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Microbial Photosynthesis Michael Kühl Marine Biological Laboratory [email protected] Check: www.mbl.ku.dk/mkuhl for publication downloads etc. 1 Halobacteria live at >20% salinity in places like the Dead Sea and in Salterns and salt lakes. Energy generation and consumption of Halobacteria Purple ”photosynthetic” membrane areas Energy consumption Red membrane areas with aerobe respiration Energy generation 2 3 Benthic diatoms 4 5 Chlorophyll a is the key photopigment in oxygenic photosynthesis Antenna pigments Other chlorophylls have the same structure but different side groups and protein-associations cause different spectral absorption properties. 6 Different algal groups have different antenna pigments Eukaryotic phototrophs evolved via endosymbiosis from prokaryotic oxygenic phototrophs. The only known oxygenic prokaryotes are cyanobacteria. 7 ”Farbstreifen Sandwatt” 5 mm Foto: Lucas Stal 8 The key enzyme in N2-fixation is nitrogenase which is inhibited/destroyed by oxygen Types and characteristics of nitrogen-fixing cyanobacteria Type I Heterocystous (e.g. Anabaena, Nostoc, Nodularia, Calothrix, Scytonema) • Exclusively filamentous species with heterocysts • Strategy: Spatial separation of N2 fixation and photosynthesis and protection of nitrogenase in the heterocyst • Diazotroph growth under fully oxic conditions • Occurence; Lakes and brackish water, paddy fields, microbial mats, in symbiosis with plants and animals Type II Anaerobic N2-fixing non-heterocystous (e.g. Plectonema, Oscillatoria, Synechococcus, many more) • Both filamentous and unicellular species • Strategy: Avoidance of oxygen. Only induction and maintenance of nitrogenase when no or low O2 • Occurence: In many different environments but unclear if always growing diazotrophically Not all N2-fixing cyanobacteria have heterocysts! Type III Aerobic N2-fixing non-heterocystous (e.g. Oscillatoria, Trichodesmium, Lyngbya, Microcoleus) • Both filamentous and unicellular species • Strategy: Not precisely known. Temporal separation of N2 fixation and oxygenic photosynthesis? Spatial organization and behavioral oxygen protective mechanisms? • Diazotrophic growth under fully oxic conditions • Occurence: Tropical ocean (Trichodesmium), paddy fields, microbial mats 9 Phenotypic characteristics of Cyanobacteria Pigments: Chlorophyll-a, ß Carotene, c-Phycoerythrin, Allophycocyanin, cphycocyanin, other chlorophylls absent Nuclear material: DNA is free in the central region of the cell (nucleoplasm) and is not enclosed in a membrane Food reserves: Cyanophycean starch, cyanophycin granules (argenine and aspartic acid) Thylakoid features: Chloroplast absent; the thylakoids are free in the cytoplasm and unstacked; phycobilisomes present Cell wall: Four-layered peptidoglycan wall in which murein is the principal component Flagella Absent Aktions-spektrum Phycoerythrin and Phycocyanin are import antenna pigments of Cyanoabacteria Fykobiliner Karotenoider Klorofyl Phycoerythrin Phycocyanin -1 -1 Fotosyntese (µmol ilt l min ) Klorofyl 200 Kiselalger 150 100 50 Cyanobakterier 0 400 450 500 550 600 650 700 Lysets bølgelængde (nm) 10 Prochloron spp. Ascidian with Prochloron symbionts Lissoclinum patella 10 µm 0.8 15 •One of 3 separate lineages of prochlorophytes in cyanobacteria. •Contains Chl a & b as major photopigments. No phycobilins. •Not cultivated – many attempts without success. •Discovered in 1975, lives in symbiosis with ascidians. Chl a 0.6 10 0.4 Chl b Absorbance 10 µm Relative absorbance Prochloron 0.2 5 0.0 300 400 500 600 700 Wavelength (nm) •Prochlorothrix •Prochlorococcus (late 1980’s) (special Chl a2 & b2) Kühl et al. in prep. Phenotypic characteristics of Prochlorophytes Pigments: Chlorophyll-a + Chlorophyll-b, ß Carotene, Zeaxanthan, Cryptoxanthin, no phycobiliprotein pigments Nuclear material: DNA is free in the cytoplasm and is not enclosed in a membrane, it is not central as in Cyanophyta but is rather diffuse throughout the cell. Kühl & Larkum 2002 Prochlorophytes as the missing link? Based on phenotypic characteristics like pigmentation and organization of thylakoids. Food reserves: Cyanophycean starch; no cyanophycin granules Thylakoid features: Chloroplast absent; the thylakoids are free in the cytoplasm and stacked in groups of two or more; phycobilisomes absent Cell wall: Four-layered peptidoglycan wall in which murein is the principal component Based on genotypic characteristics of 16S rRNA genes. Flagella Absent 11 Habitat of Acaryochloris marina Acaryochloris marina •Cyanobacterium isolated from didemnid ascidians. •Contains Chl d as the major photopigment – also in the reaction centers! •Minor amounts of Chl a and phycobilins. Absorbance (a.u.) 10 µm Chlorophyll d 400 500 600 700 800 Wavelength (nm) •Assumed a symbiont, but recently also found epiphytic on red algae, but niche unknown until recently... Can use NIR Kühl et al. 2005 Inorganic carbon is fixed in the Calvin cycle in oxygenic phototrophs b c 1.0 d 0.5 1.0 0.5 0.0 100 0.0 Fluorescence (a.u.) Acaryochloris-like cells Scalar irradiance (% of incident irradiance) Prochloron Absorbance (a.u.) Habitat of Acaryochloris marina a Murakami et al. 2004 Rubisco !! e 50 0 400 500 600 700 800 Wavelength (nm) Kühl et al. 2005 12 Photosynthesis: 2 Types • Oxygenic – Plants, algae, cyanobacteria – Light energy to generate ATP and reduce CO2 to synthesize carbohydrates and release molecular oxygen CO2 + 2H2O + light energy -> [CH2O] (carbohydrate) + O2 + H2O • Anoxygenic –Other types of photosynthetic bacteria – Light energy used to create ATP and reduced organic/inorganic compounds to generate reducing power for carbon fixation. Does not release oxygen, does not use water CO2 + 2H2A + light energy -> [CH2O] + 2A + H2O e.g. 2H2S e.g. 2S 13 Two types of reaction center are involved in photosynthesis Type 1 Type 2 Chlorophyll-based Bacteriochlorophyll a is the key photopigment in anoxygenic photosynthesis Other bacteriochlorophylls have the same structure but different side groups and protein-associations cause different spectral absorption properties. 14 The color of anoxygenic phototrophs is strongly affected by their carotenoids Type 1 Type 2 Type 1 15 Chlorosomes are efficient light collectors in green photosynthetic bacteria Group of bacteria Electron donor for photoautotrophy Chlorophylls Photoheterotrophy? Chemotrophy? Anoxygenic Photosynthetic Bacteria Purple Sulfur Bacteria of the family Chromatiaceae Purple Sulfur Bacteria of the family Ectothiorhodospiraceae Purple Non-Sulfur Bacteria (family Rhodospirillaceae*) Green Sulfur Bacteria (including family Chlorobiaceae*) Bchl a & b S– or So or H2 (So globules formed inside cell from S–) some species some species Bchl a & b S– or H2 (So globules formed outside cell from S–) possibly all species some species Bchl a & b Prob. all: H2 . Some: low levels of S–, S2O3– , So all species probably all species or (So globules formed outside cell from S–) potentially all species none ? (photoautotrophy?) all species probably all species S– mainly Bchl c, d or Multicellular Filamentous Green Bacteria (including family Chloroflexaceae) e one or more of Bchl a, c, d So Oxygenic Photosynthetic Bacteria Cyanobacteria Chl a (&d) H2O some species? some species -Prochlorophytes Chl a & b H2O ? prob. none Group of bacteria Light harvesting Reaction center Preferred growth mode Anoxygenic Photosynthetic Bacteria Green filamentous bacteria Chloroflexus-subdivision (3) Bchl a & c + car (Chlorosomes) Type II Anoxygenic photo-organo-heterotrophic Aerobic chemo-organo.heterotrophic Green Sulfur Bacteria (15) Bchl a, c, d, e + car (Chlorosomes) Type I Anoxygenic photo-litho-autotroph a-proteobacteria (31) Bchl a , b + car (Intracell. Membranes) Type II Anoxygenic photo-organo-heterotroph Aerobic chemo-organo-heterotroph Bchl a Type II Aerobic chemo-organo-heterotroph Type II Anoxygenic photo-organo-heterotroph Aerobic chemo-organo-heterotroph Aerobic a-proteobacteria (23) b-proteobacteria (4) Purple Sulfur Bacteria Chromatiaceae (31) Ectothirhodospiraceae(9) Heliobacteriaceae (5) Bchl a + car (Intracell. Membranes) Bchl a & b + car (Intracell. Membranes) Bchl g + car Type II Type I Anoxygenic photo-litho-autotroph Anoxygenic photo-organo-heterotroph Oxygenic Photosynthetic Bacteria Cyanobacteria (>1000) Prochloron, Prochlorotrhix (2 Prochlorococus (1) Acaryochloris (1) Chl a +phycobilins + car (thylakoid membranes) Chl a & b + car Type I + II Oxygenic photo-litho-autotroph Chl a2 &b2 + car (+PBS) Chl d, a + car (+PBS) 16 Inorganic carbon is fixed in the Calvin cycle by anoxygenic purple bacteria Inorganic carbon is fixed in the reverse citric acid cycle by anoxygenic green sulphur bacteria Rubisco !! Inorganic carbon is fixed in the hydroxy-propionate pathway by anoxygenic green non-sulphur bacteria. Classification Purple Bacteria (Proteobacteria) Green Filamentous (nonsulfur) bact. Green Sulfur Bacteria Heliobacteria (gram + bact.) Antenna Carotenoids in spirilloxanthin, okenone, or rohodopinal groups LH1 & LH2 complexes Bchl c arranged in chlorosomes to harvest light, Carotenoids gamma or beta carotene (isorenieratene & chlorobactene groups), LH1 Chlorosomes funnel light to RC, carotenoids (& bachl c, d, e) in isorenieratene & chlorobactene groups Carotenoids – neurosporene Chlorophyll Bacteriochlorophyll Bacteriochlorophylls c or d (sm. amt. of a) a&b Bacteriochlorophyll Bacteriochlorophyll g c, d or e, (sm. amt. of a) Electron flow Reverse e- flow (reverse Krebs Cycle) Reverse e- flow (reverse Krebs Cycle) cyclic cyclic Rubisco None None None Hydroxy-propionate pathway. Some reverse TCA Reverse TCA None, no Calvin Cycle, no reverse TCA, photoheterotrophs +Rubisco Carbon fixing Calvin Cycle (but can use reverse TCA, tricarboxylic acid cycle {citric acid cycle}, fixes C into organic molecules used for metabolites or cellular components) Believed to have common ancestor Lateral transfer of phototrophy from one to the other, or from common ancestor to descendents of both lines 17 Classification Purple Bacteria (Proteobacteria) Green Gliding (nonsulfur) bact. Green Sulfur Bacteria Heliobacteria (gram + bact.) Ecology Nonsulfur bact.: grow aerobically by respiration on organic source of carbon in dark, Sulfur bact: must fix CO2 Facultatively aerobic: aerobic- live heterotrophically, not photosynth., anaerobicphotosynthetic, do not fix N Photolithotrophic, CO2 as sole C source (can use acetate), strict anaerobes, obligate phototrophs Obligate anaerobe, sensitive to O2, photoheterotrophic Can’t tolerate sulfide, rarely aquatic, fix N Produce O2? No, anoxygenic No, anoxygenic No, anoxygenic No, anoxygenic Photosynth. Type Like photosystem II, quinone-type Like photosystem II, quinone-type Like photosystem I Fe-S Like photosystem I Fe-S Electron Donor H2S, H2 & other H2S, organics Sulfide & organic hydrogen donors Organic donors Electron acceptor Quinone, Fe Quinone, Mn between quinones between quinones Ferredoxin FeS Reaction Center P870, Bchl a P840, Bchl a Heterodimeric, adequate to reduce ferredoxin, can reduce NAD+ to NADH directly P789, homodimeric P840, Bchl a, carotenoids not in RC, homodimeric, lacks H subunit Sulfuretum in Nivå Dense blooms of anoxygenic phototrophs occur in stratified waters with a thermo- and/or halocline and anoxic bottom water. Sulfuretum in Nivå 18 ”Farbstreifen Sandwatt” 5 mm Foto: Lucas Stal Differential light utilisation governs coexistence Cyanobacterial layer Purple bacterial layer 1.0 Carotenoids Carotenoids Chlorofyll a 0.8 Absorbance Chlorofyll a 0.6 Bacteriochlorofyll a 0.4 Bacteriochlorofyll c Phycobilins 0.2 Bacteriochlorofyll a & c 0.0 400 500 600 700 800 900 400 500 600 700 800 900 Wavelength (nm) 19 Pigment Chl a Absorption maxima (nm) Fluorescence maxima (nm) Cells extract Cells 670-675 435, 663 680-685 Chl b 655 455, 645 - Chl d 714-718 400, 697 740-760 Bchl a 375, 590, 805, 830-911 358, 579, 771 907-915 Bchl b 400, 605, 835-850, 986-1035 368, 407, 582, 795 1040 Bchl c 457-460, 745-755 433, 663 775 Bchl d 450, 715-745 425, 654 763 Bchl e 460-462, 710-725 459, 648 738 Bchl g 375, 419, 575, 788 365, 405, 566, 762 - ”Farbstreifen Sandwatt” 5 mm Foto: Lucas Stal 20 Microscale light measurements Fiber-optic Microsensors Microprobes (A-D) for: - radiance, irradiance, scalar irradiance (UVNIR light) - Surface detection - Pigment fluorescence - Diffusivity/Flow Micro-opt(r)odes (E) for: - O2, pH, CO2, temperature All based on multimode graded index optical fibers 100/140 µm core/cladding N.A. = 0.22 Kühl & Revsbech 2001 Field radiance measurements Microscale light measurements Collimated light 48o 60o 0o Downwelling light Forward scattered light 140o Back scattered light 21 Light-collecting properties of fiber-optic microprobes Scalar irradiance probeA 160 Irradiance probe B 100 140 120 80 100 60 80 60 40 40 20 20 0 -180 -120 -60 0 60 120 180 0 -180 -120 Angle of incident light -60 0 60 120 180 Angle of incident light Kühl et al. 1997 Strong light attenuation due to absorption and scattering PAR (% of Ed) 0 0.01 50 0.1 1 100 150 10 100 0 K 0 (λ ) = − d ln[E0 (λ )] =− dz -1 Kühl & Jørgensen 1992. Mapping the spatial light distribution ln ⎡ ⎢⎣ E 0 ( λ )1 ⎤ E0 (λ ) 2 ⎥⎦ z 2 − z1 Collimated light K0 (mm ) 5 48o 10 60o 0o 140o 0.0 Downwelling light Depth (mm) % of average response Microscale light measurements Forward scattered light Back scattered light 0.5 1.0 1.5 2.0 A B Kühl et al. in prep. 22 Directional vs. Diffuse light From one direction Integral from all directions Collimated light 48o 60o 0o Downwelling light Spektral lysnedtrængning Fykobilin Back scattered light Experimental set-up for O2-measurements Light source Bakterieklorofyl Klorofyl Klorofyl Skalar irradians (% af indfaldende lys) Forward scattered light 140o 200 150 0.0 O2 microsensor 0.2 100 0.4 50 0.6 0.8 1.0 0 400 500 600 700 800 Lysets bølgelængde (nm) 23 Aktions-spektrum Diatoms Fykobiliner Karotenoider Klorofyl -1 -1 Fotosyntese (µmol ilt l min ) Klorofyl 200 Cyanobacteria Kiselalger 150 100 50 Cyanobakterier 0 400 450 500 550 600 650 700 Lysets bølgelængde (nm) Light and Photosynthesis Oxygen (µmol O2 l-1) -1 0 200 400 600 Scalar irradians (µmol photons m-2 s-1) 0 100 200 300 800 Water Biofilm Depth (mm) 0 Photosynthesis Oxygen 2 -1 0 Light 1 Photosynthesis in steep light gradients 1 2 3 3 0 2 4 Photosynthesis (nmol O2 cm-3 s-1) 6 8 Kühl & Jørgensen 1992 24 Carotenoids protect against photooxidation Absorption vs. Action spectrum Activated chlorophyll and oxygen forms radicals that can break down proteins, lipids and other Chl* + O2 → Chl + O2* key components of cells Chl + radiation → Chl* Chl* + carotenoids → Chl + carotenoids* O2* + carotenoids → O2 + carotenoids* Carotenoids* → carotenoids + heat Types of photobehaviour Behaviour Measured Quantity Speed Photokinesis positive negative Single Cell Effect light intensity I Colony Effect Ecological Significance Accumulation in dark areas Avoiding photo damage Accumulation in illuminated areas (Optimizing photosynthesis) Intensity Speed Intensity Photophobic Response step-up step-down change in light intensity dI dt Phototaxis positive direction of light negative I bimodal Trapping in dark areas reversal of direction Trapping in illuminated areas Moving towards light source Moving away from light source Moving perpendicular to light direction Avoiding photo damage Positioning in benthic systems Optimizing photosynthesis Positioning in benthic systems Optimizing photosynthesis Moving to surface in pelagic systems Moving to the bottom in pelagic systems Keeping depth in pelagic systems 25 ”Algography” 26 Different light optima for different phototrophs Gradient-capillary-cell-tracking-setup Motility of Microorganisms in Response to Light, Oxygen, and Sulfide Gradient Capillary Setup video camera flat glass capillary (40 x 8 x 0.8 mm3) end of tubing microsensors for pH reference electrode video recorder oxygen sulfide pH gas space sulfidic agar plug oxygen-microelectrode with Picoammeter inverted microscope medium with bacteria The setup is mounted on a light microscope which allows computer-aided cell tracking via digital video recordings 27 M. gracile: dark-light transition Marichromatium gracile Cell distribution in relation to oxygen, sulfide, and pH gradients anoxic ca. 500µm oxic pH 7 .0 50 0 D arkness pH 6 .8 40 0 6 .6 rel. ce ll de n sity 30 0 20 0 H2S O2 10 0 0 0 1 2 3 4 d ista nc e (m m ) 5 6 Thar & Kühl 2001 Thar & Kühl 2001 Marichromatium gracile Phobic responses towards increasing oxygen concentrations and darkness Response to light-dark border Response to oxygen gradient Thar & Kühl 2001 28 UV radiation and it’s effects on organisms UV-C (<280 nm) • Strongly absorbed in the atmosphere, no ecological relevance. UV-B (280-320 nm) • Direct damage on biological chromophores due to absorption in: DNA, (thymin-dimers) Enzymes Lipids Photosystems. UV-A (320-400 nm) • indirect damage via photodynamic reactions caused by free radicals, especially reactive oxygen species •Similar damage is induced by high levels of visible light. Importance of UV radiation Habitat Z(1%) UV-B mm Z(1%) Visible mm Ē0 (UV-B) %incident Max E0 (UV-B) %incident K (UV-B) mm-1 Sediment Beach sand (wet) 1.25 3.10 15 127 4.1 (dry) 0.98 2.40 23 131 6.5 Sandy sediment 0.36 0.72 24 103 17.2 Cyanobacterial mat 0.50 0.95 25 105 10.5 Muddy sediment 0.23 0.45 33 103 21.6 Sargasso Sea 25 x 103 150 x 103 6 - 0.12 x 10-3 Southern Ocean 17 x 103 120 x 103 3 - 0.26 x 10-3 1 x 103 3 x 103 9 - 4.60 x 10-3 Water Wadden Sea UV light is present in a larger part of the photic zone in sediments than in the photic zone of natural waters UV protection – sunscreen pigments, Scytonemin and MAA’s UV radiation effect on photosynthetic microbial mat Gross productivity scytonemin Cell+sheath Net productivity Sheath 29 UV-induced migration of cyanobacteria UV-induced migration of cyanobacteria + UV - UV 1 mm cyanobacteria Cyanobacteria 30