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    Introduction structure Introduction structure Presentation Transcript

    • MICROBIOLOGY FALL 2011
    • Office Hrs. MW 11-12; STC 239 Tue 10-1; NEWDL 312 Independence Rd. Laboratory M/W 12-3 Lecture MW 3-4:15
    • Syllabus
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    • Dasek et al. 2000
    • Human Pathogens
      • 1415 known human pathogens
      • 61% are zoonotic
    • Wild Animals Involved in MPXV Transmission (USA, 2003) Gambian Giant Rat (Crycetomys sp.) Prairie Dog (Cynomis sp.)
    • Ghana TX WI IL * IA 15/4 09/04 Entry and Distribution Route of Crycetomys sp . and Cynomys sp. Involved in MPXV Outbreak (USA, 2003) Source: MMWR 52 (23), CDC, 2003. 15/4
    • Index case in Marshfield, 26 May 2003: Disseminated lesions.
    • Distribution of MPXV Cases in the USA WI IL IN OH KS MO Number of cases = 87 WI = 38 IN = 24 IL = 19 OH = 4 KS = 1 MO = 1 Source: MMWR 52 (23), CDC, 2003.
    • Microbiology
      • Has been defined as the study of organisms and agents too small to be seen clearly by the naked eye – that is, the study of microorganisms
    • Scope and Relevance
      • Microorganisms are exceptionally diverse, are found almost everywhere, and affect human society in countless ways. Modern microbiology is a large discipline with many different specialties: medicine, agricultural & food sciences, ecology, genetics, and biochemistry.
    • Microbiology
    • Microbiology
    • Microbiology
      • Spontaneous Generation
      • Molecular Biology/Genetics
      • Fermentation
      • Infectious Disease
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    • Infectious Disease - History
      • 1798 Jenner, smallpox vaccine
      • 1835-1844 Bassi, silkworm disease
      • 1847-1850 Semmelweis
      • 1849 Snow, cholera
      • 1861 Pasteur disproves spontaneous generation
      • 1867 Lister antiseptic surgery
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    • Infectious Disease - History
      • 1876-1877 Koch anthrax is caused by Bacillus anthracis
      • 1881 Pasteur develops anthrax vaccine
      • 1884 Koch’s postulates published
      • Autoclave developed
      • Gram stain developed
      • 1885 Pasteur develops rabies vaccine
    • Infectious Disease - History
      • 1887 Petri dish developed
      • 1890 Von Behring prepares antitoxins for diphtheria and tetanus
      • 1899 Ross shows that mosquitoes carry malaria
      • 1910 Ehrlich – magic bullet
      • 1923 – Bergey’s Manual 1 st edition
    • Infectious Disease - History
      • 1929 Fleming discovers penicillin
      • 1935 Domagk discovers sulfa drugs
      • 1975 Lyme Disease
      • 1983 HIV
      • 1986 hepatitis B vaccine – genetically engineered
    • DISEASE Occurrence of Disease
    • Signs & Symptoms of Disease
      • Fever
      • Fatigue
      • White Blood Cell Count
      • Pain
      • Muscle Aches
      • Blood Pressure
    • Molecular Biology
      • 1941 Beadle and Tatum, one gene-one-enzyme hypothesis
      • 1944 Avery shows that DNA carries information during transformation
      • 1952 Hershey and Chase viral infection of bacteria
      • 1953 Watson & Crick
    • Molecular Biology - History
      • 1961 Jacob & Monod propose the operon model of gene regulation
      • 1961-1966 Nirenberg et al. elucidate the genetic code
      • 1970 Arber & Smith – restriction endonucleases
      • 2000 Human genome sequenced
    • Fermentation - History
      • 1857 Pasteur shows that lactic fermentation is due to a microorganism
      • 1897 Buchner prepares yeast extract that ferments
    • Spontaneous Generation
      • 1799 Spallanzani attacks spontaneous generation
      • 1861Pasteur disproves spontaneous generation
    • Kingdoms
      • Animal
      • Plant
      • Fungi
      • Protista
      • Prokaryotic
    • Classification
      • Kingdom
      • Phylum
      • Class
      • Order
      • Family
      • Genus
      • species
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    • Infectious Agents / Microrganisms
      • Bacteria
      • Fungi
      • Prions
      • Protozoans
      • Helminths
    • Toxins – Exo and Endotoxins
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    • Prokaryotes
      • Greek – before a nucleus
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    • Prokaryotes / Eukaryotes
      • There are a number of differences between bacterial cells and plants, animals, fungi and protozoans.
    • Prokaryotes / Eukaryotes Size of cell 1-10 u m diameter 10-100 u m diameter Nucleus No nuclear membrane True nucleus Membrane bound organelles absent present Flagella 2 protein building blocks complex
    • Prokaryotes / Eukaryotes Glycocalyx Slime layer Absent Cell Wall Usually present Complex When present, simple Plasma membrane No CHO and lack sterols Sterols and CHO present Cytoplasm No cytoskeleton cytoskeleton
    • Prokaryotes / Eukaryotes Ribosomes Small size 70s Large 80s; small 70s Chromosome Circular, lacks histones Linear, with histones Cell division Binary fission mitosis Sexual reproduction No meiosis meiosis
    • Bacterial Classification
      • Cellular Characteristics
      • Morphology – cell shape, cell size, arrangement of cells, arrangement of flagella, capsule, endospores
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    • Cellular Characteristics
      • Staining Reactions – Gram stain, acid-fast stain
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    • Cellular Characteristics
      • Growth and nutritional characteristics – appearance in liquid culture
    • Cellular Characteristics
      • Growth and nutritional characteristics–colonial morphology
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    • Cellular Characteristics
      • Growth and nutritional characteristics pigmentation
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    • Cellular Characteristics
      • Growth and nutritional characteristics energy sources, C, N sources,
    • Cellular Characteristics
      • Growth and nutritional characteristics fermentation products
    • Cellular Characteristics
      • Growth and nutritional characteristics modes of metabolism
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    • Cellular Characteristics
      • Biochemical Characteristics – cell wall constituents, pigment biochemicals, storage inclusions, antigens, RNA molecules
    • Cellular Characteristics
      • Physiological and Ecological Characteristics – temperature range and optimum
    • Cellular Characteristics
      • Physiological and Ecological Characteristics oxygen relationships
    • Cellular Characteristics
      • Physiological and Ecological Characteristics – pH tolerance range
    • Cellular Characteristics
      • Physiological and Ecological Characteristics –salt requirement and tolerance
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    • Cellular Characteristics
      • Genetic Characteristics- DNA G + C
      • DNA hybridization
    • Fimbriae & Pili
      • G- bacteria have short, fine, hairlike appendages that are thinner than flagella and not involved in motility
    • Fimbriae & Pili
      • slender tubes composed of helically arranged protein subunits and are about 3 to 10 nm in diameter and up to several u m long
    • FIMBRIA (s) FIMBRIAE (pl)
      • Composition varies, contain protein
      • Tendency to stick to each other and surfaces
      • Bacterial attachment in aqueous environments
      • Role in colonization  infection
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    • PILUS (s) PILI (pl)
      • Hollow, non-helical (9-10nm dia)
      • Filamentous appendages
      • Thinner than flagella, more numerous
      • Example F-pilus ( SEX PILUS )
      • entry of genetic material during conjugation
      • GRAM -VE BACTERIA ONLY
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    • E.M Pili on E. coli – N. gonorrhea
    • Pili & Fimbriae
      • Some types of fimbriae attach bacteria to solid surfaces such as rocks in streams and host tissues
      • Pili – about 1 to 10 per cell, differ from fimbriae: are larger (9 to 10 nm in diameter), they are genetically determined by sex factors or conjugative plasmids and are required for bacterial mating
    • Pili & Fimbriae
      • Some bacterial viruses attach specifically to receptors on sex pili at the start of their reproductive cycle
    • Flagella & Motility
      • Most motile bacteria move by use of flagella, threadlike, locomotor appendages extending outward from the plasma membrane and cell wall.
      • Slender, rigid structures, about 20 nm across and up to 15 to 20 u m long
    • Flagella - Arrangements
      • Monotrichous
      • Amphitrichous
      • Lophotrichous
      • Peritrichous
    • Flagellar Ultrastructure
      • Filament
      • Hook
      • Basal body
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    • ARRANGEMENT OF FLAGELLA
      • POLAR - at one or both ends
      • MONTRICHOUS - single e.g., Vibrio sp.
      • LOPHOTRICHOUS - small tufts at same site
      • e.g., Pseudomonas sp.
      • AMPHITRICHOUS - at both poles, e.g., Spirillum sp.
      • LATERAL
      • PERITRICHOUS - surrounding entire cell, e.g., Proteus sp.
    • MOTILITY
      • CHEMOTAXIS: movement towards/away from chemicals
      • MAGNOTAXIS: orientation of movement in magnetic field
      • Aquaspirillium magnetotacticum - Magnetosomes
      • (Fe 3 O 4 crystalline magnetic iron oxide)
      • PHOTOTAXIS: Differences in light intensity
      • THERMOTAXIS: heat
      • Rotation of flagellar motor: reversible
      • Clockwise (CW) or Counterclockwise (CCW)
      • Smooth swimming/running motion
      • Motor rotates CCW direction
      • Flagella sweep around cell (in common axis)
      • Tumbling motion
      • Motor reverse (CW direction)
      • Flagella disperse
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    • Flagellar Synthesis
      • Complex process – involving at least 20 – 30 genes
      • Flagellin subunits are transported through the filament’s hollow internal core.
      • When they reach the tip, the subunits spontaneously aggregate so that the filament grows at its tip rather than at the base
      • Self - assembly
    • The Mechanism of Flagellar Movement
      • The filament is in the shape of a rigid helix, and the bacterium moves when this helix rotates
    • The Mechanism of Flagellar Movement
      • Act like propellers on a boat
    • The Mechanism of Flagellar Movement
      • E. coli rotates 270 r.p.s., Vibrio alginolyticus averages 1,100 r.p.s.
      • Distance and speed:
      • 20-90  m/sec
      • Equivalent to:
      • 6ft human running 5 body lengths/second
    • AXIAL FILAMENTS
      • Modified flagellum
      • Long thin microfibril, inserted into a hook, entire structure enclosed in periplasmic space
      • ENDOFLAGELLUM
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    • CELL SURFACE
      • 3 Basic Layers -
      • GLYCOCALYX
      • CELL WALL
      • CELL MEMBRANE
      • collectively termed CELL ENVELOPE
    • Glycocalyx
      • Is a network of polysaccharides extending from the surface of bacteria and other cells
      • Aids in bacterial attachment to surfaces of solid objects in aquatic environments or to tissue surfaces in plant and animal hosts
    • GLYCOCALYX
      • External mucilaginous layer
      • Surrounds cell
      • Shows organisation
      • SLIME LAYER - abundant, easily washed off
      • (poorly organised)
      • CAPSULE - abundant, not easily washed off
    • Capsule
      • Well organized and not easily washed off
      • Composed of polysaccharides, but may be constructed of other materials
      • i.e. Bacillus anthracis has a capsule of poly-D-glutamic acid
      • Visible with the light microscope
      • Resist phagocytosis
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    • Capsules
      • Contain a great deal of water – protect cell from desiccation
      • Exclude viruses
      • Exclude most hydrophobic toxic substances
    • FUNCTIONS
      • Provide protection (drying)
      • Block attachment of bacteriophages
      • Antipathogenic (inhibit engulfment of pathogenic bacteria by WBC’s) Contributes to VIRULENCE or INFECTIVE ABILITY
      • Promote attachment to surfaces
      • Streptococcus mutans : adheres to teeth ( GLUCAN ), DENTAL CARIES
    • Complement Activation
      • Some capsules prevent formation of C3 convertase on the bacterial surface
    • Host response – Antibody
      • Subvert this type of protective host response by having capsules that resemble host polysaccharides.
    • CELL WALL
      • Important in bacterial characteristics
      • Determines shape
      • Provides support/rigidity
    • STRUCTURE
      • Composed of PEPTIDOGLYCAN (MUREIN)
      • Insoluble, porous
      • Cross-linked polymer (glycan), provides strength and rigidity
      • N -acetyl Muramic Acid (NAM)
      • N -acetyl Glucosamine (NAG)
      •  1-4 glycosodic bonds
      • Differences in structure - basic principles of
      • GRAM STAIN REACTION
      • Christian Gram 1884
      • Differential stain : ability of eubacterial cells to retain dye
      • (crystal violet) after discolouration with 95% ethanol
      • Cells retain stain: Gram +ve (thick cell walls) PURPLE
      • Cells lose stain: Gram -ve (thin cell walls) RED
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    • Peptidoglycan Structure
      • Peptidoglycan or murein is an enormous polymer composed of many identical subunits.
    • Peptidoglycan Structure
      • Most G- cell wall peptidoglycan lacks the peptide bridge.
    • Gram Negative Cell Walls
      • More complex than the G+ cell walls.
      • Peptidoglycan 5-10% of the wall weight.
      • Braun’s lipoprotein – a small lipoprotein covalently joined to the underlying peptidoglycan and embedded in the outer membrane by its hydrophobic end.
      • Lipopolysaccharides (LPSs)
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    • Lipopolysaccharide Structure
      • Known as ENDOTOXIN
      • Complex molecule:
      • Inner most LIPID (Lipid A), achors LPS to outer membrane
      • Polysaccharide portion (external to Lipid A) known as O-antigen
      • O-polysaccharide long repeating sequence of sugars
    • LPSs
      • Contain both lipid and carbohydrate
      • Consist of three parts: lipid, the core polysaccharide and the O side chain
    • LPSs
      • The lipid A region contains two glucosamine sugar derivatives, each with three fatty acids and phosphate or pyrophosphate attached.
    • LPSs
      • Lipid A is buried in the outer membrane and the remainder of the LPS projects from the surface.
    • LPS
      • The core polysaccharide is joined to lipid A. In Salmonella it is constructed of 10 sugars, many of them unusual in structure.
    • LPS
      • The O side chain or O antigen is a short polysaccharide chain extending outward from the core. It has several peculiar sugars and varies in composition between bacterial strains.
    • LPS
      • G- bacteria can rapidly change the nature of their O side chains to avoid detection.
      • Contributes to the negative charge of the bacterial surface
      • LPS helps stabilize membrane structure
      • Lipid A is toxic – endotoxin
      • Serves as a protective barrier
    • LPS – protective barrier
      • Prevents or slows entry of bile salts, antibiotics, and other toxic substances.
    • Porin Proteins
      • Cluster together and span the outer membrane to form a narrow channel through which molecules smaller than about 600 to 700 daltons can pass.
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    • Porin Proteins
      • Larger molecules such as vitamin B 12 must be transported across the outer membrane by specific carriers. The outer membrane also prevents the loss of constituents like periplasmic enzymes.
    • COMPARISON OF GRM+VE /GRM-VE CELL WALLS
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    • Periplasmic Space
      • Contains enzymes involved in peptidoglycan synthesis and the modification of toxic compounds that could harm the cell.
    • Periplasmic Space
      • Gram – contains many proteins that participate in nutrient acquisition. Ex. hydrolytic enzymes attacking nucleic acids and phosphorylated molecules, and binding proteins involved in transport of materials into the cell.
      • Denitrifying and chemolithoautotrophic bacteria – electron transport proteins in periplasm
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    • CELL/CYTOPLASMIC MEMBRANE
      • 4-5nm thick
      • composed 1 o phospholipids 30-40%
      • and protein 60-70%
      • Phospholipid bilayer:
      • Polar heads
      • (outwards into aqueous phase - membrane surface)
      • Fatty acyl tails
      • (inwards - semi/liquid phase at interior)
    • FLUID MOSAIC MEMBRANE Davison & Danelli
      • ALSO CONTAINS:
      • PROTEINS
      • INTEGRAL - removed by destruction
      • i.e., Detergents
      • PERIPHERAL - loosely attached, easily removed
      • i.e., Osmotic shock
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    • FUNCTION
      • Transport - control nutrients
      • Oxidative phosphorylation (Respiration)
      • Secretion - discharge of metabolic products
      • Anchoring DNA (during cell division)
      • Metabolism - enzyme sites
    • INTERNAL CONTENTS
      • Cell Material divided into
      • PROTOPLASM
      • Granular appearance
      • Site of biochemical activity
      • Water 70-80%
      • acts as solvent for nutrients, sugars, Aa’s & salts
      • CHROMATIN AREA
      • no distinct membrane enclosed nucleus
      • no mitotic apparatus
      • BACTERIAL CHROMOSOME
      • Typically single circular strand of DNA ( CHROMATIN BODY )
      • Exception Streptomyces & Borrelia sp (Linear)
      • Rhodobacter sphaeroides (2 separate chromosomes)
      • all genes are linked
      • Aggregated in one area ( NUCLEOID )
    • Bacterial Chromosome
      • PLASMIDS
      • Additional to chromosome
      • 1 or more, small circular macromolecules of DNA
      • Capable of self-replication
      • Types:
      • Fertility (F-plasmid): genes for mating in conjugation
      • Resistance (R-plasmids): antibiotics, metals
      • Virulence factor: enterotoxin, fimbriae, antibiotic production
      • Colicinogenic (col-plasmids): gene for protein ( COLICINS ) toxic to closely related bacteria (eliminates competitors)
      • Transformation (Ti-plasmids): plant microbiology (formation of crown gall tumors) Agrobacterium
      • Metabolic: utilization of camphor, toluene
    • OTHER FEATURES
      • RIBOSOMES
      • Located in Protoplasm
      • RNA/PROTEIN bodies
      • Composed of 2 sub units (70S)
      • Svedberg Units
      • Sites of Protein Synthesis
      • MESOSOMES:
      • Extensive invaginations (infoldings) of cyto membrane
      • Continuous with membrane
      • Function NOT KNOWN
      • Corynebacterium parvum
      • INCLUSIONS/VACUOLES
      • compensate for poor availability of nutrients
      • present in Protoplast
      • VOLUTIN GRANULES/METACHROMATIC ( coloured )
      • composed of POLYPHOSPHATE
      • energy rich storage structures
      • VOLUTIN
      • i.e., POLY-  -HYDROXYBUTYRATE
      • serve as carbon and energy source
      • METACHROMATIC
      • i.e., Aquatic bacteria - colored crystals
      • ( Blue or Red dyes)
      • e.g., Corynebacterium
    • Cyanobacterium: Microcystis (12,600x)
      • Gas vacuoles - blue
      • Storage granules - red
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