Summaries

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Summaries 1BI-311

• Ch. 1

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The Historical Roots of Microbiology The
Science

• Ferdinand Cohn
• Founded the field of bacteriology
• Recognized distinction between
• prokaryotic and eukaryotic
• cellular organization
• Discovered bacterial endospores

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• The Historical Roots of Microbiology
• Louis Pasteur
• Discredited the theory of Spontaneous
• Generation.
• Introduced control of microbial growth.
• Discovered lactic acid bacteria
• Role of yeast in alcohol fermentation
• Rabies vaccine

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• The Historical Roots of Microbiology
• Robert Koch
• Growth of pure cultures of microorganisms
• Solid growth media
• Discovered cause of tuberculosis.
• Developed criteria for the study of infectious
• microorganisms
• Kochst Postulates.

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• Kochs Postulates
• OBSERVE The presence of suspected
  pathogenic microorganism correlates positively
  with the symptoms of the diseased and negative
  with healthy control
• ISOLATE the suspected pathogen into axenic
  culture
• INFECT a healthy animal with cultured
  strain. Observe whether the same symptoms show
• RE-ISOLATE the pathogen from the new victim
  and compare both cultures

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• The Historical Roots of Microbiology
  General Microbiology - Microbial Ecology and
  Diversity
• Martinus Beijerinck
• Enrichment Culture Technique
• Concept of Virus
• Sergey Winogradsky
• Concept of Chemolithotrophy and Autotrophy

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Chapter 2 (in Brock Biology of Microorganisms
2012)

• Incident light microscopy (dissecting)
• Transmitted light microscopy (compound)
• Phase contrast
• Dark field
• Differential Interference Contrast (DIC)
• Fluorescence microscopy
• Confocal Scanning Light Microcopy (CSLM),
• Transmission electron microscopy (TEM)
• Scanning electron microscopy (SEM)
• The atomic force microscope

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Principles of Light Microscopy

• Bright-field scope
• Specimens are visualized because of differences
  in contrast (density) between specimen and
  surroundings
• Two sets of lenses form the image
• Objective lens and ocular lens
• Total magnification objective magnification ?
  ocular magnification
• Maximum magnification is 2,000?
• Resolution the ability to distinguish two
  adjacent objects as separate and distinct
• Resolution is determined by the wavelength of
  light used and numerical aperture of lens
• Limit of resolution for light microscope is about
  0.2 ? m

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• Other microscope techniques
• Differential Interference Contrast (DIC) and
  Confocal Scanning Light Microcopy (CSLM) allow
  for greater three-dimensional imaging than other
  forms of light microscopy,
• Confocal microscopy allows imaging through
  thick specimens.
• The atomic force microscope yields a detailed
  three-dimensional image of live preparations.

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Improving Contrast in Light Microscopy

• Improving contrast results in a better final
  image
• Staining improves contrast
• Positively charged dyes can be used to stain
  cells (bind to negatively charged components such
  as nucleic acids, acidic polysaccharides) to
  improve their contrast
• Dyes are organic compounds that bind to specific
  cellular materials
• Examples of common stains are Methylene blue,
  Safranin, Crystal violet
• Differential staining (Gram staining) Crystal
  violet and Safranin to differentiate Gram()ve
  and (-)ve microbes (Christian Gram-1984)

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Improving Contrast in Light Microscopy

• Differential stains the Gram stain
• The Gram stain is widely used in microbiology to
  distinguish betweenBacteria with different cell
  wall structure Gram-positive bacteria appear
  purple and gram-negative bacteria appear red
  after staining and counterstaining

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GramStaining
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Gram Staining
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Imaging Cells in Three Dimensions

• Confocal Scanning Laser Microscopy (CSLM)
• Uses a computerized microscope coupled with a
  laser source to generate a three-dimensional
  image
• Computer can focus the laser on single layers of
  the specimen
• Cells are (i) either stained with fluorescent
  dyes, or (ii) different layers in specimen are
  assigned colors to generate false color images
• Different layers are then be compiled for a 3-D
  image
• Resolution is 0.1 ?m
• Applications Thick biofilms, Microbial ecology

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Electron microscopes use
electron beams instead of light. They have far
greater resolving power than do light
microscopes, the limits of resolution being about
0.2 nm. Two major types of electron microscopy
are performed Transmission Electron Microscopy
(TEM), for observing internal cell structure down
to the molecular level, and Scanning Electron
Microscopy (SEM), useful for three-dimensional
imaging and for examining surfaces.
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2.4 Electron Microscopy

• Transmission Electron Microscopy (TEM)
• Electromagnets function as lenses
• System operates in a vacuum
• High magnification and resolution (0.2 nm)
• Enables visualization of structures at the
  molecular level
• Specimen must be very thin (2060 nm) and be
  stained with compounds such as osmic acid,
  permanganate, uranium, lanthanum or lead salts
  (these contain atoms of high Atomic weight, they
  scatter electrons well to improve contrast)

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Scanning Electron Microscopy SEM Glutaraldehyde-
fixed, critical point-dried, gold-paladium coated
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Elements of Microbial Structure

• Eukaryotic vs. Prokaryotic Cells
• Eukaryotes
• DNA enclosed in a membrane-bound nucleus
• Cells are generally larger and more complex (as
  small at 0.8 ?m to several 100 ?m)
• Contain organelles
• Prokaryotes
• No membrane-enclosed organelles, no nucleus
• Generally smaller than eukaryotic cells
• Typical prokaryotic cell is 1-5 ?m long, 1 ?m
  wide

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Eukaryotic cell Freeze-etched preparation Carbon-c
oated, Gold-shaded, TEM image
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TEMs of sectioned cells from each of the domains
of living organisms
Cytoplasmicmembrane
Nucleus
Mitochondrion
Cell wall
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Gene, Genomes and Proteins comparison

• Escherichia coli Genome
• 4.64 million base pairs
• 4,300 genes
• 1,900 different kinds of protein
• 2.4 million protein molecules
• Human Cell
• 1,000? more DNA per cell than E. coli
• 7? more genes than E. coli

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The Evolutionary Tree of Life

• Evolution
• The process of change over time that results in
  new varieties and species of organisms
• Phylogeny
• Evolutionary relationships between organisms
• Relationships can be deduced by comparing genetic
  information in the different specimens
• Ribosomal RNA (rRNA) sequencing method is
  excellent for determining phylogeny
• Relationships visualized on a phylogenetic tree

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The Evolutionary Tree of Life

• Comparative rRNA sequencing has defined three
  distinct lineages of cells called domains
• Bacteria (prokaryotic)
• Archaea (prokaryotic)
• Eukarya (eukaryotic)
• Archaea and Bacteria are NOT closely related
• Archaea are more closely related to Eukarya than
  Bacteria

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Metabolic Diversity by Energy Source

• Chemoorganotrophs
• Obtain their energy from the oxidation of organic
  molecules
• Aerobes use oxygen to obtain energy
• Anaerobes obtain energy in the absence of oxygen
• Chemolithotrophs
• Obtain their energy from the oxidation of
  inorganic molecules
• Process found only in prokaryotes
• Phototrophs
• Contain pigments that allow them to use light as
  an energy source
• Oxygenic photosynthesis produces oxygen
• Anoxygenic photosynthesis does not produce oxygen

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Metabolic Diversity by C source

• All cells require carbon as a major nutrient
• Autotrophs
• Use CO2 as their carbon source
• Sometimes referred to as primary producers
• Heterotrophs
• Require one or more organic molecules for their
  carbon source
• Feed directly on autotrophs or live off products
  produced by autotrophs

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Phylogenetic Analyses of Natural Microbial
Communities

• Microbiologists believe that we have cultured
  only a small fraction of the Archaea and Bacteria
• Studies done using methods of molecular microbial
  ecology, devised by Norman Pace
• Microbial diversity is much greater than
  laboratory culturing can reveal (Metagenome?)
• More high-throughput techniques

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Summary Microscopy

• Microscopes are essential for studying
  microorganisms
• Inherent limit of bright field microscopy can be
  overcome by use of stains, phase contrast or
  dark-filed microcopy
• DIC and CFLM allows enhanced 3D imaging
• AFM used for 3D imaging of live cells
• Electron microscopes have the best resolving
  power

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Summary Genes

• Genes govern the properties of a cell
• DNA is arranged in cells as chromosomes
• Prokaryotes (most) have single chromosome
• Eukaryotes have multiple copies
• rRNA sequencing have defined 3 domains of life

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Summary Diversiy

• All cells need C and energy for growth
• Chemoorganotrophs organic chemicals as energy
  source
• Chemolithotrophs inorganic chemicals as energy
  source
• Phototrophs Light as energy source
• Autotrophs CO2 as C-source
• Heterotrophs organic compounds as C-source
• Extremophiles Can live in extreme environmental
  conditions
• Bacterial Phyla Proteobacteria, Gram positive
  bacteria, Cyanobacteria, green bacteria
• Archaea Euryarchaeota and Crenarchaeota
• Microbial Eukarya Protists (algae and protozoa),
  fungi and slime molds, Lichens

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Cell Structure and Funtion

• Chapter 3
• (in Brock Biology of Microorganisms 2012)

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Macromolecules

• Organic chemistry chemistry of carbon
• Biochemistry chemistry of macromolecules
• Water solvent chemical bonding
• properties polarity, hidrophilic vs.
  hydrophobic
• H-bonds, glycosidic, esteric, etheric,
  peptide.
• Biogenic elements C, O, H, N, S, P
  construct
• polymers from monomers polysaccharides,
• (phospho-)lipids, polypeptides, polynucleotides

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CARBOXYL
ESTER
ETHER
ALDEHYDE
PHOSPHO-ESTER
ACID ANHYDRIDE
ALCOHOL
THIOESTER
KETO
PHOSPHO ANHYDRIDE
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• The cell walls of Bacteria contain a
  polysaccharide called peptidoglycan.
• This material consists of strands of alternating
  repeats of N-acetylglucosamine and
  N-acetylmuramic acid, with the latter
  cross-linked between strands by short peptides.
  Many sheets of peptidoglycan can be present,
  depending on the organism.
• Archaea lack peptidoglycan but contain walls
  made of other polysaccharides or of protein. The
  enzyme lysozyme destroys peptidoglycan, leading
  to cell lysis in Bacteria but not in Archaea

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• In addition to peptidoglycan, gram-negative
  Bacteria contain an outer membrane consisting of
  lipopolysaccharide, protein, and lipoprotein.
• Proteins called porins allow for permeability
  across the outer membrane.
• The space between the membranes is the periplasm,
  which contains various proteins involved in
  important cellular functions.

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Prokaryotic cells often contain various surface
structures. These include fimbriae pili
S-layers capsules slime layers. These
structures have several functions, but a key one
is in attaching cells to a solid surface.
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Prokaryotic cells often contain internal granules
such as sulfur, PHB, polyphosphate, PHAs, and
magnetosomes. These substances function as
storage materials or in magnetotaxis.
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Gas vesicles are small gas-filled structures made
of protein that function to confer buoyancy on
cells. Gas vesicles contain two different
proteins arranged to form a gas permeable, but
watertight structure Gas Vesicle Proteins GVP-a
and GVP-c.
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• The endospore is a highly resistant
  differentiated bacterial cell produced by certain
  gram-positive Bacteria.
• Endospore formation leads to a highly
  dehydrated structure that contains essential
  macromolecules and a variety of substances such
  as calcium dipicolinate and small acid-soluble
  proteins, absent from vegetative cells.
• Endospores can remain dormant indefinitely but
  germinate quickly when the appropriate trigger is
  applied.

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• Motility in most microorganisms is due to
  flagella. In prokaryotes the flagellum is a
  complex structure made of several proteins.
• Most of these proteins are anchored in the cell
  wall and cytoplasmic membrane.
• The flagellum filament, which is made of a
  single kind of protein, rotates at the expense of
  the proton motive force, which drives the
  flagellar motor.

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Prokaryotes that move by gliding motility do not
employ rotating flagella, but instead creep along
a solid surface by any of several possible
mechanisms.
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• Motile bacteria can respond to chemical and
  physical gradients in their environment.
• In the processes of chemotaxis and phototaxis,
  random movement of a prokaryotic cell can be
  biased either toward or away from a stimulus by
  controlling the degree to which runs or tumbles
  occur.
• The latter are controlled by the direction of
  rotation of the flagellum, which in turn is
  controlled by a network of sensory and response
  proteins.

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• Microbial Metabolism
• Biocatalysis Energy Generation
• Phosphorylation
• Oxidation Reduction
• Fermentation Respiration
• Chemiosmosis Proton Motive Force
• ATPase Motor
• Energy Yielding Metabolic Systems
• Biosynthesis

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?G0' versus ?Gstandard conditions pH 7, 25C
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• The chemical reactions of the cell are
  accompanied by changes in energy, measured in
  kilojoules (kJ).
• A chemical reaction can occur with the release
  of free energy (exergonic) or with the
  consumption of free energy (endergonic).
• 1 calorie 4.186 Joules

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EnergyG 0f free Energy of formationfor
elements G 0f 0 ?G 0 change in free
Energy in reactions ?G 0 of the reaction AB
? CD equals ?G 0 CD - ?G 0
ABproducts - reactantsif , the
reaction is ENDERGONICif - , the reaction
is EXERGONIC ?G 0 does not affect the rates of
reaction
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• The reactants in a chemical reaction must first
  be activated before the reaction can take place,
  and this requires a catalyst.
• Enzymes are catalytic proteins that speed up the
  rate of biochemical reactions.
• Enzymes are highly specific in the reactions they
  catalyze, and this specificity resides in the
  three-dimensional structure of the polypeptide(s)
  in the protein.

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Enzyme Biocatalysis

• Specific substrate binding
• Substrate orientation o active sites
• Lowering the activation energy

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Biological Energy Conservation

• The energy released in redox reactions is
  conserved in the formation of certain compounds
  that contain energy-rich phosphate or sulfur
  bonds. The most common of these compounds is ATP,
  the prime energy carrier in the cell.
• Long-term storage of energy is linked to the
  formation of polymers, which can be consumed to
  yield ATP.

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• Microbial Metabolism
• Biocatalysis Energy Generation
• Phosphorylation
• Oxidation Reduction
• Fermentation Respiration
• Chemiosmosis Proton Motive Force
• ATPase Motor
• Energy Yielding Metabolic Systems
• Biosynthesis

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REDOX potential

• Oxidationreduction reactions involve the
  transfer of electrons from electron donor to
  electron acceptor. The tendency of a compound to
  accept or release electrons is expressed
  quantitatively by its reduction potential, E0.

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• The transfer of electrons from donor to acceptor
  in a cell typically involves one or more electron
  carriers.
• Some electron carriers are membrane-bound,
  whereas others, such as NAD/NADH, are freely
  diffusible, transferring electrons from one place
  to another in the cell.

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• The energy released in redox reactions is
  conserved in the formation of certain compounds
  that contain energy-rich phosphate or sulfur
  bonds.
• The most common of these compounds is ATP, the
  prime energy carrier in the cell.
• Long-term storage of energy is linked to the
  formation of polymers, which can be consumed to
  yield ATP.

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• Fermentation and respiration are the two means by
  which chemo-organotrophs conserve energy from the
  oxidation of organic compounds.
• During these catabolic reactions, ATP synthesis
  occurs by way of either substrate-level
  phosphorylation (fermentation) or oxidative
  phosphorylation (respiration).

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• Glycolysis is a major pathway of fermentation and
  is a widespread means of anaerobic metabolism.
• The end result of glycolysis is the release of a
  small amount of energy that is conserved as ATP
  and the production of fermentation products.
• For each glucose consumed in glycolysis, 2 ATPs
  are produced.

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• Respiration involves the complete oxidation of
  an organic compound with much greater energy
  release than during fermentation. The citric acid
  cycle plays a major role in the respiration of
  organic compounds.

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• When electrons are transported through an
  electron transport chain, protons are extruded to
  the outside of the membrane forming the proton
  motive force.
• Key electron carriers include flavins, quinones,
  the cytochrome bc1 complex, and other
  cytochromes, depending on the organism.
• The cell uses the proton motive force to make ATP
  through the activity of ATPase.

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• Chemo
• Energy from chemical reactions
• Organo trophic
• of organic compounds feeding
• Hetero
• Carbon from organic sources

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• Electron acceptors other than O2 can function as
  terminal electron acceptors for energy
  generation. Because O2 is absent under these
  conditions, the process is called anaerobic
  respiration.
• Chemolithotrophs use inorganic compounds as
  electron donors, while phototrophs use light to
  form a proton motive force.
• The proton motive force is involved in all forms
  of respiration and photosynthesis.

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Energy from
Chemical reactions or
Light Chemo- Photo-
of inorganic or
organic compounds Litho-
Organo- Source of
carbon CO2 or
(CH2O)n Auto-
Hetero-
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• Amino acids are formed from carbon skeletons
  generated during catabolism while nucleotides are
  biosynthesized using carbon from several sources.

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LipidsFatty acids are synthesized two
carbons at a time and then attached to glycerol
to form lipids.