Summaries – 1 BI-311

1. Summaries – 1BI-311 Ch. 1

2. 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

3. 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

4. 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.

5. 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

6. 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

7. 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

8. 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

9. 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.

10. 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)

11. 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

12. GramStaining Step 1 Flood the heat-fixedsmear with crystalviolet for 1 min Result:All cells purple Step 2 Add iodine solutionfor 1 min Result:All cells remain purple Step 3 Decolorize withalcohol briefly— about 20 sec Result:Gram-positivecells are purple; gram-negativecells are colorless Unknown bacteria Step 4 G- Counterstain withsafranin for 1–2 min Result:Gram-positive(G+) cells are purple;gram-negative (G-) cellsare pink to red G+

13. Gram Staining Gram positive (S. aureus) Gram negative (E. coli) Gram positive (Streptococcus) Gram negative (E. coli)

14. 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

15. 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.

16. 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)

17. Scanning Electron Microscopy – SEM Glutaraldehyde-fixed, critical point-dried, gold-paladium coated

18. 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

19. Eukaryotic cell Freeze-etched preparation Carbon-coated, Gold-shaded, TEM image

20. TEM’s of sectioned cells from each of the domains of living organisms Cytoplasmicmembrane Nucleus Mitochondrion Cell wall

21. 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

22. 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

23. 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

24. 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

25. 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

26. Phylogenetic Analyses of Natural Microbial Communities • Microbiologists believe that we have cultured only a small fraction of the ArchaeaandBacteria • 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

27. 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

28. 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

29. 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

30. Cell Structure and Funtion Chapter 3 • (in Brock Biology of Microorganisms 2012)

31. 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

32. CARBOXYL ESTER ETHER ALDEHYDE PHOSPHO-ESTER ACID ANHYDRIDE ALCOHOL THIOESTER KETO PHOSPHO ANHYDRIDE

33. 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

34. 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.

35. 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.

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

37. 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.

38. 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.

39. 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.

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

41. 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.

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

43. ∆G0' versus ∆Gstandard conditions pH 7, 25°C

44. 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

45. EnergyG 0’f = free Energy of formationfor elements G 0’f = 0ΔG 0’ = change in free Energy in reactionsΔG 0’ of the reaction: A+B  C+D equalsΔG 0’ [C+D] - ΔG 0’ [A+B]products - reactantsif + , the reaction is ENDERGONICif - , the reaction is EXERGONICΔG 0’ does not affect the rates of reaction

46. 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.

47. Enzyme Biocatalysis • Specific substrate binding • Substrate orientation o active sites • Lowering the activation energy

48. 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.

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

50. REDOX potential 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’.