Mimm 211 Midterm

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3 kingdoms of life

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Bacteria, Archaea, Eucarya.

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Common threads of life

Back 2

Growth, Metabolism, Motion, Reproduction, Response to stimuli.

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Lucretius and Fracastoro

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Suggested invisible organisms cause disease.

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Jansen

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Invents and develops first microscopes.

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Hooke

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First description and depiction of microorganism.

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Leeuwenhoek

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Discovered bacteria and protozoa.

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Spontaneous Generation

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Formation of living organisms from inanimate matters.

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Redi

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Fly larvae can only develop in meat that fly can reach.

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Needham

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Boiled broth then sealed it. Microorganisms appeared.

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Spallanzani

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Broth in flask then sealed and boiled. No microorganisms appeared.

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Pasteur

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Boiled broth in flask, curved neck of flask so air can enter but no dust, no microorganisms appeared.

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Tyndall

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Showed dust carried microorganisms.

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Beliefs in the cause of diseases

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Supernatural forces, Poisonous Vapors, Imbalances between the four humors.

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Bassi

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Showed microorganism (fungus) can cause disease in silkworms.

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Berkeley

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Potato blight was caused by fungus.

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Lister

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Sterilized instruments with heat, phenol to prevent infections.

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Koch

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Demonstrated bacterium was causing anthrax.

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Koch’s Postulates

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The microorganism is present in every case of the disease, but absent from health organisms, the suspected microorganism must be isolated and grown in pure culture, the same disease must result when the isolated microorganism is inoculated into a healthy host, the same microorganism must be isolated again from diseased host.

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Agar and Petri Dish

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Used to develop microorganisms in the lab.

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Jenner

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Immunized people from smallpox using cowpox virus.

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Pasteur

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First attenuated vaccine from pure culture of pathogen attenuated.

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Smith and Salmon

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Killed microbial cells effective as vaccines.

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Behring

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Humoral Immunity, antibodies produced in blood against toxin to be protective against infection.

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Metchnikoff

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Cellular Immunity, Phagocytes engulfing disease-causing bacteria and provide immunological protection.

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Winogradsky

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Oxidation of iron, sulfur, ammonia by soil bacteria for energy, transformation of carbon dioxide to organic matter.

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Beijerinck

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Isolated nitrogen-fixing bacteria and sulfate-reducing bacteria.

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Taxonomy

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Artificial classification of organisms, based on visible similarities.

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Phylogeny

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Natural classification of organisms, reflects evolutionary relatedness between organisms. Uses ubiquitous gene sequence to compare microorganisms.

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Haeckel’s Proposal

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Plants, Animals, Microorganisms.

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Chatton’s Proposal

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Eukaryotes and prokaryotes.

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Whittaker’s Proposal

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Monera, Protista, Fungi, Plantae, Animalia.

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Woese’s Proposal

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Bacteria, Archaea, Eukaryotes – equally distant, used 16S ribosomal RNA gene.

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Endosymbiotic theory of evolution

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Primitive eukaryotic cell engulfed an ancient prokaryote to create the first eukaryotic cell.

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Goals of Classification

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Stability, Predictability, Build larger groups, Study a member, learn about the group.

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Linnaeus’s scheme of classification

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Based on comparison of visible characteristics.

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Hierarchical scheme of classification

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Kingdom, Phylum, Class, Order, Family, Genus, Species.

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Name of bacterial species

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Lactococcus lactis (L. lactis), consists of genus name and epithet.

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Use of bacterial species naming

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Appearance, Habitat, Characteristic property, Scientist name.

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Characteristics used to separate bacterial species

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Biovars (Biochemical/Physiological differences), Morphovars (different morphology – shapes), Serovars (different antigenic properties)

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Sj

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Similarity coefficient, # similarities shared/# similarities compared, used to make dendograms

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Phage typing

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What bacteriophages can infect, bacteriophage has host range.

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Genomic characteristics

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G+C content (melting point + density), DNA hybridization (single strands allowed to mix), sequencing genes give phylogenetic relationship.

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Major differences between prokaryotes and eukaryotes

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Eukaryotes have nucleus, organelles, size is 10x bigger, prokaryotic cells have cell wall of peptidoglycan.

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Shapes/Arrangements of cells

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Spherical Cells – Cocci, Rodlike – Bacilli, Spiral – Spirilla, Other – Irregular

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Arrangements of Cocci

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Streptococci (line of more than 2), tetrad (square), sarcinae (2 layers of squares), staphylococci (big mass of layered cocci)

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Arrangements of Bacilli

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Coccobacillus (circular bacilli), Streptobacillius (line of more than 2)

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Bacterial Colony Morphologies

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Form – Punctiform, Circular, Filamentous, Irregular, Rhizoid, Spindle, Elevation – Flat, Raised, Convex, Pulvinate, Umbonate, Margin – Entire, Undulate, Lobate, Erose, Filamentous, Curled

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Bacterial Cells vs Human Cells

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Surface Area to Volume ratio of prokaryotes larger than eukaryotes, faster growth.

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Epulopiscium Fishelsoni

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Guest at fish’s banquet, found in gut of brown surgeon fish, large

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Thiomargarita Namibiensis

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Sulfur pearl of Namibia, found in ocean sediments, larger than E. fishelsoni.

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Nanochlorum eukaryotum

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Green alga, smaller than a human cell but is a true eukaryote – nucleus, mitochondrion, chroloplast.

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Bacterial Envelopes

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Gram-negative – outer membrane, cell wall, cytoplasmic membrane, Gram-positive – cell wall, cytoplasmic membrane, Mycoplasma – cytoplasmic membrane.

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Cytoplasmic membrane

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Contains cytoplasm, regulates what comes in and out, made up of a fluid phospholipid bilayer, contains proteins (transmembrane and peripheral membrane proteins).

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Phospholipid Structure

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Structurally asymmetric, charged polar heads, nonpolar hydrophobic long lipid tails.

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Peptidoglycan Cell Wall

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Gives cell shape and rigidity, osmotic pressure, peptidoglycan composition. Very thick around gram-positive, thin around gram-negative.

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Peptidoglycan Cell Wall Anchoring

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Gram-positive has teichoic acid (structure) and lipoteichoic acid (anchorage). Gram-negative has cell wall anchored by lipoproteins.

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Outer membrane

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Gram-negative, acts as protective barrier and regulates molecules coming through, made up of a fluid phospholipid bilayer (inner layer – phospholipids, outer layer – LPS, endotoxin, phospholipids). Anchored to cell wall by lipoproteins.

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Capsule/Slime layer

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Protection, reservoir of stored food, site for waste disposal, helps keep shape and rigidity, prevents infection, aids in cell adhesion and motility.

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Flagella

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Movement of bacterial cells, made up of protein polymer

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Pili/Fimbriae

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Thinner than flagella, adherence to surfaces, sex pili for mating.

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Arrangements of bacterial flagella

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Monotrichous – single flagellum, Amphitrichous – single flagellum at each pole, Lophotrichous – two or more flagella at pole/poles, Peritrichous – flagella all over surface.

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Taxis with flagella

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Bacterial movement. Counterclockwise – forward, Clockwise – tumbling

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Chemotaxis

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Directional movement towards specific chemical.

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Cytoplasm

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Site of metabolism, 90% water, nucleoid (irregular mass of DNA), ribosomes, inclusion bodies (storage, gaz, magnetosomes).

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Endospores

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Most resistant biological structures, no metabolism, state of survival induced by unfavourable growth conditions.

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Sporulation

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Divides into 2 unequal parts. Larger part engulfs smaller part – becomes forespore. Forespore matures to become an endospore – synthesis of protective thick wall, dehydrated, lysis of cell to release endospore.

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Germination

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Endospore to vegetative cell; water enters, endospore swells and ruptures coat, germ tube grows.

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Binary fission

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Divide near midpoint to form two daughter cells.

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Budding

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Forms bubble-like structure that comes out and separates from parent cell.

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Fragmentation

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Filamentous growth, extends from main outwards like branches.

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Asynchronous cultures

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Timing of cell division is random.

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Synchronous cultures

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Timing of cell division is fixed.

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Generation time

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Period for cell to enlarge, divide, and produce 2 daughter cells. All cells in population have same generation time.

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Lag phase

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Time for cells to adjust to new/fresh medium and start dividing.

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Exponential/Log phase

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Period where nutrients are not in limiting amounts and cells divide at maximum speed.

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Stationary phase

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Culture switches from log phase to stationary phase when nutrient concentration is limiting and toxic waste accumulates so cells divide slower.

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Death phase

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Nutrients completely consumed, cells stopped growing and starting to die, but not all cells die.

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Reasons for long Lag phase

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Inoculum taken from old culture, from rich medium to poorer one, from chemically different medium, from refrigerated cells, in cold medium.

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Chemostat

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Provides constant flow of nutrients, concentration of one of the nutrients is limited to reduce growth rate, some growing culture should be removed at the same rate of growth rate = loss rate so always the same amounts of cells growing.

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Parameters affecting microbial growth

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Temperature (hyperthermophiles, thermophiles, mesophiles, psychrotrophs, psychrophiles), pH (neutrophils, acidophiles, alkalophiles), gaseous environment (aerobes, anaerobes, facultatives, capnophiles).

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Toxic molecules from oxygen

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Superoxide Radical, Hydrogen Peroxide, Hydroxyl Radical.

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Protective enzymes against oxygen

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Superoxide dismutase, Catalase, Peroxidase.

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Anaerobic jar

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Oxygen removed by combining with hydrogen to make water.

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Growth on solid media

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Cells in center – death/stationary phase, Cells on edge – exponential phase (growing), Cells at surface – aerobic conditions, Cells under surface – anaerobic conditions.

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Cell count technique

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Take diluted sample, spread colonies, then count colonies. Colonies are multiplied by dilution to get cells/mL.

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Mathematics of cell growth

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Cells number: N = N0 X 2^n, where then n = 3.3(log(N) – log(N0))

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Gene

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DNA segment that codes for protein.

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Genome

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All the genes in cell/virus.

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Genotype

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Specific set of genes of an organism.

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Phenotype

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Observable characteristics.

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Prokaryote are _ , has genes

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Haploid (1N).

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Eukaryote are _ , has genes

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Diploid (2N) .

Front 93

Griffith’s Transformation Experiment

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DNA/Proteins caused disease? DNA.

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Avery’s Experiment

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DNA contains genetic information.

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Hershey-Chase Experiment

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DNA carries genetic information for T2.

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Central Dogma

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DNA goes through replication, transcription to RNA, which then goes translation to protein.

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Nucleoside

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Sugar ring, base.

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Nucleotide

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Sugar ring, base, phosphate.

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Organization of DNA in Eukaryotes

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Inside nucleus, genome of 10^9 bp, linear chromosomes, compact and organized (chromatin), small basic proteins (histones), no plasmids.

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Organization of DNA in Prokaryotes

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No nucleus, anchored to cell membrane, genome of 10^6 bp, one circular chromosome, supercoiled, small basic proteins (nonhistone), has plasmids.

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Nucleosome

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Histone H1 binds to linker regions (14-100 bp) promoting chromatin structure which is made up of nucleosomes (histone octamer and 146 bp).

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DNA Replication

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Helicase unwinds DNA helix, uses strand as template for DNA polymerization (DNA polymerase III, 750-1000 bp/sec). Opens replication fork, one parent strand is formed with one replica (semi-conservative), synthesized 5’ to 3’, with 3’ to 5’ exonuclease activity (in DNA polymerases, proof-reading).

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Replication of bacterial DNA

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Replication forks happen and as it goes around the circle, another circle forms which is at the opposite side, end result are 2 loops.

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Rolling-Circle Replication

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From nick, DNA replication occurs along the circle as the previous strand is rolled out into a line.

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SSBs

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Single-stranded DNA binding proteins keeps two strands of DNA separated.

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DNA gyrase (Topoisomerases)

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Release tension created by helicases unwinding DNA.

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DnaA helicase

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Binds to oriC locus.

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DnaB helicase

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Replaces DnaA, replication fork unwind at 75-100 rev/sec.

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Primase (RNA Polymerase)

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Short RNA at lagging strand complementary to DNA.

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DNA Polymerase III (Lagging Strand)

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Uses RNA as primer and synthesizes 5’ to 3’.

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Okazaki fragments

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Strands of non-connected DNA on lagging strand.

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DNA ligase

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Fills gap between fragments

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Gene (Cistron)

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Nucleotide sequence that codes for mRNA, tRNA or rRNA.

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Bacterial genes

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Have 4 parts: promoter, leader, coding region, trailer. Mostly continuous.

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Eukaryotic genes

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Mostly interrupted by introns.

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Codon

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3 consecutive nucleotides that specify an amino acid.

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Genetic Code

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Degenerate, more than 1 codon for each amino acid. 61 sense codons, 3 stop codons, Trp and Met only have 1 codon.

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Wobble Pairing

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Anticodons with I, bonds G to U, I to C, I to A, I to U.

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Mutations of DNA

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Spontaneous (happens by DNA Replication), Point Mutations (silent – doesn’t change codon, missense – changes codon, nonsense – creates stop codon, frameshift – deletions and insertions).

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Forward Mutation

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Wild type allele into a new allele.

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Reversion Mutation

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Changes a mutant allele into a wild type allele.

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Suppressor Mutation

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Second change in the same gene that compensates for forward mutation.

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DNA Repair

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Proofreading of DNA polymerases

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Nucleoside Excision Repair

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UvrAB looks for thymine dimer. UvrA released, UvrC binds. UvrC cuts both sides of thymine dimer. UvrD (helicase) removes damaged region. UvrC and UvrC released. DNA polymerase fills in gap, DNA ligase seals gap.

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Base Excision Repair

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DNA glycosylase recognizes non proper base pair matching, cleaves bond between base and sugar. AP endonuclease cleaves DNA backbone on 5’ side of missing base. DNA polymerase uses 5’ to 3’ exonuclease to remove damaged region and then adds normal DNA. DNA ligase seals region.

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Mismatch Repair

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MutH, MutL, MutS goes along strand. MutH cuts the strand, exonuclease digests strand beyond base mismatch. DNA polymerase fills the gaps, DNA ligase seal the ends.

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Error Free Systems

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Direct removal of lesions (thymine dimers – photoreactivation/photolyase, methyls – methylguanine methyltransferase, alkyls – alkyltransferase).

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Recombinational Repair

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By recombination, region from another strand replaces the gap in another strand, then the region which is now missing is filled with DNA polymerase and ligase. Uses recA protein.

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Postreplication Repair

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Mismatch repair, scans newly replicated DNA.

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3 types of RNA

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mRNA (proteins), tRNA (carries amino acids to ribosome), rRNA (16S, 23S, part of ribosome)

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RNA synthesis

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5’ to 3’, adds 40 nt/sec at 37 C, moves along DNA forming 12-20 bp transcription bubble. Transcription starts at promoters and stops at terminators.

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Prokaryotic Terminators

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Two types, Rho dependent – needs rho factor, no poly-U tract, no hairpins sometimes. Rho independent – no need for rho, 6 uridines after hairpin structure.

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Eukaryotic mRNA

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Monocistronic (1 gene), Cap structure at 5’ end, PolyA tail at 3’ end, lots of introns, transcription (nucleus) and translation (cytoplasm)

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Prokaryotic mRNA

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Multicistronic (operon), no cap at 5’ end, no poly-adenylation at 3’ end, no introns, transcription and translation coupled.

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Protein Synthesis

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mRNA translated into amino acid chain (protein). Ribosome at site of translation (900 residues/min, 15 amino acids/sec), 80 nt apart. tRNAs bring amino acids to ribosome, tRNAs read mRNA codons. Ribosomes synthesize proteins from N-terminus (NH2) to the C-terminus (COOH)

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Translation Steps

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Initiation, Elongation, Termination. Ribosome goes along mRNA from Shine-Dalgamo sequence, each amino acid is attached to tRNA that comes to ribosome to drop off their amino acid after matching with respective codon.

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tRNA Structure

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73 to 93 nt, ‘cloverleaf’ secondary structure, anticodon triplet complementary to codon triplet on mRNA, amino acids attached to tRNA at 3’ end.

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Regulation of mRNA Synthesis

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Negative Control – Induction, gene is turned on when enzyme needed by having an inducer molecule either turn off or on the repressor by attaching to it. Repression – too much of end product leads to corepressor which activates the repressor. Positive Control – only turned on in presence of controlling factor, meaning it only transcribes if the factor is present.

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Tryptophan Operon Leader

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Initiation and continuation of transcription controlled by levels of tryptophan. Low level of tryptophan leads to ribosome to stop, high level of tryptophan leads to ribosome to continue.

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DNA recombination in bacteria

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Homologous recombination (Crossing-over), nonreciprocal recombination, site-specific recombination (viruses), replicative recombination (mobile genetic elements)

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Crossing-Over

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Helicase and nuclease unwinds DNA, RecA-like proteins help it stay in shape, the strands then migrate to resolve in spliced and patched DNA strands.

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Nonreciprocal Recombination

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Strands are separated, segment of donor strand is separated, the same region on the host strand is removed and replaced, and the gaps are filled and ligated.

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Bacterial Plasmids

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Circular double-stranded DNA, number of copy per cell varies, exists independently from host chromosome, and has few genes that could be integrated into host chromosome.

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Different types of plasmids and factors

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Fertility factor, Resistance factors, Col plasmids, Virulence plasmids, Metabolic plasmids.

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Bacterial Gene Transfer

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Conjugation (sex pilus), DNA transformation (naked linear DNA/plasmids taken from cell environment), Transduction (bacteriophages)

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Fertility Factor

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Conjugative plasmid, genes for cell attachment and plasmid transfer, F+ (donor) F- (recipient) cell, plasmid is episome (can replicate freely in cytoplasm), has own origin of replication (oriV).