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LECTURE PRESENTATIONS For BROCK BIOLOGY OF MICROORGANISMS, THIRTEENTH EDITION Michael T. Madigan, John M. Martinko, David A. Stahl, David P. Clark Chapter 13 Phototrophy, Chemolithotrophy, and Major Biosyntheses Lectures by John Zamora Middle Tennessee State University © 2012 Pearson Education, Inc. I. Phototrophy • • • • • 13.1 Photosynthesis 13.2 Chlorophylls and Bacteriochlorophylls 13.3 Carotenoids and Phycobilins 13.4 Anoxygenic Photosynthesis 13.5 Oxygenic Photosynthesis © 2012 Pearson Education, Inc. 13.1 Photosynthesis • Photosynthesis is the conversion of light energy to chemical energy – Phototrophs carry out photosynthesis (Figure 13.1) – Most phototrophs are also autotrophs • Photosynthesis requires light-sensitive pigments called chlorophylls • Photoautotrophy requires ATP production and CO2 reduction © 2012 Pearson Education, Inc. Figure 13.1 PHOTOTROPHS (all use light as energy source) Use CO2 Photoautotrophs © 2012 Pearson Education, Inc. Use organic carbon Photoheterotrophs 13.1 Photosynthesis • Photoautotrophy – Oxidation of H2O produces O2 (oxygenic photosynthesis; Figure 13.2) – Oxygen not produced (anoxygenic photosynsthesis; Figure 13.2) © 2012 Pearson Education, Inc. Figure 13.2 Phototrophs Purple and green bacteria Cyanobacteria, algae, green plants Oxygenic Anoxygenic Reducing power Carbon electrons © 2012 Pearson Education, Inc. Energy Light Reducing power Carbon Energy Light 13.2 Chlorophylls and Bacteriochlorophylls • Organisms must produce some form of chlorophyll (or bacteriochlorophyll) to be photosynthetic (Figure 13.3a) • Chlorophyll is related to porphyrins • Number of different types of chlorophyll exist – Different chlorophylls have different absorption spectra (Figure 13.3b) © 2012 Pearson Education, Inc. Figure 13.3a Cyclopentanone ring Phytol Chlorophyll a © 2012 Pearson Education, Inc. Cyclopentanone ring Phytol Bacteriochlorophyll a Figure 13.3b 0.9 Chl a Bchl a 0.8 360 Absorbance 0.7 0.6 805 430 480 0.5 870 680 475 525 0.4 590 0.3 0.2 0.1 340 400 500 600 700 Wavelength (nm) © 2012 Pearson Education, Inc. 800 900 13.2 Chlorophylls and Bacteriochlorophylls • Cyanobacteria produce chlorophyll a (Figure 13.4) • Prochlorophytes produce chlorophyll a and b • Anoxygenic phototrophs produce bacteriochlorophylls • Chlorophyll pigments are located within special membranes – In eukaryotes, called thylakoids (Figure 13.5) – In prokaryotes, pigments are integrated into cytoplasmic membrane © 2012 Pearson Education, Inc. Figure 13.4 Pigment/Absorption maxima (in vivo) Bchl a (purple bacteria)/ 805, 830–890 nm Bchl b (purple bacteria)/ 835–850, 1020–1040 nm Bchl c (green sulfur bacteria)/745–755 nm Bchl cS (green nonsulfur bacteria)/740 nm Bchl d (green sulfur bacteria)/705–740 nm Bchl e aNo double bond between C3 and C4; additional H atoms are in positions C3 and C4. bP, Phytyl ester (C20H39O—); F, farnesyl ester (C15H25O—); Gg, geranylgeraniol ester (C10H17O—); S, stearyl alcohol (C18H37O—). (green sulfur bacteria)/719–726 nm cNo Bchl g dBacteriochlorophylls (heliobacteria)/ 670, 788 nm © 2012 Pearson Education, Inc. double bond between C3 and C4; an additional H atom is in position C3. c, d, and e consist of isomeric mixtures with the different substituents on R3 as shown. Figure 13.5 Outer membrane Inner membrane © 2012 Pearson Education, Inc. Stroma Thylakoid membrane Stacked thylakoids forming grana 13.2 Chlorophylls and Bacteriochlorophylls • Reaction centers participate directly in the conversion of light energy to ATP • Antenna pigments funnel light energy to reaction centers (Figure 13.6) • Chlorosomes function as massive antenna complexes (Figure 13.7) – Found in green sulfur bacteria and green nonsulfur bacteria © 2012 Pearson Education, Inc. Figure 13.6 LHI LHII © 2012 Pearson Education, Inc. Reaction center Figure 13.7 Bchl c, d, or e In BP FMO Out RC Membrane proteins © 2012 Pearson Education, Inc. 13.3 Carotenoids and Phycobilins • Phototrophic organisms have accessory pigments in addition to chlorophyll, including carotenoids and phycobiliproteins • Carotenoids (Figure 13.8) – Always found in phototrophic organisms – Typically yellow, red, brown, or green (Figure 13.9) – Energy absorbed by carotenoids can be transferred to a reaction center – Prevent photooxidative damage to cells © 2012 Pearson Education, Inc. Figure 13.8 H3C CH3 CH3 CH3 © 2012 Pearson Education, Inc. H3C CH3 CH3 CH3 H3C CH3 Figure 13.9 I. Carotenes Diaponeurosporene Neurosporene Lycopene -Carotene -Carotene Chlorobactene -Isorenieratene Isorenieratene II. Xanthophylls OH-Spheroidenone Spheroidenone Spirilloxanthin Okenone © 2012 Pearson Education, Inc. Heliobacteria Purple bacteria Green nonsulfur bacteria (Chloroflexus) Green sulfur bacteria Purple bacteria (in presence of air) Green sulfur bacteria (brown-colored species) 13.3 Carotenoids and Phycobilins • Phycobiliproteins are main antenna pigments of cyanobacteria and red algae – Form into aggregates within the cell called phycobilisomes (Figure 13.10) – Allow cell to capture more light energy than chlorophyll alone (Figure 13.11) © 2012 Pearson Education, Inc. Figure 13.10 Allophycocyanin Phycocyanin Thylakoid membrane Phycocyanin © 2012 Pearson Education, Inc. Figure 13.11 0.9 0.8 Chlorophyll a peaks Absorbance 0.7 0.6 Phycocyanin peak 0.5 0.4 0.3 0.2 0.1 340 400 500 600 700 Wavelength (nm) © 2012 Pearson Education, Inc. 800 13.4 Anoxygenic Photosynthesis • Anoxygenic photosynthesis is found in four phyla of Bacteria • Photosynthesis apparatus embedded in membranes (Figure 13.12) • Electron transport reactions occur in the reaction center of anoxygenic phototrophs (Figure 13.13) © 2012 Pearson Education, Inc. Figure 13.12 Vesicles Lamellar membranes © 2012 Pearson Education, Inc. Figure 13.13 Photosynthetic membrane H M L © 2012 Pearson Education, Inc. 13.4 Anoxygenic Photosynthesis • Reducing power for CO2 fixation comes from reductants present in the environment (i.e., H2S, Fe2+, or NO2) – Requires reverse electron transport for NADH production in purple phototrophs (Figure 13.14) – Electrons are transported in the membrane through a series of proteins and cytochromes (Figure 13.15) © 2012 Pearson Education, Inc. Figure 13.14 1.0 Strong electron donor 0.75 0.5 E0 (V) 0.25 Cyclic electron flow (generates proton motive force) 0.0 0.25 Poor electron donor 0.5 Red or infrared light © 2012 Pearson Education, Inc. External electron donors Figure 13.15 Light Out (periplasm) Quinone pool ATPase Photosynthetic membrane In © 2012 Pearson Education, Inc. (cytoplasm) 13.4 Anoxygenic Photosynthesis • For a purple bacterium (Figure 13.16) to grow autotrophically, the formation of ATP is not enough – Reducing power (NADH) is also necessary – Reduced substances such as H2S are oxidized and the electrons eventually end up in the “quinone pool” of the photosynthetic membrane (Figure 13.17) © 2012 Pearson Education, Inc. Figure 13.16 © 2012 Pearson Education, Inc. Figure 13.17 Purple bacteria Green sulfur bacteria Heliobacteria 1.25 1.0 0.75 0.5 E0 (V) 0.25 0 Reverse electron flow 0.25 0.5 Light Light © 2012 Pearson Education, Inc. Light 13.5 Oxygenic Photosynthesis • Oxygenic phototrophs use light to generate ATP and NADPH • The two light reactions are called photosystem I and photosystem II • “Z scheme” of photosynthesis (Figure 13.18) – Photosystem II transfers energy to photosystem I • ATP can also be produced by cyclic photophosphorylation © 2012 Pearson Education, Inc. Figure 13.18 1.25 The Z Scheme: 1.0 PSII PSI 0.75 Cyclic electron flow (generates proton motive force) 0.5 0.25 E0 0.0 (V) 0.25 Noncyclic electron flow (generates proton motive force) Light 0.5 Photosystem I 0.75 1.0 Photosystem II © 2012 Pearson Education, Inc. Light II. Chemolithotrophy • • • • • • 13.6 The Energetics of Chemolithotrophy 13.7 Hydrogen Oxidation 13.8 Oxidation of Reduced Sulfur Compounds 13.9 Iron Oxidation 13.10 Nitrification 13.11 Anammox © 2012 Pearson Education, Inc. 13.6 The Energetics of Chemolithotrophy • Chemolithotrophs are organisms that obtain energy from the oxidation of inorganic compounds • Mixotrophs are chemolithotrophs that require organic carbon as a carbon source • Many sources of reduced molecules exist in the environment • The oxidation of different reduced compounds yields varying amounts of energy © 2012 Pearson Education, Inc. 13.7 Hydrogen Oxidation • Anaerobic H2-oxidizing Bacteria and Archaea are known • Catalyzed by hydrogenase • Calvin cycle and hydrogenase enzymes allow chemolithotrophic growth (Figure 13.20) © 2012 Pearson Education, Inc. Figure 13.20 Membrane-integrated hydrogenase Out In Cytoplasmic hydrogenase © 2012 Pearson Education, Inc. Cell material 13.8 Oxidation of Reduced Sulfur Compounds • Many reduced sulfur compounds are used as electron donors (Figure 13.22a) • Discovered by Sergei Winogradsky • H2S, S0, S2O3 are commonly used • One product of sulfur oxidation is H+, which lowers of the pH of its surroundings • Sox system oxidizes reduced sulfur compounds directly to sulfate • Usually aerobic, but some organisms can use nitrate as an electron acceptor © 2012 Pearson Education, Inc. Figure 13.22a Electron transport Sox system APS reductase sulfide Electron transport Adenosine phosphosulfate (APS) sulfur thiosulfate sulfite sulfate Sulfite oxidase © 2012 Pearson Education, Inc. Substrate-level phosphorylation 13.8 Oxidation of Reduced Sulfur Compounds • Electrons from reduced sulfur compounds reach the electron transport system (Figure 13.22b) – Transported through the chain to O2 – Generates a proton motive force that leads to ATP synthesis by ATPase © 2012 Pearson Education, Inc. Figure 13.22b Out Reverse e flow In Cell material © 2012 Pearson Education, Inc. 13.9 Iron Oxidation • Ferrous iron (Fe2+) oxidized to ferric iron (Fe3+) • Ferric hydroxide precipitates in water • Many Fe oxidizers can grow at pH < 1 – Often associated with acidic pollution from coal mining activities (Figure 13.23) • Some anoxygenic phototrophs can oxidize Fe2+ anaerobically using Fe2+ as an electron donor for CO2 reduction © 2012 Pearson Education, Inc. Figure 13.23 © 2012 Pearson Education, Inc. 13.9 Iron Oxidation • Ferrous iron oxidation begins in the periplasm, where rusticyanin oxidizes Fe2+ to Fe3+ (Figure 13.24) • Rusticyanin then reduces cytochrome c, and this subsequently reduces cytochrome a • Cytochrome a interacts with O2 to form H2O • ATP is synthesized from ATPases in the membrane • Autotrophy in Acidithiobacillus ferrooxidans is driven by the Calvin cycle © 2012 Pearson Education, Inc. Figure 13.24 Out (pH 2) Outer membrane cyt c Rusticyanin Reverse e flow In (pH 6) Cell material © 2012 Pearson Education, Inc. 13.9 Iron Oxidation • Ferrous iron can be oxidized under anoxic conditions by certain anoxygenic phototrophic bacteria (Figure 13.25) © 2012 Pearson Education, Inc. Figure 13.25 © 2012 Pearson Education, Inc. 13.10 Nitrification • NH3 and NO2 are oxidized by nitrifying bacteria during the process of nitrification • Two groups of bacteria work in concert to fully oxidize ammonia to nitrate • Key enzymes are ammonia monooxygenase, hydroxylamine oxidoreductase, and nitrite oxidoreductase • Only small energy yields from this reaction – Growth of nitrifying bacteria is very slow © 2012 Pearson Education, Inc. 13.10 Nitrification • Ammonia-oxidizing bacteria (Figure 13.26) – NH3 is oxidized by ammonia monooxygenase producing NH2OH and H2O – Hydroxylamine oxidoreductase then oxidizes NH2OH to NO2 – Electrons and protons used to generate ATP © 2012 Pearson Education, Inc. Figure 13.26 Oxidation of hydroxylamine Out In Oxidation of ammonia © 2012 Pearson Education, Inc. Reduction of oxygen 13.10 Nitrification • Nitrite-oxidizing bacteria (Figure 13.27) – Nitrite (NO2) is oxidized by enzyme nitrite oxidoreductase to nitrate (NO3) – Electrons and protons used to generate ATP © 2012 Pearson Education, Inc. Figure 13.27 Periplasm Reverse e flow to make NADH Cytoplasm © 2012 Pearson Education, Inc. Oxidation of nitrite Reduction of oxygen 13.11 Anammox • Anammox: anoxic ammonia oxidation – Performed by unusual group of obligate aerobes (Figure 13.28a and b) – Anammoxosome is compartment where anammox reactions occur (Figure 13.28c) • Protects cell from reactions occurring during anammox • Hydrazine is an intermediate of anammox • Anammox is very beneficial in the treatment of sewage and wastewater © 2012 Pearson Education, Inc. Figure 13.28a © 2012 Pearson Education, Inc. Figure 13.28b Anammoxosome © 2012 Pearson Education, Inc. Figure 13.28c Anammoxosome membrane Out © 2012 Pearson Education, Inc. In Electron transport III. Major Biosyntheses: Autotrophy and Nitrogen Fixation • • • • 13.12 The Calvin Cycle 13.13 Other Autotrophic Pathways in Phototrophs 13.14 Nitrogen Fixation and Nitrogenase 13.15 Genetics and Regulation of Nitrogen Fixation © 2012 Pearson Education, Inc. 13.12 The Calvin Cycle • The Calvin cycle (Figure 13.29) – Named for its discoverer Melvin Calvin – Fixes CO2 into cellular material for autotrophic growth – Requires NADPH, ATP, ribulose bisphophate carboxylase (RubisCO), and phosphoribulokinase – 6 molecules of CO2 are required to make 1 molecule of glucose (Figure 13.30) © 2012 Pearson Education, Inc. Figure 13.29 Ribulose bisphosphate carboxylase (RubisCO) Ribulose bisphosphate Phosphoglyceric acid Unstable intermediate Two phosphoglyceric acid (PGA) Oxygenation reaction 1,3-Bisphosphoglyceric acid Glyceraldehyde 3-phosphate To biosynthesis Cycle repeats starting with (a) Phosphoribulokinase Ribulose 5-phosphate © 2012 Pearson Education, Inc. Ribulose bisphosphate Figure 13.30 12 3-Phosphoglycerate (36 carbons) RubisCO 6 Ribulose 1,5-bisphosphate (30 carbons) 12 1,3-Bisphosphoglycerate (36 carbons) Phosphoribulokinase 12 Glyceraldehyde 3-phosphate (36 carbons) 6 Ribulose 5-phosphate (30 carbons) Sugar rearrangements 10 Glyceraldehyde 3-phosphate (30 carbons) Fructose 6-phosphate (6 carbons) To biosynthesis Overall stoichiometry: 6 CO2 12 NADPH 18 ATP 12 NADP 18 ADP 17 Pi © 2012 Pearson Education, Inc. C6 H12 O6(PO3 H2) 13.13 Other Autotrophic Pathways in Phototrophs • Green sulfur bacteria use the reverse citric acid cycle to fix CO2 (Figure 13.32a) • Green nonsulfur bacteria use the hydroxypropionate pathway to fix CO2 (Figure 13.32b) © 2012 Pearson Education, Inc. Figure 13.32 Cell material Glyceraldehyde 3-P Hexose-P Oxalacetate Phosphoenolpyruvate Malate Fumarate Pyruvate Succinate Ferredoxinred Succinyl-CoA Ferredoxinred Acetyl-CoA Citrate -Ketoglutarate Net reaction: Isocitrate 3 CO2 12 H 5 ATP C3H6O3PO32 3 H2O Cell material (Acetyl-CoA) Malyl-CoA (Hydroxypropionyl-CoA) Glyoxylate Succinyl-CoA Net reaction: 2 CO2 4 H 3 ATP (Propionyl-CoA) (Methylmalonyl-CoA) © 2012 Pearson Education, Inc. C2H2O3 H2O 13.14 Nitrogenase and Nitrogen Fixation • Only certain prokaryotes can fix nitrogen • Some nitrogen fixers are free-living and others are symbiotic • Reaction is catalyzed by nitrogenase – Sensitive to the presence of oxygen • A wide variety of nitrogenases use different metal cofactors (Figure 13.33) © 2012 Pearson Education, Inc. Figure 13.33 Protein Homocitrate Protein © 2012 Pearson Education, Inc. 13.14 Nitrogenase and Nitrogen Fixation • Electron Flow in Nitrogen Fixation (Figure 13.34) – Electron donor dinitrogenase reductase dinitrogenase N2 – Ammonia is the final product © 2012 Pearson Education, Inc. Figure 13.34 Pyruvate Electron flow to nitrogenase Pyruvate flavodoxin oxidoreductase Flavodoxin (Oxidized) Flavodoxin (Reduced) Dinitrogenase reductase (Reduced) Dinitrogenase reductase (Oxidized) Dinitrogenase (Oxidized) Dinitrogenase (Reduced) Nitrogenase enzyme complex N2 reduction © 2012 Pearson Education, Inc. 13.14 Nitrogenase and Nitrogen Fixation • Unique nitrogenase of Streptomyces thermoautotrophicus (Figure 13.36) – A structurally and functionally novel molybdenum nitrogenase – S. thermoautotrophicus nitrogenase is completely insensitive to O2 • Nitrogenase activity can be assayed using the acetylene reduction assay (Figure 13.37) © 2012 Pearson Education, Inc. Figure 13.36 Mo Mo Superoxide formation CO dehydrogenase © 2012 Pearson Education, Inc. Nitrogenase Figure 13.37 Atmosphere, 10% C2H2 in air (aerobes) or 10% C2H2 in H2 or Ar (anaerobes) Acetylene Ethylene Chart recorder for gas chromatograph C2H2 Stoppered vial containing cell suspension © 2012 Pearson Education, Inc. Sample headspace periodically and inject into gas chromatograph C2H4 C2H2 C2H4 Nitrogenase Incubation C2H2 Time 0 1h 2h 13.15 Genetics and Regulation of Nitrogen Fixation • Highly regulated process because it is such an energy- demanding process • nif regulon coordinates regulation of genes essential to nitrogen fixation (Figure 13.38) • Oxygen and ammonia are the two main regulatory effectors © 2012 Pearson Education, Inc. Figure 13.38 Nitrogenase proteins Dinitrogenase reductase FeMo-co synthesis FeMo-co synthesis Denitrogenase reductase processing Regulators Mo processing Positive Negative Flavodoxin Dinitrogenase Homocitrate synthesis FeMo-co synthesis Electron transport Pyruvate flavodoxin oxidoreductase FeMo-co insertion into dinitrogenase Metal center biosynthesis nif DNA Q B A L F M Z W V S U X N E Y T K D H J RNA © 2012 Pearson Education, Inc.