Metabolism

Nutrition and Metabolism
Metabolism is the sum of all chemical processes that occur in a cell.
anabolic reactions are the reactions that build up or synthesize molecules from others.
catabolic reactions are the reactions that break apart or tear down molecules into simpler molecules.
Bacteria place most of their metabolic activities into the pursuit of increasing cell numbers = Population growth.
To do this bacteria need:
raw materials or substrates
driving force in the form of energy and reducing power
blue print or plan = chromosome
Raw materials which microorganisms must get from the environment!!!!
Carbon sources
Classify bacteria based on carbon source:
heterotrophs which use organic molecules such as sugars, amino acids, fatty acids, organic acids, aromatic compounds, nitrogen bases, and countless other organic molecules, as their source of carbon.
autotrophs which use the inorganic molecule carbon dioxide as their source of carbon.
Bacteria are about 50% Carbon by weight.
Nitrogen
Bacteria are about 12% nitrogen by weight. Nitrogen found in amino acids, nucleotides, and other cell constituents.
Nitrogen in organic and inorganic forms. Example of organic is amino acids or nucleotides. Examples of inorganic form are NH₄ or NO₃.
A few species can take atmospheric nitrogen N₂ (80%+ of air and inert) and convert it to ammonium and ultimately amino acids or nucleotides. These bacteria are referred to as nitrogen-fixing bacteria.
Phosphorous
Phosphorous found in nucleotides and phospholipids.
Phosphorous occurs in nature as organic or inorganic as phosphates forms.
Sulfur
Sulfur found in specific amino acids and vitamins.
Chemically transformed (oxidation states change) due to microorganisms activities. Sulfur occurs in nature as organic or inorganic forms. Bacteria use sulfate or sulfide forms.
Potassium
Required as a cofactor for a number of enzymes.
Magnesium
Required to stabilize ribosomes, cell membranes, nucleic acids and required by a number of enzymes.
Calcium
Stabilizes cell wall and important for heat stability of spores.
Sodium
Required by some but not all microorganisms. Usually seawater organisms have a requirement but freshwater or terrestrial organisms do not.
Iron
Required in small amounts but still considered a macro-nutrient.
Required for cytochromes and iron-sulfur proteins that are important for electron transport.
Found as insoluble inorganic form in nature. Bacteria produce siderophores to chelate insoluble iron and transport it to the cell. Siderophores produced by Escherichia coli or S. typhimurium are called enterobactins.
Micronutrients are found in your text. These are inorganic elements that are required in trace amounts by some or all bacteria for growth. Used primarily as enzyme cofactors.
Organic growth factors
Includes vitamins, amino acids, purines or pyrimidines. Most microorganisms can synthesize these but others require them be presynthesized.
Again microorganisms must either obtain these from the environment or be able to synthesize them for themselves. Culture medium that we use must provide the necessary raw products for growth if the cell cannot synthesize it.
Types of medium:
Complex - which is chemically undefined
Defined - which is chemically defined.
Defined medium usually differs considerably between different species of bacteria. One species of bacteria (e.g., E. coli) may require relatively few ingredients for a defined medium whereas another species (e.g., Leuconostoc) requires many organic growth factors in addition to mineral salts. E.coli is said to have a greater biosynthetic capacity than Leuconostoc since E. coli can make many of the organic growth factors that Leuconostoc cannot make.
Stress - different microorganisms have different requirements for growth - no one medium will allow all bacteria to grow.
Energy - again bacteria require energy to synthesize molecules needed. The energy is derived from the environment. There is nothing magical about it.
Sources of energy
Solar - refer to these as phototrophs
Oxidation of chemicals - refer to chemotrophs
Oxidation of inorganic molecules - refer to these as chemolithotrophs
Oxidation of Organic molecules - refer to these as chemoorganotrophs.
Free energy
Energy defined as the ability to do work. Total chemical energy released during the oxidation of a substrate - either organic or inorganic - expressed as enthalpy - H. But according to the second law of thermodynamics there must be an increase in entropy which in this case is an unusable form of energy called heat. Free energy, G, is the energy released that can do useful work. Free energy at standard condition is G^o'. Reactions with a negative [Delta]G^o' are said to be exergonic and occur spontaneously and reactions with a positive [Delta]G^o' are said to be endergonic and do not occur spontaneously.
Free energy of formation or G^o_f
Using the free energies of formation, you can calculated [Delta]G^o' which is equal to the products minus the reactants after balancing the equation.
Micoorganisms need energy to assemble and put macromolecules into order. They need to have a portable source of energy.
Two sources:
Adenosine triphosphate - ribonucleoside adenosine plus three phosphates. The terminal two phosphate bonds are high energy bonds. ATP is generated during exergonic reactions and is used to drive endergonic reactions.
Coenzyme A - derivatives of CoA such as acetyl coA are high energy compounds. Hydrolysis of acetyl CoA can be used to drive the synthesis of ATP from ADP plus inorganic phosphate.
Two pathways in bacterial metabolism - both of which can generate energy storing compounds:
Aerobic metabolism - occurs where there is oxygen present and uses oxygen - there are anaerobic pathways in aerobic bacteria.
Anaerobic metabolism - occurs where oxgen is absent and obviously oxygen is not used.
Talk about anaerobic metabolism first.
Fermentation - the oxidation of organic matter in a series of oxidation - reduction reactions which are internally balanced since there is no external electron acceptor. We will come back to what oxidation - reduction reactions are. Put it another way, organic matter is oxidized during fermentation and the fermentation product is subsequently reduced. Or organic matter acts as both the electron donor and electron acceptor. During this ATP is made by substrate level phosphorylation.
Example C₆H₁₂O₆ ----> 2 C₂H₆O + 2 CO₂
Glucose is fermented to ethanol plus carbon dioxide.
Notice that some carbon, i.e., carbon dioxide, is more oxidized than glucose and other carbon, i.e., ethanol, is more reduced than glucose.
Calculate [Delta]G^o' from energy of formation values.
[Sigma] (products) - [Sigma] (reactants)
[2 (-181.75) + 2 (-394.4)] - [-917.22] = -235.1 kJoules/mole
Some of this energy is captured in the generation of ATP from ADP and inorganic phosphate.
Glycolysis or Embden - Meyerhof pathway
Glyco - sugar lysis - breakdown or breakapart.
Again, as a reminder, we are going to talk about a series of coupled oxidation - reduction reactions. Reactions where electrons are removed from an electron donor molecule and transfered to an electron acceptor molecule. The compound that loses the electron is said to be oxidized and acts as a reducing agent and the compound that accepts the electron is said to be reduced and acts as an oxidizing agent.
1/2 O₂ + 2e^- + 2H⁺ ---> H₂O
H₂ ----> 2e^- +2H⁺
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H₂ + 1/2O₂ ---->H₂O
Oygen is the oxidizing agent and is reduced and Hydrogen is the reducing agent and is oxidized.
Reduction potential or redox potential (Eo') is measure of the tendency of a substance to gain or lose electrons.
go back to our example above:
the redox potential of 2H⁺ + 2e^- ----> H₂ is -420 mVolts
the redox potential of 1/2 O₂ + 2e^- + 2H⁺ ---> H₂O is +820 mVolts
Electrons move from the more negative redox potential half equation to the more positive potential half equation. Therefore electrons move from H₂ to O₂. (see figure 4.7)
The difference is the redox potentials between the electron donor and the electron acceptor is [Delta]E_o^' which is proportional to [Delta]G^o'. In catabolism the electron donor is often referred to as the energy source.

Getting back to glycolysis!
Divide glycolysis into three stages. Specific enzymes carry out each reaction.
Enzymes are proteins composed of amino acids. Going back to our water example. [Delta]G^o' is a negative number meaning that the reaction is exergonic and occurs spontaneously. But it will not occur at an appreciable rate because the O-O bonds and H-H bonds must first be broken before new bonds can be formed. This requires energy -called activation energy. Enzymes act by lowering the activation energy of a reaction. To do this, an enzyme catalyzes a singe reaction usually. The enzyme has an active site where the substrates bind to the enzyme.

Stage I - glucose is phosphorylated and converted to fructuose 1 phosphate.
Fructose 1 phosphate is phosphorylated to fructose 1,6 bisphosphate. Phosphorylation reactions often precede oxidation reactions.
Fructose 1,6 bisphosphate is split into glyceraldehyde 3 phosphate and dihydroxyacetone phosphate - 2 3 carbon molecules. Notice that no oxidation - reduction reactions occurred and ATP was consumed!
Stage II - Glyceraldehyde 3 phosphate is oxidized and phosporylated to yield 1,3 bisphospho glycerate and NAD⁺ is reduced to NADH.
What is NAD⁺?
Nicotinamide adenine dinucleotide (NAD⁺) is a coenzyme of dehydrogenase enzymes and a soluble electron carrier/acceptor.
ATP is generated when 1,3 bisphosphate glycerate is converted to 3 phosphoglycerate. Another ATP is generated when phosphoenol pyruvate is converted to pyruvate.
Net: 2 NADH +H⁺
2 ATPs = 4 generated - 2 consumed
Cell has a problem now! What to do with the reduced NADH since there is a limited supply of oxidized form of NAD⁺. The cell must regenerate NAD⁺ or it will not continue to oxidize glucose.
Stage III - to overcome the problem of regenerating NADH to NAD+, a number of organic molecules can act as electron acceptors - remember that this is our definition of fermentation.
Pyruvate is reduced to ethanol by yeasts
Pyruvate is reduced to lactic acid by lactic acid bacteria or in your muscles.
Summary:
1 x 6 carbon molecule - glucose
2 x 3 carbon molecules - pyruvate which is further metabolized e.g., ethanol fermentation.
2 ATPs
No where was oxygen used either - glycolysis is an anaerobic pathway.
Respiration
Aerobic respiration where oxygen is the terminal electron acceptor not an organic molecule. More energy released because:
the organic molecule is further oxidized than during fermentation.
oxygen is at a much higher redox potential than organic compounds thus a greater difference in potentials between the electron donor and electron acceptor.
Electron Flow
membrane associated electron carriers that accept electrons and donate electrons while conserving energy which will be used to make ATP.
NADH dehydrogenases - accept hydrogen atoms from NADH generated from cell metabolism.
Flavoproteins - includes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) - Flavin prosthetic group accepts hydrogen atoms and passes on electrons - what happens to the protons that are left over?
Cytochromes - iron containing proteins that accept and donate a single electron. The iron alternates between Fe⁺² (ferrous) and Fe⁺³ (ferric iron). There are many different types of cytchromes with different redox potentials.
Nonheme iron-sulfur proteins - no cytochromes where iron is bound to sulfur and cysteine residues of the protein. Ferridoxin is a nonheme iron-sulfur protein. They carry only electrons and not protons.
Quinones - extremely hydrophobic molecules that act as hydron acceptors and electron donors like flavoproteins.
How does the electron flow conserve energy to generate ATP?
Flow of electrons is from a carrier with a lower redox potential to one with a higher redox potential. (Figure 4.18 for example)

See Figure 4.19 - cytoplasmic membrane
Electron transport chain is set up to separate electrons from protons. For example the hydrogen atoms from NADH are separated into electrons and protons by the electron transport chain. The electrons are moved down the electron transport chain and the protons are extruded outside the cell environment (periplasm space in gram negative cells and outside the cell in gram positive cells. Flavoprotein, Coenzyme QHow do we know this? the periplasm becomes more acidic and the cytosol becomes more basic. The electron is passed ultimately to oxygen and oxygen is reduced to water.
IMPORTANT OUTCOME The important outcome of this is a pH gradient and a electrochemical gradient, inside the cell is more negative and alkaline and outside the cel is more positive and acidic or potential across the membrane. This represents potential energy much like a battery and is referred to as the proton motive force and measured in mVolts. This can be used to transport molecules across the membrane or drive the flagella or drive the generation of ATP. The idea of a proton motive force driving ATP synthesis was first proposed as the chemiosmotic theory by Peter Mitchell in 1961.
Oxidative Phosphorylation
The proton motive force is used to drive ATP synthesis.
ATP synthase - a big multisubunit protein that has a large head and a tailpiece that spans the membrane. Protons flow through the tailpiece and headpiece where ATP is made from ADP plus inorganic phosphate. About 4 protons move through for every ATP synthesized.
This can be reversed and ATP is consumed to generate a proton motive force. The PMF can be used to drive motility or transport processes. So organisms that do not carry out oxidative phosphorylation still require an ATP synthase to generate PMF.
Uncouplers and inhibitors
Uncouple the PMF from oxidative phosphorylation by dissipating the PMF.
dinitrophenol and dicumarol - both lipid soluble substances that increase the permeability of the membrane to protons.
Inhibitors block the flow of electrons
carbon monoxide, cyanide, hydrogen sulfide, and azide.
Back to carbon flow in the cell
Glycolysis yielded two molecules of pyruvate which were used as electron acceptors in fermentation but what about in respiration? What happens to pyruvate?
Pyruvate is decarboxylated and the acetate is coupled to Coenzyme A to form acetyl-CoA and CO₂ and NADH + H⁺ are formed. The acetyl CoA than integrates into the Citric Acid Cycle or Kreb's cycle or Tricarboxylic acid (TCA) cycle.
Walk through this (Figure 4.21).
Citric acid cycle is extremely important since many key intermediates for biosynthesis come from this cycle. alpha keto glutarate (amino acid precursor) and oxaloacetate (amino acid precursor), acetyl-CoA (fatty acid biosynthesis) are key intermediates.
Roughly 40% of the carbon from glucose is converted to cell biomass in an aerobically grown E. coli. The rest is burned off as CO₂.
Summary of aerobic respiration
oxidation of pyruvate yields 3 carbon dioxides, 4 molecules of NADH, 1 molecule of FADH
oxidation of NADH yields 12 ATP molecules by oxidative phosphorylation
oxidation of FADH yields 2 molecules of ATP
One GTP made by substrate level phosphorylation
For a total of 15 ATPs made from TCA cycle products times 2 since there were two pyruvates from glucose = 30 ATPs.
2 ATPs from glycolysis
and 6 ATPs from the oxidation of NADH generated during glycolysis.
Grand total of 38 ATPs / molecule of glucose contrast to 2 molecules made from fermentation. These 38 ATPs represent about 40% of the total energy in glucose - the difference in energy is lost as heat.
Alternate modes of energy generation
Anaerobic respiration - oxidized compounds or elements act as electron acceptors in anaerobic environment. Examples include SO₄⁼, NO₃^-, Fe³⁺, and CO₃^- , which all have a more negative redox potential than O₂/H₂O and therefore less energy is released when these alternative electron acceptors are used. Electron donor and energy source are organic compounds.
chemolithotrophy - rock eaters - oxidize inorganic compounds, = energy source, such as H₂S, H₂, NH₃, and Fe⁺². The removed electrons move down an electron transport chain to oxygen to generate a PMF. Distinction between these and chemoorganotrophs is their source of carbon. Chemolithotrophs are usually autotrophs whereas organotrophs are heterotrophs.
Phototrophs - Light drives electrons through the electron transport chain to generate a PMF to generate ATP. These may be autotrophs or heterotrophs.
Diversity of energy sources
organic molecules
reduced inorganic molecules
light
All used to generate a PMF. We will look at these more closely later.
Anabolic reactions

Sugars - (See figure 4.26) Key intermediates are glucose 6 phosphate, from glycolysis, and uridine diphosphoglucose.
Hexoses synthesized by gluconeogenesis - by reversing glycolysis from phosphoenol pyruvate from glycolysis. PEP can come from the decarboxylation of oxaloacetate which comes from the TCA cycle.
Pentoses come from the removal of a carbon from a hexose such as glucose.
Amino acids - (See figure 4.27) Precursors from glycolysis or TCA cycle for the carbon skeleton.
Amino group can be obtained from another amino acid such as glutamate in a transamination reaction.
Ultimate source of amino group? (See figure 4.28)
ammonia from the cell's environment. Ammonia can be incorporated into alpha keto glutarate to make glutamate using the enzyme glutamate dehydrogenase. The amino group can be transfered from glutamate to other carbon skeletons via a transamination reaction carried out by transaminases.
Purine and pyrimidine biosynthesis - (see figure 4.29)
Purine biosynthesis begins with the ribose phosphate to which various atoms are attached.
Note: carbons from formyl groups are from reactions involving folic acid - a vitamin. Folic acid is involved in a number of single carbon transfer reactions. Many species of bacteria cannot synthesize folic acid and therefore it must be provided. Sulfonamides - are drugs that inhibit folic acid biosynthesis.
The first purine is inosinic acid which is converted to either adenylic acid or guanylic acid.
Pyrimidine biosynthesis begins with the ring formation in orotic acid before the addition of the sugar phosphate to form a nucleoside.
Fatty acid biosynthesis (See figure 4.31)
Saturated fatty acids - intermediate begins with acetyl - CoA from glycolysis - TCA cycle. The acetyl is transfered to acyl carrier protein. Acetyl - ACP is carboxylated to malonyl - ACP. Fatty acids are synthesized by adding the acetyl group from malonyl-ACP to the acetyl group of acetyl-ACP. The ketones are reduced by NADPH to y ield butyryl-ACP. Then another malonyl-ACP donates its acetyl group to yield crotonylACP and so on.
Unsaturated fatty acids - cis configuration double bonds in the hydrocarbon chain.