LECTURE 3 MICROBIAL METABOLISM 1 Outline of metabolism

LECTURE 3. MICROBIAL METABOLISM 1. Outline of metabolism 2. Energy and carbon sources 3. Metabolic categories 4. “Our” metabolism: chemoorganoheterotrophy 5. Fermentation 6. Electron transport (respiratory) chains 7. Oxidative phosphorilation 8. The potential tower: donors and acceptors 9. Inorganic donors: chemolithotrophy 10. Main chemolithotrophy types 11. Main anaerobic respiration types 12. Photosynthesis 13. Autotrophy 14. N 2 fixation L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

1. GENERAL OUTLINE CARBON SOURCE ENERGY SOURCE Organic carbon CO 2 Organic matter Inorganic matter Light ATP REDUCING POWER OTHER NUTRIENTS PRECURSOR METABOLITES Fatty acids Sugars Aminoacids Nucleotides CELL WALL STORAGE MATERIALS RIBOSOMES Lipids Polysacharides Protein Nucleic acids MEMBRANES FLAGELLA, PILI DNA, RNA N, P, S… BIOSYNTHESIS POLYMERIZATION ASSEMBLAGE L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

2. ENERGY AND CARBON SOURCES An ENERGY SOURCE is a molecule (either organic or inorganic) that can be oxidized using its electrons to obtain energy in the form of ATP (or PMF) and reducing power. The energy source is thus an ELECTRONIC DONOR. It always needs an ELECTRONIC ACCEPTOR to obtain energy. When an organism uses chemical compounds as an energy source, it is called a CHEMIOLITHOTROPH; when it uses light, a PHOTOTROPH. As we shall see, the light is used to create an electronic donor. A CARBON SOURCE is either CO 2 or an organic molecule and its role is to provide C atoms needed to build the cell molecules. When an organism uses CO 2 as a carbon source, it is called an AUTOTROPH; when it uses organic matter, a HETEROTROPH. 3. METABOLIC CATEGORIES L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

4. “OUR” METABOLISM: CHEMOORGANOHETEROTROPHY PROTEINS AMINOACIDS NH 4+ CARBOHYDRATES MONOSACCHARIDES FATTY ACIDS PIRUVATE COMPEX MOLECULES (REDUCED) CATABOLISM (PRODUCES ENERGY) ACETIL-Co. A SIMPLE MOLECULES (OXIDIZED) CO 2 PRECURSORS CELL COMPONENTS ANABOLISM (USES ENERGY) CARBON AND ENERGY SOURCES? L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

4. “OUR” METABOLISM: CHEMOORGANOHETEROTROPHY CARBON AND ENERGY SOURCES ENERGY ELECTRONIC TRANSPORT CHAIN COMPLEX MOLECULES ADP ATP COMPLEX EXTERNAL NAD+ FAD MOLECULES CELLULAR e- NADH FADH 2 CATABOLISM (OXIDATION) H 2 0 e. REDUCING POWER O 2 ANABOLISM (REDUCTION*) ELECTRONIC ACCEPTOR EXTERNAL MOLECULE e. NADPH “SIMPLE” MOLECULES NAPD+ PRECURSORS … In this case, organic molecules are being used as energy source. This is typical of chemo-organotrophic organisms L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

4. “OUR” METABOLISM: CHEMOORGANOHETEROTROPHY Example: glucose metabolism NADH FADH 2 RIBULOSE NAPD+ e- NADPH GLUCOSE PYRUVATE ATP e- NAD + FAD ee- H 2 0 e- ACETIL-Co. A m. o. AMINOACIDS O 2 CO 2 m. o. + O 2 INTERM. KREBS cycle POLYSACCHARIDES ADP CO 2 + H 2 O + ATP Cellular material LIPIDS L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

5. FERMENTATION Example: lactic fermentation of glucose NAD+ e- e. NADH GLUCOSE 2 G-3 -P ADP Oxidation state ENERGY SOURCE PYRUVATE 2 P-ENOLPIRUVATE LACTATE ATP SUBSTRATE LEVEL PHOSPHORILATION ACCEPTOR REDUCED ACCEPTOR WASTE PRODUCTS L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

5. FERMENTATION CHEMO-ORGANOTROPH ORGANIC COMPOUND (REDUCED) e. NAD+ FAD NADH FADH 2 e- ORGANIC C. PARTIALLY OXIDIZED EXTERNAL e- ACCEPTOR e. ATP ORGANIC C. PARTIALLY REDUCED NADH FADH 2 NAD+ FAD H+ c. t. e- ADP e- e- H+ Acceptor (oxidized) Acceptor (reduced) FERMENTATION ATP synthesis only by substrate level phosphorilation ATP NADPH NO EXTERNAL e- ACCEPTOR ADP NADP+ RESPIRATION L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

5. FERMENTATION TYPES OF FERMENTATIONS L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

QUESTIONS TO BE ANSWERED BY THE STUDENTS What are the main characteristics of fermentations? Can you think of their ecological implications? L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

6. (RESPIRATORY) ELECTRON TRANSPORT CHAINS PROTON MOTIVE FORCE (PMF) DONOR (A MOLECULE ABLE TO GET OXIDIZED) OXIDIZED DONOR OUTSIDE*… MEMBRANE (What membrane? ) INSIDE*… PROTON PUMP ACCEPTOR (A MOLECULE ABLE TO GET REDUCED) REDUCED ACCEPTOR TRANSPORTADORES: proteínas de membrana capaces de aceptar electrones desde el transportador anterior y ceder electrones al transportador siguiente. Están ordenados en la membrana según su potencial red-ox. En el transporte de electrones a favor de gradiente se libera energía; algunos transportadores (bombas de protones) usan esta energía para transportar protones de un lado a otro de la membrana. tbtbs L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

6. (RESPIRATORY) ELECTRON TRANSPORT CHAINS DONOR: ORGANIC INORGANIC EXCITED PHOTOPIGMENT DONOR (A MOLECULE ABLE TO GET OXIDIZED) TYPE OF METABOLISM? CHEMO-ORGANOTROPHY CHEMO-LITOTROPHY PHOTOTROPHY OXIDIZED DONOR ACCEPTOR (A MOLECULE ABLE TO GET REDUCED) ACCEPTOR: INORGANIC: OXYGEN OTHERS ORGANIC NADP+ OR OXIDIZED PIGMENT REDUCED ACCEPTOR TYPE OF RESPIRATION? AEROBIC ANAEROBIC PHOTOSYNTHESIS L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

6. (RESPIRATORY) ELECTRON TRANSPORT CHAINS H 2 S, NO 2 -, NH 4+, Fe 2+, … CHEMO-LITOTROPHY SO 42 -, NO 3 -, NO 2 -, Fe 3+, … Organic molecules Photo-excited pigment Oxidized C (CO 2) CHEMO-ORGANOTROPHY Non-excited pigment PHOTOSYNTHESIS e. DONOR OXIDIZED DONOR ACCEPTOR REDUCED ACCEPTOR e. AEROBIC RESPIRATION ANAEROBIC RESPIRATION PHOTOSYNTHESIS O 2 H 2 O SO 42 -, NO 3 -, NO 2 -, CO 2, Fe 3+, … Organic molecules Oxidized pigment / NADP+ H 2 S, NO 2 -, NH 4+, CH 4, Fe 2+, … “Reduced” organic molecules Reduced pigment / NADPH L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

7. OXIDATIVE PHOSPHORILATION La ATPasa o ATP SINTETASA utiliza la energía liberada en el transporte de protones (la energía de la fuerza protón-motriz) para unir un Pi a una molécula de ADP, obteniendo ATP. Este proceso es una FOSFORILACIÓN. Puesto que la fuerza protón-motriz se origina mediante un proceso de oxidación-reducción de transportadores en la cadena respiratoria, a esta fosforilación se le denomina FOSFORILACIÓN OXIDATIVA. En la fotosíntesis, se denomina específicamente FOTOFOSFORILACIÓN OXIDATIVA o FOTOFOSFORILACIÓN. Existe otro mecanismo generador de energía en forma de ATP, que es la FOSFORILACIÓN A NIVEL DE SUSTRATO, que NUNCA está acoplado a una cadena de transporte de electrones ni a una ATPasa y que, por tanto, no se considerará una fosforilación oxidativa. tbtbs L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

IMPORTANCE OF PROTON MOTIVE FORCE L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

8. POTENTIAL TOWER: DONORS AND ACCEPTORS (reduction potentials) Eo (-) Eo (+) The energy released by the redox reaction depends on the distance in the tower between the pairs involved, Go’= -n. F Eo’ L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

For each pair: OXIDIZED/REDUCED FORM Where are the best acceptors? And the best donors? And the best energy sources? “Bad” acceptors/ “Good” donors “Good” acceptors/”Bad” donors L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

9. INORGANIC DONORS: CHEMO-LITOTROPHY ATP AND REDUCING POWER Donor NAD(P)+ FPM/ATP NAD(P)H Uses PMF Donor FPM/ATP O 2 Electronic donors: H 2, HS-, S 0, NH 4+, NO 2 -, Fe+2 H 2 O L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

9. INORGANIC DONORS: CHEMO-LITOTROPHY REVERSE ELECTRON TRANSPORT Uses FPM Produces FPM Reducing power Acceptor Energy source (donor) L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

10. TYPES OF CHEMOLITOTROPHY 10. 1. NITRIFICATION Donors: NH 4+, NO 2 - Nitrification NH 4+ Nitrosomonas NO 2 - Nitrobacter NO 3 - Acceptor: O 2 Thaumarquea L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

10. TYPES OF CHEMOLITOTROPHY 10. 1. ANAMMOX Anaerobic ammonia oxidation: ANAMMOX NH 4+ + NO 2 donor N 2 + 2 H 2 O acceptor Brocardia anammoxidans L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

10. TYPES OF CHEMOLITOTROPHY 10. 2. SULFUR OXIDATION (NON PHOTOSYNTHETIC) Donors H 2 S, S 0, S 2 O 32 -, metallic sulfides S 0 can accumulate (inside or outside) as a storage material L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

10. TYPES OF CHEMOLITOTROPHY 10. 3. HYDROGEN OXIDATION Donor: H 2 2 hydrogenases (soluble and transmembrane) – Cofactor: Ni Microaerophilic Good or bad donor? L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

10. CHEMOLITHOTROPHY TYPES 10. 4. METAL OXIDATION (IRON, COPPER, MANGANESE, …) Metal oxidizers must consume large amounts of substrate (energy source)… WHY? L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

10. CHEMOLITHOTROPHY TYPES L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

11. ANAEROBIC RESPIRATIONS 11. 1. DENITRIFICATION L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

11. ANAEROBIC RESPIRATION 11. 2. SULFUR AND SULFATE REDUCTIONS 1º/ Sulfate activation to form APS 2º/ APS acts as acceptor Donors H 2 m. o. L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

11. ANAEROBIC RESPIRATIONS 11. 3. METHANOGENESIS (ONLY IN ARCHAEA-METHANOGENS) Three substrates: -CO 2 type (plus H 2) -Methyl compounds -Acetate L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

11. ANAEROBIC RESPIRATIONS 11. 4. IRON REDUCTION Acceptors Donor Acceptor Donor L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

12. PHOTOSYNTHESIS 12. 1. OXYGENIC PHOTOSYNTHESIS REACTION CENTRE PIGMENT: CHLOROPHYLL a L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

12. PHOTOSYNTHESIS 12. 1. OXYGENIC PHOTOSYNTHESIS FPM/ATP Reducing power Water photolysis ATP / NADPH: Non-cyclic photophosphorylation ATP: Cyclic photophosphorilation (only PS I) Electronic donor Proton motive force L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

12. PHOTOSYNTHESIS 12. 1. OXYGENIC PHOTOSYNTHESIS Non cyclic photophosphorylation Eo (-) Cyclic photophosphorylation EXCITED PHOTOSYNTHETIC PIGMENTS Eo (-) e- EXCITED PHOTOSYNTHETIC PIGMENTS e- ATP ADP Eo (+) NON-EXCITED PHOTOSYNTHETIC PIGMENTS e- T. C. NADPH e- T. C. NADP+ Eo (+) NON-EXCITED PHOTOSYNTHETIC PIGMENTS e. LIGHT O 2, SO NO EXTERNAL e- DONORS H 2 O, H 2 S L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

PHOTOSYNTHESIS vs RESPIRATION CHEMICAL COMPOUND Eo (-) (REDUCED) e- PHOTOSYNTHETIC PIGMENTS EXCITED e- ENERGY SOURCE e- ATP NADPH NADP+ ADP c. t. e- Eo (+) Acceptor (reduced) Acceptor (oxidized) Eo (+) NADPH NADP+ NON-EXCITED LUZ Electron transport chain Reducing power Proton motive force ATP by oxidative phosphorylation PHOTOSYNTHETIC PIGMENTS ENERGY SOURCE Electron transport chain Reducing power Proton motive force ATP by oxidative photophosphorylation L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

12. PHOTOSYNTHESIS 12. 2. ANOXYGENIC PHOTOSYNTHESIS ATP: cyclic photophosphorylation REACTION CENTRE PIGMENT: BACTERIOCHLOROPHYLL L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

12. PHOTOSYNTHESIS 12. 2. ANOXYGENIC PHOTOSYNTHESIS Generation of reducing power (DONOR NEEDED): - Direct transfer from H 2 (HYDROGENASE) - Non-cyclic electronic flow (from the oxidation of reduced S compounds) - Reverse electronic flow (from S and Fe compounds) L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

12. PHOTOSYNTHESIS 12. 3. RETINAL-BASED PHOTOSYNTHESIS BACTERIORHODOPSIN / PROTEORHODOPSIN WARNING!! Bacteriorhodopsin in Archaea; Proteorhodopsin in Bacteria L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

13. AUTOTROPHY ENERGY SORCE CO 2 Inorganic matter Light Los organismos AUTÓTROFOS fijan CO 2. La fijación es un reducción de CO 2 a carbono orgánico, por lo que se necesita ATP y poder reductor en el proceso. Esta energía y estos electrones son aportados por la fuente de energía, que únicamente será la luz o la materia inorgánica. Sólo los organismos FOTOTROFOS Y QUIMIOLITOTROFOS fijan CO 2 (aunque no todos son necesariamente autótrofos). tbtbs CO 2 fixation paths in prokaryotes: CALVIN CYCLE FIXATION ATP REDUCING POWER PRECUSOR METABOLITES Cyanobacteria, “purple bacteria” and chemolithotrophs Product: sugar REVERSE CITRIC ACID CYCLE Green sulfur bacteria Fatty acids Sugars Aminoacids Nucleotides Product: acetyl-Co. A HYDROXYPROPIONATE CYCLE Green non-sulfur bacteria Lipids Polysaccharides Proteins Nucleic acids Product: glyoxylate ACETYL-Co. A PATHWAY Sulfate-reducing bacteria and other anaerobes Product: acetyl-Co. A L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

Ru. Bis. CO: 13. AUTOTROPHY CALVIN CYCLE Ribulose 1, 5 -bisphosphate carboxylase/oxygenase L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

13. AUTOTROPHY REVERSE CITRIC ACID CYCLE L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

13. AUTOTROPHY HYDROXYPROPIONATE CYCLE L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

13. AUTOTROPHY ACETYL-Co. A PATHWAY L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014

IN SUMMARY… (BBE) 1 2 CHEMIOLITHOHETEROTROPH (1) CHEMIOLITHOATOTROPH (2) CHEMOORGANOHETEROTROPH Respiration (aerobic/anaerobic) or fermentation Respiration (aerobic/anaerobic) 2 1 PHOTOHETEROTROPH (1) PHOTOAUTOTROPH (2) L. 3: Metabolic diversity in prokaryotes. Microbiology. 2 nd Biology ARA 2013 -2014