Summaries – 1BI-311

Ch. 1

Ferdinand Cohn • Founded the field of bacteriology

• Recognized distinction between

prokaryotic and eukaryotic

cellular organization

• Discovered bacterial endospores

The Historical Roots of Microbiology: The Science

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

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.

Koch’s 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

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

• 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

Chapter 2 (in Brock Biology of Microorganisms 2012)

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

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.

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)

Improving Contrast in Light Microscopy

• Differential stains: the Gram stain• The Gram stain is widely used in microbiology to distinguish between

Bacteria with different cell wall structure: Gram-positive bacteria appear purple and gram-negative bacteria appear red after staining and counterstaining

Step 1

Result:All cells purple

Result:All cells remain purple

Result:Gram-positivecells are purple;gram-negativecells are colorless

Result:Gram-positive(G+) cells are purple;gram-negative (G-) cellsare pink to red

Flood the heat-fixedsmear with crystalviolet for 1 min

Add iodine solutionfor 1 min

Decolorize withalcohol briefly— about 20 sec

Counterstain withsafranin for 1–2 min

G+

G-

Step 2

Step 3

Step 4

GramStaining

Unknown bacteria

Gram positive(S. aureus)

Gram negative(E. coli)

Gram positive(Streptococcus)

Gram negative(E. coli)

Gram Staining

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

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.

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 (20–60 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)

Scanning Electron Microscopy – SEMGlutaraldehyde-fixed, critical point-dried, gold-paladium coated

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

Eukaryotic cellFreeze-etched preparationCarbon-coated, Gold-shaded, TEM image

TEM’s of sectioned cells from each of the domains of living organisms

Cytoplasmicmembrane Nucleus

Cell wall Mitochondrion

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

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

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

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

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

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

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

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

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

Cell Structure and Funtion

Chapter 3

•(in Brock Biology of Microorganisms 2012)

Macromolecules

• Organic chemistry = chemistry of carbon

• Biochemistry = chemistry of macromolecules

• Water = solvent & chemical bondingproperties: polarity, hidrophilic vs. hydrophobicH-bonds, glycosidic, esteric, etheric, peptide.

• Biogenic elements = C, O, H, N, S, P constructpolymers from monomers: polysaccharides, (phospho-)lipids, polypeptides, polynucleotides

CARBOXYL

ALDEHYDE

ALCOHOL

KETO

ESTER

PHOSPHO-ESTER

THIOESTER

ETHER

ACID ANHYDRIDE

PHOSPHO ANHYDRIDE

• 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

• 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.

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.

Prokaryotic cells often contain internal granules such as sulfur, PHB, polyphosphate, PHAs, and magnetosomes. These substances function as storage materials or in magnetotaxis.

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.

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.

• 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.

Prokaryotes that move by gliding motility do not employ rotating flagella, but instead creep along a solid surface by any of several possible mechanisms.

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.

Microbial Metabolism

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

∆G0' versus ∆G

standard conditions pH 7, 25°C

• 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

Energy

G 0’f = free Energy of formation

for elements G 0’f = 0

ΔG 0’ = change in free Energy in reactions

ΔG 0’ of the reaction: A+B C+D equals

ΔG 0’ [C+D] - ΔG 0’ [A+B]products - reactants

if + , the reaction is ENDERGONICif - , the reaction is EXERGONIC

ΔG 0’ does not affect the rates of reaction

• 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.

Enzyme Biocatalysis

• Specific substrate binding

• Substrate orientation o active sites

• Lowering the activation energy

• 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.

Biological Energy Conservation

Microbial Metabolism

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

Oxidation–reduction 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’.

REDOX potential

• 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.

• 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.

• 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).

• 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.

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.

• 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.

• Chemo – Energy from chemical reactions

• Organo – – trophic

of organic compounds feeding

• Hetero – Carbon from organic sources

• 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.

Energy from:

Chemical reactions or Light

Chemo- Photo- of:

inorganic or organic compounds

Litho- Organo-

Source of carbon :

CO2 or (CH2O)n

Auto- Hetero-

Amino acids are formed from carbon skeletons generated during catabolism while nucleotides are biosynthesized using carbon from several sources.

Lipids

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