Metabolic Diversity
Spend more time talking about phototrophs, anoxygenic and oxygenic phototrophs, chemolithotrophs and anaerobic respiration.
Phototrophs - eukaryotic and prokaryotic microorganisms.
Chlorophyll - heterocyclic compound with a Mg atom in the middle. Chlorophyll a is the common chlorophyll of higher plants. It absorbs red (680 nm) and blue (430 nm) light wavelengths and reflects green - Why plants are green! Other chlorophylls have different absorption spectra - absorb different wavelengths of light.
Bacteriochlorophyll - analogous to higher plants chlorophyll but have a absorption maximum in the neighborhood of 700 nm and greater. Different phototrophs with different bacteriochlorophylls can coexist since they are not competing for the same light wavelengths - increase biodiversity.
Bacteriochlorophyll located in (1) invaginations of the cytoplasmic membrane, (2) the cytoplasmic membrane, or (3) the cytoplasmic membrane and chlorosomes. Recall in eukaryotic phototrophs the chlorophyll is found in the thylakoid membranes inside the chloroplasts. In these membranes there are special chlorophyll molecules called reaction centers associated with other light-harvesting chlorophyll molecules whose purpose is to harvest quanta of light and transfer their energy to the reaction center.
Purple bacteria
Invagination of cytoplasmic membrane called chromatophores. Contains reaction center, light-harvesting I and light-harvesting II components plus bc1 complex which is common to the respiratory electron transport chain. The function of light harvesting I and II also referred to as B870 and B800-850 respectively, is to channel absorbed light energy to the reaction center.
Electron flow ( Figure 13.10) - Light harvesting antennae bacteriochlorophyll a molecules funnel their energy to the reaction center bacteriochlorophyll a molecules - the special pair. The special pair becomes excited and is converted to a good electron donor with sufficiently low E_o' (-1000 mVolts) to reduce a very electronegative acceptor molecule - bacteriopheophytin. Solar energy has now been converted to chemical energy.
Bacteriopheophytin reduces quinones in the reaction center which reduce quinones in the quinone pool before the electrons flow to Cyt bc1 to iron sulfur proteins, through Cyt c2 back to P870 of the reaction center.
How does this drive ATP synthesis?
The quinones move electrons down the electron transport chain but also move protons from inside the cell to outside the cytoplasmic membrane thus establishing a PMF which can be used to drive ATP synthesis via photophosphorylation - cyclic photophosphorylation since the electrons flow in a circular fashion.
How are reducing equivalents (NADPH) generated? NADPH is used to reduce CO₂ to organic carbon used by the cell- auxotrophy!
Need a source of electrons since in cyclic photophosphorylation there is no net gain or loss of electrons from the transporters. The ultimate souce of electrons for the reduction of NADP⁺ is a reduced inorganic molecule like H₂S, elemenal sulfur, thiosulfate, H₂ or organic acids like butyrate or malate.
Two ways to reduce NADP⁺ to NADPH.
Direct reduction if the reducing agent has a redox potential more negative than NADP⁺. H₂ has a redox potential of -420 mVolts and NADP⁺ is -320 mVolts.
Reverse electron flow - reduced compounds that have a more positive redox potential than NADP⁺. The membrane potential is used to push electrons from the quinone pool to NADP+ - where the quinones have a more positive redox potential than NADP+.
Anoxygenic phototrophs - by definition these are organisms that obtain their energy from the sun by converting solar energy to chemical energy and do not produce oxygen during photosynthesis as higher plants and some eukaryotic and prokaryotic microorganisms do.
Compare Figure 13.9 with 13.13
We have been talking about energy but what about carbon? How do autotrophs obtain their carbon if not from organic sources?
Calvin cycle or reductive pentose cycle (see 13.18)
Ribulose bisphosphate carboxylase is the enzyme responsible for the first reaction in the cycle involving CO2 and ribulose bisphosphate - a 5 carbon sugar. The product is 2 molecules of phosphoglyceric acid.
Phosphoglycerate is phosphorylated and reduced by NADPH to 3-phosphoglyceraldehyde.

Steps:
6 molecules of ribulose bisphosphate accepts 6 molecules of CO2 for a total of 36 carbons among 12 3-carbon molecules (phosphoglyceric acid). These 12 molecules are than rearranged to make 6 5-carbon molecules (ribulose bisphosphate) and 1 6-carbon (hexose) molecule. A total of 12 NADPHs and 18 ATPs are used during the reduction of 6 CO2s to hexose.
Chemolithotrophs - obtain their energy from the oxidation of reduced inorganic molecules. ATP generated from oxidation of inorganic molecules like chemoorganotrophs (aerobic respiration process) and reducing power (NAD(P)H) is generated either directly if the inorganic molecule has a redox potential sufficiently low or by reverse electron transport reactions. Recall the greater the difference between the redox potentials of two reactions, the greater the amount of energy released.
Hydrogen oxidizing bacteria - most are facultative chemolithotrophs capable of growing either chemoorganotrophically or chemolithotrophically.
Chemolithotrophic growth - hydrogenase enzyme oxidizes H2 and the electrons are transfered to quinones in the electron transport chain ultimately moving to oxygen as the terminal electron acceptor. A PMF is generated by the movement of the electrons and ATP is made via membrane bound ATPases.
Reducing equivalents in the form of NADPH
some species reduce NADP+ directly
most species use the reverse electron transport mechanism discussed earlier
Carbon fixation - use the Calvin cycle but organic carbon will repress the synthesis of key enzymes of the Calvin cycle.

H2 --------> 2H+ 2e (-0.420 V)
1/2O2 +2H+ +2e ----------> H2O (+0.820 V)
H2 + 1/2 O2 ------->H2O
[Delta]G^o = -nF[Delta]E_o´
= -2 (96.5kJ/V) (0.820 -(-)0.420)
= -240 kJ/reaction

Sulfur oxidizing bacteria - These were some of the first chemolithotrophs described by Winogradsky. Use reduced sulfur such as H²S, S^o, and S₂O₃^2-(thiosulfate).
ATP formation - Electrons enter the electron transport chain at various points depending on the redox potential of the source. Electrons flow to oxygen as the terminal electron acceptor and generate a PMF which is used to drive ATP synthesis.
Reducing equivalents - NADPH is formed by the reverse electron transport mechanism.
Carbon fixation - Calvin cycle
Note: they grow poorly on elemental sulfur or thiosulfate since these compounds have a relatively high redox potential.

HS^- ---------> S^o + 2e + H⁺ (-0.270 V)
1/2O₂ +2H+ +2e ----------> H2O (+0.820 V)
HS^- + 1/2O₂ + H⁺ -----> S^o + H₂O
= -2 (96.5 kJ/V) (0.82 -(-)0.270)
= -210.37 kJ/reaction
S^o + 4H₂O----------> SO₄^2- + 6e + 8H⁺ (-0.180 V)
3/2O₂ +6H⁺ +6e ----------> 3H₂O (+0.820 V)
S^o + H₂O +3/2O₂ -------> SO₄^2- + 2H⁺
=-6 (96.5 kJ/V) (0.82 -(-)0.180)
=-587.1 kJ/reaction

Note:H+ is generated which means that this is an acidifying reaction - sulfuric acid is generated. Some sulfur-oxidizing bacteria can live at pHs in the 1-2 range.
Iron-oxidizing bacteria - Ferrous iron, Fe+2, is oxidized to ferric iron, Fe+3. Iron has an interesting chemistry about it: Ferrous iron rapidly oxidizes to ferric iron in air at neutral pH - stable in anoxic conditions. At acidic conditions, ferrous iron is stable. Best known iron-oxidizing bacteria is Thiobacillus ferroxidans. Found in acid polluted waters like coal mining dumps.
Energy production - ATP formation
Very little difference between the redox potential of Fe+3/Fe+2 and 1/2O2/H2O compare +0.77 V at pH 3 for the former to +820 V for the latter. The redox potential of Fe+3/Fe+2 is too positive to reduce NAD+, FAD, and many of the electron transport chain.
Unique ecology allows these bacteria to take advantage of the natural pH gradient in which they live. The live in acidic environments such that the pH outside the cell might be 2 and inside the cell the pH is around 6 - this difference is a preformed PMF. To maintain a constant pH inside the cell, the oxidation of Fe+2 to Fe+3 is a proton consuming reaction - 2Fe⁺³ + 1/2O₂ +2H⁺ -----> 2Fe⁺³ + H₂O.
Carbon fixation - Calvin cycle as usual. Generate NADPH by the reverse electron transport process using the PMF to drive electrons uphill. Because of the very positive redox potential of Fe+3 / Fe+2 the reverse electron process requires a lot of energy, therefore there is very little biomass generated by these organisms in the habitat but there is lots of ferric iron precipitates.
There is some iron oxidation occuring near neutral pH specifically at the oxic and anoxic interfaces in nature. Recall that at neutral pH ferrous iron is rapidly oxidized to ferric iron in the presence of oxygen.
Neat phenomenon - large deposits of ferric iron have been found in ancient sediments which were originally thought to be the result of abiotic oxidation of ferrous iron where the oxygen came from oxygenic photosynthesis. Perhaps these deposits were formed by anoxygenic phototrophs oxidizing ferric iron in anoxic environments.
Ammonium and nitrite-oxidizing bacteria - referred to as the nitrifying bacteria. They use ammonia and nitrite as their energy source - electron source. Nitrosomonas oxidize ammonia and Nitrobacter oxidize nitrite. These bacteria are widespread in soils.
Growth yields from the oxidation of NH₃ or NO₂^- are very small since there is very little energy available from the oxidation of these two compounds. The redox potentials of ammonia to nitrite is 0 volts and nitrite to nitrate is +0.43 Volts and therefore they donate electrons to the electron transport chain at relatively high redox potential electron carriers.
Carbon fixation - Calvin cycle
Summary - chemolithotrophs growing autotrophically require ATP and NADH. NADH generated either by the direct reduction of NAD⁺ by H₂ for the hydrogen oxidizing bacteria or the energy driven reverse electron flow for the other chemolithotrophs.
ATP production - the chemolithotrophs contain cytochromes and quinones like the chemoorganotrophs. With the exception of the hydrogen oxidizing bacteria, the other chemolithotrophs cannot feed electrons into the electron transport chain at NADH level but instead at some intermediate level between NADH and oxygen.
Mixotrophy - chemolithotrophs that can grow on organic carbon as heterotrophs.
Anaerobic respiration - uses an electron acceptor other than oxygen - could be organic or inorganic in nature.
facultative aerobes - are aerobes that can use either oxygen or another electron acceptor such as nitrate.
obligate anaerobes - are anaerobes unable to use oxygen.
Nitrate reduction and denitrification - nitrate as an electron acceptor and reduced to nitrous oxide or dinitrogen gas. This process is called denitrification which is detrimental to agriculture since it represents a loss of nitrogen from the ecosystem.
Enzyme is nitrate reductase which is synthesized under anoxic conditions only. Produces nitrite which is reduced using nitrite reductase.
Nitrite may be reduced to ammonia or reduced to nitrous oxide or dinitrogen gas. (See figure 13.29).
See figure 13.30 for comparison of nitrate reduction with aerobic respiration. The point is less energy from nitrate reduction than aerobic respiration.
Common denitrifiers include species of pseudomonads such as P. fluorescens and P. aeruginosa.

Sulfate - reducing bacteria - sulfate is reduced to hydrogen sulfide. An organic molecules including organic acids, fatty acids, alcohols and H₂ as an electron source = source of energy. H2 produced during the oxidation of lactate diffuses out of the cell and is subsequently oxidized by a hydrogenase leaving the protons outside the cell and transporting the electrons to sulfate. This generates the PMF which can be used to make ATP. There are other types of sulfate reducing bacteria that we will not get into.
Methanogenesis - methane formers. These use carbon dioxide as an electron acceptor

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⁺
__________________________________
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.

Cell Biology - Chapters 3 & 4

Today we want to begin to exam some of the structure and function relationships in prokaryotic cells. Remember that cells are essentially well organized assemblages of macromolecules with many common features shared among diverse types of cells.
Begin the discussion with the microscope since it has been instrumental in microbiological studies.

Microscopes

Two features:
Magnification - pretty much increase the magnification of an image indefinitely. Magnification is the product of the eye piece magnification times the objectives magnification.
Resolution - the ability to separate or resolve to close objects. There is a limit which is a function of the wavelength of the light source. Resolution equals 0.5 times [lambda] divided by the numerical aperture. Blue light gives the greatest resolution.
Two types of microscopes:
Compound light microscope - resolution of about 0.2 micron.
Electron microscope which we will come back to later.
Compound light microscopes
Bright field microscope - Microscope you used in class the other day. Usually visualize specimens by contrasting the cells against the light background. We can use dyes to stain the cells.
Types of dyes used are cationic, meaning that they are positively charged since the cell and much of the macromolecules in a cell are negatively charged. Methylene blue, saffarin, and crystal violet are common dyes used in the laboratory.
Phase contrast microscope - view living cells and don't need to stain the cells. How? Light passing through a cell is retarded relative to the light passing through the liquid medium. The changes in the phase of the light waves is translated into changes in the intensity of light seen through the ocular lens.
Dark field microscope - A dark field ring placed in the condenser ring causes the light to be focused into a hollow cone with the specimen at the apex of the cone. The light is moving in a direction such that it would not enter the objective lens. Therefore without anything on the stage the field of observation would be dark. A specimen on the stage will cause the light to bend and enter the objective lens causing a bright image on a dark background when viewed through the ocular lens. Very high resolution and good to view flagella.
Fluorescence microscope - a specialized microscope which illuminates the specimen with light of one wavelength, e.g., ultraviolet, and the specimen emits light at a higher wavelength which is observed. Detect fluorochromes that may be attached to antibodies. The fluorochromes have resonating double bonds that when hit by high energy light such as ultraviolet causes the electrons to become excited. The excited electrons return to ground state and emit light rather than simply heat.
Electron microscope - very high resolution microscope which used electrons rather than light. Can see proteins and nucleic acids using the electron microscope.
Transmission electron microscope - Thin sections of cells impregnated in a matrix are made. The thin sections are then viewed using the transmission electron microscope. To enhance contrast, the sample is coated with an electron dense stains such as osmic acid, uranium, or lead. These stains cause the electrons to be scattered thus increasing the contrast between sub-cellular constituents.
Scanning electron microscope - Used to observe intact cells. The cells are coated with something like gold. Electrons are scattered by the gold plated cells which allows them to be viewed. Magnify up to 100,000X.

Specimen preparation -

in most situations the specimen is heat fixed to the slide before all staining procedures. This is to bind the cells to the slide so that they will not be subsequently washed off in the staining procedure.

Dyes are ionic chromophores meaning that they carry an electronic charge and are colored. basic dyes are cations with positive charges. Most bacteria have a slightly negative charge at neutral pH. Therefore basic dyes will be attracted and bind to the cells. acidic dyes are anions with a negative charge. They are not attracted to cells and usually are used to provide a dark background with the cells highlighted.
Simple stains are used to simply provide contrast to see the cells. Cells are mostly water and therefore would be very difficult to see with a light microscope. A simple stain such as methylene blue binds to the cell and highlights the whole cell.
Differential stains are used to differentiate between microorganisms. The most obvious differentiation based on staining is the Gram positive versus Gram negative species of bacteria. Gram staining was devised by Hans Christian Gram. Cells are first stained with crystal violet, followed by a mordant such as iodine, followed by a alcohol wash and subsequently stained with a counter stain such as safranin.
Special stains include the negative stain, capsule stains and flagella stain.

Common features of a prokaryotic cell

Cell wall - rigid structure that provides strength and shape to cell.
Cytoplasmic membrane - semi-permeable barrier between outside and inside cell.
Ribosomes - small particles in cell which are responsible for the translation of mRNA into a polypeptide.
Inclusion bodies - storage "organelles" found in prokaryotic cells.
Nucleoid - nuclear region of the prokaryotic cell. Remember there is no true nucleus like in a eukaryotic cell.
Size of prokaryotes
Average 1 x 3 microns but there is lots of variation.
Smallness affects their biological activity: Small cocci have a greater surface to volume ratio than bigger cocci. This effect exchange of nutrients and waste products in and out of cell. This in turn allows small prokaryotes to have more rapid growth rates and larger population sizes than most eukaryotes in a microbial habitat.
Basic shapes include coccus (spheres), bacillus (rod), and spiral.

Cell structure outside in Flagella -

used for motility. Not found in all bacteria, i.e., there are nonmotile bacteria. Arranged differently on different species of bacteria - polar flagella are attached to one (monotrichous) or both ends of the cell (amphitrichous). Lophotrichous flagella are tufts of flagella attached to one end of the cell. Peritrichous flagella are attached at many places around the cell.

Structure
Flagella composed of subunits of a protein called flagellin.
Base of the flagellum is the hook which is composed of a single protein and serves to attach the flagellum to the motor apparatus.
The motor, called the basal body, is anchored in the cytoplasmic membrane and the cell wall. Consists of a rod inserted through two rings in gram positive cells and three rings in gram negative cells. The inner two rings of all prokaryotes are anchored in the cytoplasmic membrane and peptidoglycan layer of the cell wall. The third ring of the gram negative cells is anchored in the outer membrane.
Mot proteins - proteins that surround the inner ring in the cytoplasmic membrane. These are the actual motor proteins. They cause the flagella to rotate.
Fli proteins - proteins associated with the inner ring. They are responsible for switching the direction in which the flagella rotates.
Overall there are some 40 genes required to make an operational flagella.
Growth
Flagella grow at the tip not the base. Flagellin is passed up through the hollow core of the flagella. They than self assemble at the tip of the flagella.
Function
Used for motility. Flagella is a semirigid structure with a helical shape- like a cork screw - that actually rotates. Proton motive force drives the motor as the protons move through the mot proteins. Cells can move about 60 cell lengths per second - faster than a cheetah which moves at 25 body lengths per second.

Cells are always in gradients and need to be able to respond to gradients either positively or negatively by moving into or away from signal molecules respectively.
chemotaxis - bacterial response to a chemical gradient. Bacteria sense the gradient temporally not spatially since they are so small. In the absence of a gradient, the bacteria move randomly about in a series of runs and tumbles and moves nowhere. Runs are due to the flagellar motor moving counterclockwise and tumbles occur when the motor is moving clockwise. As they move into a gradient of an attractant, movement becomes biased with longer runs and fewer episodes of tumbling. Similarly as the cell moves away from a repellent, there are longer runs and fewer tumbles.
The actual sensing mechanism is complex and will be discussed when we talk about gene regulation. Suffice it to say that the cell has chemoreceptors for different molecules and these proteins interact with cytoplasmic proteins that affect the motor direction.
phototaxis - analogous to chemotaxis but the cells are responding to light gradients. This type of taxis is found in phototrophic bacteria which may want to orient themselves in the best position to carry out photosynthesis. Photoreceptors rather than chemoreceptors sense the light gradients.
other taxes - response to other types of gradients such as oxygen, magnetic fields, or ionic strength. Similar processes as discussed above.

Fimbriae and pili -

Fimbriae are similar to flagella in that they consist of protein. There are many more fimbriae than flagella. Their function is not always known, but they are used for attachment in the case of some pathogens or forming pellicles or scums on surfaces.
Pili are structurally like fimbriae but there are fewer per cell. Function in gene transfer = sex pili and attachment to host tissues.

Glycocalyx

A general term referring to the slime layer or capsule of a cell. Made up of polysaccharide and proteins which is highly variable depending on the bacterial species.
Function to aid in attachment to surfaces, resist phagocytosis -a virulence factor for pathogens, and resist desiccation since the glycocalyx binds lots of water.

Outer membrane of gram negative cells
Structure
Based on a differential stain, called the Gram stain, prokaryotes can be divided into gram positive and gram negative microbes. The outer membrane is found only in gram negative prokaryotes. Referred to as lipopolysaccharide layer or simple LPS.

Composed of phospholipids, polysaccharides and protein.
Polysaccharides consists of core polysaccharides and O-polysaccharides.
Core polysaccharides contains ketodeoxyoctonate (KDO) and various sugars.
O-polysaccharides consist of repeating units of sugars and are linked to the core polysaccharides. These are linked to Lipid A.
Lipid A is not a glycerol lipid but rather fatty acids are linked to a disaccharide of N-acetyl-glucosamine phosphate via an ester amine linkage. The O-Polysaccharide is linked to lipid A via KDO.
Lipoproteins are found in the inner half of the outer membrane. These anchor the outer membrane to the peptidoglycan of the cell wall.
Other proteins are found in the outer half of the outer membrane.
Notice that the lipids of the outer surface of the outer membrane is made up primarily of LPS and the inner surface is made up of phospholipids.
Function
The outer membrane of many gram negative bacteria is toxic to animals. Referred to as endotoxin - lipid A is the toxic portion. Includes Salmonella, Shigella, and Escherichia coli.
Provides resistance to some toxins and antibiotics.
Acts as a second lipid bilayer. Contains porin, protein channels, that allow hydrophilic, low molecular weight molecules to move through the bilayer. Porins are responsible for some antibiotic resistance properties of gram negative bacteria.

Periplasm

-

space between the outer and inner (cytoplasmic) membranes - gram positive lack a well defined periplasm. Contains numerous proteins of hydrolytic enzymes, which break down food, binding proteins, which bind molecules and transport them to the inner membrane carrier proteins, and chemoreceptors.

Cell wall

Cell wall found in both gram positive and gram negative prokaryotes.
Function
Resist turgor pressure due to dissolved solutes inside the cell - pressure about that of a car tire.
Provide shape and rigidity of cell.
Cocci - spherical shape
Rod or bacilli - rod shaped
Spirilla - twisted rod about an axis
Spirochetes - tightly coiled
Appendaged bacteria - possess extensions such as stalks or long tubes
Filamentous bacteria - form long chains of cells
The basis of gram positive vs gram negative prokaryotes. The cell wall of these two are distinctly different.
Structure
Peptidoglycan composed of two sugars, N-acetyl-glucosamine (NAG) and N-acetyl-muramic acid (NAM) and a small group of amino acids including L-alanine, D-alanine, D-glutamic acid, and either lysine or diaminopimelic acid (DAP). Rare instance where you find D forms of amino acids used in biological systems.
Thin sheet of alternating NAG and NAM linked by glycosidic linkages. NAM has a short peptide of 4 amino acids linked to it.
In gram negative bacteria, there is a direct linkage between the amino group of DAP of one NAM and the carboxyl group of the terminal D-alanine of a second NAM.
In gram positive prokaryotes, there is a oligopeptide linker between the amino acid side chains of adjacent NAMs. There is lots of variation on this theme
90% of the gram positive cell wall consists of peptidoglycan. There may be up to 25 layers of it in the cell wall. In gram negative cell only about 10% of the cell wall is peptidoglycan. The majority is the complex outer membrane layer.
Diversity of peptidoglycans due to differences in the tetrapeptide - though these are small differences, and more differences in the crosslinking interbridge between the tetrapeptides of adjacent NAMs.
Summary - what is constant is the two sugars are always NAG and NAM linked by beta 1,4 linkages and NAM is always the sugar that is crossed linked.
Differences
Gram positive cell wall contains teichoic acids. Not found in gram negative prokaryotes.

Protoplast formation
Protoplasts lack or have their cell wall integrity disrupted by a treatment.
Lysozyme breaks the beta 1,4 glycosidic linkage between NAM and NAG. Water may enter into the cell and cause it to burst in a process called lysis. Lysozyme found in tears, saliva and other bodily fluids presumably as a defense against bacteria.
Protoplasts are cells where the cell wall is degraded with lysozyme but placed in an isotonic solution so water does not enter the cell and cause it to burst.
There are some bacteria that lack cell walls and peptidoglycan layers - these are the mycoplasmas. Essentially free-living protoplasts that are able to survive under specific conditions.

Inner membrane or Cytoplasmic membrane - thin structure that surrounds the cell's cytoplasm. Highly selective barrier allowing concentrating of nutrients inside the cell and excretion of wastes.

Composition
Phospholipids forming a phospholipid bilayer with the hydrophilic phosphate groups facing outwards and the hydrophobic hydrocarbon tails pointing inwards to form a hydrophobic core. Referred to as unit membrane.

Proteins associated with the membrane:
Integral proteins that span the membrane. They contain a hydrophobic core region which is embedded in the hydrophobic core of the membrane and hydrophilic regions that are stick out of the membrane.
Peripheral proteins are associated with either the internal or external surface of the membrane. Frequently associated with integral proteins. Some of them associated with lipids and are referred to as lipoproteins. The lipids function to anchor the protein in a certain position on the membrane.
Fluid mosaic - though the membrane looks rigid it is fluid - viscosity like light motor oil. And the proteins move about the membrane like ships on water - hence the fluid mosaic model for the cytoplasmic membrane.

Function
Selectively permeable - allows specific ions, and molecules to move across.

Small, non polar and fat-soluble substances can move across the membrane freely. Water moves across the membrane freely.

Polar solutes, charged molecules, e.g., amino acids, organic acids, and inorganic salts, do not move across the membrane. Hydrogen anions do not since they hydrate to H3O+ which is charged.

Membrane transport proteins
3 classes
Uniports
Symports
Antiports

Facilitated diffusion
Protein allows or moves molecules across the membrane down the molecules concentration gradient - a source of potential energy. Still requires a protein. Some proteins are non specific and move related molecules and others are specific as to what they let cross the membrane.

Active transporters
General features
1. Requires a carrier protein
2. Requires energy to move the molecule. The hydrolysis of ATP or the dissipation of a proton gradient generated during energy releasing reactions in the cell.
3. Can move the molecule up its concentration gradient (i.e., against a concentration gradient) - which is why it requires energy.
Group translocation - the transported molecule is modified as it is transported across the membrane. The phosphotransferase system or PTS is the most intensely studied group translocation system. A complex system of many cytoplasmic and membrane bound proteins. Phosphoenol pyruvate is the intermediate which donates the phosphate group.
Active transport - the molecule is not modified as it is transported across the membrane. Energy derived from hydrolysis of ATP or the dissipation of a proton gradient -a so called proton motive force.

DNA
A chromosome with few associated proteins. No nuclear membrane. In addition, there are extrachromosomal elements called plasmids. Chromosome normally circular molecule of a couple of thousand kilobase pairs (1000 bps). Tends to aggregate and form a nucleoid. Linearized chromsomes of 4000 kilobase pairs would be about 1 mm in length while the bacteria is only 3 microns. To compact the chromsome, the DNA is supercoiled with 50 or more domains maintained by proteins that interact with each domain.
Because a cell can divide faster than the chromsome can replicate there are usually more than one copy or partial copy of the chromosome in a cell. We call bacteria haploids since they contain only on chromosome.
Genetic exchange - conjugation, transduction, and transformation

.Endospores
Differentiated cells that are extremely resistant to a number of harsh treatments and remain dormant yet viable for long periods of time. Very important in how we preserve food since several food borne pathogens form spores including Clostridium botulinum which causes botulism - potentially lethal.