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University of Manitoba
MBIO 1010
Christopher Rathgeber

MBIO 1010: Microbiology I ← Monday May 2, 2011 ← Microbiology: • Study of organisms too small to be seen with the naked eye o Ex. Bacteria, viruses, and single celled eukaryotes • Some microorganisms however are visible with the human eye: o Ex. Fungi, algae, mold • Some microbes are multicellular o Myxobacteria, Slime mold • Therefore microbiology is defined by differentiation (the organisms under study are not as differentiated as higher organisms) and techniques: o Sterilization o Culture Media o Isolation and growth of organisms o Genetics and genomics ← ← 3 Categories of Microorganisms (based on cell structure): 1. Eukaryotes • Membrane bound nucleus and organelles • Complex internal organization (very well known complex cytoskeleton) • Division by mitosis and meiosis (terms only used for eukaryotic cells) • 2 groups of eukaryotic cells: o Protists: Unicellular or multicellular without differentiation into tissues  Protozoa: animal-like microorganisms  Algae: photosynthetic plant-like microorganisms  Slime mold and water mold: filamentous (fungi-like) o Fungi: Unicellular (yeast), filamentous (mold), or multicellular (mushrooms) 2. Prokaryotes • No membrane bound nucleus and organelles • Generally smaller than the eukaryotes (approx. 1 μm diameter) • Simple internal structure • Divide by binary fission • Most are unicellular • 2 Types: o Bacteria (old name: eubacteria)  Extremely diverse metabolic styles  Includes pathogens (disease causing) and non-pathogens o Archaea (old name: Archaebacteria)  Distinct cell structure  Often found in extreme environments (high temp or acidic) 3. Viruses • Acellular infectious particles • Extremely small • Obligate intracellular parasites (can only perform their function when they enter the host cell) • Lack independent metabolism as a result • No ribosomes and no ribosomal RNA (rRNA) o Cannot be classified with other microbes ← ← Microbial Genomes: • Genome: Full set of genetic information present in an organism o i.e. full set of genes Gene: A section of DNA that encodes for a product (protein, RNA) • • DNA: a long polymer of nucleotides o Stores genetic information ← • The sequence of nucleotide in a genome provide an enormous amount of information • First complete genome: virus ΦX174, 5000 base pairs, 10 genes o Infects E. coli bacteria • First cellular organism: bacterium Haemophilus influenzae, 2,000,000 base pairs (~1700 genes) • First eukaryote: Saccharomyces cerevisiae (yeast), 12,100,000 base pairs (~6000 genes) • Human genome, 3,200,000,000 base pairs (~23000 genes) ← Metagenome • Sequence all genes from the environment • Ex. Sequence of all genes from the human colon o Gives information on all organisms as a whole living in the colon ← Comparative Genomics • The science of comparing available genomes to look for similarities and differences ← Microbial Systematics: • Wittaker’s 5 kingdom system of classification (Fig. 1-2) o Really old, good only for higher organisms (Plantae, Fungi, Animalia) o Did the classifications based on morphological similarities and differences ← Classifying Organisms based on Evolutionary Relationships: • Comparing small subunit (SSU) rRNA gene sequences • Ribosome: Site of protein synthesis o Composed of proteins and rRNA • All cellular organisms have ribosomes • In Prokaryotes: 70S ribosomes o 16S SSU rRNA used • In Eukaryotes: 80S ribosomes o 18S SSU rRNA used ← • rRNA genes change slowly overtime • Examines genetic differences rather than morphological differences • Basic Steps involved in sequencing rRNA genes (Fig. 1-3) • Step 1: DNA is collected from the a pure culture • Step 2: the SSU rRNA gene is amplified using the polymerase chain reaction (PCR) o PCR: used to synthesize many identical copies of a short sequence of DNA • Step 3: gene is sequenced • Step 4: Sequence is aligned with sequences from other organisms o Number of differences is used to calculate evolutionary distance o Then make a Phylogenetic Tree: a graphic representation of the evolutionary distance between organisms ← ← Evolutionary Tree of Life (Fig. 1-4) • Relatedness of organisms is based on the 16S (or 18S) ribosomal DNA sequences • Suggests that all organisms evolved from a common ancestor • Different from Wittaker’s 5 kingdom system of classification ← ← Hierarchical Classification • Groups of organisms are placed in successively larger groups • Fig. 1-5 ← ← Binomial Nomenclature • Binomial System ← Escherichia coli Species: both names ← ← Genus (Capitalized) Specific Epithet ← (not capitalized) • Strains can be identified by symbols after the species name o Ex. E. coli K12 ← Rules: 1) Name is latinized 2) Italicized or underlined 3) Genus is capitalized, epithet is not 4) Genus name maybe abbreviated (ex. E. coli) (Note: write the full genus name in the beginning, then abbreviate after) 5) Trivial names can be used, but don’t follow the rules ← ← Discovery of Microorganisms ← Robert Hooke – 1665 • Invented the first compound microscope (2 lenses) • Allowed magnification up to 30x • He used to observe o Cells in cork o Bread mold filaments – 1 microbe seen • Beginning of cell theory – all living things are composed of cells ← ← Antoni van Leeuwenhoek – 1673 • Built microscopes that magnifies specimen by 50 – 300x • Observed the first bacteria – called them Animalcules. ← ← Spontaneous Generation vs. Biogenesis ← Spontaneous Generation: Life can develop from non-living matter • Ex. Meat left unattended quickly gets covered in maggots • Therefore maggots must come from meat Biogenesis: life arises only from pre-existing life Francesco Redi – 1688 • Did an experiment where he put meat into 3 containers 1. Uncovered: attracted flies and produced maggots 2. Covered: no maggots 3. Covered in gauze: flies laid eggs on the gauze • Skeptics proposed that microbes arose by spontaneous generation even though higher organisms do not ← The early Microbiologists ← ← John Needham – 1748 • Boiled mutton broth in flasks – nutrient broth • After boiling, the flasks were sealed • Flasks became cloudy with microbes • Proposed that organic matter contained a vital force that could confer life to nonliving matter ← ← Lazzaro Spallanzani – 1765 • Prepared broth in pre-sealed flasks • As long as the flask remained closed, no growth occurred • Proposed that air carried germs • Important development toward sterilization • Skeptics proposed that heating the air destroyed its ability to support life ← ← Louis Pasteur – 1861 (Fig. 1-6) • Devised an unsealed flask with a long bent “swan-neck” • Allowed air to enter but prevented the entry of dust and germs • Broth was boiled inside the flask • As long as the flask remained upright – it remained free of microbes • If flask was tilted, growth commenced immediately • Development of aseptic technique ← ← The controversy over spontaneous generation was solved using the scientific method (Fig. 1-7) ← ← John Tyndall – 1877 • Repeated Pasteur’s experiment • Found that it was not always reproducible • If broth was made from meat, it remained free of microbes • If broth was made from hay, it did not • Discovered exceptionally heat resistant bacteria that survived boiling by forming endospores • Solution: repeated cycles of boiling and cooling causes endospore to germinate and be killed by next boiling – Tyndallization • Today has been replaced by heating to 121 °C under pressure using a machine necessary for any microbiology lab called an autoclave ← ← May 3, 2011 ← ← The Germ Theory of Disease ← ← Louis Pasteur – 1857 – 1860 • Studied wine and beer production • Yeast converted sugar to alcohol in the absence of oxygen o Fermentation: “La vie sans air” • Bacteria can sour wine by converting alcohols into acid • Developed a method of gentle heating to kill unwanted bacteria, sealed it, and it was free from bacteria – a process called Pasteurization • This led to the discovery that microorganisms are responsible for changes of the things around them. ← ← Joseph Lister – 1865 (a surgeon) • Noted that half of his patients died of sepsis (rotting of flesh) after amputation • Used phenol to treat wounds and surgical tools • Drastically lower the incidence of disease after surgery • Led to modern antiseptic surgical techniques – antisepsis (to kill or destroy microbes in or on the body) ← ← Robert Koch – 1876 • Studied anthrax – responsible for epidemics in livestock • He isolated bacteria from the carcass of a diseased animal – Bacillus anthracis • Injected healthy rabbits with the bacterium • Rabbits became ill with the bacterium • Re-isolated bacteria from diseased rabbits – identical – B. anthracis • Established a set of criteria for relating a specific microbe to a disease o Koch’s Postulates (Fig. 1-8) Pure Culture Techniques Pure Culture: a population of cells that are identical • Arise from a single cell • Easily achieved using a solid growth media ← Broth medium solidifies with agar • Polysaccharide derived from fairly rare marine algae • Melts at ~97 °C and polymerizes (solidifies) at ~45 °C • Cannot be degraded by most microorganisms • Typical Petri plate – nutrient broth medium +1.5% agar Two common lab media ← Nutrient Broth g/L of water Nutrient Agar Peptone 5 Beef Extract 3 NaCl 5 Bring up to 1L with2dH O • Same as Nutrient Broth but add 15g/L of agar Immunology ← Edward Jenner – 1796 • Development of the smallpox vaccine ← Smallpox: serious disease • Affected ~60% of people • Mortality ~1/3 • Left survivors with disfiguring scars ← Investigated belief that anyone who had cowpox never contracted smallpox ← Cowpox: mild disease • Not fatal • No disfiguring scars Jenner’s experiment • Extracted pus from a cowpox pustule • Injected it into the arm of an 8 year old boy • The boy became ill – fever and mild illness • When the boy recovered, Jenner inoculated him with pus from a smallpox pustule • The boy did not get sick with smallpox! ← ← Louis Pasteur – 1879 • Isolated a bacterium that caused fowl cholera in chickens o Went on vacation for three months • When he returned, inoculated healthy chickens with the bacterium o Chickens remained healthy • He then inoculated chickens with a fresh isolate o New chickens: contracted the disease and died o Previously inoculated chickens: remained healthy • Proposed that exposure to a weakened, non-pathogenic strain allowed the chickens to develop immunity ← Vaccine: Preparation of weakened or killed microorganisms (or parts of the microorganisms) used to induce an immune response. ← ← Chemotherapy ← Paul Erhlich – 1910 • Discovered that dyes react differently with microbes than they do with animal cells • Searched for a chemical that would destroy bacteria but not do harm to body tissues - “Selective Toxicity” ← ← Salvarsan – arsenic derivative that could be used to treat syphilis • Later led to the development of sulfonamides (Sulfa drugs) by the Bayer company ← ← Alexander Fleming – 1928 • Noticed that mold contaminating a Petri plate inhibited the growth of bacteria • Proposed that the mold was excreting an antibacterial chemical – penicillin – the first antibiotic • Antibiotic: a chemical produced by bacteria or fungi that inhibits the growth of other microbes. ← ← The Rise of Cell Biology • Electron microscope: used a beam of electrons instead of light o Allows for greater magnification o Direct visualization of cell components • Biochemistry: chemical reactions mediated by enzymes are responsible for cell function o Ex. Glycolysis and the Krebs cycle (TCA) • The ultracentrifuge: uses high centrifugal forces used to separate cell components o Ex. Bacteria have 70S ribosomes o Svedberg unit (S): a measurement to the rate at which a particle sediments in an ultracentrifuge ← ← The DNA Revolution ← ← Griffith’s Experiment – 1928 • Streptococcus pneumoniae (pneumonia) • S strain o Produced capsule smooth colony o Causes disease • R strain o No capsule rough colonies o Does not cause disease • Did the experiment Fig. 1-9 o Injected live S strain intdisease o  Injected live R strain intno disease o Injected heat killed S strain inno disease o Injected live R strain with dead diseasen  Re-isolated bacteria from dead mouse, found that it was live S strain • Transformation: free genetic material is taken up and changes the characteristics of the cell ← ← Avery’s Experiment – 1944 • Prepared an extract from S strain • Used enzymes to destroy specific cell constituents – protein, RNA, and DNA • Fig. 1-10 • When DNA is destroyed, transformation does not work o Therefore DNA is the genetic material ← ← DNA sequencing – 1970’s • Involves an in vitro DNA synthesis reaction (Fig. 1-11) • Automated – an entire genome can be sequenced in days (Fig. 1-12) ← ← ← Chapter 2: Observing Microorganisms ← The Compound Light Microscope (Fig. 2-1) • Uses visible light to observe a specimen • Image is formed by action of 2 lenses: ocular lens, objective lens • Condenser focuses light from a light source to a beam into the specimen • Aperture (Iris) diaphragm controls the amount of light going through the lens o Helps see color and contrast ← ← Calculating Magnification • Magnification = ocular x objective o Ex. Ocular = 10xObjective = 40x o Magnification = 10x40 = 400x ← ← Resolution • The ability of a lens to distinguish small objects that are close together • Two points can be distinguished if they are at least 0.2 μm apart • Light must pass between two points for them to be viewed as separate objects • As wavelength decreases, resolution improves (Fig. 2-2) o Gamma rays would give the best resolution since they have the smallest wavelength ← ← Refraction of Light • Bending of light as it travels from one substance into another (Fig. 2-3) • When light travels from glass into air, isome light misses the objective, and resolution decreases • Refractive index: measure of how greatly a substance slows the velocity of light • Immersion Oil: o Has the same refractive index as glass (Fig. 2-4) o Connects the specimen to the objective o Light is not refracted o Allows resolution o Use only with the 100x objective, the lower objectives gets damaged with immersion oil. ← ← May 4, 2011 ← ← Staining Specimens • Increase visibility of colorless microbes • Can reveal specific features – differential staining • Simple stain: one dye used to color specimen o Chromophore: colored portion of a dye (absorbs light at a specific wavelength) o Two types:  Basic Dye: Positively charges chromophore  Binds to negatively charged molecules on cell surface  Acidic Dye: Negatively charged chromophore  Repelled by cell surface  Used to stain background of slide  Also called Negative stain ← ← Differential Staining: • Used to distinguish groups of organisms based on cell structure • The Gram Stain: o Separates bacteria into 2 groups based on cell wall structure o Fig. 2-5 o Mordant: anything that makes the primary stain work better by sticking to some cells irreversibly o Gram Positive: cells that retain primary stain  Purple o Gram Negative: cells that lose the primary stain  Take the color of the counterstain  Pink o Note: both Crystal Violet and Safranin are basic dyes • Acid Fast Stain: o Detects mycolic acid in the cell wall of the genus Mycobacterium o Mycobacterium – retains primary stain  Fuchsia (pink) o Destaining by acid NOT alcohol o Anything else on the slide – color of counterstain  Blue o Ex. Sputum containing Mycobacterium tuberculosis (Fig 2-6) • Endospore Stain: o Endospores retain primary stain  Green o Cells are counterstained  Pink o Ex. Bacillus anthracis spores (Fig. 2-7) ← ← Light Microscopy ← Bright Field Microscopy (Fig. 2-8) • Produce a dark image against a bright background • Excellent for Observing colored specimen • Ex. Bacteria stained with dyes, some colored microbes • Aperture (Iris) diaphragm o Controls contrast to see unstained cells  Open  more light, allows you to see color   Closed less light, gives contrast  Ex. Colorless yeast against a bright background ← ← Phase Contrast Microscopy (Fig. 2-9,10) • Works on the cells that refract light • Light refracted by specimen is removed • Specimen appears dark on a light background • Excellent for observing unstained cells • Examples: o Organelles of eukaryotic cell structure o Live bacteria o Some internal cell structures of bacteria ← ← Dark Field Microscopy • Specimen is illuminated with a hollow cone of light • Only refracted light enters the objective • Specimen appears as a bright object on a dark background • Used to observe bacteria that does not stain well • Ex. Treponema pallidum – the causative agent of syphilis (Fig. 2-11) ← ← Differential Interference Contract (DIC) Microscopy • Two beams of polarized light o One beam passes through specimen o Other beam passes through clear area of slide • Beams are combined to form an image • Interference between the two beams gives the illusion of shadowing • Makes clearly defined images • Examples: o Bacteria (Fig. 2-12) o Internal structure of eukaryotes (Fig. 2-13) ← ← ← Fluorescence Microscopy • Light is absorbed by molecules in the specimen – Fluorophores (or Fluorochromes) o Fluorophores are excited o Re-emit light at lower energy (ie lower wavelength) o (Fig. 2-14), this is an episcopic microscope which means that light shines down on the specimen, then back up to the objective at a higher wavelength. • Fluorophores can be: o Natural  Ex. Photosynthetic cyanobacteria have chlorophyll (Fig. 2-15)  Absorbs light at 430 nm (blue – violet)  Emits at 670 nm (red) o Fluorescent Stains  Used to detect living cells  Specific cell components  Ex. DAPI specifically binds to DNA (fluoresces blue) (Fig. 2-16)  Since DNA is found in living cells only, if we find DNA, we can assume the cell observed is living o Gene Conferring Fluorescence  Introduced into the cell  Ex. E. coli is transformed with a gene for green fluorescent protein (GFP)  Gene is expressed  GFP is produced o Chimeric Proteins  GFP gene is added to a gene of interest (gene fusion)  Gene is expressed, it produces a protein attached to GFP  Can be used to locate structural proteins in the cell  Ex. Gene for Bacillus subtilis fused to GFP  Protein forms a helix around cell – cytoskeleton (Fig. 2- 17) o Fluorophore Labeled Antibodies  Antibody: produced by any animal. Targets very specific antigens found on another cell surface (ex. Found on bacteria)  Fluorescent dye is attached to antibody binds to specific cell components  Immunofluorescence: (Fig. 2-18) the red color, antibodies that stuck to the outside of the cell o DNA Hybridization  Fluorophore is attached to a piece of DNA  That piece of DNA attaches to complementary sequence on chromosome  Ex. B. subtilis (Fig. 2-18)  Green: DNA bound to chromosome  Red: antibody attached to membrane ← ← ← Confocal Microscopy • Uses a laser to illuminate the specimen • Removes light from above and below the plane of focus o Forms very sharp images • Several planes of focus used to construct a 3D image • Ex. Pseudomonas aeruginosa biofilm (Fig. 2-19) o Biofilm is the red layer of cells o Living cells: stained green o Dead cells: stained red ← ← Electron Microscopy • Electrons used as the illuminating beam instead of light • Wavelength of electrons is much shorter than light o Therefore higher resolution • Allows magnification greater than 100,000x • Used to study cell structures • Fig. 2-20 • Fig. 2-21: electron microscope, bacterial endospore • Fig. 2-22: same image but with phase contrast microscopy ← ← Transmission Electron Microscopy (TEM) • Electron beam focused on specimen by a condenser o Magnet used instead of lenses • Electrons that pass through the specimen are focused by two sets of lenses o Compound microscope • Electrons strike a fluorescent viewing screen, photographic film, or electron detector • Fig. 2-24 ← ← Staining a Specimen for TEM • Unstained cells do a poor job of scattering electrons • Must be stained with metallead or uranium (not chromophores) • Bind to cell structures to make them more electron dense • Structures that bind to more sdarker • Nuclear material does not pick up the stain, making a light area in the image ← ← Negative Staining and Shadowing • Background is stained with uranium o Specimen appears light on a dark background (Fig. 2-25 a) • Shadowing: specimen is coated with platinum from one side o Side coated with metal appears darker o Uncoated region acquires a shadow (Fig. 2-25 b) ← ← ← Scanning Electron Microscopy (SEM) • Specimen is coated with a thin layer of metal Electron beam scans the surface of the specimen • o Surface atoms release secondary electrons  Which gets trapped by a detector • Allows for an accurate 3D image of specimen’s surface • Scanning vs. Transmission (Fig. 2-26) ← ← Electron Crytomography • Specimen is frozen quickly (ex. Liquid ethane, -181 °C) o Preserves the native state of cell structures • Images are recorded from many directions o Used to form 3D images o Allows imaging of fine ultrastructures o Ex. Elements of the bacterial cytoskeleton ← ← Atomic Force Microscopy • Sharp probe moved over surface of specimen o Atomic force between probe and surface o Deflection of probe is measured by a laser o Used to construct an image • Magnification greater than 1,000,000x • Allows a view of atoms on an object surface • Ex. Structure of DNA at 2,000,000x (Fig. 2-28) ← ← X-ray Crystallography • Used to visualize molecules • A crystal is bombarded with X-rays o X-rays are diffracted • Diffraction patterns are analyzed to develop structural models of the molecule • Determines position of every atom • Fig. 2-29 ← ← May 5, 2011 ← ← Prokaryotic Cell Structure ← Prokaryote (before kernel) • No membrane bound nucleus or organelles • Generally smaller than eukaryotes, exception: Fig. 2-30 • Simple internal structure • Divide by binary fission • Most are unicellular • Two Domains: o Bacteria (eubacteria)  Diverse metabolism  Live in broad range of ecosystems  Pathogens and non-pathogens o Archaea (Archaebacteria)  Diverse metabolism (distinctly different than bacteria in some cases)  Live in extreme environments, as well as everywhere else  Non-pathogens ← ← Cell Shape ← Coccus (pl. Cocci) • Roughly spherical • Ex. Streptococcus pyogenes (Fig. 2-31) ← Bacillus (pl. bacilli) • Rod shaped • Ex. E. coli (Fig. 2-32) ← Spirillum (pl. Spirilla) • Spiral shaped • Ex. Spirillum volutans (Fig. 2-33) ← Other Shapes ← Vibrioid • Comma shaped • Ex. Vibrio ← Prosthecate Have a prostheca (stalk-like appendage) • • Ex. Caulobacter crescentus (Fig. 2-34) ← Spirochetes • Ex. Treponema pallidum • Tightly wound spiral (cork screw) ← Coccobacilli • Fig. 2-35 ← Filamentous • Ex. Streptomyces griseus • Grow like thread-like cells, fungi-like ← Pleomorphic • Variable in shape • Ex. Mycoplasma (Fig. 2-36) • Don’t have a cell wall Cell Size Average • E. coli ~1.0 μm (diameter) x 3.0 μm (length) • Stapphylococcus aureus ~ 1.0 μm diameter ← Very Small • Mycoplasma genitalium ~ 0.3 μm ← Very Large • Epulopiscium fishelsoni ~ 80 x 600 μm ← ← Arrangement • Occurs when cells remain attached after division (Fig. 2-37) • Diplococcus: cells divide and remain together in pairs • Streptococcus: cells divide in one plane (forms chains) • Tetrad: cells divide in 2 planes • Sarcina: cells divide in three planes (cuboidal) • Staphylococcus: cells divide in random planes (grape-like structures) • Note: not all bacteria have arrangements • Fig. 2-38: Staphylococci • Fig. 2-39: Tetrad ← ← Cell Organization • Fig. 2-40 • Capsule: “optional structure” • Cell envelope: o The plasma membrane is an all surrounding layer o Encloses the cytoplasm o Selectively permeable barrier  Nutrient and waste transport o Location of many metabolic processes o Composed of phospholipids (20 – 30%), neutral lipids and proteins (>50%)  However basic structure depends on phospholipids ← ← Esterphospholipids • Consists of glycerol, fatty acids, phosphate group, and polar head group. Fig. 2-41 • Glycerol: a carrier molecule with 3 locations for bonding • Amphipathic: has both polar and non-polar characteristics • Polar: molecule carries a differential charge o Hydrophilic • Non-polar: molecule is uncharged o Hydrophobic ← ← Phospholipids in Water • Tends to form micelles: Fig. 2-42 • Can form bilayer: Fig. 2-43 • Thermodynamically favorable structure ← ← Membrane Structure (Fig. 2-44) • Phospholipids held together by hydrophobic interactions • Peripheral Proteins: o 20 – 30 % of membrane proteins o Loosely associated with the membrane o Soluble in water – polar • Integral Proteins: o 70 – 80% of membrane proteins o Amphipathic  Hydrophilic region lies outside the membrane  Hydrophobic region buried inside membrane ← ← Fluid Mosaic Model • Lipid membrane exists in a semi-fluid state • Lipids in the outer leaflet are different from lipids in inner leaflet o Ex. Different head groups, different fatty acid tails • Integral Proteins can move laterally but do not flip-flop sides • Overall composition may change to suit conditions Cell Membrane of Eukaryotes • Similar to membrane of bacteria • Phospholipid bilayer – ester linked fatty acids o Fluid mosaic model • Differences o Tends to have less proteins  Since membrane bound organelles perform same metabolic functions o Different neutral lipids (Fig. 2-45)  Eukaryotic membranes contain sterols  Bacterial membranes contain hopanoids Neutral Lipid Function Modulate the fluidity of membrane • • Stabilize the membrane • Fig. 2-46 ← ← Archaeal Phospholipids • Fats linked to glycerol backbone by ether bonds o Branched chain alcohols – phytanols • Diglycerol tetraether lipids o 2 glycerol units joined by long branched hydrocarbons o Fig. 2-47 ← ← Archaeal Membranes • Composed of phospholipids, proteins, and neutral lipids o Forms a bilayer (Fig. 2-48 a) o Fluid mosaic model o Tetraethers can form a monolayer membrane (Fig. 2-48 b)  More stable  Remains fluid at high temperatures ← ← The Prokaryotic Cell Wall • The outside of the cell membrane • Rigid o Helps determine cell shape • Not a major permeability barrier o Porous to most small molecules • Protects the cell from osmotic changes o Some toxic molecules o Plays a role in pathogenicity ← ← Bacterial Cell Walls • Contains a complex polymer – Peptidoglycan (murein) • Divided into two broad groups o Gram positive cell wall – thick peptidoglycan o Gram negative cell wall – thinner, more complex ← ← Archaeal Cell Walls • Do not contain peptidoglycan • Composed of protein, polysaccharide, or other complex polymers (pseudomurein) • Stain Gram Negative ← ← Peptidoglycan – E. coli (Gram Negative) • Chain of 2 alternating sugar derivatives (Fig. 2-49) o N-acetylmuramic acid (NAM) o N-acetylglucosamine (NAG) o Linked by β-1,4 glycosidic bonds • Glycosidic Bond: Covalent bond linking a sugar to another molecule • Attached to a carboxyl group of NAM is a tetrapeptide (chain of 4 amino acids) • Amide Bond: covalent bond between an amino group and a carboxyl group. ← ← May 6, 2011 ← • Tetrapeptide: o On NAM o Four different amino acids  L-Alanine  D-Glutamic acid  meso-diaminopimeric acid (DAP)  D-Alanine o Amino acids are joined by peptide bonds o Peptide Bond: an amide bond between two amino acids o 3 unusual amino acids  D-Glu, DAP, and D-Ala not found in proteins o Defensive strategy  Resistance to most peptidases ← ← Cross-linking in Peptidoglycan • COOH terminus of tetrapeptide can be joined to free NH2 of DAP • Fig. 2-50 • Forms a “cross-link” between peptidoglycan strands o Occurs in ~10 – 20% of tetrapeptides o Forms a mesh-like structure surrounding the cell (Fig. 2-51) ← ← ← Peptidoglycan – Staphylococcus aureus (Gram Positive) • Structures vary • Some gram positives have the same PG as E. coli • 2 amino acid in tetrapeptide is D-Glutamine rd • 3 amino acid is L-Lysine • Crosslinks involve in pentaglycine peptide interbridge (Fig. 2-52) o Links D-Ala to L-Lys on adjacent strand ← ← Model of Peptidoglycan surrounding the cell (Fig. 2-53) • Backbone formed by NAM and NAG connected by glycosidic bonds • Crosslinks are formed by a peptide (pentaglycine) • Peptidoglycan strand is helical (Fig. 2-54) o Allows 3 dimensional crosslinking • E. coli has one layer • Some cell walls can be 50 – 100 layers thick ← ← The Gram Positive Cell Wall – S. aureus • Composed of thick layers of peptidoglycan o 50 – 100 layers (Fig. 2-55) o >90% of tetrapeptides cross-linked • Responsible for strength and rigidity ← The Gram Stain • Ethanol causes dehydration of peptidoglycan o Shrinks wall • Pores close, trapping crystal violet – iodine complex • Therefore remains purple ← ← Model of Gram Positive Cell Wall (Fig. 2-56) • Mostly peptidoglycan • Also contains teichoic acid and lipoteichoic acid • Teichoic acid: o Polymer of glycerol phosphates o Attached to peptidoglycan o Extends outward from the cell o Helps give the cell surface its net negative charge • Lipoteichoic acid: o Teichoic acid connected to a lipid at one end o Links peptidoglycan to the plasma membrane by the phosphate groups in it The Gram Negative Cell Wall – E. coli (Fig. 2-57) • Thinner than gram positive • More complex • 1 – 2 layers of peptidoglycan o 10 – 20% cross-linked • Surrounded by a 2 lipid membrane o Outer Membrane (OM) ← The Gram Stain • Ethanol disrupt the outer membrane • Thin peptidoglycan cannot retain crystal violet – iodine complex • Cells become clear • Take on color of counter stain o Pink (or red) ← ← Model of Gram Negative Cell Wall (Fig. 2-58) ← ← Peptidoglycan • 5 – 10% of wall weight • Provides rigidity • 1 – 2 layers thick ← ← Outer Membrane ← True unit membrane – Lipid Bilayer • Ester phospholipids: forms inner leaflet • Lipopolysaccharides: forms outer leaflet • Outer Membrane Proteins: differ from PM proteins ← ← Lipopolysaccharide (LPS) (Fig. 2-59) • Made of 3 parts • Lipid A: o Composed of sugars, phosphates, and fatty acids  Amphipathic • Core Oligosaccharide: o Chain of ~10 sugars o 2 unusual sugars  Keto-deoxyoctionic acid (C8)  Heptose (C7) o Some sugars modified by phosphates – polar part o Composition is relatively uniform among gram negative bacteria O antigen • o Long polysaccharide o Repeating units 4 – 5 sugars o Highly selective  Even strains of the same species can have different O antigens  Antigens: a molecule that can trigger a response by the immune system  Often used to type strains • Ex. E. coli 0157:H7 refers to the specific O antigen  Phosphates and sugars – net negative charge ← ← Outer Membrane Structure • Inner leaflet – ester phospholipids • Outer leaflet – lipopolysaccharides o O antigen extends to the exterior ← ← Outer Membrane Proteins ← Different from Proteins in the plasma membrane ← Braun’s Lipoproteins • Proteins with a modified end • Carries a glycerol esterified to fatty acids • Fatty acids interact with core of the membrane • Proteins forms bonds with DAP in peptidoglycan • Anchors the outer membrane to the peptidoglycan ← Porin (Omp C, Omp F) • Omp: Outer Membrane Proteins • Fig. 2-60 • Forms a bilayer • Spans the outer membrane o Forms a water filled channel o Allows hydrophilic molecules to pass through OM based on size ← Specific Transport Proteins • Specific for certain substrates or classes of substrate o Ex. Lam B allows transport of maltose ← Periplasmic Space (or Periplasm) • Space of between OM and PM • Area of high metabolic activity • Contains proteins distinct from OM or PM proteins ← ← Periplasmic Proteins: • Binding Proteins o Bind substrates that have passed across OM o Movement to PM • Hydrolytic Proteins o Degrade foreign substances (ex. DNA) • Biosynthetic Enzymes o Ex. For cell wall synthesis (Crosslinking) ← ← Role of the Cell Wall • Fig. 2-61 • Cell Wall prevents cell expansion – protects from osmotic lysis • Protects against toxic substances – large hydrophobic molecules o Ex. Detergents, antibodies • Pathogenicity o Helps evade host immune system o Helps bacterium sticks to surfaces • Partly responsible for cell shape ← ← Ways to Attack the Cell Wall ← ← Lysozyme: enzyme found in animal tissues and secretions • ex. Tears, saliva, egg whites, etc o Breaks β-1,4 glycosidic bonds between NAG and NAM • When treated with lysozyme: o Gram Positive Cells  Lose most of cell wall  Forms protoplasts  Enveloped by plasma membrane o Gram Negative Cells  Lose peptidoglycan but OM remains  Called spheroblasts  Enveloped by PM and OM o Both are osmotically fragile  Lysis in hypotonic solution  Stable in isotonic solution ← ← Penicillin: • Inhibits enzymes for biosynthesis of peptidoglycan o Most effective against fast growing cells  Cells lyse due to osmotic pressure o Little effect on slow growing cells  No effect on Mycoplasms – no cell wall  Archaea – no true peptidoglycan in cell wall ← ← Capsules and Slime Layers • May surround the cell wall • Usually composed of polysaccharide – glycocalyx • Capsule: well organized not easily removed o Protective function  Prevents drying  Excludes hydrophobic detergents  Resists phagocytosis • Slime Layer: disorganized, removed by washing o Helps cells stick to surfaces o Ex. Streptococcus mutans (cavities) o Slime layer from sugar – sticks to teeth • S Layer: o Protein layer may surround the cell wall o Protects against:  Foreign enzymes  Predacious bacteria  Host immune cells o Contributes to cell shape o Helps cells stick to surfaces o Spontaneously associates: self-assembling ← ← Archaeal Cell Walls • Lack Peptidoglycan • Wide variety of structures: Fig. 2-62 a. S – layer b. S – layer and protein sheath c. S – layer, protein sheath, and methanochondroitin o Similar to protein in animal connective tissue d. Pseudomurein – surrounded by S – layer (no NAM) e. Single thick layer of polysaccharide or pseudomurein o May stain gram positive ← ← May 9, 2011 ← The Cytoplasm • Cytoplasm: material bounded by PM • Protoplasm: PM and everything within • Cytoplasm as a bag of water: o Proteins o Macromolecules – amino acids, nucleotides, etc. o DNA and RNA o Ribosomes o Inclusions • Fig. 3-1 ← ← Proteins • Serve many functions • Enzymes: Catalyze chemical reactions • Transport Proteins: Move other molecules across membranes • Structural Proteins: Helps determine shape of the cell o Involved in cell division • Proteins are made of polypeptides • Polypeptide: a long polymer of amino acids joined by peptide bonds o 20 amino acid commonly found in proteins o Fig. 3-2 Protein Organization Primary Structure • Linear sequence of amino acids • Determined by genetics – DNA code specifies amino acid sequence o One gene  codes for one polypeptide o Fig. 3-3 + - • Each polypeptide has an –NH2(–NH 3 and –COOH (or –COO ) terminus ← Secondary Structure • Regular patterns that repeat over short sections of the polypeptide • Two most common secondary structures: o α-helix o β-sheet o Stabilized by H bonds between amino acids (carboxyl groups and amino groups) o Fig. 3-4 o In α-helix, H bonds are formed between every 4 amino acid ← Tertiary Structure • A polypeptide’s unique 3D shape • Determined by amino acid side chains • Involves H bonds, ionic bonds, and covalent bonds • Fig. 3-5 ← Quaternary Structure • Multiple polypeptides aggregate to form functional protein units o Subunits o Olgomeric protein • Fig. 3-6,7 • Proteins have moving parts • Changes in shape (conformation) are often necessary for activity Internal Membrane Systems • Invaginations of the PM • Fig. 3-8 • Forms tubules, vesicles, flattened stack • Greater surface area for metabolic processes • High concentrations of proteins • Anammoxasome: membrane bound organelle o Site of anaerobic ammonia oxidation o Fig. 3-9 ← ← Inclusion Bodies • Aggregates of organic or inorganic materials o Note: in biology, organic means has both carbon and hydrogen • Maybe surrounded by a non-unit membrane (does not include a lipid bilayer) o Lipid or protein • Often used for storage ← Organic Inclusions • Poly-β-hydroxybutyrate • Starch or glycogen granules o Carbon and energy storage ← Inorganic Inclusions • Polyphosphate granules – volutin o Storage of phosphate and energy • Sulfur globules o Storage of sulfur used in energy generation o Fig. 3-10: dark circles in cells (since sulfur is insoluble) ← ← The Nucleoid • The region that contains the genome (Fig. 3-11) ← The Typical bacterial genome: • Single circular double stranded (ds) DNA chromosome • May have one or more plasmids o Smaller circular dsDNA o Self-replicating (separate from the Chromosome) o Carry non-essential genes  Selective advantage o Ex. Genes for antibiotic resistance ← DNA • Carries genetic information of all living cells • Made of building blocks – deoxyribonucleotides (Fig. 3-12) ← ← Deoxyribonucleotides: Consists of three parts: ← • Nitrogen Base o Either a purine or a pyrimidine  Purines adenine or guanine  Pyrimidines cytosine or thymine • Pentose (5-carbon) sugar o Deoxyribose o Number 1’ – 5’ • One to three phosphates o Linked to 5’ C of pentose ← ← Ribonucleotides • Pyrimidine: uracil instead of thymine • Pentose: Ribose instead of deoxyribose (OH in the 2’ carbon) ← ← RNA Structure (Fig. 3-13) • Polymer of ribonucleotides monophosphates o AMP, GMP, CMP, UMP, (but not TMP) o Joined by 3’ – 5’ phosphodiester bonds • N bases extend away from sugar phosphate backbone • Designation 53’, indicates direction of synthesis ← ← DNA Structure (Fig. 3-14) • Polymer of deoxyribonucleotides monophosphates o dAMP, dGMP, dCMP, dTMP • DNA is double stranded (ds) o Two strands run anti-parallel o Oriented in opposite directions  If one is oriented 5’ to 3’  Other is oriented 3’ to 5’ • Two strands held together by hydrogen bonds between N bases o Bonding is specific o Watson-Crick base pairs ← Watson-Crick base pairs (bp) ← ← Adenine forms 2 H-bonds with thymine • A=T pair ← ← Guanine forms 3 H-bonds with cytosine • G≡C pair ← ← Fig. 3-15 ← ← *Two strands are complementary, not identical* ← ← ← ← The DNA Double Helix (Fig. 3-16) • Strands twist about each other • There are 10 bp per turn o Each rotates 36° relative to the previous bp o Bp are inside the structure o Sugar-phosphate backbones are outside forming double helix • Strands are not directly opposite each other o Results in major and minor grooves DNA Organization • Typical bacterial chromosome ~400 kbp • Overall length ~1.4 mm • Packed into cell ~3 μm long • Packaging types: o DNA is folded into 50 or more domains or loops (Fig. 3-17)  Done by DNA binding proteins o DNA in loops is supercoiled (Fig. 3-18) o Fig. 3-19, 3-20 ← ← RNA Structure • Single stranded (ss) o But there are ds regions o Intramolecular base pairing  Ex. Hairpin (Fig. 3-21) ← ← Cytoplasmic RNA ← ← Messenger RNA (mRNA) • Carries information about protein structure ← Ribosomal RNA (rRNA) • Component of the ribosome • Fig. 3-22 ← Transfer RNA (tRNA) • Activation/transport of amino acids • Fig. 3-23 ← Small RNA (sRNA) • Regulate stability of other RNA ← ← Bacterial Ribosomes (Fig. 3-24) • Site of protein synthesis • 70S ribosomes • 2 parts: o 30S subunit (small subunit)  Protein  16S rRNA o 50S subunit (large subunit)  Protein  23S and 5S rRNA ← Cytoplasmic Ribosomes • Synthesize cytoplasmic proteins ← PM associated Ribosomes • Synthesize membrane proteins • Proteins to be exported from the cell ← ← Cytoskeleton • Bacteria have homologs of eukaryotic cytoskeleton proteins • Homologs: proteins with similar amino acid sequence o Studied by comparative genomics o Structurally similar • Involved in: o Cell division o Cell shape o Localizing proteins to specific sites inside cell ← ← FtsZ • Forms ring at center of dividing cell • Required for formation of spetum • Fig. 3-25 (FtsZ is attached to GFP) ← ← MreB • Maintains cell shape • Involved in peptidoglycan synthesis • Fig. 3-26 (MreB attached to GFP) ← ← ← Mutation: a change in the DNA sequence of a gene • Can result in a protein with o Modified function o No function • Ex. Bacillus subtilis mreB mutant o Mutation in mreB gene o Not able to produce MreB gene (Fig. 3-27 C) ← ← Crescentin: found only in Caulobacter crescentus • Responsible for vibirioid shape • Fig. 3-28 ← ← May 10, 2011 ← ← Other Structures on Outside of Cell ← ← Fimbriae • Straight hair-like appendages o Made from protein o Anchored to PM, extend through CW o Allows adhesion to solid surfaces o Ex. Used by some bacteria to adhere to red blood cells o Protects from phagocytic white blood cells o Fig. 4-1 ← ← Pili • Longer protein appendage • Fig. 4-2 • Involved in gene transfer between bacteria • Ex. E. coli F-pilus • Genes located on F plasmid o If a strain of E. coli has the F plasmid, it can make the F-pilus • Allows cell to bind strains that do no have F plasmid • Transfer of plasmid through pilus • Can be thought of the F plasmid as an infectious particle that infects E. coli ← ← Flagella • Hollow protein filaments o Impart motility • Must be stained to view – Flagella stain ← Can be used for identification: • Monotrichous – single flagellum o Polar or subpolar • Amphitrichous – two flagella at opposite end • Lophotrichous – Multiple flagella in tufts • Peritrichous – flagella distributed around cell ← ← ← Flagellum Structure – 3 parts ← ← Filament • Rigid helical protein ~20 μm long (10x longer than the bacteria itself) • Composed of identical protein subunits – flagellin o Subunits travel through hollow tube o Fig. 4-4 o Filament capping protein adds them to tip o Growth occurs at tip, not at base ← Hook Flexible coupling between filament and basal body • • Fig. 4-5 ← Basal Body • Anchors hook to the cell envelope o Differs between gram positives and gram negatives ← ← Gram Negative Basal Body • Rod – protein cylinder to which hook is anchored o Passes through cell wall o Terminates in plasma membrane • Four Protein Rings: o L – Ring: in OM  Named for lipopolysaccharide o P – Ring: bound to peptidoglycan o MS – Ring: membrane Stator ring  Located in the PM o Note, L, P, and MS – Rings are though to be stationary C – Ring: Located in cytoplasm ← ← Gram Positive Basal Body • Only two protein rings • Inner Ring: bound to PM (like MS – ring) • Outer Ring: attached to peptidoglycan ← ← Flagellar Motility (Fig. 4-6) • Flagellum turns counter-clockwise o Swims in a straight line o Short period of time o Run Flagellum turns clockwise after each run • o Cell stops o Reorients itself in a random new direction o Tumble • Random walk: a series of runs and tumbles (Fig. 4-7 a) o No net movement of cells o The group as a whole doesn’t go anywhere • Chemotaxis: movement toward an attractant or away from a repellent (Fig. 4- 7 b) o Note bacteria can delay a tumble, making it run for a longer time.  It can control when it wants to delay, example delay run when going towards attractant and doesn’t delay when its moving away from it o Also called Random biased walk. ← ← Bacterial Endospore • Produced only by some gram positive rods • Ex. Bacillus sp – aerobic gram positive rods • Clostridium sp – anaerobic gram positive rods • Extremely Resistant to: o Temperature o UV and gamma radiation o Disinfectants o Dessication (drying) o Age • Fig. 4-8 ← ← Lifecycle of a Spore Forming Bacterium (Fig. 4-9) • Vegetative Cell: capable of normal growth o Metabolically active • Endospore: dormant cell, formed inside of a mother cell o Metabolically inactive o Endo – since formed inside the mother cell Eukaryotic Cell Structure Eukaryote (true kernel) • Fig. 4-10 • Genetic material is housed in a nucleus • Generally larger than prokaryotes • Complex internal structure o Membrane bound organelles o Intracytoplasmic membranes used for transport • Divide by Mitosis and meiosis • Unicellular, multicellular, or coenocytic o Coenocytic Fig. 4-11  A) No separate cells, just multiple nuclei in one filament  B) Incomplete septum, separate nuclei, but shared cytoplasm ← Size of Cells ← • From Fig. 4-12 • Eukaryotes have: o Lower surface area to volume ratio  Need more sophisticated transport mechanisms  Grow slower ← ← The Nucleus (Fig. 4-13) • Holds the genetic information o Multiple linear dsDNA chromosomes o Chromatin: complex of DNA and proteins – histones • Bounded by the nuclear envelope o Double membrane ← Nuclear Envelope • Spanned by nuclear pore complex • Transport of molecules into and out of the nucleus ← ← The Eukaryotic Cytoplasm ← Cytosol: liquid component • Location of many biochemical processes • Highly organized ← Cytoskeleton • Network of protein filaments • Organize cytoplasm • Fig. 4-14 • Microfilaments (actin) o Involved in amoeboid movement o Movement of cell structures around the cell o Homolog = MreB in bacteria • Intermediate Filaments o Structural role, provide shape to cells o Ex. Anchor the organelles o Homolog = crescentin • Microtubules o Thin cylinders o Form tracks for moving organelles around the cell  Ex. Separate chromosomes during cell division o Homolog = FtsZ ← ← Cilia and Flagella • Whip-like structures allowing motility • Fig. 4-15 • Membrane bound cylinders o Microtubules impart structure and function ← ← Secretory Endocytic Pathway (Fig. 4-16) • Organelles and vesicles that move materials in and out of the cell ← Endoplasmic Reticulum • Network of Internal membranes • Rough ER o Coated in ribosomes o Transports proteins • Smooth ER o No ribosomes o Synthesizes and transports lipids  Ex. New membranes ← ← Golgi Apparatus • Flattened membrane stacks • No ribosomes • Prepares material for secretion from the cell o Ex. Extracellular enzymes ← ← Lysosomes • Spherical membrane bound structures • Involved in endocytosis • Cells takes up particles through invaginations of the cell membrane ← ← Eukaryotic Ribosomes • Larger – 80S • Composed of protein and rRNA o Small subunit = 40S subunit o Large subunit = 60S subunit ← Cytoplasmic Ribosomes • Synthesize soluble cytoplasmic proteins (hydrophilic proteins) • Some nuclear and organelle proteins ER-bound Ribosomes ← • Integral membrane proteins (hydrophobic proteins) • Proteins for secretion ← ← Membrane Bound Organelles • Solutes can be concentrated • Fig. 4-17 • Reactive molecules can be separated from the rest of the cell • Membrane surface area is increased • Mitochondria o Energy generation o Site of respiration • Chloroplasts o Energy generation o Photosynthesis ← Semi-autonomous • Organelles are not produced by construction, but rather by division • Contain their own DNA (mtDNA or ctDNA) o No separate membrane o A circular dsDNA chromosome o No histones ← ← Mitochondria (Fig. 4-18) • Size of a large bacterium • Bounded by two membranes • Inner Membrane o Folded to form cristae  Electron Transport Chain: in the membrane  TCA Cycle: in the matrix • Outer Membrane o Contains porins, similar to gram negative bacteria Ribosomes: 70S • o Streptomycin: antibiotic that inhibits protein synthesis in bacteria  Mitochondria respond by dying since it is toxic to them ← ← Chloroplast (Fig. 4-19) • Site of photosynthesis o Contain chlorophyll • Surrounded by 2 membranes • Stroma (matrix) o DNA and ribosomes (70S) o Dark Reaction enzymes – carbohydrate synthesis • Thylakoids o Infoldings of inner membrane o Enzymes, electron carriers, and chlorophyll involved in light reactions – energy generation ← ← May 11, 2011 ← ← The Endosymbiotic Hypothesis (Fig. 4-20) • Mitochondria and chloroplasts evolved from bacterial cells • Evidence o Semi autonomous o Circular chromosomes  Lack histones o 70S ribosomes o Two membranes o Outer membrane has porin ← ← Comparison of 16S rRNA Gene Sequences • Fig. 4-21 ← Mitochondria • Mostly closely related to Rickettsia o Proteobacteria • Obligate intracellular pathogens o Ex. Rocky-mountain spotted fever ← Chloroplasts • Most closely related to Cyanobacteria o Blue-green algae ← ← Cyanophora paradoxa • Symbiotic relationship between a eukaryote and a cyanobacterium • Begins life as non-photosynthetic protozoa o Engulfs a cyanobacterium o Cyanelle – photosynthetic organelle  Does not divide • Fig. 4-22 Viruses • Acellular • Obligate intracellular pathogens o Reproduces only inside of living cells o Lacks independent metabolism ← Composed of at least 2 parts • Genome: Nucleic acid (DNA or RNA) • Capsid: Proteins o Together = Nucleocapsid o Fig. 5-1 a • Some viruses have an envelope o Layer of lipid surrounding the nucleocapsid o Fig. 5-1 b ← ← Virion: a complete virus particle ← The Discovery of Viruses ← Dimitri Ivanowski – 1892 • Bacteria not the cause of tobacco mosaic disease • Prepared extracts from infected leaves o Tilted to remove bacteria o Cell free extract was infectious o Fig. 5-2 ← Martinus Meijerinck – 1898 • Recognized that the agent multiplied • Named it Virus – Latin for poison ← Felix d’Herrel – 1920’s • Discovered viruses that infected bacteria – bacteriophage • Isolated viruses from patients sick with dysentery o Spread the virus on top of layer of bacteria  Clear areas formed – Plaques  Fig. 5-3 (plaques = the clear circles on petri dish) ← Host Range • Viruses infect all domains of life o Eukaryotes: Animals, plants, fungi, protozoa, algae o Prokaryotes: Bacteria and Archaea ← Most viruses are specific to a single host species • Virus must attach to specific receptors on the host cell surface o Only host with these receptors can be infected o Ex. HIV binds to CD4 (a receptor)  Chemoreceptor on surface of some human immune system cells  HIV only infects humans ← Some Viruses infect more than one species • Ex. Influenza attaches to a glycoprotein o Found on surface of several animal cells  Infects humans, pigs, chickens, seals, etc. ← ← Size • Pass through most filters • Smallest ~size of a ribosome • Largest ~ size of small bacterium • Fig. 5-4 • Electron microscope is needed to view viruses ← ← Viral Genome • DNA or RNA, NOT both • Single-stranded or double-stranded • Circular or linear • Can be in several pieces – segmented o Fig. 5-5 a • Ex. Influenza virus o Genome is made up of RNA (8 pieces or segments) Genome Size Smallest ~ 3.6 kbp for some ssRNA viruses • 3 genes • Fig. 5-5 b o gag for core proteins (capsid) o pol for enzymes in virus o env for the viral envelope ← Largest ~ >150kbp for some dsDNA viruses • >100 genes ← ← Viruses are essentially inert • Lack ribosomes and almost all enzymes Use host metabolic machinery • o Replicate genome o Express viral genes ← ← Capsid • Protein coat surrounds the genome • Allows transfer of viral genome between host cells • Made of identical polypeptides – protomers ← Helical Capsids • Fig. 5-6 • Protomers form a spiral cylinder • Ex. TMV capsid made of ~2100 identical protomers ← Icosaherdral Capsid • Fig. 5-1 • Regular geometric shape – 20 triangular faces • Protomers aggregate to form capsomeres o Capsomeres used to build the capsid o Pentamer – 5 protomers – form vertices o Hexamer – 6 protomers – form faces o Fig. 5-7 • Other regular and irregular polyhedral shapes possible ← ← Envelope • Consists of a lipid bilayer + proteins • Surrounds the nucleocapsid • Fig. 5-8 ← Lipid Bilayer • Acquired from the host membrane • Most common in animal viruses • Used only for viruses to protect themselves from the host’s immune cell Proteins ← • Specified by viral genes – spikes • Ex. Influenza virus o Flexible helical capsid o Surrounded by an envelope ← ← Complex Capsids • Binal structure • Polyhedral head with an attached helical tail • Fig. 5-9 • Ex. T-even bacteriophages of E. coli o T2, T4, T6 coliphages • Genome is carried in polyhedral capsid • Helical tail used to inject DNA into a host cell ← ← One Step Growth Experiment • Concentrated culture of bacteria • Bacterial cells are infected with phage o Time is given for phage to initiate infection o Excess phage washed away • Count the number of infective phage particles present at timed intervals • Results plotted as number of phage over time • Fig. 5-10 o Ignore blue line for now o The bottom plateau is equal to 0  i.e. phage count = 0 o At about 20 min  Phage count increases very quickly  ~300 for every bacterium in culture o Now decide to break open the cells before every count  This is where the blue line comes in  At about 17 minutes  Find ~100 phage  After another minute  ~ 200 phage  After another 2 minutes  ~ 300 phage  Then it stops ← ← Viral Multiplication • Fig. 5-11 • Adsorption o Attachment to the host cell o Interacts with specific receptors  Often proteins or glycoproteins • Entry – 3 basic methods o Injection of genome into the cell  Phage o Fusion with plasma membrane  Common for enveloped animal virus  Fig. 5-12 a o Endocytosis  Naked (non-enveloped) or enveloped animal viruses  Fig. 5-12 b & c • Uncoating o The capsid is removed o Genome is released • Biosynthesis o Viral genes are expressed to make viral proteins o Viral genome is replicated o Almost all functions performed by host machinery • Assembly o Viral proteins and genomes are combined to make nucleocapsids • Release – 2 basic mechanisms o Cell Lysis – in naked viruses o Budding – in enveloped viruses  Fig. 5-13  Viral proteins inserted into the host membrane  Spikes (green proteins in diagram)  Nucleocapsid associates with the spikes  Membrane buds to form the envelope 
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