MCB3020 Exam 1 Study Guide PDF

**MCB3020 Study Guide (Dr. Maksym Bobrovskyy)** **Module 1** **Lecture 1: Introduction and Origin** **1) What is a Microbe?** - - - Fred Sanger - developed the first method of DNA sequencing that was fast enough to sequence large genomes - First sequenced genomes - - - **...

**MCB3020 Study Guide (Dr. Maksym Bobrovskyy)** **Module 1** **Lecture 1: Introduction and Origin** **1) What is a Microbe?** - - - Fred Sanger - developed the first method of DNA sequencing that was fast enough to sequence large genomes - First sequenced genomes - - - **2) Impact of Microbes on Human History** Microbes shaped human history and culture - - - - Florence Nightingale - Recognized the significance of disease in warfare during the Crimean War; more soldiers died of microbial infections than battle wounds - - - Robert Hooke - Built the first compound microscope and published the first manuscript illustrating microbes: Micrographia - Antonie van Leeuwenhoek - Built single-lens magnifiers, complete with sample holder and focus adjustment - - Louis Pasteur - Discovered the microbial basis of fermentation - - - Spontaneous generation - theory that microbes could arise spontaneously, without a parental organism **3) Origins of Medical Microbiology** Robert Koch - Founded the scientific method of microbiology - - [Miasma vs Germ theory] - - Koch's Postulates - criteria for establishing a causative link between an infectious agent and a disease 1. 2. 3. 4. Pure culture - Culture of a singular bacteria species; used to prove a specific bacteria caused a certain disease - - Edward Jenner - discovered that cowpox protects patients from smallpox - Ignaz Semmelweis - ordered doctors to wash their hands with chlorinated lime solution, an antiseptic agent - Alexander Fleming - discovered that Penicillium mold created a substance that kills bacteria - Dmitri Ivanovsky - Discovered that the agent of transmission for tobacco mosaic disease could pass through a Pasteur-Chamberland filter that blocked all known microbes Martinus Beijerinck - Discovered that the agent of transmission was not a bacterium, but rather a contagious living fluid Wendell Stanley - Purified and crystallized the agent of transmission for tobacco mosaic disease Viruses are not cells, rather, viral particles assembled from proteins and nucleic acids - - - - - - **4) Microbial Ecology and Environment** Lithotrophs - organisms that use inorganic materials for biosynthesis - - Sergei Winogradsky - Developed enrichment cultures (the use of a certain growth media to favor the growth of a particular microorganism over others) - Extremophiles - organisms able to live in extreme environments Endosymbionts - microbes living symbiotically inside a larger organism, usually mutualistically - Biofilms - community of microorganisms that stick together, forming a slimy layer **5) The Microbial Family Tree** [Early taxonomists faced two challenges as they attempted to classify microbes:] 1. - 2. - - Ernst Haeckel referred to microbes as neither plant nor animal, but a third kingdom classified as Monera - - - - - Lynn Margulis - modified the five kingdom classification, proposing theory of endosymbiosis - - - Monophyletic - organisms descend from a common ancestor Polyphyletic - organisms do not share common ancestors Carl Woese - Discovered prokaryotes that live in hot springs and produce methane which belong to a unique group: Archaea -  **6) Cell Biology and the Amazing DNA Revolution** Frederick Griffith - discovered transformation in bacteria - **Lecture 2: Discovery and Observation** **1) Observing Microbes**. Resolution - the smallest distance by which two objects can be separated and still be distinguished - - [Microbes differ in size over a range of a few orders of magnitude:] - - -  **2) Optics and Properties of Light** Understand the nature of electromagnetic radiation spectrum and the range of visible light. Electromagnetic radiation is composed of electrical and magnetic waves [perpendicular] to each other - - - For [electromagnetic radiation to resolve an object], certain conditions must be met: - - - Magnification requires the bending of light rays, as in [refraction] - - Parabolic lens - refracts parallel rays such that all of the rays meet at a certain point , called the focal point  Resolution of detail in microscopy is limited by the wave nature of light -  - - - - **3) Light Microscopy** Numerical aperture (NA) - determines magnification - - - Minimum resolution distance (R) - half the wavelength of light (λ/2) and NA - - Compound microscope - a system of multiple lenses designed to correct or compensate for aberration - - - Total magnification - magnification of ocular multiplied by the objective Aberration - defect in an image caused by light rays not focusing properly on the lens, resulting in blurring or distortion [Types of microscopy] Bright-field microscopy - Generates a dark image of an object over a light background - - - Dark-field microscopy - Generates a light image over a dark background - - Phase-contrast microscopy - Uses light to enhance the contrast of a transparent, unstained specimen - - **4) Fixation and Staining for Bright-field microscopy** Wet mount - placing microbes in a drop of water on a slide with a cover slip - - - - - - Fixation - cells are made to adhere to a slide in a fixed position Staining - cells are given a distinct color - - [Types of staining] Simple stain - adds dark color to cells, but not to the surrounding tissue (e.g. methylene blue) Differential stain - uses multiple dyes to differentiate between different type of microorganisms (e.g. gram stain)  Gram stain - used to differentiate between two groups, gram-positive and gram-negative, and identify the type of bacteria present in a sample Acid-fast stain - carbolfuchsin used to stain Mycobacterium species Spore stain - malachite green used to detect spores of Bacillus and Clostridium Negative stain - colors the background, which makes capsules more visible **5) Fluorescence microscopy** Understand how fluorophores work and their properties, and why filters are used in fluorescent microscopy. Fluorescent microscopy - utilizes the physical properties of fluorophores (molecules that fluoresce when excited by light of a certain wavelength) - - - [Color filters...] - -  Fluorophore specificity can be determined by... - - - Fluorescence In Situ Hybridization (FISH) - a technique that uses fluorescent DNA probes that bind specifically to the complementary sequence of a chromosome - Green fluorescent proteins (GFPs) - fluorescent proteins that emit green light when exposed to blue or ultraviolet light - - **6) Electron microscopy (EM)** Electrons behave like light waves - - - [Electron microscopy mechanism:] - - - - - The specimens for electron microscopy can be prepared in several ways: - - - Transmission electron microscopy (TEM) - electrons pass through the specimen, revealing internal structures - - - Scanning electron microscopy (SEM) - electrons scan the specimen surface, revealing external structures in 3D - - Cryo-Electron microscopy - specimen is suspended in water and rapidly frozen in a refrigerant - - Cryo-Electron tomography - images are combined digitally to visualize the entire object in 3D - - Understand the general principle of AFM and X-ray crystallography. Contrast with EM methods. Scanning probe microscopy (SPM) - enables nanoscale observation of cell surfaces - - - [EM vs. AFM] - - - X-ray diffraction analysis - sample is crystallized to fix position of specimen atoms in a symmetrical crystal structure - - - **Lecture 3: Structure and Function** **1) The Bacterial Cell: An Overview** Cytoplasm - consists of a viscous gel-like substance/network Cell membrane - encases the cytoplasm Cell wall - covers the cell membrane Nucleoid - non-membrane-bound area of the cytoplasm that contains the chromosome in the form of looped coils Flagellum - external helical filament whose rotary motor propels the cell [Biochemical and genetic analyses:] Cell fractionation - cells must be broken up by techniques that allow subcellular parts to remain intact - - - - Ultracentrifuge - separates subcellular components - Genetic analysis - utilizes strains with different genotypes - - - Cell membrane - semi permeable phospholipid bilayer that behaves as a 2D fluid - - A phospholipid is composed of a glycerol molecule with two uncharged, hydrophobic fatty acid tails attached by ester linkages, and a polar, negatively charged phosphate head group which can have a variable side group, making it amphipathic (both hydrophilic and hydrophobic) in nature. - Unsaturated fatty acids - [increase] membrane fluidity, improving function at low temps Saturated fatty acids - [decrease] membrane fluidity, improving function at high temps Cyclized fatty acid - fatty acid where the carbon chain forms a ring structure, creating a closed loop instead of a linear chain, causing a [decrease] in membrane fluidity Cardiolipin - double phospholipid linked by a glycerol - - - Hopanoids - pentacyclic lipids that modify membrane fluidity in response to environmental stress - - Bacterial phospholipids - have [ester] linkages connecting their fatty acid chains to glycerol Archaeal phospholipids - have [ether] linkages connecting their isoprenoid chains to glycerol **2) Membrane Molecules and Transport** The cell membrane is semipermeable - - Solutes move along a concentration gradient, from high to low concentration Passive transport - molecules move along their concentration gradient - Active transport - molecules move against their concentration gradient - Know the details of different active transport mechanisms: [Types of active transport mechanisms:] 1. ATP-binding cassette (ABC) - largest family of active transport systems - - i. ii. iii. 2. Phosphotransferase system (PTS) - type of group translocation mechanism - - 3. **3) Cell Envelope** Know the function of the cell wall, its composition and the structure of peptidoglycan (murein): Cell wall - gives the cell rigidity and shape, and helps it withstand turgor pressure - - - - - [How PG synthesis is impacted by Penicillin and Vancomycin:] Penicillin inhibits the transpeptidase that cross-links the peptides Vancomycin prevents cross-bridge formation by binding to the terminal D-Ala-D-Ala dipeptide [Bacterial murein vs. archaeal pseudomurein] Pseudomurein found in Archaea is [functionally] same to bacterial peptidoglycan - - - -  [Gram-Positive Cell Envelope: S-Layer] - - - - - - - [Mycobacterial Cell Envelope] - - - [Gram-Negative Cell Envelope] - - - - - - - Lipopolysaccharide (LPS) - - - Braun\'s lipoprotein (Lpp) tethers the outer membrane to the peptidoglycan (PG) by acting as a covalent link between the two layers; its N-terminal end is anchored in the outer membrane via lipid modifications, while its C-terminal end is covalently attached to a diaminopimelic acid (DAP) residue in the peptidoglycan, effectively holding the two structures together Eukaryotic microbes possess their own structures to avoid osmotic shock - increase in internal pressure due to influx of water into the cell via osmosis - - - - **4) Bacterial Cytoskeleton and Cell Division** Prokaryotes divide by binary fission - 1 mother cell splits to form 2 daughter cells - - - [Cell division proteins and how they contribute to cell shape:] - - - Know general steps of cell division starting with initiation of chromosome replication and ending with daughter cells separation Septation **5) Cell Asymmetry** Polar aging - a process where cells exhibit asymmetry during division, with older cellular components preferentially accumulating at one pole of the cell (considered the \"old pole\"), while the newer components are distributed to the newly formed pole, essentially leading to a \"generational\" difference between the daughter cells during division - Caulobacter crescentus - bacteria that generates two kinds of daughter cells as a result of cell asymmetry (motile and stalk types) **6) Specialized Structures** Flagella - specialized structure used for swimming [Flagella number and location can vary:] - - - - Chemotaxis - the movement of a bacterium in response to chemical gradients - - - - - - - **Lecture 4: Genomes and Replication** **1) DNA: The Genetic Material** Understand Griffith and Avery experiments and how they demonstrated that DNA is the transforming substance Frederick Griffith - discovered that mice could be killed by live harmless bacteria when co-injected with the dead pathogenic bacteria - -  Oswald Avery - demonstrated that DNA, and not protein or RNA, was responsible for the phenomenon observed by Griffith - Hershey-Chase experiment - established that DNA encoded hereditary information Rosalind Franklin - discovered that DNA forms a double helix using x-ray crystallography Watson and Crick - discovered the complementary pairing between bases of DNA and the antiparallel form of the double helix [DNA: Nucleotides] DNA is a polymer of nucleotides - - - - - - [DNA: Base Pairing] Nucleic acid is composed of nucleotides linked via phosphodiester bond between the 5' phosphate group of one deoxyribose and the 3' OH group of another - - - - - [DNA: The Double Helix] Double-helix has a wide major groove and a narrow minor groove determined by the angles of complementary bases - **2) Genome Organization** Understand genome organization, gene structure and transfer mechanisms Genome - all of the genetic information that defines an organism - - - [Microbial genomes:] - - - - - - [Genes can be organized into:] - - [Genes can be transferred:] - -  Understand chromosome organization- nucleoid, NAPs, DNA supercoiling (positive vs negative) Nucleoid - compacted bacterial chromosome; supercoiled DNA-loop domains - Negative supercoiling - DNA is twisted in the opposite direction of the double helix, making it [unwound] - Positive supercoiling - DNA is twisted in the same direction as the double helix, making it [overwound] - Know the mechanisms of how topoisomerases introduce supercoiling, Topo I vs Topo II (DNA Gyrase) Topoisomerase - DNA nuclease enzymes that introduce or remove supercoils, changing the topology of the chromosome Topo I - enzyme that cuts one of the two strands of DNA, relaxes the strand, and reanneals the strand -  Topo II - creates double strand break which creates negative supercoiling - - **3) DNA Replication** Know the mechanism and steps of DNA replication [Steps:] 1. 2. 3. [Key proteins involved in DNA replication:] - - - - - - DNA replication is semiconservative - each new double helix is composed of one parental strand and one newly synthesized strand Bidirectional replication - two replication forks progressing in opposite direction around the chromosome After initiation, the restrictions imposed by DNA polymerases establish two modes of synthesis at each of the two replication forks within the bubble - - - know the enzymes (e.g. DNA Pol III, Helicase etc) and DNA elements (e.g. oriC, ter) involved in the replication process DNA Pol III- 5' to 3' synthesis, phosphodiester bonds  Initiation **4) Plasmids and Secondary Chromosomes** [Plasmids vs. Secondary Chromosomes] Extrachromosomal DNA elements that replicate autonomously from the chromosome: - - [Rolling circle vs bidirectional plasmid replication:] Rolling circle replication - unidirectional; initiated by RepA, encoded by the plasmid gene  Bidirectional plasmid replication - proceeds in two opposite directions from a single origin, replicating both strands simultaneously, resulting in two identical copies of the plasmid DNA