Metabolism of Microbes
I. A Model for Metabolism (using E. coli as an example)
What is metabolism? All the biochemical reactions that take place in a cell.
The Model: Metabolism leading to the synthesis of a new microbial cell has 3 requirements:
1. Raw Materials – nutrients composed of carbon (carbohydrates, proteins, etc.)
2. Driving Force
a. energy – (ATP – adenosine triphosphate) – Some chemical produce ATP; some chemical reactions use ATP.
b. reducing power – Many biochemical reactions involve oxidation (removal of electrons from a compound) & reduction (addition of electrons to a compound; E. coli stores electrons in coenzymes called NAD (nicotinamide adenine dinucleotide) & NADP (nicotinamide adenine dinucleotide phosphate). These compounds capture electrons in the form of hydrogen atoms from compounds that are being oxidized, thus forming NADH & NADPH). NAD & NADP stores the cell’s reducing power. Bacteria will then use this reducing power to build its cellular components (it will reduce other compounds in this process).
Hint: O.I.L. = oxidation is loss; R.I.G. = reduction is gain
3. A Plan – contained in the cell’s DNA; this is “the genetic code.”
II. Metabolism: An Overview
A. Flow of Materials – Sequential Steps:
1. Entry Mechanisms – Raw materials from the environment are transported into the cells by various mechanisms.
2. Catabolic Reactions – reactions in which raw materials are broken down into smaller molecules (precursor molecules); there are 12 precursor molecules that are required to synthesize building blocks (amino acids, monosaccharides, nucleotides, fatty acids) that will build a new cell.
3. Anabolic Reactions – reactions that build up larger molecules from smaller ones:
a.) biosynthesis – 12 precursor molecules are put together to produce building blocks.
b.) polymerization – building blocks are joined together to form macromolecules (proteins, nucleic acids, lipids, polysaccharides, peptidoglycan); ex. amino acids are put together to form a protein, nucleotides are put together to form DNA.
c.) assembly – macromolecules are assembled into biological structures; ex. peptidoglycan forms a cell wall.
raw materials à precursor molecules à buliding blocks àmacromolecules
B. Driving Force – Where is ATP & Reducing Power Produced & Used in the Above Reactions?
1. Entry Mechanisms – Many materials that move into the cell are moving from low to high concentration; this requires ATP; remember the bacterial is usually hypertonic to its environment.
2. Catabolic Reactions – In general, the catabolic reactions transform raw materials into precursor molecules, reducing power, & ATP.
3. Anabolic Reactions – In general, these reactions use precursor molecules, ATP, & reducing power.
III. Aerobic Metabolism (We’ll use E. coli as the example)
(Aerobic means that this type of metabolism requires oxygen)
A. Catabolic Reactions – Supply the cell with the 12 precursor molecules, reducing power (NADH & NADPH), & ATP.
1. Precursor Molecules – A minimum of 3 different pathways are required to produce all 12 precursor molecules: (We’ll look at these later in this handout)
a. glycolysis – produces 6
b. tricarboxylic acid (TCA) cycle – produces 4
c. pentose phosphate pathway – produces 2 (we won’t discuss this one)
2. Reducing Power – Many steps in a catabolic pathway involve oxidation (loss of electrons)/reduction (gain of electrons) reactions. The electrons (along with hydrogens) are usually transferred from the molecule being oxidized to the coenzymes NAD or NADP, to form NADH & NADPH. NADH & NADPH are used to reduce other molecules or are used to generate new ATP molecules.
a. Stored Energy – The principal compound that stores chemical energy in all cells is ATP (adenosine triphosphate). The energy-storage capabilities of ATP depend on the 2 bonds that join the 3 phosphate groups in the ATP molecule. These bonds are among the most highly reactive bonds found in biochemicals. They are called high-energy bonds. The phosphate groups that are joined in ATP by high-energy bonds are readily donated to other compounds. These compounds that receive a phosphate group from ATP are termed phosphorylated compounds. After receiving a high-energy phosphate, phosphorylated compounds can then participate in chemical reactions that would not occur if the compounds were unphosphorylated. This is why we say that the energy “stored” in the bonds of ATP is used to drive other metabolic reactions.
b. ATP Formation from ADP [most of the ATP is made by chemiosmosis]
1.) Chemiosmosis & the Electron Transport System– The electron transport system is made up of a series or chain of compounds. Some of these compounds are hydrogen-carriers and some are electron-carriers. NADPH/NADH transfers hydrogen atoms to a hydrogen-carrier in the chain. This hydrogen-carrier then passes hydrogen electrons to an electron-carrier in the chain; the hydrogen ions are pumped out of the cell (or out of the inner mitochondrial compartment, if you’re talking about eukaryotes). Each compound in the chain will then alternately accept & then release hydrogen electrons; as electrons are passed along the chain they drop to lower and lower energy levels. At the end of the chain, electrons are accepted by oxygen to form water. (The final electron acceptor is oxygen!! This is why this process is called aerobic respiration!).
ATPase is an enzyme located in the cell membrane of prokaryotes; in eukaryotes it’s located in the inner mitochondrial membrane. In E. coli, the pumping of hydrogen ions (H+) out of the cell creates a H+ concentration gradient & an electrical gradient across the cell membrane (there are more H+ outside the cell than inside). H+ then flows back into the cell (H+ ions want to move from a high concentration to a low concentration). The ions move through the membrane-bound ATPase, converting ADP to ATP. This process is called chemiosmosis.
4. Catabolic Pathways (breaking down) – Chemical reaction sequences that transform raw materials into the 12 precursor molecules, store energy in the form of ATP, & store reducing power in the form of NADPH/NADH. Central metabolism (described below) begins with the monosaccharide glucose. Most organisms have other catabolic pathways, which use substrates other than glucose (ex. humans can use protein & lipids, if glucose concentrations are low). These pathways all eventually feed into central metabolism at various points.
a. Glycolysis (= “glucose splitting”) – This pathway begins with glucose (a 6 carbon sugar) and ends with 2 pyruvic acids or pyruvates (3 carbon molecules). The initial reactions in glycolysis require ATP. Later reactions in glycolysis produce a small amount of ATP. A small amount of NADH is also produced, which will result in the production of more ATP in the electron transport system. The main function of glycolysis is to split glucose.
b. Kreb’s Cycle – Some of the pyruvate formed by glycolysis is used in biosynthesis, the rest is oxidized to another precursor molecule, acetyl CoA (coenzyme A). Acetyl-CoA then enters the Kreb’s Cycle by combining with a precursor molecule oxaloacetate to form citrate. In a series of 6 reactions, carbon dioxide is released & oxaloacetate is regenerated (this is where most of the carbon dioxide you exhale comes from!). In this cycle, only a small amount of ATP is formed (called substrate level phosphorylation). However, considerable reducing power is stored in the form of NADH, NADPH, & FADH2 (flavine adenine dinucleotide), which are used to produce ATP in the electron transport system.
c. Pentose Phosphate Pathway – we won’t discuss.
5. Anabolic Pathways (building up) – use precursor molecules, ATP, & reducing power produced in above catabolic reactions.
a. Biosynthesis – E. coli converts precursor molecules produced in catabolism into building blocks (amino acids, monosaccharides, nucleotides, fatty acids) of macromolecules (proteins, nucleic acids, lipids, polysaccharides, peptidoglycan). Organisms that can’t make a given building block grow only if that molecule is provided in the medium (or diet). Ex. E. coli can make all 20 amino acids. Humans are unable to make 9 of the 20, so these nutrients must come from our diets.
b. Polymerization – In these reactions, building blocks produced in biosynthesis are joined to form macromolecules. The major cellular polymerization reactions are DNA replication, RNA synthesis, protein synthesis, & polysaccharide & peptidoglycan synthesis. For most macromolecules, building blocks must be joined in a specific order. For ex., amino acids must be arranged in the proper order to produce the right proteins. If you change the amino acid sequence, you change the protein’s structure and therefore change its function.
Some polymerization is determined directly by the organism’s DNA (ex. DNA replication, RNA synthesis, protein synthesis). Other reactions are indirectly determined by DNA (ex. polysaccharide & peptidoglycan synthesis). In the latter, building blocks are ordered by the enzymes that catalyze the polymerization reactions. Because enzymes are proteins & protein structure is determined by DNA, the reactions the enzymes catalyze are indirectly determined by DNA.
c. Assembly – Assembly of macromolecules into cellular structures (ex. flagella, ribosomes, cell wall) may occur spontaneously (self-assembly) or as a result of reactions catalyzed by enzymes.
IV. Anaerobic Metabolism
Anaerobic metabolism allows cells to grow in the absence of oxygen.
Strict anaerobes are capable of only anaerobic metabolism.
Facultative anaerobes are capable of both aerobic & anaerobic metabolism. (ex. E. coli)
A. Anaerobic Respiration – Involves an electron transport chain, but uses a compound other than oxygen as the final electron acceptor, allowing the cell to generate ATP by chemiosmosis. Compounds that can be used as final electron acceptors include sulfate & nitrate.
1. Nitrate users: These organisms, including E. coli, play a role in the nitrogen cycle (removing nitrogen from terrestrial & aquatic environments & returning it to the atmosphere). Some microbes reduce nitrate to nitrite. Some microbes reduce nitrite further to nitrogen gas. We’ll see this in lab!
2. Sulfate users: These organisms, called sulfur reducers, play a role in the sulfur cycle. Sulfate is reduced to hydrogen sulfide gas. Sulfate reducers typically grow in marine & river mud flats, giving these environments a rotten-egg odor & turning the mud black. We’ll see this in lab!
B. Fermentation – This type of anaerobic metabolism uses no electron transport chain.
Some differences between fermentation & respiration (aerobic or anaerobic):
1. Fermentation generates fewer ATP per molecule of substrate. (ex. E. coli can produce 38 ATP/molecule of glucose by aerobic respiration, but only 3 ATP by fermentation.)
2. Because many molecules of substrate must be metabolized to supply a cell’s ATP requirements, the substrate must be in abundance in order for the microbe to grow.
3. Sugars are usually the only substrate that can be used in fermentation.
1. Lactic Acid Fermentation – This type of fermentation is carried out by lactic acid bacteria, the microbes that cause milk to sour (used to produce yogurt & buttermilk). Muscle tissue of animals also carries out lactic acid fermentation, when deprived of oxygen (during strenuous exercise – this is what makes your muscles sore). Steps:
1. Glycolysis – Glucose is metabolized to produce 2 molecules of pyruvic acid (a little ATP & NADH is also produced).
2. Pyruvic Acid Oxidation – pyruvic acid is oxidized to lactic acid using NADH, thus using up the reducing power stored in glycolysis.
Parts of the Kreb’s cycle & Pentose Phosphate pathway are used to help generate the 12 precursor metabolites; they cannot all be formed in fermentation.
2. Alcoholic Fermentation
This type of fermentation is typical of yeast, a type of fungi. In this pathway, pyruvic acid is converted to carbon dioxide and ethanol.
glucose à pyruvic acid à carbon dioxide + ethanol
V. Nutritional Classes of Microorganisms
Microbes are classified according to nutritional class, which depends on 2 factors:
1.) How it generates ATP & reducing power: chemo- (from oxidation of inorganic compounds like sulfur nitrite, ammonia, iron) or photo- (sun)
2.) The source of carbon atoms it uses to make precursor metabolites: auto- (obtain carbon from carbon dioxide) or hetero- (obtain carbon from organic compounds like carbs, proteins, lipids, etc.)
Can have combinations of all of these:
chemoautotrophs, chemoheterotrophs, photoautotrophs, photoheterotrophs.