Unit 2 2/19/17 7:00 PM

What is Organic?

Microbial Nutrition

Nutrition: the process of acquiring chemical substances or nutrients from the  environment and using them in cellular activities

• Essential Nutrients

o Macronutrients: elements that make up the majority of the  cell, CHONPS  

▪ Carbon

▪ Hydrogen

▪ Oxygen

▪ Phosphorus

▪ Sulfur

o Micronutrients: elements that are required in small amounts  and involved in cell function. Ex: Mg used in chlorophyll.  

• Nutrients can be:

o Organic: carbon, carbohydrates, proteins

o Inorganic: CO2, water, gases like oxygen

• Microbial cytoplasm made up of  

o 70% water

o Proteins  

o 96% of cell made up of 6 elements (CHONPS) – this is in a  Dry Weight Perspective

• Chemical Composition of E. Coli

o Dry Weight

▪ Proteins: 50%

▪ RNA: 20%

▪ DNA: 3%

What is Inorganic?

▪ Carbohydrates: 10%

▪ Lipids: 10%

o Wet Weight

▪ Proteins: 15%

▪ RNA: 6%

▪ DNA: 1%

▪ Carbohydrates: 3%

▪ Lipids: 3%

• Sources of Essential Nutrients

o Carbon Sources

▪ Heterotroph: must obtain Carbon from organic sources  (proteins, carbohydrates, lipids, nucleic acids)  

???? Chemoheterotrophs: Microorganisms that use  organic compounds for both energy and carbon  If you want to learn more check out psy 402 midterm
If you want to learn more check out isqs

(Chemotroph get energy from chemical sources)  

▪ Autotroph: organism that gets carbon through inorganic  sources, make organic compounds from CO2 –

(photosynthetic organisms = photoautotroph)  

???? Chemoautotroph: microorganism that uses CO2  as a source and catabolizes organic molecule for  

energy  

o Nitrogen Sources

▪ Reservoirs

▪ It is a component of air and chitin

▪ Some organisms can use it as Nitrate and other can  make nitrogen through the nitrogen cycle  

Microbial cytoplasm made up of what?

o Oxygen Sources

▪ It is a component of the air, carbohydrates, lipids,  nucleic acids, proteins, salts

▪ It can be directly used

???? Anaerobes don’t get oxygen in the same form  (gas) but still use it.  

o Hydrogen Sources

▪ Can get it from water, gases and salts  

▪ Roles of Hydrogen are to maintain pH and is an  acceptor of oxygen during cellular respiration

o Phosphorus/Phosphate Sources

▪ Component of nucleic acids (essential to genetics) ▪ Used in energy transfers (ATP)  

o Sulfur Sources

▪ Found in amino acids and vitamins  

▪ It is widely distributed in the environment

▪ Used in protein stability

o *Other Nutrients that are Important in Microbial Metabolism ▪ Potassium

▪ Sodium

▪ Calcium

▪ Magnesium Don't forget about the age old question of penn state math 41

▪ Iron

▪ Zinc, copper, nickel, manganese, etc.  

• Classification of Nutritional Types

o Carbon Source

▪ Autotroph: organisms that use inorganic sources of  carbon as their only source of carbon and make organic  compounds from CO2

▪ Heterotroph: catabolize organic molecules they get from  other organisms.

o Energy Source  

▪ Chemotroph: organisms that get energy from inorganic  and organic chemicals through redox reactions  

???? Chemoautotroph: microorganism that use CO2 as  

their carbon source but chemical compounds as  

their energy source  

???? Chemoheterotroph: Microorganisms that use  

organic compounds for the carbon source and  

chemical compounds as their energy source  

▪ Phototroph: organisms that use light as a source of  energy

???? Photoautotroph: use CO2 as source of carbon and  

light as source of energy

???? Photoheterotroph: use organic compounds as  

source of carbon and light as source of energy  We also discuss several other topics like sjsu bus5 187

• Transport Mechanisms

o Passive Processes – no energy used  

▪ Simple Diffusion: net movement of chemical down its  concentration gradient, requires no spending of energy  ▪ Facilitated Diffusion/Transport: proteins act as channels  or carriers to transport down the concentration  

gradient, into or out of the cell, no energy spent,  

???? Non-specific channel proteins don’t have a  

preference for what they transport

???? Permeases: channels that carry only specific  

chemicals  

▪ Osmosis: diffusion of water across semipermeable  Don't forget about the age old question of bbh 119 exam 2

membrane, down concentration gradient  

o Active Processes – uses energy, can move against or with the  concentration gradient

▪ Active Transport: use transmembrane permease  

proteins to move molecules across the membrane, has  

to use energy to do so, use gated channels or ports

???? Uniport: one thing moving in one direction

???? Antiport: move two things at the same time but in  

opposite directions

???? Symport: move two things at the same time in  

the same direction  

▪ Group Translocation: The substance transported across  the membrane is also chemically changed during the  

transport  

???? Ex: when take glucose into the cell and it is  

phosphorylated at the same time

• Solution Types

o Isotonic: when solutions on both sides of the semipermeable  membrane have the same concentration of solutes

o Hypertonic: concentration of solutions unequal, the solution  with higher concentration of solutes is called this

o Hypotonic: when concentration of solutions unequal, the  solution with the lower concentration of solutes is called this  Microbial Growth  

*Prokaryotes do not perform phagocytosis, only Eukaryotes do, Prok. send  enzymes out to break down things and then import them via active transport  and facilitated diffusion  

• Population Growth

o Generation Time: the time required for a cell to double We also discuss several other topics like math 101 notes

▪ Cells divide by binary fission (form of exponential  

growth, to calculate the number of cells that one cell  

can produce = 2^n, n being the number of generations,

to calculate the total number of cells = multiply number  of original cells by 2^n )

???? Exponential Growth: regular spaced division, very  rapid increase in growth

▪ Bacteria Cells synthesize a new cell wall while  weakening the outside cell wall to form two new cells  from the original.  

o Growth Curves: graph that plots # of organisms in a growing  population over time  

▪ Lag Phase: time period where the cells are adapting to  the new environment, do not reproduce right away,  synthesize enzymes to use nutrients in the medium

▪ Log Phase: phase when cell population increases  logarithmically

▪ Stationary Phase: nutrients become depleted and the  number of dying cells equal the number of cells being  produced, population remains constant  

▪ Death Phase: the number of cell deaths rises about the  number of cells produced

o Diauxic Shift: Discovered by Jacques Monod 1941. different  sugars produce different growth curves, the growth curves  are typically based off of how the bacteria would be grown in  a laboratory setting, with a single carbon source. The true  growth curves would be different than these standard ones  because the organism would have many different carbon  sources in real life.  

o Methods for Analyzing Growth:  

▪ Hemocytometer: sample under a glass slide with an  etched grid on the bottom, the grid is 25 squares and  designed to suspend the microbes in 0.02mm of liquid,  volume of suspension of microbes is 1mm x 1mm x  0.02mm = 0.02mm^3

???? Not good for motile things  

▪ Coulter Counter: as cells pass through triggers an electronic sensor that tallies their numbers

???? Useful to count larger cells (yeast, unicellular  

algae, protozoa), and for bacterial counts because  

of debris in the media.

▪ Flow Cytometer: uses a light sensitive detector, detects  changes in light transmission through the tube as cells  pass, used to tell between differentially stained cells  

that have been stained with fluorescent dyes or tagged  w/fluorescent antibodies

???? Detect and sort different populations of cells from  

blood  

???? Counts bacteria and host cells  

???? Used to look at fungal cells to determine if they  

are haploid or diploid

▪ Pour Plate Technique: step-wise dilution of a culture,  dilution factor the same every time, scientist plates set  amount of dilution onto agar plate and counts the  

number of colonies resulting on a plate.  

▪ Turbidity: cloudiness of the solution, more bacteria the  cloudier the solution

???? Spectophotometer measure the amount of light  

transmitted through a culture under standard  

conditions. Higher concentration of bacteria in a  

broth more light will be absorbed and scattered,  

less passing through and hitting back sensor.  

???? Technique only useful density above 1 million per  

mL.

• Environmental Factors Influencing Growth

o Environment – enzymes adapt to whatever condition it grows  at

▪ Temperature

???? Psychrophile: microorganism requiring cold  

temperatures below 20°C

???? Mesophile: microorganism that requires  

temperatures ranging from 20°C to 40°C

• Optimum Growth (can grow at other  

temperatures but grows best at optimum  

temp) = 37°C

???? Thermophile: grows at temperatures above 45°C • In compost piles and hot springs

▪ Oxygen Requirements

???? Aerobic: organism that uses oxygen as a final  electron acceptor in ETC

• Obligate Aerobe: they are obliged to use  

oxygen, they can’t survive below depth that  oxygen penetrates

???? Microaerophilic: Microorganism that requires low  levels of oxygen

???? Facultative Anaerobe: Can live with/without  oxygen, prefers oxygen  

???? Aerotolerant Anaerobe: microorganisms that  prefer anaerobic conditions but can tolerate  

exposure to low levels of oxygen.

• Expect relatively even growth until it  

reaches its point of oxygen tolerance.  

???? Anaerobe (Strict): An organism that cannot tolerate oxygen.

▪ pH

???? Acidophile: organisms that grow best in acidic  conditions

???? Neutrophile: grow best in a narrow range around  a neutral pH (between 6.5 and 7.5, which is also  the pH range of tissues and organs in the human  body)  

???? Alkalophile: organisms that live in alkaline soils  and water up to 11.5pH

▪ Osmolarity  

???? Halophile: need high salt concentration

???? Osmotolerant: organism can tolerate salt, doesn’t need it to survive  

• Ex: Staphylococcus epidermidis

• Ecological Associations  

o Symbiotic: organism lives in close nutritional relationship, one  or both of the organisms involved require the relationship.  ▪ Mutualism: both organisms involved require the  

relationship and both benefit from it.  

???? Ex: bacteria in cow’s rumen & interaction between  

pea plants and Rhizobium & E.coli

▪ Commensalism: one organism benefits from the  

relationship, the other is not harmed or benefitted.  

???? Ex: Satellitism between Staphylococcus aureus

and Hemophilus influenza

▪ Parasitism: the relationship is not required by both  

organisms, the one that requires it is doing damage to  

the other  

???? The host is always harmed – levels of damage  

may differ

???? Ex: any pathogen that can get you sick  

o Non-Symbiotic: organisms are free-living, the relationships  are not required for survival

▪ Synergism: members benefit from each other but are  

still able to survive on their own  

???? Works in either biosynthesis of a certain end  

product or the breakdown of waste

• Provide extra things to get to the end  

product  

▪ Antagonism: relationships don’t help survival, compete  for nutrition and space  

???? Some members are inhibited or destroyed by  

others  

???? Organisms that produce antibiotics, one microbe  

makes antibiotic and the other is hurt by it

• Led to penicillin  

Metabolism of Microbes

• Metabolism: chemical and physical workings of a cell

o Anabolism: biosynthesis, forms larger molecules, requires  energy input  

o Catabolism: degradative, breaks bonds to form smaller  molecules, releases energy  

o *The intermediates can be used for other reactions at  multiple stages in the reaction  

• Enzymes (biological catalysts, lower energy of activation to increase  the rate of chemical reaction) – in biological reactions  o Energy of Activation: the resistance to a reaction, amount of  energy needed to trigger a chemical rxn

o Enzymes are Not permanently altered in the rxn  

o They promotes rxn by having a physical site for substrates to  bind to  

o Most are made of protein and may have cofactors

▪ Cofactors could be metallic cofactor like iron in  

Hemoglobin or non-metallic cofactors

o They are organic catalysts  

▪ Catalyst: chemicals that increase the likelihood of a rxn  but are not permanently changed in the process.

o Have unique characteristics (shape, function, specificity) that  doesn’t allow them to react with just any type of substrate o Have an active site – where the substrate binds and the  reaction happens

o The enzyme itself is not part of the reaction substrate or  products  

o It is not used up in the reactions or recycled  

o The rxns are often controlled mechanisms  

o Lock and Key Model: shape of the substrate is complementary  to the shape of the active site, describes the enzyme

substrate specificity.  

o Induced-fit Model: lock and key analogy not entirely correct  because the enzyme’s active site changes shape when the  substrate binds, describes enhanced enzyme-substrate  specificity

▪ Shape of the substrate is kind of specific to the active  site but not exact  

o Structure

▪ Apoenzyme: the protein portion of enzyme, enzyme  without its cofactor, is inactive unless bound to one or  more cofactors.  

▪ Holoenzyme (Conjugated): binding of an apoenzyme  and its cofactor that makes an active enzyme.

???? Cofactors: inorganic ions or organic molecules  that are essential for the enzyme action.

• Coenzymes are an example of organic  

cofactors, organic cofactors can also be  

vitamin based

o three important ones are the electron  

carriers (NAD+, NADP+, FAD)

• Metallic Cofactors

▪ Exoenzymes: enzyme that functions outside the cell  that produced it

???? Can secrete things and damage things  

???? Eukaryotes and Prokaryotes can secrete things so  there are proteins that end up outside the cell for  specific reasons  

▪ Endoenzymes: enzyme that functions inside the cell  that produced it

???? Most enzymes are these, intracellular

▪ Constitutive Enzymes: enzymes that are always  present, they are always produced regardless of  

physiological demand because their function in cell  processes is indispensible.

▪ Induced Enzymes: molecules induce or enhance the  production of these enzymes

▪ Synthesis or Condensation Reactions: anabolic rxns,  form covalent bonds between smaller substrate  

molecules, require ATP, release one molecule of water  for each bond formed

▪ Hydrolysis Reactions: Catabolic reactions, breakdown substrates into smaller molecules, need water to break  bonds  

o Environmental Sensitivities – activity influenced by  environment  

▪ Labile: describes some enzymes when they are  chemically unstable  

▪ Increased temperature = Denatured Protein: protein  heated up = bonds are broken. If cooled it won’t go  back to normal because it didn’t start out complete,  they fold as they are made  

???? Functional Protein: needs structure to get  

catalytic site in place  

▪ pH: denatures protein when ions released from acids  and bases interfere w/h-bonding and disrupt secondary  and tertiary structure

▪ Enzyme/Substrate Concentration: As concentration of  substrate increases the enzyme activity increases until  all active sites are bound = saturation point, more  substrate will not increase rate of activity

o Regulation of Enzymes  

▪ Competitive Inhibition: inhibitory molecules, similar in  shape to the substrate molecules, compete for & block  the active sites. This can be permanent or reversible, if  it is reversible it can be overcome by increasing the  concentration of substrate = increasing the likelihood  that substrate will bind before inhibitor.  

▪ Noncompetitive Inhibition: Allosteric, regulatory  inhibition  

???? Allosteric Inhibition: The regulatory site is  

separate from the active site, when the inhibitor  binds it distorts the active site and doesn’t allow  the substrate to bind  

???? Allosteric Activation: the activator reverses this  ▪ Feedback Control: when end product of a series of  reactions is an allosteric inhibitor of an enzyme in an

earlier part of the pathway, prevents synthesis of more  of this end product.  

???? There is enough product already made, signals  that they don’t need anymore

▪ Control of Gene Synthesis – this is a slower response ???? Enzyme Repression: controls the synthesis of key  enzymes and inhibits at the genetic level

???? Enzyme Induction: enzymes are made only when  the right substrates are present  

o Pursuit and Utilization of Energy  

▪ Energy: the capacity of a system to do work

???? Forms of energy: Thermal, radiant, electrical,  

mechanical, atomic, chemical  

▪ Exergonic: get energy out  

???? X + Y ????(Enzyme)???? Z + Energy  

▪ Endergonic: not going to happen unless you put energy  in

???? Energy + A + B ????(Enzyme)???? C  

▪ ATP is the energy currency in the Cell  

???? 3 part molecule

• Adenine – nitrogenous base

• Ribose – 5-carbon sugar

• 3 phosphate groups attached  

???? using and replenishing of ATP is constantly  

happening in the cell

???? Remove terminal phosphate = energy released  ???? Formation of ATP through three different  

mechanisms:  

• Substrate Level Phosphorylation: transfer  

phosphate from phosphorylated compound  

(substrate) directly to ADP

• Oxidative Phosphorylation: series of redox  

reactions occur during respiratory pathway  

• Photophosphorylation: ATP is made by using  

the energy of sunlight

o Biological Oxidation and Reduction

▪ Redox Reactions: reduction/oxidation reactions, always  in pairs (electron donor and acceptor involved), the  

process saves electrons and their energy, the energy  

that is released can be used to phosphorylate ADP or  

another compound

???? Oxidation: rxn where electrons are donated (often  in the form of Hydrogen)  

???? Reduction: rxn where electrons are gained

???? (OIL RIG = oxidation is loss & reduction is gain)

???? Reduction Potential: measure of tendency of a  

chemical species to acquire electrons therefore  

being reduced (measured in Volts)  

▪ Electron and Proton Carriers: repeatedly accept and  release electrons and hydrogen to help transfer redox  energy  

???? These are Coenzymes (NAD+, NADP, FAD,  

coenzyme A, and compounds of respiratory chain)  

• Aerobic Respiration: series of enzyme-catalyzed reactions, electrons  are passed from fuel molecules to the last electron acceptor,  oxygen.

o Glycolysis: glucose is split and produces two molecules of  pyruvic acid, while at the same time producing small amounts  of ATP, NADH is also generated  

▪ Step 1: Glucose is phosphorylated by ATP into glucose 6-phosphate (G6P)

▪ Step 2: G6P is rearranged and another phosphate is  added to make Fructose-1,6-bisphosphate (F1,6P)

▪ Step 3: F-1,6-P is divided into DHAP (which is  

converted to G3P) and G3P

▪ Step 4: An inorganic phosphate is added to the two G3P  molecules to make two molecules of 1,3BPG and a  

NAD+ is reduced to NADH (two times)

▪ Step 5: (two molecules so this step happens two times)  ADP steals a phosphate from the 1,3BPG and that  

becomes 3PG

▪ Step 6: 3PG is rearranged to become 2PG

▪ Step 7: Water molecule released when 2PG is converted  to Phospho-enolpyruvic acid (happens 2 times for the  two molecules)  

▪ Step 8: ADP steal the phosphate off the P-enolpyruvic  acid and make pyruvate, and ATP (rxn happens 2x for 2  molecules)  

▪ NET TOTAL: 2 ATP and 2 NADH

▪ ALTERNATE PATHWAYS (Instead of Glycolysis)

???? Pentose-Phosphate Pathway

• The enzymatic formation of phosphorylated  

pentose sugars from glucose-6-phosphate

• alternate way to breakdown glucose

• yields NADPH instead of NADH

• has different intermediates such as Ribose

5-phosphate which feeds into the synthesis  

of nucleotides  

• Yields less ATP than glycolysis

???? Entner-Dourdoroff Pathway

• Produces NADPH instead of NADH

• Yields half ATP than glycolysis (1 ATP)

• Pseudomonads use this pathway to  

catabolize glucose

o Synthesis of Acetyl-CoA: Decarboxylation, enzymes remove a  carbon from the pyruvic acid as CO2. Enzyme then joins the  remaining two-carbon molecule (acetate) to coenzyme A to  make acetyl-CoA and also produced a molecule of NADH (From NAD+).

▪ Because glycolysis made 2 molecules of pyruvic acid  this is done 2 times 

o TCA/Citric Acid Cycle/Krebs Cycle: processes pyruvic acid and  generates 3 CO2 molecules , NADH and FADH2 are generated ▪ Occurs in the cytosol of prokaryotes and matrix of  mitochondria. in eukaryotes. Cells also use for  

catabolism of lipids and proteins

▪ Step 1: oxaloacetate combines with acetyl-CoA

▪ Step 2: citrate is converted to isocitrate so the right  isomer is in place

▪ Step 3: isocitrate is converted to α-ketoglutarate  (NAD+ is converted to NADH and a CO2 molecule is  released)

???? *Isocitrate can also take the Glyoxylate

???? Bypass which produces 2 carbon compounds,  without this there would not be enough carbon  

left to survive at the end of the cycle  

▪ Step 4: α-ketoglutarate interacts with CoA to form  succinyl-CoA (NAD+ and CoA are used to form Succinyl CoA and NADH and CO2 are released)

▪ Step 5: succinyl-CoA releases CoA to form succinate  (two molecules of acetyl-CoA pass through the Krebs  cycle from every one glucose molecules creating two  pyruvates, from these two molecules two molecules  of ATP are produced by substrate level  

phosphorylation, GTP is an intermediary at this step) ▪ Step 6: Succinate is converted to fumarate (FAD is  reduced to FADH2)

▪ Step 7: fumarate gains water to generate malic acid ▪ Step 8: malic acid is converted to oxaloactetate (NAD  +is converted to NADH)

▪ SUMMARY: overall the two molecules of acetyl-CoA that  are gained from the original glucose molecule, end up  forming: 6 NADH, 2 FADH2, 2 ATP

???? Glycolysis and TCA created ATP, Carbon  

intermediates to make other molecules, reduced  

electron carriers (NADH, FADH)

o Electron Transport Chain: Final processing of electrons and  hydrogen and the major generator of ATP  

▪ occurs in the cytoplasmic membranes of prokaryotes  and the inner mitochondrial membranes of eukaryotes ▪ Accepts electrons from NADH and FADH2, this  generates energy through redox reactions and this

energy is captured by ATP synthase to produce ATP =  oxidative phosphorylation

???? Oxidative Phosphorylation: protons flow down  their electrochemical gradient through protein  channels called ATP synthases and these channels  phosphorylate molecules of ADP to ATP.

• Specifically, Hydrogen ions diffuse back  

through the ATP synthase complex causing  

it to rotate, causing a 3-dimensional change  

resulting in the production of ATP  

o Form of Chemiosmosis:

▪ as the electron transport  

carriers shuttle electrons, they  

actively pump hydrogen ions  

(protons) across the membrane  

setting up a gradient of  

hydrogen ions – Proton Motive  

Force

▪ Membrane Set Up – membrane includes  

???? flavoproteins: integral proteins, FMN is an  example of these and is the first carrier molecule  of the transport chain that NADH passes electrons  to.  

???? Ubiquinones are lipid-soluble nonprotein carriers  found in the membrane, Coenzyme Q is an  

example here.

???? Metal containing proteins are a mix of integral  proteins. Cytochromes are integral proteins  

associated with hemes  

▪ Bacterial Process – involves 7 enzymes that rapidly  undergo redox reactions  

???? 1. Complex I – NADH dehydrogenase/FMN ???? 2. Complex II  

???? 3. Coenzyme Q

???? 4. Complex III

???? 5. Cytochrome C

???? 6. Complex IV – cytochrome a/a3 and ATP  

Synthase  

???? The final step: Oxygen accepts 2 electrons from  

ETS and forms water with 2 hydrogen ions from  

the solution. Oxygen is the final electron acceptor  

???? OUTPUT:

• From the 2 NADH from Glycolysis through  

the ETS = 6 ATPs

• From the 6 NADH from the TCA through the  

ETS = 18 ATPs

• From the 2 FADH2 from the TCA through  

the ETS = 4 ATPs

• TOTAL from ETS: 28 ATP

o Complete aerobic oxidation of one molecule of glucose  by a prokaryote is 38 molecules of ATP. Eukaryotes have  to use 2 ATP to get NADH from the cytoplasm to the  

mitochondria so their net ATP is 36.

▪ 10 molecules of NADH from glycolysis, synthesis  of acetyl-coA, and TCA = 30 ATP

▪ 2 molecules of FADH2 from TCA = 4 ATP

▪ 2 ATP netted from glycolysis

▪ 2 ATP from TCA

• Anaerobic Respiration: use inorganic chemicals (oxygen containing  ions) rather than oxygen as the final electron acceptor in the ETC  o Examples of what is used as the final electron acceptor =  Nitrate (NO3-), Nitrite (NO2-), Sulfate, Methanogens may use  CO2 and end up with methane, some use methyl  

o Most obligate anaerobes use H+ made during glycolysis and  Krebs cycle to reduce the compound  

• Fermentation: partial oxidation of sugars (glucose or other  carbohydrates when there is no oxygen present) to release energy o Final electron acceptors in these pathways are organic  molecules

o These yield a small amount of ATP

o Examples:

▪ Ethanol Pathway: Decarboxylation of pyruvic acid  

produces Acetaldehyde, with oxidation of NADH to  

NAD+ ethanol is produced.

???? Yeasts ferment glucose to produce ethanol (then  

complete aerobic respiration)

▪ Lactic Acid Pathway: NADH reduces pyruvic acid to  create lactic acid

???? Various bacteria acting on pyruvic acid forms acid,  gas and other products

▪ Both pathways recycle the electron carrier NAD+  to be used again in Glycolysis to produce more  

energy, benefit to fermentation is ATP production  without a final electron acceptor or cellular  

respiration.

• Amphibolic Pathways: pathways can be catabolic and anabolic o Catabolic pathways can have molecular intermediates that are  diverted into anabolic pathways  

▪ Pyruvic Acid converted into amino acids through  

amination

▪ Amino acids converted into energy sources through  deamination

???? Polypeptides can be broken down into their  

component amino acids by Proteases, the amino  

acids can then be deaminated to create an  

intermediate that feeds into the Krebs Cycle

???? Reactions that make and convert amino acids

• Amination: add a free amine to a molecule  

to produce an amino acid  

o Ex: NH2 + Oxaloacetic Acid ????

Aspartic Acid/Aspartate

• Transamination: switch an amine group  

from one molecule to another

o Ex: Glutamic Acid + Oxaloacetic Acid  

???? α-ketoglutaric acid + Aspartic  

Acid/Aspartate

▪ Glyceraldehyde-3-phosphate converted into precursors  for amino acids, carbohydrates, and fats  

???? Side note: energy that comes from 6 carbon fatty  

acid is about 50 ATP while energy that comes  

from 6 carbon sugar is about 38 ATP

Microbial Genetics  

• Genome: all of the genetic material in a cell

o Made up of

▪ Chromosomes: A molecule of DNA that is associated  

with proteins  

???? Prokaryotes: circular and in the nucleoid

???? Eukaryotes: threadlike (chromatin structure,  

when packed it is wrapped around histones)  

???? Genes: divisions of the chromosome, the unit of  

heredity responsible for a given trait, are a  

specific sequence of nucleotides that code for RNA  

or polypeptide molecules  

• Three Categories

o Structural Genes: genes that code for  

proteins

o Regulatory Genes: genes that control  

gene expression

o RNA Genes: Genes that code for RNA  

• Genotype: the actual set of genes in an  

organisms genotype

• Phenotype: the physical features &  

functional traits of an organism

▪ Plasmids: small circular molecule of DNA, replicates on  its own with its own genes, has genes for nonessential  

functions like resistance to antibiotics  

▪ Mitochondria & Chloroplasts: eukaryotes have  

extranuclear DNA in these & plasmids  

o Prokaryotic Genome Structure: haploid, chromosomal DNA,  circular, all extrachromosomal DNA in the form of plasmids.

o Eukaryotic Genome Structure: nuclear chromosomal DNA,  one or more linear chromosomes, plus all extranuclear DNA in  mito, chloroplasts, and plasmids

o The genome is a thousand times longer than the cell and  must package the DNA efficiently  

• DNA: composed of two strands twisted into a double helix, the basic  unit is a nucleotide

o Nucleotide has three parts:

▪ 5 carbon sugar - deoxyribose

▪ Phosphate Group

▪ Nitrogenous Base – adenine, guanine, thymine, cytosine o The backbone: the sugar-phosphate linkages

▪ Each sugar attaches to a phosphate at its 5’ carbon and  at its 3’ carbon

o The Nitrogenous Bases hydrogen bond with their  

complements in the center

▪ Adenine bonds to Thymine with 2 hydrogen bonds, a  section of DNA that is AT rich is weaker and easier to  

separate

▪ Guanine bonds to Cytosine with 3 hydrogen bonds, a  section that is GC rick is stronger and harder to  

separate  

o As the helix winds it creates a Minor and Major Groove ▪ Minor Groove: information poor

???? Here the proteins can only detect what the base is

▪ Major Groove: information rich  

???? Here proteins interact and based on their ability  

to form bases can tell the difference between A or  

T and G or C, they can also methylate the strand  

and use base flips to find a specific sequence  

without ever having to separate the strands.  

▪ In these grooves there are h-bond donators and h-bond  acceptors they are labeled as D and A respectively.

o Watson and Crick – 1953: These are the two scientists given  credit for discovering the structure of DNA, however, they

would never have gotten it right if it weren’t for Roslin  Franklin’s crystallography data.  

o Significance of this structure:

▪ Code must be maintained during reproduction, this  constancy of base pairing makes sure the code will be  kept (if don’t have constancy then we have mutation)  ▪ Order of base pairs provides variety which is  

responsible for the unique qualities of organisms  

• DNA Replication – takes 30 different enzymes to make exact  duplicate

o Begins at Origin of Replication – this is usually an AT rich  region because of the weaker hydrogen bonding  

▪ Prokaryotes: one origin

▪ Eukaryotes: multiple sites present  

o Helicase unwinds & unzips the DNA by breaking hydrogen  bonds between complementary bases – creates Replication  Forks and Replication Bubble  

▪ Theta Structure: when circular DNA is being replicated  the fork is opening/replicating in both directions until  meeting and forming two new DNA Molecules

o Stabilizing proteins (ssb protein) keep strands from coming  back together

o At origin of replication the enzyme RNA Primase makes a  short RNA sequence that is complementary to the DNA strand  being copied – Primer – provides hydroxyl group for DNA  Polymerase III to add nucleotides in the 5’ to 3’ direction (DNA Polymerase III also proofreads – nuclease activity - as  it adds bases and can back up to delete incorrect ones and  replaces with correct)  

▪ Leading Strand: DNA is replicated towards the  

replication fork, 5’ to 3’ continuously starting from one  primer

▪ Lagging Strand: DNA is replicated away from the  

replication fork, synthesized in short 5’ to 3’ segments  with many primers (overall the direction is 3’ to 5’)

???? Okazaki Fragments: fragments of replicated DNA,  

must be replicated this way on the lagging strand  

to maintain 5’ to 3’ direction  

o DNA Polymerase I: removes RNA primers and replaces with  DNA segments  

o DNA Ligase: link the DNA fragments of the lagging strand to  complete the synthesis  

o The two strands of the original DNA molecule have now  separated into two daughter strands each with one original  parent strand and one new strand – Semiconservative  

• From DNA to Protein – Transcription (DNA into RNA) and  Translation (RNA into Polypeptides/Proteins)  

o Rules for the Standard Code:  

▪ Redundant but not ambiguous: Many codons can code  for one amino acid, but each codon only codes for one  amino acid.  

▪ Code is Universal

o RNA – single stranded molecule made of nucleotides (instead  of deoxyribose the 5 carbon sugar is Ribose, and uracil  replaces thymine)

▪ mRNA: molecules which carry genetic information from  chromosomes to ribosomes as codons (mRNA contains  nucleotides and a set of three nucleotides makes up one  Codon)

???? In Prokaryotes:

• mRNA carries start codon, codons for other  

amino acids in a polypeptide, and one of 3  

stop codons

???? In Eukaryotes:  

• The mRNA is first called pre-mRNA because  

it has to be processed first  

• Contains instructions for only one  

polypeptide

▪ tRNA: molecules that deliver the right amino acids to the ribosomes based on the sequence of nucleotides in  the mRNA.

???? Anticodon: bottom loop of the tRNA that is  

complementary to the codon in the mRNA

???? Acceptor Stem: holds the specific amino acid  

designated by the anticodon

???? tRNA can recognize more than one codon because  of Wobble – a change in the angle of the molecule  can cause the third nucleotide to bond with  

another besides its normal complement

▪ rRNA: part of ribosomes where protein synthesis occurs o Transcription: Information stored in DNA transcribed into  mRNA  

▪ Initiation: 

???? RNA Polymerase binds to promoter region  

???? RNA Polymerase made of 3 subunits: Alpha, Beta,  Beta prime  

• In Prokaryotes: RNA binds to the promoter  

with the help of RNA subunit called Sigma  

Factors

o Sigma factors bind to the promoter so  

RNA polymerase can recognize it  

easier, how tightly the sigma factor  

binds varies, this can lead to variation  

in amount and kind of polypeptide  

formed.  

• In Eukaryotes:  

o Many Transcription Factors (TFIIH,  

TFIID) are used to bind RNA  

Polymerase to the promoter

o Have 3 types of RNA Polymerase (kind  

to transcribe mRNA, rRNA, tRNA and  

other small rRNA)  

▪ Elongation: 

???? RNA Polymerase adds nucleotides complementary  to the template strand of DNA in 5’ to 3’ direction

???? Uracil placed as adenine’s complement

???? Only one DNA strand is transcribed

▪ Termination: 

???? RNA polymerase recognizes signals and releases  the transcript (100 – 1,200 bases long)  

???? In Prokaryotes:

• Self-Termination: RNA polymerase reaches  

terminator sequence (rich in GC followed by  

rich in AU) , when reaches GC portion

transcription slows and gives time for the  

RNA transcript to bind together and form a  

hairpin, this causes tension that the AU  

portion of the terminator cannot withstand  

and the transcript breaks off.  

• Rho-dependent Termination: Rho protein  

binds to end of RNA transcript and works its  

way towards the growing 3’ end, the Rho  

protein wedges in between polymerase and  

DNA strand and forces them apart,  

releasing the transcript, the Rho protein and  

the RNA Polymerase.

???? In Eukaryotes:

• After termination and before Translation the  

pre-mRNA must be processed into  

mRNA(helps export the RNA from the  

nucleus, stabilizes the RNA in cytoplasm,  

and aids in translation) – Eukaryotic  

transcript more stable than prokaryotic  

o Capping: adds Guanine nucleotides to  

the 5’ end of mRNA

o Polyadenylation: after termination,

proteins add 100-250 adenine  

nucleotides to the 3’ end  

o Splicing: spliceosome acts as  

ribozyme and removes introns,  

splicing together exons  

o Translation: mRNA is used by ribosomes to translate the  genetic code into polypeptides

▪ Initiation: Initiation complex forms (2 ribosomal  subunits, mRNA, several protein factors, the initiator  tRNA)

???? In Prokaryotes:  

• Might happen while cell is still transcribing  RNA from DNA  

• Steps:

o Smaller ribosomal subunit attaches to  

mRNA at ribosome-binding site near  

start codon (AUG)  

o Anticodon of initiator tRNA aligns  

w/start codon on mRNA, bound in  

place w/energy from GTP

o Larger subunit attaches to form  

complete initiation complex

???? In Eukaryotes:

• Same process but initiation happens when  small ribosomal subunit binds to the 5’ cap  

not a specific nucleotide sequence

▪ Elongation:  

???? tRNAs deliver amino acids to the A site

(accommodates a tRNA delivering an amino acid)  as directed by the codons of the mRNA  

???? rRNA (enzymatic RNA molecule – ribozyme) in the  large subunit makes a peptide bond between  amino acid at A site & growing polypeptide in P  site (holds tRNA and growing polypeptide)

???? the ribosome moves ahead one codon & the tRNA  that was in the P site exits through the E site,  leaving the polypeptide attached to the tRNA that  was in the A site but is now in the P site, the A  site is now open for the next amino acid to be  delivered  

???? Elongation Factors escort the tRNA along with a  GTP molecule

???? When the empty tRNA is released back into the cytosol an enzyme recharges it with another  

molecule

???? As elongation goes on and the ribosome passes  over the start codon, it is exposed for other  

ribosomes to attach and begin translating  

identical polypeptides (a group of these ribosomes  = polyribosomal complex)

▪ Termination:  

???? Termination factors recognize the stop codon

(UAA, UAG, UGA codons that don’t have a tRNA)  

and modify the large subunit to activate another  

ribozyme that cuts the polypeptide from the final  tRNA – ribosome dissociates

???? The polypeptides released can function alone as  proteins or w/others in quaternary proteins  

???? *Eukaryotic Note: ribosomes attached to  

endoplasmic reticulum can synthesize  

polypeptides into the cavity of the RER

▪ Protein Folding  

▪ Processing

o Regulation of Protein Synthesis

▪ Rules of Genetic Regulation

???? If the cell doesn’t need it, turn it off

???? If the cell needs it, get an enzyme there as quick  as possible

???? The rate of synthesis can be controlled w/DNA  sequence

???? Enzymes needed only under certain conditions

???? In prokaryotes regulation pathways can be  

controlled as operons  

▪ Operons: set of genes that are regulated as a single  unit

???? made up of promoter, set of genes that code for  enzymes and structures, & an operator

???? Inducible Operons: (Enzyme Induction) operon  not normally transcribed must be turned on by  inducers

• Ex: Lac Operon – normally off

o Made up of 3 segments  

▪ Regulator: gene that codes for  

repressor

▪ Control Locus: contains  

promoter & operator

▪ Structural Locus: 3 genes that  

each code for an enzyme that  

catabolizes lactose

???? β-galactosidase:

hydrolyzes lactose

???? Permease: brings lactose  

across the cell membrane

???? β-galactosidase  

transacetylase: uncertain  

fxn

o Activated by:

▪ 1. positive regulation by protein  

called CAP

???? When glucose is low 

increases [cAMP] which  

binds to CAP (catabolite  

activator protein), CAP  

binds to CAP-binding  

sites, RNA polymerase  

more attracted to the lac  

operon promoter =  

increased transcription of  

lactose catabolizing genes

▪ 2. deactivation of repressor  

molecule  

???? When lactose is present 

and glucose is absent,

allolactase acts as the  

inducer, binds to the  

repressor, changes its  

shape and causes it to fall  

off the operator so RNA  

polymerase can bind to  

the promoter & lactose  

catabolism genes can be  

transcribed  

???? Absence of lactose =  

repressor binds the  

operator locus and blocks  

RNA Polymerase  

transcription of lactose  

catabolism genes

o Lac Operon inactivation/activation a  

form of Catabolite Repression

(inducible operons involved in  

controlling catabolic pathways whose  

polypeptides are not needed unless a  

particular nutrient is available)

▪ Following is repetitive:

▪ Low lactose & High glucose =  

low cAMP = few CAP-cAMP  

complexes = inactivation of lac  

operon

▪ High lactose & low glucose =  

high cAMP = many CAP-cAMP  

complexes = activation of lac  

operon

???? Repressible Operons: operons that are continually  transcribed unless repressor deactivates it