Biol 3400 Tortora et al- Chap 5 Microbial Metabolism I. Metabolism • Metabolism is all of an organism's chemical processes (an emergent property that arises from interactions of molecules in the orderly environment of the cell) • Metabolism is very important for the management of cellular material and energy resources Metabolic reactions • Metabolic reactions are organized into pathways of enzyme controlled chemical reactions. • Cells need a supply of molecules and energy • Cells need to get rid of waste products Catabolic pathways • Break down complex molecules into simple molecules • Energy stored in complex molecules is made available to do work or transformed into readily usable chemical forms (i.e., ATP) • small molecules resulting from the catabolism of complex energy rich molecules may be used by the cell to build new molecules • e.g., cellular respiration Energy stored in compounds can be used to perform cellular work • mechanical - movement of cilia, chromosomes, organelles • transport - movement of substances across membranes • chemical - endergonic reactions Anabolic pathways • Use energy for the biosynthesis of complex molecules from simple molecules. • Energy is obtained from usable chemical forms of energy (i.e., ATP) produced during catabolic processes or from energy released during catabolic processes • e.g., synthesis of macromolecules Note: some pathways may function both catabolically and anabolically – these pathways are known as amphibolic pathways Review the Enzymes Section on your own (Pages 115 to 121) 1 Biol 3400 Tortora et al- Chap 5 II. Metabolic diversity among microorganisms • Life is based on organic molecules made of carbon skeletons • Oxygen and hydrogen are important elements of organic compounds • Electrons are needed i) for processes that provide energy (e.g., movement of electrons along energy transport chains and during oxidation reduction reactions) for cellular work and ii) to reduce molecules during biosynthesis • Molecules that serve as a source of carbon may also provide a source of oxygen and hydrogen • Microbes show an incredible ability to use organic molecules as carbon sources Organisms can be classified based on their sources of carbon, energy and electrons Carbon Source o Autotroph – CO2 is the sole or principal carbon source o Heterotroph – reduced, preformed, organic molecules from other organisms Energy Source o Phototrophs – Light o Chemotrophs – oxidation of organic or inorganic compounds Electron Source o Lithotrophs – reduced inorganic chemicals o Organotrophs – Organic molecules Major Nutritional Types of Microorganisms Carbon Energy Electron Examples Source source source 1. Photolithoautotrophy CO2 Light Inorganic Cyanobacteria, • aka - Photoautotrophs e- donor Purple sulfur bacteria 2. Photoorganoheterotrophy Organic Light Organic Purple nonsulfur • aka - Photoheterotrophs carbon but e- donor bacteria and Green CO2 may be nonsulfur bacteria used 3. Chemolithoautotrophy CO2 Inorganic Inorganic Sulfur oxidizing • aka - Chemoautotrophs chemicals e- donor bacteria, methanogens, nitrifying bacteria 4. Chemolithoheterotrophy Organic Inorganic Inorganic Some sulfur - carbon but chemicals e donor oxidizing bacteria CO2 may be Used 5. Chemoorganoheterotrophy Organic Organic Organic Most nonphotosynthetic • aka – Chemoheterotrophs carbon chemicals e- donor microbes including often the often the most pathogens, fungi, same as same as many protist and many C-source C-source archaea 2 Biol 3400 Tortora et al- Chap 5 III Heterotrophic (Chemoorganotrophic) Metabolism • Conversion of organic substrate molecules to end products by a metabolic pathway that releases sufficient energy for it to be coupled to the formation of ATP. • Chemoorganotrophs have three options for generating ATP from organic molecules; the electron acceptor used differentiates these processes: i) aerobic respiration, ii) anaerobic respiration and iii) fermentation i. Respiration • An external terminal electron acceptor is present and is not derived from the organic substrate • Involves the activity of an electron transport chain, proton motive force (PMF) is generated and ATP produced predominantly by oxidative phosphorylation a) Aerobic - O2 is the terminal electron acceptor. C6H12O6 + 6 O2 → 6 CO2 + 6 H2O + (ATP + Heat) - 2- b) Anaerobic - compounds other than O2 serve as electron acceptor (e.g., NO3 , SO4 , CO2, fumarate,…) *Some microbes can carry out both aerobic and anaerobic respiration – dependent upon the conditions ii. Fermentation • An external terminal electron acceptor is absent • Fermentation does not use an electron transport chain or the generation of a PMF • Fermentations are internally balanced oxidation-reduction reactions – i.e., the terminal electron acceptor is derived from the initial substrate or electron donor (e.g., glucose) • The terminal electron acceptor is required to balance redox reactions • Net result is energy production and an internally balanced redox reactions • ATP produced predominantly by substrate-level phosphorylation 3 Biol 3400 Tortora et al- Chap 5 A. Respiration 1. Glycolytic pathways • Breakdown of sugars to pyruvate and similar intermediates • Some production of ATP (substrate-level phosphorylation) and reducing power (reduced coenzymes; NADH) • Several pathways by which a cell can break down a sugar (sugars are the major substrates of catabolic energy releasing reactions used in heterotrophic metabolism). • Glycolytic pathways are typically anoxic processes that do not require oxygen • The end-product of glycolysis is commonly pyruvate COOH | C = O | CH3 Glycolytic pathways i). Embden-Meyerhof pathway (EMP) • Most common pathway - Central metabolic pathway for eukaryotic cells and many bacteria Net reaction 10 enzymatic steps Glucose + 2 ADP + 2 Pi + 2 NAD+→ 2 pyruvate + 2 ATP + 2 NADH + 2 H2O • ATP production - Substrate level phosphorylation • Phosphofructokinase is key enzyme in regulating this process, catalyzing the conversion of fructose – 6-P to fructose 1,6-bisphosphate ii). Entner-Doudoroff pathway • Mainly used by Gram negative soil bacteria and a few other Gram-negative bacteria as well as some Archaea • Lacks 6-phosphofructokinase (i.e., a key enzyme in Embden-Meyerhof pathway) Net reaction 9 enzymatic steps Glucose + ADP + Pi + NADP+ + NAD+ → 2 pyruvate + 1 ATP + NADPH + NADH + 1 H2O *NADPH is usually used in biosynthetic pathways 4 Biol 3400 Tortora et al- Chap 5 iii). Pentose Phosphate pathway • Can occur at the same time as the Embden-Meyerhof or the Entner-Doudoroff pathways • connects the metabolism of 6-C and 5-C sugars • Consumes 1 ATP • Products = reducing power (NADPH) and small molecules required for biosynthesis • 5-C sugars produced (e.g., ribose 5-phosphate; xylulose 5-phosphate) • Erythrose 4-phosphate is used to synthesize aromatic amino acids and vitamin B6 • Intermediates of the pathway may be fed into the EMP to produce ATP iv). Methylglyoxal pathway • Alternative to Embden-Meyerhof pathway during conditions of low phosphate availability • Consumes 2 ATP but produces pyruvate that can be used to generate ATP 2. Oxidation of pyruvate to 3 CO2. i) Initial Step + Pyruvate + NAD + CoA → Acetyl-CoA + NADH + CO2 • Three step process mediated by multienzyme pyruvate dehydrogenase complex • Acetyl CoA is a very unstable and reactive product. • Acetyl CoA feeds it's acetate → TCA cycle • Carbohydrates, fatty acids and amino acids may be converted into acetyl CoA during aerobic respiration ii) Tricarboxylic acid cycle (TCA cycle; also known as Citric acid cycle, Krebs cycle) • Discovered by Hans Krebs - 1930s • 8 steps involved in the TCA cycle • 2 C enter in a relatively reduced form – acetate and two different C leave in a completely oxidized form (CO2) • acetate joins the cycle by enzymatic addition to oxaloacetate (4 C) → formation of citrate • cyclical process resulting in the regeneration of oxaloacetate by the decomposition of citrate and evolution of 2 CO2 per acetate + • oxidation steps (transfer of electrons) of one acetate results in reduction of 3 NAD to 3 - NADH and 1 FAD to FADH2 (like NADH it donates its e to the electron transport chain but at a lower energy level) • One step for the production of GTP (substrate level phosphorylation; GTP can be converted to ATP) Review with Figure 5.13 5 Biol 3400 Tortora et al- Chap 5 + pyruvate + 4 NAD + FAD → 3 CO2 + 4 NADH + 1 FADH2 + 1 GTP • TCA cycle source of key biosynthetic intermediates e.g., oxaloacetate and α-ketoglutarate are precursors to a number of amino acids acetyl-CoA → starting material for fatty acid biosynthesis 3. Oxidative phosphorylation • Reducing power (NADH and FADH2) is used to generate a proton gradient (proton motive force) • NADH - FADH2 are oxidized – electron transport carrier proteins are reduced and in the process H+ are moved across the plasma membrane (prokaryotes) or inner mitochondrial membrane (eukaryotes). This results in a proton gradient or proton motive force across the membrane. The movement of H+ across the membrane is not completely understood Chemiosmosis • Proton Gradient driving ATP synthesis • Peter Mitchell (Nobel prize in Chemistry in 1978) ATP synthase (ATPase) • F0 subunit – multimeric membrane spanning proton conducting channel • F1 – multimeric headpiece → inside of the membrane • Catalyze ADP + Pi → ATP (oxidative phosphorylation) • Highly conserved throughout all domains of life • Can also drive reverse reaction ATP → ADP + Pi • explains why some obligate fermenters have ATPase - creates proton gradients that can be used to drive