Test 3 Lecture Objectives

Proton Gradient in Prokaryotes

proton gradient is across the plasma membrane

Proton Gradient in Eukaryotes

proton gradient is across the inner mitochondrial membrane

Substrate-level phosphorylation

phosphate group is directly transferred from glucose to ADP, forming ATP; does not require oxygen; produces small amount of ATP

Aerobic Respiration

AYP is generated via oxidative phosphorylation (ETC) uses oxygen as final electron acceptor; highly efficient 36-38 ATP

Anerobic Respiration

Similar to aerobic respiration, but uses another electron acceptor (nitrate, sulfate, or CO2, NOT O2); not as efficient as aerobic respiration, but more efficient than fermentation

Glycolysis (Occurs in the Cytoplasm)

• Glucose (6-carbon) is broken down into two molecules of pyruvate (3-carbon each).

• A small amount of ATP is made via substrate-level phosphorylation (2 ATP per glucose).

• NAD⁺ is reduced to NADH, carrying high-energy electrons to the next stage.

Pyruvate Processing (Occurs in the Mitochondrial Matrix in Eukaryotes)

• Each pyruvate is converted into Acetyl-CoA (2-carbon).

• Carbon dioxide (CO₂) is released.

• More NADH is produced for later use in the electron transport chain (ETC).

Citric Acid Cycle (Krebs Cycle, Occurs in the Mitochondrial Matrix)

• Acetyl-CoA enters the cycle, breaking down into CO₂.

• More NADH and FADH₂ (another electron carrier) are generated.

• A small amount of ATP is made via substrate-level phosphorylation (2 ATP per glucose).

Electron Transport Chain (ETC) & Chemiosmosis (Occurs in the Inner Mitochondrial Membrane)

• NADH and FADH₂ donate electrons to the ETC.

• Electrons move through protein complexes, pumping protons (H⁺) into the intermembrane space, creating a proton gradient.

• Oxygen (O₂) acts as the final electron acceptor, forming water (H₂O)

ATP Synthesis (Occurs via ATP Synthase in the Inner Mitochondrial Membrane)

• Protons (H⁺) flow back into the mitochondrial matrix through ATP synthase (a molecular turbine).

• This powers ATP synthesis via oxidative phosphorylation.

• About 34 ATP are produced here per glucose

Final Energy Yield from One Glucose Molecule

• Glycolysis → 2 ATP

• Krebs Cycle → 2 ATP

• Electron Transport & Oxidative Phosphorylation → ~34 ATP

• Total: ~36-38 ATP per glucose

Inhibitors

block electron transport and prevents proton gradient

Uncouplers

allow protons to pass across membrane (“leaky)

Catabolic reactions

break down large molecules into smaller ones, releases/generated energy

Anabolic reactions

Anabolic reactions: build larger molecules from smaller ones, require energy

Polysaccharides construction

gluconeogenesis from citric acid cycle intermediates; Needed for cell structure and storage

Amino acids construction

come from the intermediates of energy synthesis (Citric acid cycle and glycolysis)

Nucleotides (Nucleic Acids)

DNA precursors are formed by reduction of RNA precursors (pentoses)

Purines are made from

inosine

Pyrimidines are made from

orotate

Fatty Acids (lipids)

acyl carrier protein (ACP) holds elongating fatty acids, adds two at a time form 3-carbon malonyl-ACP; Lipids needed for membranes and energy storage

Pentoses (ribose and deoxyribose)

are essential for nucleic acid synthesis, forming the sugar backbone of RNA and DNA

Hexoses (glucose)

key building blocks for polysaccharide synthesis, forming structural and storage molecules

How is glucose produced?

Gluconeogenesis: pyruvate and amino acids are converted into glucose

Polymerization of glucose

activated into UDPG/ADPG, which acts as a high-energy donor for building polysaccharides

steps in simple cell division

DNA replication, Cell elongation, Septum formation, Completion of septum with formation of district walls, Cell separation

Divisome

a multiprotein complex that forms a new septum for cell division

FtsZ

forms a ring (z-ring) at the site of cell division

FtsA and ZipA

anchor the z-ring to the cell membrane

FtsI

crosslinks peptidoglycan for cell wall synthesis and division

FtsK

chromosome segregation for cell division

MinC

proteins that prevent cell division and z-ring formation until cell center has been located

MinE

proteins that inhibit MinC, localize the center, and recruit FtsZ

MreB

forms polymers like actin filaments that make up the cytoskeleton in eukaryotic cells, Dictates cell shape

Autolysins

break glycosidic bonds in peptidoglycan at point of new synthesis

Bactoprenol

hydrophobic, shuttles precursors across the membrane, and interacts with assembly proteins to catalyze incorporation of new glycosidic bonds

Peptidoglycan transpeptidase

catalyzes cross-linking and is the target of penicillin

Generation

the doubling of a bacterial population n =3.3(logN – logNo)

Generation Time (doubling Time)

time required for one generation to occur; inversely related to growth rate g = t/g

Exponential Growth

pattern of growth during which the population doubles per unit of time

Number of cells at a given time

N = No2n (No=starting number of cells)

steps of batch culture growth

Lag, Exponential, Stationary, and Death Phase

Lag Phase

cells adjust to the new medium and prepare for division; no significant increase in cell number, but high metabolic activity

Exponential Phase

cells divide at a constant, maximum rate; high metabolic activity, balanced growth, and optimal replication conditions

Stationary Phase

nutrient depletion and waste accumulation slow cell division and there is an equilibrium between growth rate and death rate; population stabilizes

Death Phase

cells begin to die at an increasing rate due to lack of nutrients and toxic waste buildup; cell lysis, and exponential decrease in cell count

Batch culture

finite nutrient supply leading to a short period of exponential growth followed by a stationary and death phase

Chemostat growth

replenishing nutrient supply leading to a maintained growth rate depending on the nutrient supply; extended exponential growth

experimental uses of chemostat growth

• Constant supply of cells in a stable, unvarying condition

• Ecological studies to measure long-term relationships among mixed populations

• Enrichment and isolation of stains with specific growth characteristics

• Measurement of genetic variation over extended generations

Total Cell Counts

Direct measurement, number /mm2, number / mm3, number /cm3 (mL), all cells counted

Viable Counts

Direct measurement, number /mm2, number / mm3, number /cm3 (mL), only viable cells counted

Turbidimetric Methods

Indirect measurement, Optical Density (OD) and Klett units, all cells counted

Cardinal Temperatures of Growth

minimum, optimum, and maximum temperatures

Psychrophilic

optimal growth below 15C, max growth 20C, min grow 0C or lower

Psychrotolerant

organisms can grow at 0C, but have optimal growth at 20-40C

Adaptations to Psychrophily

• Enzymes active in the cold

• more unsaturated lipids in membrane

• cryoprotective molecules reduce dehydration and ic-crystal formation

Thermophiles

optimal growth over 45C

Hyperthermophiles

optimal growth over 80C

Adaptation to Thermophily

• Amino acid substitutions at key places in enzymes to increase stability at high temperatures

• more ionic bonds and denser hydrophobic protein cores

• cytoplasmic solutes help stabilize proteins

• high saturation rates of fatty acids in membranes

• use of lipid monolayers in Archaea

Acidophile: Example

Picrophilus oshimae

Acidophiles: Definition

optimum pH 0.7, lyses above pH 4, grows in volcanic soils

Alkaliphile: Example

Bacillus firmus

Alkaliphiles: Definition

up to pH 11, uses NA+ gradient to dribe transport and locomotion, halophilic Archaeal strains

Non-halophile

prefer low salt concentrations

Halotolerant

can tolerate moderate salt concentrations, but prefer low salt conditions

Halophile

required moderate to high salt concentrations

Extreme Halophile

requires very high salt concentrations

Adaptation to Osmolarity

some organisms produce or accumulate intracellular compatible solutes that function to maintain a positive water balance in the cell

Obligate Aerobes

require O2; aerobic respiration

Facultative Aerobes

O2 not required, but grow better with O2; aerobic respiration, anaerobic respiration, fermentation

Microaerophilic Aerobes

O2 required but at low levels; aerobic respiration

Aerotolerant Anaerobes

O2 not required, and grow no better with O2; fermentation

Obligate Anaerobes

O2 harmful or lethal; fermentation, anaerobic respiration

Toxic Forms of Oxygen

Superoxide, Hydrogen Peroxide, Hydroxyl Radical

Sterilization

must eliminate the most heat-resistant organisms, usually bacterial endospores

Sterilization Example

autoclave

Pasteurization

does not sterilize liquids, but reduces microbial load, killing most pathogens and inhibiting the growth of spoilage microorganisms

Pasteurization Examples:

Milk and eggs

Decimal reduction time

the time required to reduce a microbial population by 90% under specific conditions

Decimal reduction time: Higher temperature

accelerates microbial death, reducing the time needed for a 90% reduction

Decimal reduction time: Lower temperature

requires more time for a 90% reduction

UV Radiation

causes DNA damage; surface decontamination

Ionizing Radiation

breaks DNA strands and damages protein and membranes; high penetration

Lethal radiation does

used to kill organisms and reduce population by a factor of 10^-12

Decimal reduction radiation dose

the amount of radiation required to reduce a microbial population by 90%

Depth Filters

fibrous nature, used to pre-filter liquids, sterilization of air

• HEPA filters

Standard Membrane Filter

~80% open pore, traps filtrate on surface, common heat-sensitive liquid sterilization filter

Nucleopore Membrane Filter

formed by etching polycarbonate film after nuclear radiation

Bactericidal

Kills bacteria

Bacteriostatic

Prevents bacterial growth without killing

Bacteriolytic

Causes bacterial death by cell lysis

Minimum Inhibitory Concentration (MIC) assay

the lowest concentration of an antimicrobial that inhibits visible bacterial growth; can be seen with test tubes

Disc Diffusion assay

Measures the zone of inhibition around an antimicrobial disc on an agar plate

Antiseptics

chemical agents applied to living tissues to reduce or eliminate microbial growth

Chemotherapeutic Agents

synthetic or naturally derived drugs that act inside the body to kill or inhibit microbial growth

Synthetic antimicrobial agents

growth factor analogs and quinolones

Antibiotics

natural and semisynthetic; can be produced by fungi and bacteria, semi-synthetic types are chemically modified

Sulfa drugs

bacteriostatic, inhibits enzymes and uses folic acid

Penicillin

bactericidal, inhibits cell wall synthesis

Rifampin

bactericidal, inhibits RNA synthesis