MICR 221 Lecture 11 Metabolism and Transport - PDF

Lecture 11: Metabolism and Nutrient Transport Jan. 30, 2025 1 Lecture Learning Outcomes After this lecture, students will be able to describe… The different carbon, energy, and electron sources used by bacteria The general steps of aerobic respiration...

Lecture 11: Metabolism and Nutrient Transport Jan. 30, 2025 1 Lecture Learning Outcomes After this lecture, students will be able to describe… The different carbon, energy, and electron sources used by bacteria The general steps of aerobic respiration, anaerobic respiration, and fermentation Why bacterial metabolism is a difficult antibiotic target, and how sulfa drugs and trimethoprim work The different uptake systems used by bacteria to allow nutrients through the cytoplasmic and outer membranes 2  Carbon Cells mostly built from organic molecules (carbon backbones) Microbes classified based on carbon source Heterotrophs use organic molecules Autotrophs use CO2 Carbons used to make precursor metabolites (simple organic molecules) Intermediates in metabolic pathways Used to build more complex organic molecules 3 Image from: Prescott’s Microbiology, 12th Edn Building a Bacterium Energy and electrons are needed to make precursor metabolites, and to assemble them into macromolecules (e.g., proteins, nucleic acids) 4 Energy and ATP Energy is used to power chemical processes Energy also needed for transport (e.g., importing nutrients), mechanical work (e.g., flagellar rotation) Classify microbes based on energy source: Chemotrophs: organic or inorganic molecules Phototrophs: light Energy from energy source is commonly conserved in the form of ATP 5  Reducing Power (Electrons) Building some organic molecules requires reducing power Also can be used to make ATP (electron transport chain) Classify microbes based on electron source: Organotrophs: reduced organic molecules Lithotrophs: reduced inorganic molecules Electrons funneled to electron carriers NAD+, FAD Makes NADH and FADH2 (reduced forms) 6 Image from: https://commons.wikimedia.org/wiki/File:NAD_oxidation_reduction.svg Other Nutrients Elements other than carbon needed Macronutrients (large amounts) C, O, H, N, S, P Building blocks (amino acids, etc) K, Ca, Mg, Fe (cations) Co-factors Stabilize cellular structures (e.g., cell wall) Micronutrients (small amounts) Aka trace elements Co, Cu, Mo, Mn, Zn, Ni (cations) Co-factors 7  Microbial Metabolism Most bacteria on human body are chemoorganohetero- trophs Get carbon, energy, electrons from organic molecules Make ATP by oxidizing reduced organic molecules Aerobic respiration Anaerobic respiration Fermentation Make ATP with electron transport chain Part of aerobic, anaerobic respiration 8 Image from: https://doi.org/10.1016/B0-08-045044-X/00088-2 Aerobic Respiration During aerobic respiration: Energy conserved as ATP Electrons collected in NADH, FADH2 Precursor metabolites are made Glycolytic pathways (glycolysis) Glucose oxidized, makes pyruvate Makes ATP, NADH Tricarboxylic acid (TCA) cycle Acetyl-CoA oxidized, makes CO2 Makes ATP, NADH, FADH2 Electron transport chain (ETC) Electrons from NADH, FADH2 used to make ATP 9 Glycolytic Pathways Embden-Meyerhof pathway Most common (also in animals) Makes pyruvate, ATP, NADH Entner-Doudoroff pathway Occurs in some bacteria Makes glyceraldehyde 3- phosphate (G3P), pyruvate, NADPH G3P enters Embden-Meyerhof Pentose phosphate pathway Often used for biosynthesis Makes NADPH Makes precursor metabolites for nucleotides, amino acids 10 Tricarboxylic Acid Cycle Pyruvate dehydrogenase complex converts pyruvate into acetyl-CoA Acetyl-CoA enters tricarboxylic acid (TCA) cycle Acetyl-CoA oxidized, producing CO2 Makes GTP (ATP), NADH, FADH2 Some intermediates used as precursor metabolites 11  Electron Transport Chain (ETC) Electrons in NADH, FADH2 used to make more ATP Transferred to membrane-bound electron carriers Ubiquinone (coenzyme Q, CoQ) Cytochromes (proteins with heme co-factors) Lower redox potential = more likely to give up electrons Each carrier is reduced by preceding carrier, oxidized by following carrier Increasing redox potentials 12 Image from: Molecular Cell Biology, 6th Edn  Electron Transport Chain in E. coli Electrons transferred from NADH to ubiquinone (Q) Electrons pass through series of cytochromes E.g., b562, o Finally, passed to terminal electron acceptor Aerobic respiration: O2 Electron transfers drive transport of protons into periplasm Generates proton-motive force (PMF) 13 Image from: Prescott’s Microbiology, 11th Edn  Proton Motive Force and ATP Synthase PMF: potential energy due periplasm/ to proton, charge gradient extracellular across membrane Cytoplasm is relatively alkaline, negatively charged cytoplasm Protons flow down gradient into cytoplasm, performing work E.g., travel through ATP synthase, making ATP 14 Image from: Prescott’s Microbiology, 11th Edn  Anaerobic Respiration Similar to aerobic respiration Glycolysis, TCA cycle, ETC Makes ATP, NADH, and FADH2 Except, terminal electron acceptor is not O2 Instead: nitrate, sulfate, CO2, others E.g., Paracoccus denitrificans reduces nitrate to N2: 15 Image from: Prescott’s Microbiology, 11th Edn  Fermentation Some bacteria lack (or repress) an ETC Have to use NADH for something else Fermentation: pathway in which substrate is partially oxidized (e.g., by glycolysis) Makes ATP But, no ETC and no exogenous electron acceptor Electrons usually transferred from NADH to pyruvate or a pyruvate derivative Regenerates NAD+ 16 Image from: Prescott’s Microbiology, 11th Edn  Bacterial Metabolism and Antibiotics We are chemoheterotrophs Much of human microbiota are chemoheterotrophs Use our food as nutrients Use us as nutrients Humans and human microbiota often use similar metabolic pathways Difficult to selectively target bacterial metabolic enzymes Toxicity 17 Image from: https://www.nature.com/articles/d42859-019-00015-1 Tetrahydrofolate Synthesis Tetrahydrofolate is a cofactor Needed to make purines and pyrimidines (DNA/RNA), methionine (amino acid) Humans acquire folates from diet (vitamin B9) Some bacteria have to make their own tetrahydrofolate Folate biosynthetic enzymes are antibiotic targets 18 Sulfa Drugs and Trimethoprim Sulfa drugs (e.g., SMX) inhibit dihydropteroate synthase Resemble p-aminobenzoic acid Competitive inhibitors Trimethoprim inhibits dihydrofolate reductase Resembles dihydrofolic acid Block tetrahydrofolate production, inhibit growth Can be combined for synergy (e.g., TMP/SMX) 19 Nutrient Uptake Nutrients must be collected from environment Often limited abundance, lots of competition Cell envelope is a good barrier Hydrophilic nutrients can’t pass through Protein channels and transporters often needed 20 Energy Independent Transport Diffusion mechanisms don’t require input of energy Substances move down concentration gradient Not very useful (concentration usually lower outside) Some substances diffuse across cytoplasmic membrane, others diffuse through carrier proteins 21  Primary Active Transport Active transport: transport against concentration gradient Requires energy input Primary active transporters use energy from ATP hydrolysis ATP-binding cassette (ABC) transporters Common primary active transporters in bacteria Importers used for sugars, other nutrients Exporters expel substances from cell 22 Image from: Prescott’s Microbiology, 11th Edn  ABC Transporter Solute Binding Proteins Most ABC transporters work with solute-binding proteins (SBPs) SBPs deliver a specific substrate to transporter Gram-negatives: SBP is in periplasm Gram-positives: SBP is lipoprotein or associated with PG 23 Image from: Prescott’s Microbiology, 11th Edn  Secondary Active Transport Potential energy from an ion gradient can power transport of a substance against its own gradient Ion gradient can be generated in several ways: Electron transport chain (PMF) V-type ATPases Energy from ATP hydrolysis creates H+ or Na+ gradient Reverse of ATP synthase Other ion gradients E.g., H+/Na+ antiporter 24 Image from: https://doi.org/10.1038/nrmicro1767 Secondary Active Transport E.g., H+/Na+ antiporter ETC generates proton motive force (PMF) Transport of H+ down its gradient through antiporter powers export of Na+ up its gradient New Na+ gradient can then power Na+/nutrient symporter 25  Group Translocation Active transport where substrate is chemically modified E.g., sugar phosphotransferase system (PTS) Transports sugars across cytoplasmic membrane Sugar is phosphorylated during transport Phosphate from phosphoenolpyruvate (PEP) transferred to PTS components, then to sugar 26 Image from: Prescott’s Microbiology, 11th Edn  Outer Membrane Transport Gram-negative outer membrane contains porins β-barrel proteins Solutes travel through porins to reach periplasm Allow for diffusion Cannot directly concentrate substrates Size of water-filled channel determines which substrates can enter Some porins have substrate- binding sites Helps attract certain substrates to porin 27 Image from: Prescott’s Microbiology, 11th Edn  Porins and Antibiotic Resistance Lack of specificity can be detrimental E.g., antibiotics Mutations to porin genes can confer antibiotic resistance Changes number and/or structure However, porin loss exerts a fitness cost Decreases nutrient uptake Impairs membrane structure 28 Image from: https://doi.org/10.1128/CMR.00043-12 TonB-Dependent Receptors Active transporters in outer membrane β-Barrel proteins Channel blocked by plug Substrate specific Substrate binding exposes TonB box TonB box binds to TonB TonB pulls out plug and substrate is transported Energy provided by PMF 29  Iron and Siderophores Bacteria need iron Cytochromes (ETC) Enzyme co-factors Ferric iron (Fe3+) very enterobactin insoluble, limited availability Bacteria scavenge iron using siderophores E.g., enterobactin Bind very tightly to Fe3+ Released into environment, where they compete for Fe3+ 30 Image from: Prescott’s Microbiology, 11th Edn Iron and Siderophores Fe3+ must reach cytoplasm Different paths possible Secreted siderophore binds to Fe3+ Transported to periplasm by TonB-dependent receptor Binds to periplasmic SBP Delivered to ABC transporter Siderophore-Fe3+ complex transported to cytoplasm 31 Reminders Bacteriology Quiz 3 opens Jan. 30 at 3 PM Lectures 9 - 11 Lab 1 Assignment Section 004: due Jan. 30 at 2:30 PM Sections 003, 005: due Jan. 31 at 2:30 PM Lab 2 Section 004: Jan. 30 at 2:30 PM Sections 003, 005: Jan. 31 at 2:30 PM Complete Lab 2 Pre-Lab Quiz before 32