BIOL 3332 Spring 2016
Unit 1/Exam 1 Review (Chapters 1, 3, 6, and part of 4)
A. Chapter 1 Microbial Life: Origin & Discovery
a. Impact of microbiology on our lives
i. Ecology, public health, biotechnology, knowledge of cell biology and genetics
b. Defining what a microbe is: * a microbe is a living organism that requires a microscope to be seen
c. Where did microbes come from? Evolutionary origins, fossil evidence; prokaryotic cells  eukaryotic cells * bacteria is the oldest known life form. Oldest datable geographical evidence was 3.8 bya * eukayotic cells arose from divergent prokaryotic lines. Endosymbioses came from mitochondria and chloroplasts. Unicellular types came from simple multicellular forms metazoans; significance of cyanobacterial ancestors * presence of cyanobacteria-like chains of cells in stomatolite fossils represent growth of cyanobacteria * cyanobacteria photosynthesize like plants; they use H2O to synthesize O2.
d. Microbial taxonomy & phylogeny (3 domains: Archaea, Bacteria, & Eukarya ) * the 3 domains (bacteria, archaea, and eukaryotes) evolved from a common cell * Archaea and bacteria include prokaryotes * Eukarya includes algae, plants, fungi, animals, and protists eukaryotes * Monera includes all 3 domains
i. Taxonomic groupings: microbes in the different kingdoms of those domains ii. Similarities & differences: eukaryotic & prokaryotic cells; genomes iii. Metagenomics
e. Who are the microbes: bacteria/archaea/fungi/protists/viruses; characterize/describe these * Bacteria: cells lacking a nucleus * Fungi: are in eukarya domain. A heterotrophic eukaryote with chitinous cell walls. * Protist: single-celled eukaryotic microbe, usually motile. Not a fungus * Virus: consists of a noncellular particle containing genetic material that takes over the metabolism of a cell to generate more virus particles. We do consider viruses as microbes; they are their own entities but are not unicellular.
f. Historical perspective of microbiology
i. Exploitation of microbial activity for different processes ii. Medical: infectious disease/antibiotics/vaccination iii. Advances in biotechnology, agriculture, food, energy/environment, fighting disease iv. Microscopy (Hooke, van Leeuwenhoek); Spontaneous generation * Robert Hooke: first microscopist to publish a systematic study of the world as seen under a microscope * Built the first compound microscope. Was able to observe biological materials such as nematode “vinegar eels,” mites, etc. * Was the first to observe distinct units of living material, which he called “cells.” * Antonie van Leeuwenhoek: first individual to observe single-celled microbes * “small animals” * Spontaneous generation: the theory that living creatures such as maggots could arise spontaneously, without parental organisms
v. Pasteur’s contributions: including refuting spontaneous generation & Germ Theory of Fermentation * Pasteur reveals the biochemical basis of microbial growth * Discovered the fundamental chemical property of chirality. * Discovered that fermentation (a process by which microbes gain energy by converting sugars into alcohol), is actually caused by living yeast, a single celled fungus. * Pasteur disproved that lack of oxygen was reason for failure of spontaneous generation in Spallanzani’s flasks. Showed providing oxygen did not enable spontaneous generation vi. Robert Koch’s contributions: germ theory of disease, pure culture technique, Koch’s postulates * Germ theory of disease: theory that many diseases are caused by microbes * Koch tried to work with tuberculosis after their experimental logic of chain of infection (transmission of disease, pg 20). Needed to isolate a pure culture of microorganisms, a culture grown from a single “parental” cell. * Koch’s postulates: criteria for establishing a causative link between an infectious agent and disease 1. The microbe is found in all cases of the disease but is absent from healthy individuals 2. The microbe is isolated from the diseased host and grown in pure culture 3. When the microbe is introduced into a healthy, susceptible host, the same disease occurs 4. The same strain of microbe is obtained from the newly diseased host. When cultured, the strain shows the same characteristics as before vii. Principle of immunization & vaccination; Edward Jenner. Your body’s immune response * Edward Jenner (physician), deliberately infected patients with matter from cowpox lesions to prevent from smallpox. The practice of cowpox inoculation was called vaccination * Pasteur was first to recognize significance of attenuation and extend the principle to other pathogens. * Immunization: the stimulation of an immune response by deliberate inoculation with an attenuated pathogen. viii. Application of aseptic technique, antiseptics/disinfectants; discovery of antibiotics * Antiseptic agent: a chemical that kills microbes, such as chlorine * In 20th century, surgeons developed fully aseptic environments for surgery meaning environments completely free of microbes * Antibiotics: killed only microbes, but leaves host unharmed. * Important step in the search for antibiotics was the realization that microbes themselves produce antibiotic compounds. accidental discovery of penicillin by Alexander Fleming ix. Microbial ecology: Winogradsky, Biejerink & enrichment culture; importance of microbes in geochemical cycling; nitrogen cycle * Microbes support natural ecosystems. Winogradsky studied microbes in natural habitats (saw microbes with metabolisms quite alien from human digestion) * i.e. saw bacterium Beggiatoa oxidize H2S to sulfuric acid (H2SO4) * fixes CO2 into biomass without consuming organic food * Organisms that feed solely on inorganic minerals are known as chemolithotrophs, or lithotrophs. * Instead of isolating pure colonies, Winogradsky built a model wetland ecosystem containing regions of enrichment for microbes of diverse metabolism called Winogradsky column. Has a glass tube containing mud. At the top, cyanobacteria conduct photosynthesis, using light energy to split water and produce molecular oxygen. * Nitrogen fixation: bacteria and archaea fix nitrogen (N2) by reducing it to ammonia (NH3), the form of nitrogen assimilated by plants.
g. Microbes & Human Welfare: bioremediation, wastewater treatment, biotechnology
i. Recombinant DNA technology
B. Chapter 3 Prokaryotic Cell Structure & Function
a. The prokaryotic cell and its structures ‐ model of a bacterial cell * Cell membrane: contains the cytoplasm * In gram negative bacteria, cell membrane is called the inner membrane to distinguish from outer membrane * Cell wall: between inner and outer membranes. Is a cage like structure composed of polysaccharides linked covalently by peptides (peptidoglycan) * Limits the expansion of the cytoplasm, keeps in tact the cell membrane when water flows in * Envelope: constituted by the cell wall and outer membrane * Flagellum: external structure. Is a helical protein filament whose rotary motor propels the cell in search of a more favorable environment. * Nucleoid: chromosome organized within thee cytoplasm as a system of looped coils * Not bound by membrane loops of DNA from nucleoid are transcribed by RNA polymerase to form mRNA, rRNA, and tRNA. As mRNA transcripts grow, they bind ribosomes to start synthesizing polypeptide chains
b. Features all cells have in common; basic differences btwn prokaryotes vs. eukaryotes * All cells share: * Water * Essential ions (K, Mg, and Cl) * Small organic molecules (lipids and sugars provide nutrition) * Macromolecules (nucleic acids and proteins that contain information, catalyze reactions, and mediate transport)
c. Cell membrane structure: phospholipids & proteins; hopanoids; mycobacterial & archaeal membrane structure * Hopanoids: add strength to membranes. They fit between fatty acid side chains of membranes and limit their motion, thus stiffening membrane *
i. Functions of cell membrane proteins * Structural support: anchor together different layers of the cell envelope. Other proteins attach the membrane to the cytoskeleton, or form the base of structures extending out from the cell, like flagella. * Detection of environmental signals: membrane protein ToxR detects acidity and elevated temperature- signs in the host digestive tract * Secretion of virulence factors and communication signals: membrane protein complexes export toxins and cell signals across envelope. i.e. symbiotic nitogen-fixiing rhizobia require membrane proteins NodI and NodJ to transport nodulation signals out to host plant roots * Ion transport and energy storage: transport proteins manage ion influx between cell and exterior, and store energy in ion gradients
d. Transport: passive (simple & facilitated diffusion), active transport ; osmosis; membrane‐permeant acids; ion gradients * Passive diffusion: small unchanged molecules (O2, CO2, H2O) easily permeate the membrane. Molecules like ethanol, also disrupt the membrane, toxic to cells. Large polar molecules such as sugars, and charged molecules like amino acids generally cannot penetrate the hydrophobic interior of the membrane (require transport by specific proteins) * Use of transport proteins facilitated diffusion * With an ion gradient, can use to transport to another molecule * Active transport: often in conjugation with hydrogens * Aquaporins: are water specific transport proteins * Osmosis: because water can cross the membrane but charged solutes cannot, water tends to diffuse across the membrane into the cell, causing the expansion of cell volume, in this process * Resulting pressure is osmotic pressure can cause a celle to burst in the absence of a countering pressure provided by the cell wall. That is how bacteria are killed by penicillin, which disrupts cell wall synthesis. * Membrane- permeant weak acids and bases: exist in equilibrium beween charged and undcharged forms * Weak acid: HA H+ +A- * Weak base: B + H2O BH+ + OH- * Ion gradients: an ion gradient (ratio of ion concentrations) across the cell membrane can store energy for nutrition, or to drive the transport of other molecules * Inorganic ions require transport through specific transport proteins. * In passive transport, molecules accumulate or dissipate along their concentration gradient. * Active transport: transport from low concentration to high concentration, requires cells to spend energy
e. Cytoplasm/nucleoid/plasmids: transcription & translation (polysome formation); DNA replication (replisomes; bidirectional; septation)
f. Bacterial cell wall structure ‐ know/understand these:
i. Peptidoglycan layer: structure * Most bacterial cell walls are composed of peptidoglycan, a polymer of peptide-linked chains of amino sugars. The molecule consists of parallel polymers of disaccharides called glycan chains cross linked with peptides of 4 amino acids ii. Gram positive cell envelope (teichoic acids, S layer) * Teichoic acids reinforce the multiple layers of peptidoglycan in the membrane of gram positive cells * Are chains of phosphodiester- linked glycerol or ribitol, with sugars or amino acid linked to middle OH groups. * The negatively charged cross-threads of teichoic acids help retain gram stain * S layer: is the surface layer, found on the outside of cell walls. Is a crystalline layer of thick subunits consisting of protein or glycoprotein * each of its subunits contains a pore large enough to admit a wide range of molecules and form a smooth layer on cell wall or outer membrane. Is rigid, but also flexes and allows substances to pass through it. iii. Mycobacterial cell envelope: mycolic acids * mycolic acids: contain a hydroxyl acid backbone with 2 hydrocarbon chains. * Mycobacterial cell wall includes peptidoglycan linked to chains of galactose, called galactans * Mycolic acids provide the basis for acid-fast staining. * Picture found on pg 96

iv. Archaea: pseudomurein cell wall; ether linked membrane lipids (terpenoids)
v. Gram negative cell envelope
1. Murein lipoprotein; diaminopimelic acid * Murein lipoprotein consists of a protein with an N terminal cysteine attached to 3 fatty acid side chains * What happens to a mutant cell that fails to make murein lipoprotein? As the cell grows and divides, it fails to attach its outer membrane to the growing cell wall, causing the outer membrane to balloon out in the region where daughter cells separate
2. LPS layer structure; O polysaccharide * Main outward facing phospholipids are called lipopolysaccharides. The LPS lipids have shorter fatty acid chains than those of the inner cell membrane, and some are branched. LPS is of crucial importance because it acts as an endotoxin a cell component that is harmless as long as the pathogen stays intact; but when released by a lysed cell, endotoxin overstimulates host defenses, inducing potentially lethal endotoxin shock. * O polysaccharide: a chain of as many as 200 sugars. May extend longer than the cell itself. These chains form a layer that helps bacteria resist phagocytosis by white blood cells.
3. Outer membrane proteins, periplasmic space * Outer membranes contain a class of transporters called porins, which permit the entry of nutrients such as sugars and peptides. * Outer membrane porins may lack specificity, allowing uptake of various molecules- including antibiotics such as ampicillin. form of penicillin which must get through outer membrane to access the cell wall in order to block the formation of peptide cross bridges. * Porin called OmpF * Periplasm: the region between the inner and outer membrane of gram negative cells, including cell wall, defines a separate membrane enclosed compartment of the cell known as the periplasm. * Periplasmic proteins are subjected to fluctuations of in pH and salt concentration, because the outer membrane is porous to ions. Some help refold proteins unfolded by oxidizing agents or by acidification.
g. Bacterial cytoskeleton: what is its function? Examples: FtsZ, MreB, CreS proteins * Bacterial cytoskeletal proteis were discovered through gene defects that confer loss of cell shape, and by fluorescent labeling of the corresponding gene products in wild type cells. * FtsZ: polymerizes around the circumference of cell division plate and forms a Z ring. Z ring determines cell diameter. * MreB: localizes in arc-shaped patches, just beneath cell membrane of the rod shaped cell. Arcs of MreB driven by elongating strands of the cell wall * CreS: polymerizes along the inner curve of the crescent.
h. Specialized structures:
i. thylakoids; carboxysomes, gas vacuoles * Thylakoids conduct only the “light reactions” of photon absorption and energy storage. The energy obtained is rapidly spent to fix carbon dioxide- a process that occurs within carboxysomes. * Carboxysomes are protein covered bodies packed with the enzyme Rubisco for CO2 fixation * Gas vesicles used to increase buoyancy and keep the cell high in the water column, near the sun. Gas vesicles trap and collect gases such as hydrogen or CO2 produced by the cell’s metabolism ii. various type storage granules: metachromatic granules, starch, glycogen, PHB, sulfur granules * During times of starvation, phototrophs may digest their thylakoids for energy and a source of nitrogen * Cell may also digest enery rich materials from storage granules composed of glycogen or other polymers, such as PHB and PHA. * Another storage device is sulfur- granules of elemental sulfur.
i. Magnetosomes * Are microscopic membrane-embedded crystals of the magnetic mineral, magnetite, Fe3O4. Found in anaerobic pond-dwelling organisms * Can generate a magnetic dipole moment along length of bacterium, constraining it to swim along a magnetic field. This magnetic orientation of swimming is called magnetotaxis (ability to sense and respond to magnetism)
j. Pili/fimbriae; stalk; flagellum & mechanism of its action; chemotaxis; flagellar arrangements * Pili: also called fimbrae, is an adherence structure. * Stalk: another attachment organelle is a membrane embed extension of the cytoplasm. Tip of the stalk secretes adhesion factors called holdfasts, which firmly attach bacterium in an environment that is favorable. * Rotary flagella: helical propellers that drive the cell forward like the motor of a boat. * Chemotaxis: the attractants and repellents are detected by a polar array of chemoreceptors. Requires a mechanism for the rotary flagella to propel the cell toward attractants or away from repellents. Accomplished by flagellar rotation either clockwise or counterclockwise relative to cell. * When the cell is swimming toward an attractant chemical, the flagella rotates counterclockwise (CCW) * When the cell veers away from the attractant, receptors send a signal that allows one or more flagella to switch rotation clockwise (CW) against the twist of the helix * The switch in directions of rotation disrupts the bundle of flagella, causing the cell to tumble briefly, ending up pointed in a random direction.
k. External to cell envelope: slime layer, capsule
C. Chapter 6 Viruses, Viroids, & Prions
a. What is a virus? Describe/define a virus ‐ virion; acellular; phages, animal viruses * A virus is a noncellular particle that infects a host cell, where it reproduces * The virus particle, or virion, consists of an infective nucleic acid (either DNA or RNA) contained within a protective shell made of protein, called a capsid.
b. Viral infectivity ‐ host range; specificity; what is the basis for this?
c. Viral genomes
d. Describe virus structure: capsid; symmetrical (icosahedral, filamentous) vs. asymmetrical
i. envelope/glycoprotein spikes; tegument
e. Classification: ICTV & Baltimore classification (genome type & route to express mRNA; groups I‐VII)
i. RNA viruses: (+) ss RNA, (‐) ssRNA, dsRNA  have RNA dependent RNA polymerase ii. Retroviruses [(+) ss RNA]: reverse transcriptase
f. Viral life cycles: steps of any viral life cycle
i. Bacteriophage cycles: lytic (virulent) & lysogenic (temperate)  be able to describe/characterize
1. Phage exit from host: lysis or slow release (filamentous phages)
2. Bacterial host defenses: genetic resistance; CRISPR; restriction enzymes ii. Animal virus cycles: tropism; uncoating mechanisms
1. DNA virus v. RNA virus v. Retrovirus
a. RNA‐dependent RNA polymerase; reverse transcriptase
2. Envelope protein insertion; release of progeny (lysis, budding)
3. Life cycle examples: HPV (DNA virus); Picornavirus (RNA); HIV retrovirus (RNA)
4. Animal/Plant host defenses
g. Describe/characterize viroids and prions  what are these?
i. Viroids: infectious RNA; lack protein capsid; are not considered viruses ii. Prions: infectious protein
D. Chapter 4 Bacterial Culture, Growth, & Development
a. Microbial Growth
i. purpose/importance of studying growth; requirements for growth (macro‐. micro‐nutrients, growth factors); essential nutrients * Importance of understanding microbial growth: 1. Optimizing growth achieve high cell density 2. Biochemical analysis 3. Determining growth rates 4. Controlling growth * Essential nutrients: those compounds a microbe cannot itself make but must gather from its environment if the cell is to grow and divide * Required macronutrients: CHONPS. (carbon, nitrogen, phosphorus, hydrogen, oxygen, and sulfur), which make up the carbohydrates, lipids, nucleic acids, and proteins of the cell. * 4 other macronutrients are Mg2+, Fe2+, K+, and Ca2+ * All cells require very small amounts of certain trace elements called micronutrients * Co, Cu, Mn, Mo, Ni, Zn. ii. Prototroph/auxotroph/axenic growth * Nutritional types: * Prototroph: wild-type (one that is first discovered) * Auxotroph: is a mutant; generally deficient in formation of a nutrient (often in the context of an amino acid…for example, alanine auxotroph cannot synthesize alanine) * Axenic growth: growth outside a host cell pure culture iii. Growth Media:
1. Complex v. Defined (minimal) media – what is each & how are they different? Semi‐defined media;
Fastidious microbe * Defined minimal medium contains only those compounds needed for an organism to grow; the chemical composition is known; cells must synthesize all required molecules * Fastidious bacteria: enriched media have a long list of items they need to grow, because they can’t synthesize everything.
2. Selective and differential media * Selective media employs components to inhibit certain bacterial types over others * Differential media allows for visualization of metabolic differences between types.
b. Classifying microbes by carbon & energy sources: heterotroph, autotroph, chemotroph, phototroph, chemoheterotroph (chemoorganotroph), photoheteroroph, chemoautotroph (chemolithotroph), photoautotroph  how are each of these defined? Carbon source/energy source/electron source * Heterotroph: rely on other organisms to form the organic compounds (like glucose) that they use as carbon sources * Autotroph: assimilate CO2 as a carbon source, reducing it (adding H atoms) to make complex constituents made up of C, H, and O. * Are classified as photoautotrophs or chemoautotrophs by how they obtain energy * Photoautotroph: use light for photosynthesis * Chemoautotrophs (also known as chemolithotrophs or lithotrophs) gain energy by oxidizing inorganic substances such as iron or ammonia. Both photoautotrophs and chemoautotrophs carry out the autotrophic process of CO2 fixation, which forms part of the carbon cycle between autotrophs and heterotrophs.
c. Culturing Bacteria:
i. Growth on rich v. minimal media; enrichment culture;