An overview of Microbial metabolism What is Metabolism? ▪ The sum of controlled chemical reactions that occur within cells ▪ Metabolism includes catabolism and anabolism What is Metabolism? ▪ Catabolism (breakdown of nutrients) Provides energy and precursor metabolites for anabolism and other cellular functions ▪ Anabolism (biosynthesis) Uses energy and precursor metabolites of catabolism to make and assemble subunits of macromolecules for cellular structures Cells use redox reactions to extract energy from nutrient molecules such as glucose ▪ Redox reactions involve the transfer of electrons from an electron donor to an electron acceptor ▪ Redox reactions are oxidation-reduction reactions ▪ Most biological oxidations involve the transfer of hydrogen atoms (one electron plus one proton-H+) - dehydrogenation reactions Cells use redox reactions to extract energy from nutrient molecules such as glucose ▪ Cells transfer electrons and protons from the energy source (i.e., glucose) to electron carriers called coenzymes ▪ The energy of the reduced coenzyme NADH (or FADH2) is used to make ATP in later reactions ▪ ATP is made by ▪ Substrate-level phosphorylation ▪ Oxidative phosphorylation ▪ Photophosphorylation ▪ ATP synthesized during catabolism is used for: ▪ biosynthetic reactions or ▪ processes such as active transport across the plasma membrane The role of ATP as an intermediate between catabolism and anabolism Metabolic pathways and enzymes ▪ Chemical reactions are organized into series of sequential reactions called metabolic pathways ▪ Each step of a metabolic pathway is catalyzed by an enzyme, and cells control their synthesis and activity (allosteric enzymes) Enzymes are the catalysts of biological reactions ▪ Specific for a given substrate/chemical reaction ▪ The shape of the enzyme provides a distinctive site called the active site or catalytic site of the enzyme ▪ The substrate specifically fits into the enzyme’s active site Enzymes can use cofactors and coenzymes to carry out the catalytic activity ▪ Cofactors (i.e., magnesium, manganese, iron, copper, zinc, calcium, cobalt) ▪ Coenzymes (i.e., NAD+, NADP+, and FAD - derived from vitamins) function as electron carriers Enzymes lower the activation energy of chemical reactions ▪ The substrate specifically interacts with the active site of the enzyme to form an enzyme-substrate complex ▪ The substrate is converted into product(s), which is/are then released ▪ The enzyme is recovered unchanged Factors that affect the activity of enzymes ▪ Temperature ▪ pH ▪ Enzyme and substrate concentration ▪ Presence/absence of inhibitors ▪ Temperature and pH can denature proteins, including enzymes ▪ Denatured proteins are not functional!!! Enzyme inhibitors ▪ Inhibition can be reversible or irreversible ▪ Competitive inhibition ▪ Sulfa drugs are competitive inhibitors ▪ Sulfanilamide competes against PABA, an intermediate of the pathway for synthesis of folic acid (needed to make nucleic acids) ▪ A competitive inhibitor “competes” against the substrate for binding to the active site of the enzyme Enzyme inhibitors ▪ Inhibition can be reversible or irreversible ▪ Noncompetitive inhibition ▪ Poisons such as fluoride are noncompetitive inhibitors ▪ Non-competitive inhibition is also a mechanism cells use to regulate the activity of allosteric enzymes - regulators bind to the allosteric site(s) of an enzyme and can inhibit or activate it Catabolism of carbohydrates through cellular respiration or fermentation ▪ Most microorganisms oxidize carbohydrates as the primary source of cellular energy ▪ Two general processes are used ▪ Cellular respiration (aerobic/anaerobic respiration) ▪ Fermentation ▪ Cellular respiration and fermentation can share a common pathway called glycolysis or Embden-Meyerhof pathway Aerobic cellular respiration ▪ Glucose (C6H12O6) is completely oxidized to CO2 and water in presence of O2 (terminal electron acceptor) ▪ Steps involve ▪ Glycolysis ▪ Transition step ▪ Krebs cycle ▪ Electron transport chain ▪ ATP is made by ▪ substrate level phosphorylation ▪ oxidative phosphorylation Aerobic cellular respiration ▪ Glycolysis oxidizes glucose to two molecules of pyruvate, NADH and ATP are made ▪ Each pyruvate is converted to acetyl CoA/NADH is produced ▪ Acetyl group of acetyl CoA is oxidized to CO2 by the Krebs cycle, reducing NAD+ to NADH, FAD to FADH2, and producing ATP ▪ NADH and FADH2carry electrons to the electron transport chain - the energy released is eventually used to produce ATP How is ATP made during glycolysis or Krebs cycle? Substrate-level phosphorylation ▪ Direct enzymatic transfer of a phosphate group from a substrate molecule such as PEP to ADP Synthesis of ATP by oxidative phosphorylation ▪ NADH/FADH2 transfer electrons to the ETC ▪ Certain carriers in the ETC (as they transfer electrons down to O2) pump protons across the membrane E. coli ▪ The proton gradient built across the plasma membrane is a form of potential energy, which is called proton motive force ▪ The proton motive force powers synthesis of ATP by ATP synthase, (enzyme that catalyzes synthesis of ATP from ADP and P) ▪ The proton motive force powers also active transport and rotation of flagella The oxidase test you will learn in the lab!! E. coli ▪ The oxidase test is based on detecting presence or absence of the cytochrome c oxidase in the ETC and it enables us to distinguish between species: E. coli is oxidase negative, Pseudomonas and Neisseria are oxidase positive ▪ The mechanism of synthesis of ATP shown in the figure was proposed by P. Mitchell in 1961 (Nobel Prize in 1978) - Chemiosmotic theory How many molecules of ATP are made during aerobic respiration in bacteria? ▪ “Assuming” 3ATP/NADH and 2ATP/FADH2 ▪ Theoretical ATP yield/molecule of glucose oxidized to CO2/H2O ▪ 38 ATP ▪ Only 4 ATP produced by substrate level phosphorylation Fermentation ▪ Harvests energy from oxidation of organic molecules such as sugars ▪ Does not involve the Krebs cycle or the electron transport chain ▪ Glycolysis is the only pathway that produces ATP in fermentation ▪ Pyruvate/derivative is the final electron acceptor from NADH, regenerating NAD+for glycolysis Lactic acid and alcohol Fermentation ▪ Lactic acid bacteria include Streptococcus and Lactobacillus ▪ Alcohol fermentation carried out by Saccharomyces cerevisiae (yeast) End-Products of Fermentation ▪ Chemical analysis of the end-products of fermentation help identify microbes, including pathogens in clinical specimens Butyric acid Mixed acid Butanediol MR & VP tests How do we apply our knowledge of microbial metabolism to differentiate bacteria in the lab? ▪ Phenol red lactose broths after incubation Durham tube Control Positive Negative fermentation Proteus/Salmonella fermentation no gas production Positive fermentation gas production E.coli/Klebsiella/Enterobacter Microbes release enzymes to hydrolyze complex molecules ▪ Products are then transported into the cell for metabolism ▪ Proteases breakdown proteins to amino acids ▪ Amylases and cellulases degrade starch and cellulose (carbohydrates) ▪ Lipases hydrolyze lipids to glycerol and fatty acids Anabolic pathways and their link to catabolic pathways ▪ Most of the ATP produced during catabolism is used to synthesize new cellular components ▪ Anabolic pathways require also the precursor metabolites from glycolysis, Krebs cycle, transition step ▪ Precursor metabolites are intermediates of the catabolic pathways from which subunits of macromolecules or polymers can be made ▪ Amino acids for proteins, enzymes ▪ Carbohydrates for polysaccharides, peptidoglycan ▪ Glycerol/fatty acids for lipids in cell membranes ▪ Purines and pyrimidines for nucleotides of DNA/RNA Anabolic pathways and their link to catabolic pathways ▪ The figure provides one example of how cells use the precursor metabolites for anabolism ▪ The biosynthesis of lipids Dual metabolic role of the central catabolic pathways ▪ Pathways that function in both anabolism and catabolism are called amphibolic pathways ▪ Glycolysis ▪ Krebs cycle Catabolic versatility and energy yields!!! ▪ Aerobic respiration uses O2as terminal electron acceptor ▪ Anaerobic respiration uses nitrate, nitrite, or sulfate as a terminal electron acceptor ▪ Fermentation uses pyruvate or its derivatives as terminal electron acceptor to regenerate NAD+