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Fermentation: Catabolism of carbon in the absence of a terminal electron acceptor (like O 2 ) for electron transport chain Slide 2 Compare the E h for putting electrons onto O 2 vs. lactate Slide 3 The unusual fermentation of oxalate by Oxalobacter formigenes Thank goodness for this hard-working anaerobe in your gut: it degrades oxalate from amino acid catabolism, coffee, tea, fruits, veggies and helps prevent kidney stones!! You can lose it by taking doxycycline and other antibiotics, but can regain it by guess how? Slide 4 And now for something completely different! Slide 5 Photosynthesis and Autotrophy I.Photosynthesis A.General Aspects B.Classes of Photosynthetic Bacteria C. Mechanism of Photosynthesis 1. Anoxygenic Photosynthesis 2. Oxygenic Photosynthesis D.Halobacterium (light-driven H+ pump) II.Autotrophy A.General Aspects B.Types of Autotrophic Pathways Slide 6 Slide 7 PHOTOSYNTHESIS (Photoautotrophy) X CO 2 CH 2 O NADP + NADPH e- photon Excited state Ground state Slide 8 PHOTOAUTOTROPHY: 2 reactions 1. LIGHT CHEMICAL ENERGY (ATP) 2. CO 2 reduction Organic compounds Slide 9 Phototrophic Prokaryotes: the metabolic menu GroupReducing powerOxidized product Purple nonsulfur bacteriaH 2, reduced organicOxidized organics Purple sulfur bacteriaH 2 SSO 4 -2 Green sulfur bacteriaH 2 SSO 4 -2 Green non sulfur bacteria*H 2 SSO 4 -2 Heliobacteria**Lactate, organicsOxidized organics CyanobacteriaH 2 OO 2 Prochlorophytes***H 2 OO 2 *Most ancient? **Gram positive, heterotrophs ***Related to cyanobacteria Slide 10 Three types of photochemical energy capturing systems in microorganisms: 1.Carotenoid-based light-capturing system that is structurally similar to rhodopsin in eyes. In halophilic Archaea. 2.Anoxygenic (uses chlorophyll, no O 2 made) 3.Oxygenic (uses chlorophyll, splits water, generates oxygen) Slide 11 Carotenoid-based (bacteriorhodopsin) -no chlorophyll, no metals: protein with G-protein coupled receptor-like structure plus chromophore (retinal) -chromophore is a long-chain hydrocarbon with extensive conjugation -ancient protection for oxygenic phototrophs against toxic O2 -light-powered ion transfer Nagel et al. 2005. Mechanics of Biolenergetic Membrane Proteins 33: 863 Slide 12 Photosystems do not absorb at short enough wavelengths to split water, so must get e - s somewhere else. Cyclic: electrons run in closed circuit Slide 13 Photosystems can take light energy strong enough to split water. Non-cyclic (although cyclic can occur) Slide 14 Chlorophyll: Light Harvesting Molecule Porphyrin (like heme in cytochromes, but Mg instead of Fe) Bacteriochlorophyll: Absorbs at ~700 nm; allows light harvesting at depths where light is low and environment is anoxic Not enough energy to extract e - from H 2 O; must use H 2 S instead Eventually, chlorophyll evolved. Utilizes a short enough wavelength (680 nm) to split H 2 O and generate O 2. Slide 15 Consequence of oxyenic photosynthesis in evolution: *DNA absorbs UV at 260 nm; mutations occur *Some exant organisms are resistant to damaging radiation (e.g. Deinococcus radiodurans: survives 100 rad while 10 rads kills us D. radiodurans is resistant to chromosome shattering and mutation) -O 2 is a reactive molecule: O 2 - H 2 O 2 OH -At first, protected by Fe +2 (ferrous iron): Fe +2 + O 2 FeOH 3 Banded iron formations from Wittenoom Gorge in Australia Slide 16 Consequence of oxyenic photosynthesis in evolution: -Bacteria began evolving carotenoids: protection against singlet oxygen; convert to less toxic state -Eventually (at least 2 billion years ago), used up ferrous iron -Accumulation of O 2 in atmosphere -O 2 + sun (UV radiation) O 3 (ozone) -Ozone screened out wavelengths below 290 nm -Life could evolve on land, because water no longer necessary to screen out damaging/mutagenic UV radiation Slide 17 Production of Reactive Oxygen Species (ROS) During normal cellular respiration, oxygen is reduced to water and highly reactive superoxide ( O2- ). Reactive oxygen species react with nucleic acids, sugars, proteins and lipids - eventually leading to molecular degradation. Slide 18 Cellular Defense Mechanisms Prevent ROS Buildup. -Due to the oxygen rich environment in which proteins exist, reactions with ROS are unavoidable. -Superoxide dismutase, catalase, and glutathione peroxidase are natural antioxidants present in organisms which eliminate some ROS. Other molecules are antioxidants too (e.g. ascorbic acid, or Ignose/Godnose!) -Glutathione peroxidase catalyzes the reduction of peroxide by oxidizing glutathione (GSH) to GSSG. Slide 19 Detection of algal blooms from satellites via remote sensing: relies on reflected spectral properties of chlorophylls. Nutrient upwelling (El Nino) = phytoplankton blooms Slide 20 Compared to freshwater, nutrients (N, P, Fe) are limiting. Fewer cells found than in freshwater (only 10 6 /mL prokaryotes and 10 4 eukaryotes) Because oceans are huge, collective O 2 production and CO 2 fixation there is a major contributor to Earths carbon balance. Influence food chain, global climate Many marine microbes use light to drive ATP synthesis. Photic zone = upper 300 meters Oxygenic and anoxygenic photosynthesis Chlorophylls a and b (cyanobacteria and relatives; algae) Proteorhodopsin (very similar to bacteriorhodopsin but Bacteria, not Archaea) Photosynthesis in the open oceans Slide 21 Phototrophic Primary Producers (red = chlorophyll) Slide 22 Phototrophic Prokaryotes: 1.Purple nonsulfur bacteria 2.Green nonsulfur 3.Purple sulfur bacteria (sulfur inside cell) 4.Green sulfur bacteria (sulfur outside cell 5.Heliobacteria (G+ relatives of Clostridium, endospores, N 2 - fixation) 5.Cyanobacteria 6.Prochlorophytes 7.Halobacterium-type 1 group of photocapable prokaryotes in the Domain Archaea (the halobacteria = extreme halophiles [salt-loving]) Domain Bacteria Slide 23 Slide 24 Photosynthetic Prokaryotes GroupReducing powerOxidized product Purple nonsulfur bacteriaH 2, reduced organicOxidized organics Purple sulfur bacteriaH 2 SSO 4 -2 Green sulfur bacteriaH 2 SSO 4 -2 Green non sulfur bacteriaH 2 SSO 4 -2 Heliobacteria*Lactate, organicsOxidized organics CyanobacteriaH 2 OO 2 Prochlorophytes**H 2 OO 2 *Gram positive, heterotrophs **Related to cyanobacteria Slide 25 Chlorophyll Diversity Different absorbance maxima = different niches e.g. lower or higher in water column. Chlorophyll (cyanobacteria) = 680 nm Bchl a (purple bacteria) = 805, 870 Slide 26 Structure of bacteriochlorophylls Slide 27 Slide 28 Accessory pigments: Carotenoids Slide 29 Accessory pigments: Phycobilins Slide 30 Photosynthetic Membranes Reaction center chlorophyll -few -convert light energy to ATP Light harvesting chlorophyll -many - antenna -captures faint signal of low light environments Accessory pigments Carotenoids Phycobilins Slide 31 light harvesting complex in cyanobacteria, plants Slide 32 Mechanism of Photosynthesis 1) Anoxygenic Photosynthesis Cyclic Your text: Fig. 17.14, 17.15, and 17.18 Purple Bacteria Green Bacteria Heliobacteria Slide 33 Purple Bacteria (within phylum Proteobacteria) photosynthetic membranes are lamellae or tubes with the plasma membrane bacteriochlorophyll a or b accessory pigments are purple colored carotenoid pigments (see Fig. 12.5 in your text) may live as photoheterotrophs two types: 1. sulfur 2. nonsulfur Slide 34 Green Bacteria photosynthetic membranes are vesicles attached to but not continuous with the plasma membrane bacteriochlorophyll c, b, or e (small amount of a in LH and RC) accessory pigments are yellow to brown-colored carotenoids two types: 1. sulfur (green sulfur bacteria phylum) 2. nonsulfur (green nonsulfur bacteria phylum) Slide 35 Heliobacteria plasma membrane only (no specialized photosynthetic membranes) bacteriochlorophyll g Photoheterotrophs: require organic carbon These are the only Gram-positive photosynthetic bacteria Slide 36 Electron donors: H 2 S, Fe 2+, S 0, etc. Slide 37 Anoxygenic Photosynthesis Purple bacteria strong e - donor Slide 38 Cyclic NAD(P)H and ATP can be generated by PMF Purple bacteria Slide 39 Elemental sulfur globules outside filamentous cyanobacterium Oscillatoria limnetica Many cyanobacteria can use H 2 S as an electron donor for anoxygenic photosynthesis. Slide 40 Green bacterium (Chlorobium): external sulfur deposits Purple bacterium (Chromatium): internal sulfur deposits Slide 41 Variation on the Theme ATP onlyATP & NAD(P)H * * * Off to supply reducing power for CO 2 fixation via reverse citric acid cycle ATP only Slide 42 Green Sulfur Bacteria (Chorobium, Chlorobaculum, Prosthecochloris) Aquatic, anoxic environments Most are facultative heterotrophs; strict autotrophy requires reverse TCA cycle Have chlorosomes: very efficient at light harvesting so live at great depths May form consortia aggregates of cells that have differing metabolic duties; chemotrophic and phototrophic (epibiont) components. Example: Chlorochromatium aggregatum (not a formal taxonomic name because not a single species) Slide 43 Green Non Sulfur Bacteria (Choroflexus) Filamentous, form microbial mats with cyanobacteria in neutral to alkaline hot springs Like Green Sulfur Bacteria: has chlorosomes But reaction center of in cell membrane is like purple bacteria Earliest known photosynthetic bacterium: perhaps reaction center first, chlorosome later by HGT Most are facultative heterotrophs; CO 2 fixation requires hydroxypropionate pathway (unique to very ancient organisms) Slide 44 Light harvesting complex in green photosynthetic bacteria (both sulfur and non-sulfur) Chlorosome is a giant antenna: Bchl c, d, or e BP = baseplate (proteins) LH = light harvesting complex (Bchl a) RC = reaction center (Bchl a) Slide 45 Chlorosomes (EM, stained dark) -in green sulfur bacteria -lie along the inside of cytoplasmic membrane -proteinaceous (nonlipid) membrane -each vesicle contains ~ 10,000 bacteriochlorophyll c molecules in tubes/rods -chlorosomes transmit energy via subantenna of bacteriochlorophyll a. Slide 46 Mechanism of Photosynthesis Oxygenic Photosynthesis Photosystems I & II Noncyclic Your text, Fig. 17.19 Cyanobacteria Algae (protists) Plants Slide 47 Cyanobacteria (phylum contains cyanobacteria and prochlorophytes) Synechococcus, Oscillatoria, Nostoc, Anabaena photosynthetic mebranes are multilayered lamellae formerly called blue-green algae but now known to be prokaryotic and possess peptidoglycan chlorophyll a only accessory pigments are carotenoids and phycobilin proteins Photosystem I and II are present (oxygenic photosynthesis) Autotrophs Gas vesicles frequent Some are filamentous, N 2 fixing (heterocysts) Slide 48 Lake Mendota up close: eutrophic (nutrient-rich) lake algal blooms July through September (ag runoff) Slide 49 Slide 50 Electron donor: H 2 O Slide 51 Slide 52 Halobacterium-type Use light-driven proton pump consisting of patches of the pigment bacteriorhodopsin in cytoplasmic membrane bacteriorhodopsin resembles rhodopsin, the visual pigment Absorbs light near 570 nm (green region of spectrum) Extreme halophile (2-4M NaCl = 12-23%): balances Na + outside with K + inside to maintain osmotic equilibrium Heterotrophs (use amino acids and organic acids for growth) Most are obligate aerobes; some can do anaerobic respiration or fermentation Slide 53 Solar Salt Evaporation Ponds (salterns) in CA Red coloration due to carotenoids of halobacteria Slide 54 Colonies of halobacteria isolated from Portsmouth salt piles. Plates contain 25 % NaCl ! Slide 55 Halobacteria Domain Archaea Not autotrophs - grow as chemoheterotrophs but can function as phototrophs Bacteriorhodopsin, proteorhodopsin = cytoplasmic membrane-associated photopigment similar to rhodopsin of mammalian eye. Bacteriorhodopsin is a light driven ion (proton) pump... Homologous protein in Halobacteria is called halorhodopsin; a chloride pump Oops, wrong, outdated hypothesis Slide 56 Light at 570 nm excites the retinal chromophore of bacteriorhodopsin, converting it from its normal all-trans conformation to a cis form. Conversion instigates the movement of a proton across the membrane. Proton loss returns retinal to its all-trans form. Light + H + = cis Loss of H + = trans Correct; see next slide Chloride ions flow across membrane in reverse direction for halorhodopsin Slide 57 Arrangement of bacteriorhodopsin in the cytoplasmic membrane: Purple structures are proteins (opsin) that hold the chromophore (retinal) Slide 58 Current model for how bacteriorhodopsin and halorhodopsin work Biochemical studies show that rather than transporting H + out, bacteriorhodopsin (BR) may actually transport OH - in and halorhodopsin (HR) may transport in a Cl - (from all that NaCl in its environment) Bacteriorhodopsin and its retinal chromophore. Yellow arrow indicates direction of ion transfer. Bacteriorhodopsin in the cell membrane. CP = cytoplasm, EC = extracellular space. Arrows indicate direction of ion transfer. Slide 59 Autotrophy General Aspects Heterotrophs: organisms requiring organic compounds as a carbon source Autotrophs: organism capable of biosynthesizing all cellular material from CO 2 ; CO 2 as a sole carbon source Slide 60 Autotrophy Types of Autotrophic Pathways 1. Calvin Cycle Fig. 17.21 & 17.22 2. Acetyl-CoA Pathway Fig. 17.41 3. Reverse TCA Cycle Fig.17.24a 4. Hydroxypropionate Pathway Fig. 17.24b Slide 61 Calvin-Benson Cycle Fig. 17.21 & 17.22 Key enzymes: A. Ribulose biphosphate carboxylase (RuBisCo) carboxyosomes : Inclusion bodies B. Phosphoribulokinase Slide 62 Calvin-Benson Cycle Cyanobacteria Key enzymes: ribulose biphosphate carboxylase (RuBisCo) = first enzyme, phosphoribulokinase = final enzyme in cycle Slide 63 Requires ATP and reducing power Slide 64 Reverse TCA Cycle some methanogens Green Sulfur bacteria (Chlorobium) Slide 65 Hydroxypropionate Pathway Green Non-Sulfur Bacteria (Chloroflexus)