Microbial Metabolism: Catabolism and Anabolism

• Microbial Metabolism: An overview

Metabolism

• The sum of the controlled chemical reactions within a cell
• Includes catabolism and anabolism

Catabolism

• The breakdown of nutrients
• Provides energy and precursor metabolites for anabolism and other cellular functions

Anabolism

• Biosynthesis
• Uses energy and precursor metabolites from catabolism
• Assembles subunits of macromolecules for cellular structures

Redox Reactions

• Cells use redox reactions to extract energy from nutrient molecules, such as glucose
• Involve the transfer of electrons from a donor to an acceptor
• Oxidation-reduction reactions
• Most biological oxidations involve the transfer of hydrogen atoms (one electron plus one proton-H+) - dehydrogenation reactions
• Cells transfer electrons and protons from an energy source (i.e., glucose) to coenzymes
• The energy of the reduced coenzyme NADH (or FADH₂) is used to make ATP in later reactions

ATP

• An intermediate between catabolism and anabolism
• Made by: substrate-level phosphorylation, oxidative phosphorylation and photophosphorylation
• Synthesized during catabolism and employed for biosynthetic reactions or processes like active transport

Metabolic Pathways

• Chemical reactions are organized into series of sequential reactions
• Each step in a metabolic pathway is catalyzed by an enzyme, and cells control their synthesis and activity (allosteric enzymes)

Enzymes

• Biological reaction catalysts
• Specific for a substrate or chemical reaction
• Provides a distinctive active or catalytic site
• Substrates specifically fit into the enzymes active site
• May use cofactors (magnesium, manganese, iron, copper, zinc, calcium, cobalt)
• May use coenzymes (NAD+, NADP+, FAD)
• Function as electron carriers

Enzyme Activity

• Lower the activation energy of chemical reactions
• The substrate interacts with the active site to form an enzyme-substrate complex
• The substrate is converted into products, and then released
• The enzyme is recovered unchanged
• Affected by temperature, pH, enzyme and substrate concentration, presence or 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 compete against PABA, an intermediate of the pathway for synthesis of folic acid
• Noncompetitive Inhibition allosteric
• Non-competitive inhibition is a mechanism for cells to regulate the activity of allosteric enzymes
• Regulators bind to the allosteric site(s), and can inhibit or activate it

Catabolism

• Most microorganisms oxidize carbohydrates as the primary source of cellular energy
• Two general processes are used; Cellular respiration (aerobic/anaerobic respiration) and Fermentation
• Cellular respiration and fermentation can share a common pathway called glycolysis or Embden-Meyerhof pathway

Cellular Respiration

• Aerobic: Glucose (C6H12O6) is completely oxidized to CO₂ and water in presence of O₂
• Process occurs in stages of Glycolysis, a Transition step, Krebs cycle, and finally the Electron transport chain
• ATP is commonly made by substrate level phosphorylation and oxidative phosphorylation
• Glycolysis oxidizes glucose to two molecules of pyruvate, NADH and ATP
• Each pyruvate is converted to Acetyl CoA/NADH
• Acetyl CoA is oxidized to CO₂ by the Krebs cycle, reducing NAD + to NADH, FAD to FADH2
• NADH/​FADH2 carry electrons to the electron transport chain-used to produce ATP

ATP Production

• NADH produces 2.5 ATP, and each molecule of FADH2 accounts for 1.5 ATP
• 32 ATP generated in bacteria
• Only 4 ATP produced via substrate-level phosphorylation

Substrate-Level Phosphorylation

• Direct enzymatic transfer of a phosphate group
• A substrate molecule such as PEP to ADP

Oxidative Phosphorylation

• NADH/FADH₂ transfer electrons to the electron transport chain (ETC)
• Carriers in the ETC transfer electrons to O₂, pumping protons across the membrane
• A proton gradient across the plasma membrane is a form of potential energy proton motive force
• Proton motive force powers ATP synthesis by ATP synthase, which catalyses synthesis of ATP from ADP and Pi
• Proton motive force also fuels active transport and rotation of flagella
• The oxidase test determines presence or absence of cytochrome c oxidase in the ETC
• E. coli is oxidase negative, but Pseudomonas and Neisseria are oxidase positive
• ATP Synthesis Mechanism: proposed by P. Mitchell in 1961

Fermentation

• Harvests energy from the 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
• Pyruvate or a derivative serves as the final electron acceptor from NADH, regenerating NAD+ for glycolysis

Lactic Acid Bacteria

• Includes Streptococcus and Lactobacillus

Alcohol Fermentation

• Carried out by Saccharomyces cerevisiae (aka yeast)

Fermentation End-Products

• Chemical analysis may identify microbes, including pathogens in clinical specimens

Phenol Red Lactose Broths

• Differentiates Bacteria
• Positive fermentation with no gas production.
• Positive fermentation with gas production.
• Negative fermentation.

Hydrolyzing Enzymes

• 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

• Most ATP produced during catabolism is used to synthesize new cellular components
• Requires precursor metabolites from glycolysis, Krebs cycle, and transition step
• Precursor metabolites are intermediates of the catabolic pathways from which subunits of macromolecules or polymers are made
• Some major constituents of macromolecules are: Amino acids for proteins, enzymes, Carbohydrates for polysaccharides, peptidoglycan, Glycerol/fatty acids for lipids in cell membranes and Purines and pyrimidines for nucleotides of DNA/RNA

Dual Metabolic Role

• Anabolic Pathways
• Amphibolic Pathways
• Glycolysis and Krebs cycle

Catabolism Versatility

• Aerobic respiration uses O₂ as 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+