Microbial Metabolism and Classification

• Lecture topic is Metabolism and Nutrient Transport

Lecture Learning Outcomes

• Describe the different carbon, energy, and electron sources used by bacteria
• Describe the general steps of aerobic respiration, anaerobic respiration, and fermentation
• Explain why bacterial metabolism is a difficult antibiotic target, and how sulfa drugs and trimethoprim work
• Describe the different uptake systems used by bacteria to allow nutrients through the cytoplasmic and outer membranes

Carbon

• Cells are mainly built from organic molecules, specifically carbon backbones
• Microbes are classified based on their carbon source
• Heterotrophs use organic carbon molecules
• Autotrophs use CO2 as a carbon source
• Carbons are used to make precursor metabolites, which are simple organic molecules
• Precursor metabolites are intermediates in metabolic pathways
• Precursor metabolites are used to build more complex organic molecules

Building a Bacterium

• Energy and electrons are needed to make precursor metabolites
• Energy and electrons are needed to assemble precursor metabolites into macromolecules like proteins and nucleic acids

Energy and ATP

• Energy powers chemical processes
• Energy is needed for transport, like importing nutrients, and mechanical work like flagellar rotation
• Microbes are classified based on their energy source
• Chemotrophs use organic or inorganic molecules
• Phototrophs use light
• Energy from an energy source is commonly conserved in the form of ATP

Reducing Power (Electrons)

• Building some organic molecules requires reducing power
• Reducing power can be used to make ATP via the electron transport chain
• Microbes are also classified based on their electron source:
• Organotrophs use reduced organic molecules
• Lithotrophs use reduced inorganic molecules
• Electrons are funneled to electron carriers NAD+ and FAD
• This makes NADH and FADH2, which are reduced forms of the electron carriers

Other Nutrients

• Elements besides carbon are needed for cell function
• Macronutrients are needed in large amounts
• These include C, O, H, N, S, and P
• They are the building blocks (amino acids, etc)
• K, Ca, Mg, and Fe, as cations and co-factors
• They stabilize cellular structures like the cell wall
• Micronutrients are needed in small amounts
• They are also known as trace elements
• Co, Cu, Mo, Mn, Zn, and Ni are cations and co-factors

Microbial Metabolism

• Most bacteria on the human body are chemoorganoheterotrophs
• These bacteria get their carbon, energy, and electrons from organic molecules
• ATP is made by oxidizing reduced organic molecules, such as through:
• Aerobic respiration
• Anaerobic respiration
• Fermentation
• ATP can be generated with an electron transport chain
• This is part of aerobic and anaerobic respiration

Aerobic Respiration

• During aerobic respiration, energy is conserved as ATP, and electrons are collected in NADH and FADH2 forming precursor metabolites
• Glycolytic pathways (glycolysis) oxidize glucose to make pyruvate, ATP, and NADH
• The tricarboxylic acid (TCA) cycle oxidizes acetyl-CoA to produce CO2, ATP, NADH, and FADH2 Electrons from NADH and FADH2 are used in the electron transport chain (ETC) to generate more ATP

Glycolytic Pathways

• Embden-Meyerhof pathway
• This is the most common pathway and is also found in animals
• Makes pyruvate, ATP, and NADH
• Entner-Doudoroff pathway
• Occurs in some bacteria
• Makes glyceraldehyde 3-phosphate (G3P), pyruvate, and NADPH
• G3P enters the Embden-Meyerhof pathway
• Pentose phosphate pathway
• Often used for biosynthesis
• Makes NADPH
• Makes precursor metabolites for nucleotides and amino acids

Tricarboxylic Acid Cycle

• The pyruvate dehydrogenase complex converts pyruvate into acetyl-CoA
• Acetyl-CoA then enters the tricarboxylic acid (TCA) cycle
• Acetyl-CoA is oxidized, producing CO2, GTP (ATP), NADH, and FADH2
• Some intermediates from the TCA cycle are used as precursor metabolites

Electron Transport Chain (ETC)

• Electrons in NADH and FADH2 are used to make more ATP
• These electrons are transferred to membrane-bound electron carriers
• Ubiquinone, also known as coenzyme Q (CoQ)
• Cytochromes made from proteins with heme co-factors
• A Lower redox potential means a greater likelihood of giving up electrons
• Each carrier is reduced by the preceding carrier and oxidized by the following carrier
• There are increasing redox potentials in the electron transport chain

Electron Transport Chain in E. coli

• Electrons are transferred from NADH to ubiquinone (Q)
• Electrons pass through a series of cytochromes and the terminal electron acceptor
• For example, b562 and o
• Aerobic respiration uses O2 as a terminal electron acceptor
• Electron transfers drive the transport of protons into the periplasm, generating a proton-motive force (PMF)

Proton Motive Force and ATP Synthase

• PMF refers to potential energy due to proton and charge gradient across a membrane
• Cytoplasm is relatively alkaline and negatively charged
• Protons flow down the concetration gradient into the cytoplasm
• Protons travel through ATP synthase to make ATP

Anaerobic Respiration

• Similar to aerobic respiration, including glycolysis, TCA cycle, and ETC
• Also makes ATP, NADH, and FADH2, but the terminal electron acceptor is not O2
• Instead, nitrate, sulfate, and CO2 are the final electron acceptor
• Paracoccus denitrificans reduces nitrate to N2

Fermentation

• Some bacteria lack or repress an ETC, meaning NADH has to be used for something other than energy production
• Fermentation is a pathway by which a substrate is partially oxidized, such as by glycolysis
• The process makes ATP
• There is no ETC and no exogenous electron acceptor
• Electrons are transferred from NADH to pyruvate or a pyruvate derivative
• This process regenerates NAD+

Bacterial Metabolism and Antibiotics

• Humans are chemoheterotrophs
• Human microbiota are chemoheterotrophs, using our food and us as nutrients
• Humans and human microbiota use similar metabolic pathways
• It is difficult to selectively target bacterial metabolic enzymes
• This creates a toxicity concern

Tetrahydrofolate Synthesis

• Tetrahydrofolate is a cofactor
• Needed to make purines and pyrimidines (DNA/RNA), and methionine (amino acid)
• Humans acquire folates from diet (vitamin B9)
• Some bacteria have to make their own tetrahydrofolate
• Folate biosynthetic enzymes are therefore antibiotic targets

Sulfa Drugs and Trimethoprim

• Sulfa drugs (e.g., SMX) inhibit dihydropteroate synthase
• They resemble p-aminobenzoic acid and act as competitive inhibitors
• Trimethoprim inhibits dihydrofolate reductase, resembling dihydrofolic acid
• Blocking tetrahydrofolate production inhibits bacterial growth
• Combination of sulfa drugs and trimethoprim is often used for synergistic effect (e.g., TMP/SMX)

Nutrient Uptake

• Nutrients must be collected from environment
• Often limited abundance, leading to competition
• Cell envelope is a good barrier, but hydrophilic nutrients can't pass through
• Protein channels and transporters are thus often needed

Energy Independent Transport

• Diffusion mechanisms do not require an input of energy
• Substances move down a concentration gradient
• Not very useful, as concentration is usually lower outside of the cell
• Some substances diffuse across the cytoplasmic membrane, others through carrier proteins

Primary Active Transport

• Active transport involves moving substances against their concentration gradient, requiring energy input
• Primary active transporters use energy from ATP hydrolysis
• ATP-binding cassette (ABC) transporters are common primary active transporters in bacteria
• Importers are used for sugars and other nutrients
• Exporters expel substances from the cell

ABC Transporter Solute Binding Proteins

• Most ABC transporters work with solute-binding proteins (SBPs)
• SBPs deliver a specific substrate to the transporter
• In Gram-negatives, SBP is in the periplasm
• In Gram-positives, SBP is lipoprotein or is associated with PG

Secondary Active Transport

• Potential energy from an ion gradient can power the transport of a substance against its concentration gradient
• The ion gradient generation includes the electron transport chain (PMF)
• The ion gradient can also stem from V-type ATPases
• Energy from ATP hydrolysis creates a H+ or Na+ gradient
• This reverses ATP synthase
• Other ion gradients
• E.g., H+/Na+ antiporter

Secondary Active Transport

• H+/Na+ antiporter is a form of secondary active transport
• ETC generates a proton motive force (PMF)
• Transport of H+ down its gradient powers the export of Na+ up its gradient via the antiporter
• The new Na+ gradient then powers the Na+/nutrient symporter

Group Translocation

• This is an active transport process where the substrate is chemically modified
• An example is the sugar phosphotransferase system (PTS)
• Sugars are transported across the cytoplasmic membrane and are phosphorylated during transport
• Phosphate from phosphoenolpyruvate (PEP) is transferred to PTS components, then to the sugar

Outer Membrane Transport

• The Gram-negative outer membrane contains porins
• These are beta-barrel proteins
• Solutes travel through porins to reach the periplasm, allowing for diffusion
• This process cannot directly concentrate substrates
• The size of the water-filled channel determines which substrates enter
• Some porins have substrate-binding sites, which helps attract certain substrates to the porin

Porins and Antibiotic Resistance

• Lack of specificity can be detrimental, for example, with antibiotics
• Mutations to porin genes can confer antibiotic resistance
• Changes to number and/or structure of porins
• Porin loss exerts a fitness cost
• Decreases nutrient uptake
• Impairs membrane structure

TonB-Dependent Receptors

• These are active transporters in the outer membrane
• They contain Beta-Barrel proteins
• The channel is blocked by a plug
• Substrate specific and substrate binding exposes the TonB box
• The TonB box binds to TonB
• TonB pulls out the plug, and the substrate id transported
• Energy is provided by PMF

Iron and Siderophores

• Bacteria need iron for cytochromes (ETC) and enzyme co-factors
• Ferric iron (Fe3+) is very insoluble, limiting its availability
• Bacteria scavenge iron using siderophores
• E.g., enterobactin
• Binds very tightly to Fe3+
• Siderophores are released into the environment, where they compete for Fe3+

Iron and Siderophores

• Fe3+ must then reach the cytoplasm
• There are different paths to do this
• Secreted siderophore binds to Fe3+
• Transports to periplasm by TonB-dependent receptor
• Then binds to periplasmic SBP
• Next delivered to the ABC transporter
• Where the siderophore-Fe3+ complex is transported to cytoplasm

Explore microbial classification based on energy, carbon, and electron sources. Key topics include chemotrophs, organotrophs, macronutrients, glycolysis, and aerobic/anaerobic respiration. Learn about the role of acetyl-CoA, fermentation, and sulfa drugs in microbial processes.