygen access. Also strip mines must be promptly covered up once mining is done to block access of microbes and oxygen to sulfur.
Value of this reaction:
(a) farmers or gardeners can dump free S on alkaline soil, bacteria will produce acid
(b) miners can use process to recover Cu from low grade ores, where smelting is not economical. Pile up mine "tailings" with copper ore; scrap shallow hole and fill with water. If tailings contain S, microbes will produce H₂SO₄. Now pump the acid over the tailings, Cu will be leached out and accumulate as soluble ions in acid pool. Eventually process the acid, recover Cu.

Nitrifying Bacteria

Very important soil organisms -- process all ammonia, nitrite in soils, break down amino acids, nitrogen bases ---> ammonia (NH₃)
Two different groups: one oxidizes ammonia, one oxidizes nitrite
Ex. 1: Nitrosomonas: 2 NH₃ (ammonia) + 3 O₂ -----> 2 HNO₂ (nitrite) + 2 H₂O
Ex. 2: Nitrobacter: 2 HNO₂ (nitrite) + 2 O₂ -----> 2 HNO₃ (nitrate)
Note potential problem: redox potential for nitrite as electron donor is + 0.42 v., so can easily pass electrons down to oxygen at + 0.82 v., reaction will be spontaneous. Electrons can be passed through an electron transport system, make ATP by chemiosmotic phosphorylation.
BUT --- how to make NADPH? (remember, this an autotroph, needs both ATP and NADPH to grow). How to get NADPH? The redox potential is much higher than nitrite.
Solution: Reverse electron transport. Accumulate enough proton gradient by oxidation of nitrite to force electrons back to carriers with higher redox potentials, all the way back to NADH ---> NADPH. This works as long as concentrations of reduced forms are kept very low, and NADPH is used up immediately to make glyceraldehyde-3-phosphate. See handout
This is very inefficient process. Nitrobacter can have 18 hour generation time. But it has no competition, so what's a little extra time?

Iron Bacteria

Curious discovery: Ferrobacillus ferrooxidans. Carries out oxidation of iron: Fe⁺⁺ (ferrous) ----> Fe⁺⁺⁺ (ferric) + e^-
Originally thought bacteria get energy from oxidation, make ATP. But redox potential of Fe oxidation is + 0.78 v., and redox potential for oxygen is + 0.86 v., so delta E_o' for aerobic respiration is only -0.08 v., calculated delta G_o' is much less than the 7.3 kcal/mole needed to make ATP. How does this organism grow?
Answer: it only grows in very acidic habitats, pH less than 3. Found with Thiobacillus thiooxidans, bacterium that produces sulfuric acid. Ferrobacillus lives off the pH gradient created by acidic pH. This maintains very high proton gradient. As H⁺ flows in, ATP gets made. But need to get rid of H⁺ inside, keep internal pH at 7. Use Fe⁺⁺ as electron donor to oxygen, combine with H⁺ to form water, get rid of outside cell. Iron functions as electron supplier to get rid of protons.
Cells process an enormous amount of iron for very small yields of energy. Fe⁺⁺⁺ reacts with OH^- ions to form insoluble precipitate, Fe(OH)3, reddish yellow color.
View acidic stream containing iron bacteria

Phototrophs

Use energy from sunlight to get high energy electrons (attached to carriers high on redox tower). Use CO₂ and Calvin-Benson cycle to make all organic molecules.
Critical molecules: photon absorbers = bacteriochlorophylls. Several different varieties. Light is trapped by a patch of pigments = "antenna field", gets passed around to a "reaction center" where an electron is released from Mg⁺⁺ ion with high energy, passed to electron transport system -- from this point, can use electron transport systems to generate proton gradients, make ATP.
Problem: need to make not only ATP (available from proton gradient), but also NADPH. How to obtain?
Two solutions:
use a reduced molecule with high redox potential like hydrogen gas (H₂) or hydrogen sulfide (H₂S) to pass electrons to NADP⁺. Light not needed for this.
use a reduced molecule with low redox potential like water to release electrons and H⁺ ions. Need lots of energy to drive this reaction, so need an extra step. Light is needed for this.

Anaerobic photosynthetic bacteria

Three common groups:

Purple bacteria Exs: Chromatium vinosum, Thiospirillum jenense
Purple nonsulfur bacteria. Exs: Rhodospirillum rubrum, Rhodobacter sphaeroides vannielii
Green sulfur bacteria (many are actually brown) Exs: Chlorobium limicola, Prosthecochloris aestuarii,

To see slides of these bacteria, visit Bacteriology 303 at U. Wisconsin, scroll down page to Figs. 6-8.
See handout on "Anoxygenic Photosynthesis"
View animated movie of anoxygenic photosynthesis (from Cornell University)
Notes: in both groups, electrons released by light travel through electron transport systems back to the original photosystem = cyclic electron flow. Proton gradient is produced, ATP is made as protons flow back through ATP synthase molecules. Specific carriers are different.
To make NADPH, need reduced electron donor.
(1) in purple bacteria, can use organic molecules (e.g. fumarate), or H₂ for non-sulfur bacteria; or can use H₂S or H₂ for purple sulfur bacteria. Sulfur accumulates inside cells when H₂S is used, hence the name.
(2) in green sulfur bacteria, can use H₂S, or H₂. Sulfur accumulates outside cells.

Aerobic photosynthetic bacteria = cyanobacteria

See handout on Oxygenic Photosynthesis.
includes both prokaryotes (cyanobacteria, formerly called blue-green bacteria) and eukaryotes (algae, green plants)

View light micrograph of Anabaena x1000, a cyanobacterium. The longer, clearer cells are heterocysts, specialized cells that fix nitrogen.
View light micrograph of Oscillatoria x1000, another filamentous cyanobacterium.
View light micrograph of Nostoc x1000, yet another cyanobacterium.

View animated movie of oxygenic photosynthesis (from Cornell University)
Notes: Two photosystems are needed, not one as in anoxygenic photosynthesis. Why?
Source of NADPH = electrons removed from photosystem I ---> excited by light to high redox potential, passed to ferredoxin, then directly to NADP⁺ -----> NADPH
But wait!!! Now photosystem I has + charge, can't supply any more electrons. Can't have this, so replace electrons from another photosystem II (see handout diagram), also energized by light. During this process, electrons flow through ETS system and make a proton gradient ( ------> ATP by chemiosmotic phosphorylation). But electrons aren't flowing back to same place they started from --- this is non-cyclic electron flow. Path resembles a letter "Z", so often called "Z-scheme" photosynthesis
Problem: we've "borrowed" electrons from system I to make NADPH, then "borrowed" electrons from system II to repay system I. How to repay system II?
Solution: Split water: H₂O (+ light energy) ------> 2e^- + 2H⁺ + 1/2 O₂ (waste gas)
Note: this is extremely important evolutionary step, took place billions of years ago. Freed phototrophs from need to wait for production of hydrogen, hydrogen sulfide, etc. until they could grow --- now they could grow whenever there was light and water. Early earth was totally anaerobic --- but once oxygenic phototrophs evolved, oxygen gas began to appear in atmosphere. Now new evolution possible: use of oxygen as electron acceptor, big boost in ATP yields. Result: oxygen concentration stabilized at 20%, constantly produced by phototrophs, consumed by respiration.

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