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Robert Hooke

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Examined cork slices through his rudimentary microscope and described "cella."

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Anton van Leeuwenhoek

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Produced a microscope with 300X magnification, which allowed him to visualize for the first time "animalcules."

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Francesco Redi disputes Spontaneous Generation

answer

In the open jar, maggots appeared on the meat. In the covered jaw, no maggots appeared.

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John Needham's experiment that disputed Redi's claim

answer

He boiled tubes of chicken broth for a few minutes, each sealed with corks.

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Lazzaro Spallanzani's experiment that disputed Needham's work

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He boiled seeds and water in several sealed flasks for 45 minutes. Some of the flasks were left open to the air, while others were covered loosely with cork stoppers.

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He put spontaneous generation to rest

answer

Louis Pasteur

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Agostini Bassi

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Showed that a disease of silkworms was caused by a fungus

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MJ Berkeley

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Demonstrated that the great Potato Blight of Ireland was caused by a water mold

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Heinrich de Bary

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Showed that smut and rust fungi caused cereal crop diseases

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Joseph Lister

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Provided indirect evidence that microorganisms were the causal agents of disease (Germ Theory of Disease), developed a system of antiseptic surgery designed to prevent microorganisms from entering wounds as well as methods for treating instruments and surgical dressing, his patients has fewer postoperative infections. Instructed carboxylic acid to clean instruments and even hands.

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Louis Pasteur

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Demonstrated protection against cholera disease in chickens. Developed protection for anthrax. Showed that the pebrine disease of silk worms was caused by a protozoan. Proposes that germs cause infectious disease = germ theory of disease

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Robert Koch

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Formalizes standards to identify germs causing infectious diseases; anthrax & tuberculosis. Proof of the germ theory. Development was Koch's Postulates.

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Koch's laboratory developments

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Development of solid growth media (agar). Pure cultures made disease ID possible

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Demonstration of Koch's Postulates

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Took healthy host and injected with microorganisms of dead animals.

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Edward Jenner

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Used a vaccination procedure to protect individuals from smallpox. This preceded the work establishing the role of microorganismal disease.

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Pasteur ; Emile Roux

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Discovered that incubation of cultures for long intervals between transfers caused pathogens to lose their ability to cause disease.

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Pasteur ; how coworkers (1885)

answer

Developed vaccines for chicken cholera, anthrax ; rabies

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Emil von Behring and Shibasaburo Kitasato

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Developed antitoxins for microbial toxins, diphtheria and tetanus. Evidence for humoral immunity.

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Elie Metchnikoff

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Discovered bacteria-engulfing, phagocytis cells in the blood. Evidence for cellular immunity.;

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Louis Pasteur

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Demonstrated that alcohol fermentations and other fermentations were the result of microbial activity. Developed the process of pasteurization to preserve wine during storage.

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Sergei Winogradsky ; Martinus Beijerinck

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Studied soil microorganisms and discovered numerous intersting metabolic processes (nitogren fixation). Pioneered the use of enrichment cultures and selective media.

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Modern light microscopes

answer

Allow us to visualize "animalcules" that are micrometers in length.

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Electron microscopy

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Can visualize objects that are nanometers in length.

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Challenges of microbiology

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Antimicrobial resistance, emerging ; re-emerging infectious diseases, bioterrorism.

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Pathogen

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Disease-causing microorganism

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Light microscopy

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Uses visible light to resolve objects

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Light properties

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Large wavelengths have low energy and low resolution. Light energy increases the resolution.

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Phase-contrast microscopy

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Objective lenses list the contrast on the lens.

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Dark-field microscopy

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Dark background, light given off by the specimen.

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Fluoescence microscopy

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Some specimens can absorb or emit light. Using UV light source.

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Differential Interference Contrast (DIC)

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Two beams of light passing through a specimen. Illuminates unstained, live organisms with color and 3D detail.

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Special Stain

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Staining procedure illuminates structures (flagella, capsules).

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Differential Stain

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Bacteria can be distinguished by dyes. Gram-stain, acid-fast, and endospores are examples.

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Gram-stain

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Essential first step in bacterial ID. Bacteria are classified into one of two categories based upon their finished product to these reactions.

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Electron Microscopy

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Provides detailed images of cells and cell parts. TEM ; SEM.

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Transmission Electron Microscope (TEM)

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Shows structures and organelles.

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Scanning Electron Microscopy (SEM)

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Shows surfaces.

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Confocal Scanning Laser Microscopy

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Laser illuminates 3D structure lost be traditional light microscopes.

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Scanning Probe Microscopy

answer

Scanning tunneling and atomic force are two examples. Both new microscopy instruments use a probe to scan the surface of the specimen. Can achieve molecular resolution.

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Shapes of Prokaryotes

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Cocci - round, helical - spiral ; bacilli - rod.

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Chains.

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"Strepto-"

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Clusters

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"Staph-"

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Pleomorphic

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Organisms that are variable in shape

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The Cell Membrane of Prokaryotes

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AKA the inner/plasma/cytoplasmic membrane; located at the boundary of cell's internal compartment. Selectively permeable barrier. Some molecules are allowed to pass into or out of the cell. Transport systems aid in movement of molecules. Location of crucial metabolic processes. Detection of and reponses to chemicals in surroundings with the aid of special receptor molecules in the membrane.

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Plasma Membrane Composition

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Amphipathic - lipid bilayer in which proteins float. Polar ends - interact with water, hydrophilic; nonpolar ends - insoluble to water, hydrophobic.

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Peripheral proteins

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Loosely associated with the membrane and easily removed

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Integral Proteins

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Embedded within the membrane and not easily removed

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Hopanoids, sterol-like molecules

answer

Eukaryotes contain sterols, like cholesterol

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Plasma membrane infoldings

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Observed in many photosynthetic bacteria and in prokaryotes with high respiratory activity

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The Cell Cytoplasm

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Contains internal structures (nucleiod, ribosomes, and inclusion bodies). Lacks organelles bound by unit membranes. Composed largely of water. Is a mjor part of the protoplasm (the plasma membrane + everything within).

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Nucleoid

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The nucleoid represents a subcompartment containing the DNA chromosome. Usually a closed, circular, double-stranded DNA molecule. Usually one DNA molecule per cell. Looped and coiled extensively, nucleoid proteins probably aid in folding.

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Plasmids

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USually small, closed circular DNA molecules. Exist and replicate independently of chromosome. Have relatively few genes present (genes on plasmids are not essential to host but may confer selective advantage).

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Classification of plasmids

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Based on their mode of existence, spread & function

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Ribosomes

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Complex structures consisting of protein and RNA. Prokaryotic ribosomes - 70S. Sites of protein synthesis.

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Cytoskeleton

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Functions include roles in cell division, protein localization and determination of cell shape

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Inclusion Bodies

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Granules of organic or inorganic material that are stockpiled by the cell for future use (glycogen, poly-B-hydroxybutyrate, polyphosphate, sulfur). Some are enclosed by a single-layered membrane.

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Gas vesicles

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Aquatic bacteria use these to float on the water's surface.

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Magnetosomes

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Contain crystals of magnetite or greigite, allowing cells to respond to magnetic fields

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Functions of Cell Wall

answer

Provices characteristic shape to cell, protects the cell from osmotic lysis, may also contribute to pathogenicity.

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Osmotic Lysis

answer

Can occur when cells are in hypotonic solutions. Movement of water into cell causes swelling and lysis due to osmotic pressure.

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Chemical Lysis

answer

Lysozyme breaks the bond between N-acetylglucosamine and N-acetylmuramic acid. Penicillin inhibits peptidoglycan synthesis.

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Gram-positive bacteria cell walls

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Cell walls composed primarily of peptidoglycan (meshlike polymer composed of identical subunits of N-acetylglucosamine and N-acetylmuramic acid and several different amino acids). Contain large amounts of teichoic acids. Some have a layer of proteins on surface of peptidoglycan.

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Gram-negative cell walls

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Thicker periplasmic space. Thinner peptidoglycan within the periplasmic space. More enzymes in PS and coming into. Outer membrane composed of lipopolysaccharies.

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Adhesion Sites

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Sites of direct contact (possibly true memrbane fusions) between plasma membrane and outer membrane. Substances may move directly into cell through these.

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Lipoploysaccharides (LPS)

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Protection from host defenses (O antigen). Contributes to negative charge on cell surface (core polysaccharide). Helps stabilize outer membrane structure (Lipid A) to form a permeability barrier. Can act as an endotoxin (Lipid A).

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Outer membrane of bacteria

answer

More permable than plasma membrane due to presence of porin proteins and transporter proteins.;

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Porin proteins

answer

Form channels through which small molecules can pass.

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Gram-positive cell walls

answer

Less selectivity, don't diffuse as easily, more sensitive to antibiotics.

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Gram-negative cell walls

answer

Stronger, more selective, LPS produces TSS, more vulnerable.

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Capsules

answer

Layer of material laying outside of the cell wall. Usually composed of polysaccharides, well organized and not easily removed from cell.

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Slime layers

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Layers of material laying outside the cell wall. Similar to capsules except diffuse, unorganized and esily removed. Thinner & roped around the organism.

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S-layers

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Layer of material lying outside the cell wall. Regularly structured layer of protein or glycoprotein.

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Glycocalyx

answer

Capsules, slime layers & S-layers

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Functions of glycocalyx

answer

Protection from host defenses (phagocytosis), protection from harsh environmental conditions, attachment to surface. Protection from viral infections or predation by bacteria. Protection from chemicals in environment. Facilitate motility of gliding bacteria. Protection against osmotic lysis (S-layers).

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Functions of the Cell Wall

answer

Provides characteristic shape of cell, protects the cell from osmotic lysis, may also contribute to pathogenicity.

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Periplasmic Space

answer

Lies between the plasma membrane and cell wall. More pronounced in gram-negative bacteria. It may constitute 20-40% of the cell volume with many enzymes.

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Exoenzymes

answer

Enzymes secreted by bacteria through the periplasm.

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Gram-negative cell walls

answer

Outer membrane lies outside the thin peptidoglycan layer. Braun's lipoproteins connect outer membrane peptidoglycan.

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Spirochete motility

answer

Exhibit flexing and spinning movements of axial filaments hiwch are composed of periplasmic flagella

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Twitching motility

answer

Short, intermittent jerky motions using fimbriae. Occurs when cells are in contact with each other.

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Gliding Motility

answer

Cells coast along solid surfaces. Variety of mechanisms exists to propel cells. This is movement used mostly by stationary organisms.

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Chemotaxis

answer

Movement towards a chemical attractant of away from a chemical repellent. Concentrations of chemical attractants and chemical repellents detected by chemoreceptors in the surfaces of cells.

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Bacterial endospores

answer

Formed by some bacteria, usually gram-positive. They are dormant and highly resistant to numerous environmental conditions (heat, radiation, chemicals, desiccation).

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What makes an endospore so resistant?

answer

Calcium complexed with dpipcolinic acid, small acid-soluble DNA-binding proteins (SASPs), dehydrated core, spore coat, and DNA repair enzymes.

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Sporogenesis

answer

Also called endospore formation of sporulation. Normally commences when growth ceases because of lack of nutrients. Complex multistage process.

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Steps for a dormant spore to transform to an active vegetative cell...

answer

Activation (prepare spores for germination, often results from treatments like heating), germination (spore swelling, rupture or absorption of spore coat, loss of resistance to heat and other stresses, increased metabolic activity), and outgrowth (emergence of vegetative cell).

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Fimbriae

answer

Short, thin, hair-like proteinaceous appendages. Up to 1,000 per cell. Mediate attachment to surfaces. Some (type IV) required for twitching motility or gliding motility that occurs in some bacteria.

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Sex pili

answer

Similar to fimbriae except longer, thicker, and less numerous (1-10 per cell). Required for mating.

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Speciaized pili

answer

Conjugation pii - aid in transfer of DNA between bacteria.

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Flagella

answer

Long appendages extending from the cell surface used for motility. Contain helical filament, a hook, and a basal body.

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Monotrichous

answer

One flagella

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Polar

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Flagellun at end of the cell

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Amphitrichous

answer

One flagellum at each end of cell

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Lophotrichous

answer

Cluster of flagella at one or both ends

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Peritrichous

answer

Flagellum spread over the entire surface of the cell

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Flagellar movement

answer

It rotates like a propeller -- in general, counterclockwise rotation causes forward motion (run) and clockwise rotation disrupts run and causes a tumble

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Examples of eukaryotes

answer

Protozoa, fungi, parasitic worms

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Eukaryotic cell structure

answer

Membrane-delimited nuclei, membrane-bound organelles that perform specific functions, more structurally complex than a prokaryotic cell, generally larger than a prokaryotic cell

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Eukaryotc membranes

answer

The fluid mosaic model is based on eukaryotic membranes. Major membrane lipids include phosphoglycerides, sphingolipis, and cholesterol. Contains microdomains called lipid rafts.

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Lipid rafts

answer

Enriched for certain lipids and proteins. They participate in a variety of cell processes such as cell movement and transduction.

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Eukaryotic cytoplasm

answer

The many organelles of eukaryotic cells lie in the cytoplasmic matrix.

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Eukaryotic cytoskeleton

answer

Vast network of interconnected filaments, within the cytoplasmic matrix (microfilaments, microtubules and intermediate filaments).

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Cytoskeletal filaments role

answer

In cell shape and cell movement

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Microfilaments

answer

Minute protein filaments, 4 to 7 nm in diameter. They are composed of actin protein. They are scattered within cytoplasmic matrix or organized into networks and parallel arrays. They are involved in cell motion and shape changes.

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Microtubules

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Shaped like thin cylinders, around 25 nm in diameter. They are composed of alpha- and beta-tubulin. They help maintain cell shape. They are involved with microfilaments in cell movements. They participate in intracellular transport processes. They are more concerned with structure and shape and transport of molecules to the nucleus.

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Intermediate filaments

answer

Heterogenous elements on the cytoskeleton. They are around 10 nm in diameter. Their role in the cell is unclear. Studies in animals have shown that some form a nuclear lamina and others help link cells together to form tissues.

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The endoplasmic reticulum

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Irregular network of branching and fusing membranous tubules and flattened sacs, called cisternae. Smooth or rough. They transport proteins, lipids, and other materials within the cell. It is the major site of synthesis of some proteins that it transports. It is the major site of cell membrane synthesis.

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Rough endoplasmic reticulum

answer

Ribosomes are attached, and the synthesis of secreted proteins by the endoplasmic reticulum-associated ribosomes.

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Smooth endoplasmic reticulum

answer

Devoid of ribosomes, and the synthesis of some proteins that it transports

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The Golgi apparatus

answer

Membranous organelle made of cisternae stacked on each other. Involved in modification, packaging, and secretion of materials.

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Dictyosomes

answer

stacks of cisternae

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Lysosomes

answer

Involved in intracellular digestion. Contains hydrolases, enzymes which hydrolyze molecules and function best under slightly acidic conditions. They maintain an acidic environment by pumping protons into their interior.

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The Nucleus

answer

Membrane-bound structure that houses genetic material of eukaryotic cell

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Nuclear structure

answer

Chromatin and nuclear envelope

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Chromatin

answer

Dense fibrous material within the nucleus. It contains DNA and it condenses to form chromosomes during cell division.

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Nuclear envelope

answer

Double membrane structure that delimits nucleus. It is penetrated by nuclear pores. These pores allow materials to be transported into and out of nucleus.

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The Nucleolus

answer

Not membrane-enclosed. Important in ribosome synthesis -- it directs the synthesis and processing of rRNA, and it directs assembly of rRNA and ribosomal proteins to form ribosomes.

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Eukaryotic ribosomes

answer

80S in size - 60S + 40S subunits
They may be attached to ER or free in cytoplasmic matrix. Proteins made on ribosomes of ER are often secreted or inserted into ER membrane as integral membrane proteins. Free ribosomes synthesize nonsecretory and nonmembrane proteins (some proteins are inserted into organelles).

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Mitochondria

answer

Site of tricarboxylic acid cycle activity (TCA). Site where ATP is genreated by electron transport and oxidative phosphorylation.

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Mitochondrial structure

answer

Outer membrane, inner membrane and matrix

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Mitochondrial outer membrane

answer

Contains porins similar to the outer membrane of gram-negative bacteria.

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Mitochondrial inner membrane

answer

Highly folded to form cristae. The location of enzymes and electron carriers for electron and oxidative phosphorylation = energy production.

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Mitochondrial matrix

answer

Contains ribosomes, mitochondrial DNA, and large calcium phosphate granules. Contains enzymes of the tricarboxylic acid cycle and enzymes involved in catabolism of fatty acids.

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Chloroplasts

answer

Type of plastid - pigment-containing organelles observed in plants and algae. They are the site of photosynthetic reactions. The have stroma.

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Stroma

answer

Contains DNA, ribosomes, lipid droplets, starch granules, and thylakoids.

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Thylakoids

answer

Site of light reactions (trapping of light energy to generate ATP, NADPH, and oxygen).

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External eukaryotic cell covering

answer

Cell wall and pellicle.

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External eukayotic cell wall

answer

Rigid variable make-up with algae (cellulose and pectin), diatomes (silica) and fungi (chitin, cellulose, and glucan).

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Pellicle

answer

Relatively rigid layer of components just beneath the plasma membrane. They are common in protozoe. Tey are not as strong or rigid as cell wall. They provide characteristic shape to cell.

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Cilia and Flagella in Eukaryotes

answer

Cilia are 5-20 micrometeres long and beat with two phases (working like oars). Flagella is 100-200 micrometers long and they move in an undulationg fashion.

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Tensile structures

answer

Flagellum with cilia.

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Molecular unity of Prokaryotes and Eukaryotes

answer

Same basic chemical composition, same genetic code and same metabolic processes.

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Protists

answer

Contractile, secretory, and food vacuoles exist in the cytoplasm (divided into endo- and ectoplasm). The cytoplasm is surrounded by plasmalemma. Food is captured by a cytosome, dugested, and expelled by the cytoproct. Mobile - cilia/flagella.

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Plant morpholoy

answer

Pyrenoids synthesize and store starch; often associated with chloroplast of photosynthetic protists. Hydrogensomes release molecular H2 as a by-product of energy generation under anaerboic conditions.

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Protozoa

answer

Exist in two forms; feeding (trophozoite) and dormant (cyst)

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Dormant protozoa

answer

Protects again environmental changes, site of nuclear reorganization and cell division, means of heat transfer, stay for long periods of time, helps prevent desiccation, more stable and resistant forms.

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Protozoans reproduction

answer

Some can reproduce in both of the folling ways; asexually (binary fission - genetic idential progeny from replicated genomic material) or sexually (sexual conjugation - genetically variable progeny).

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Fungi Morphology

answer

Two cell body types (thallus) - Yeast (unicellular oval) and mold (multicellular filamentous hyphae). They possess nuecli and organelles. Their cell walls are made of chitin, but contain things other than cithin.

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Fungal growth

answer

Absorb nutrients; mycelia increases SA (saprobes). Optimum temperature is room temp (25oC); human pathogens (mycoses) thrive at body temperature (37oC); psychrophili fungus can grow in your fridge (4oC). They are dimorphic (two phases), prefer mildly acidic conditions and live in mutualism with plants.

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Saprobes

answer

Live off of dead animals and plants; secrete enzymes that can digest the substrate.

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Zygomycota

answer

Reproduces with a sporangiospore, sexually and asexually and are common molds.

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Ascomycota

answer

Reproduce by an arthrospore, sexually & asexually and are yeasts, lichens, and molds.

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Basidiomycota

answer

Reproduce by a conidiospore, seuxally & asexually and are edible and poisonous mushrooms, smut and rust.

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Deuteromycota

answer

They have imperfect reproductive structures, are strictly asexual and consist of mycotic or pathogenic fungi.

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Yeast reproduction

answer

large, oval-shaped; form bacteria-like smooth colonies on laboratory agar. They can be dimorphicl exist in oval form of hyphae form. They can reproduce sexually and asexually or asexually only by budding. Saccaromyces species most common -- baker's yeast; alcohol production.

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General properties of viruses

answer

Acellular, infectious agents. Virion - complete virus particle. Consists of more than one molecule of DNA or RNA enclosed in a coat of protein. It may even have additional layers. They cannot reproduce independent of livign cells nor carry out division as prokaryotes and eukaryotes do. They do not metabolize.

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Viral Terminology

answer

Viruses are host-specific & tissue-specific. Those that infect bacteria only are known as bacteriophages or just phages. Those that infect animal cells are just viruses.

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Viral Components

answer

Genome is composed of DNA or RNA. Capsid is composed of protein subunits and are immunogenic. Envelope loosely overs some viruses (dervied from its host; lipids and carbohydrates), and they may have spikes (pelpomers) which may aid in the attachment of viruses to host cells.

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Virion Enzymes

answer

It was first erroneously thought that all viruses lacked enzymes. Not known a variety of virions have enzymes (some associatd with the envelope or capsid but more are within the capsid), and these enzymes do not provide energy or participate in metabolic processes. Enzymes can be associated with the spikes on the envelope.

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Steps of Bacteriophage Replication

answer

Attachment, entry/penetrtion, biosynthesis, assembly, release.

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Animal Viral Entry into Cells

answer

Can occur by fusion with the membrane, endocytosis, or enveloped endocytosis.

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Latent lifestyle

answer

DNA of virus integrates into the host genome and becomes silent. Sometimes, new properties are given to the host of the provirus

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DNA virus/retrovirus

answer

Can activate later and become aggressive. Sometimes they integrate in the same spot, not allowing the cell to shut down division, leading to cancer.

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The choice of viruses

answer

Between the lysogenic and lytic lifestyles.

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The four outcomes of viral infection

answer

Acute infections ("cold" - immedaite symptoms of viru), latent infections, chronic infections (long-term), and transformation into a malignent cell (cancer).

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Viral cultivation

answer

Cultivating methods - chick embryos and cell cultures. Allowed for the creation of some of the first vaccines.

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Cytopathic effect

answer

Microscopic or macroscopic degenerative changes or abnormalities in host cells and tissues (syncytia, Negri bodies).

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Plaque formation

answer

Lysis of bacteria growing confluently on agar by bacteriophage. The bacteria that lyses undergoes the lytic cycle.

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Detection & ID of viral cultures

answer

Cytopathic effects, plaque formation, and electron microscopy, hemagglutination assay

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Hemagglutination Assay

answer

Indirect detection; hemagglutinating titer - determining relative quantities of a virus. More dilution = more virus (beucase you don't have to dilute things that don't need it).

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Viruses and Cancer

answer

Oncoviruses cause caner. Can inactivate genes responsible for suppressing tumor formation (cell cycling, DNA monitor and repair proteins); bring oncogene to healthy cell. Viruses may cause 25% of all cancers.

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Prions

answer

Infectious proteins. Protein only hypothesis - abnormal PrPsc converts normal PrPc of the brain. Mad Cow disease, transmisable spongiform encephalopathies (TSEs), variant Creutzfeldt-Jakob degenerative diseases (sponge-like holes in brain tissue; loss of coordination appetite, dementia).

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Human Prion Disease

answer

Creutzfeldt-Jakob Disease (CJD) and variant Creuzfeldt-Jakob disease (vCJD), Gertsmann-Straussler-Scheinker syndrome, fatal familial insomnia, and Kuru

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Animal Prion Disease

answer

Bovine spongiform encephalopathy (BSE), Chronic wasting disease (CWD), scrapie, transmissable mink encephalopathy, feline spongiform encephalopahty, and ungulate psongiform encephalopathy.

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Production of new viruses

answer

Genetic recombination, viral mutations, environment, social conditions, specie juming, increasing deforestation, agricultural practices, and increasing animal populations

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Macroelements

answer

Those required by organisms in large amounts: C O H N S P K Ca Mg and Fe.

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Micronutrients

answer

Those required by organisms in minute amounts. They are often supplied in water or in media components. Mn Zn Co Mo Ni and Cu

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Role of electrons

answer

1. energy production & 2. biosynthesis of macomolecules.

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Heterotrophcs

answer

Use organic molecules as carbon sources which often also serves as energy source.

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Autotrophs

answer

Use carbon dioxide as their primary or sole source of carbon and must obtain energy from other sources.

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Photolithoautotroph

answer

Carbon source is carbon dioxide, light is the energy source, and the electron source is an inorganic electron donor

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Photoorganoheterotroph

answer

Organic carbon is their carbon source, light is their energy source, and organic electron donors are their electron sources.

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Chemolithoautotroph

answer

Carbon dioxide is their carbon source, inorganic chemicals is their energy source, and inorganic electron donors are their soruces of electrons.

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Chemolithoheterotroph

answer

Organic carbon is their carbon source, inorganic chemicals is their energy source and inorganic electron donors are their electrons sources.

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Chemoorganoheterotroph

answer

Organic carbon is their carbon source, organic chemicals (often the same as their carbon source) is their eneergy source & organic electron donors (also, often the same as their carbon source) is their electron source

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Requirements of Nitrogen

answer

It is a component of amino acids

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Requirements of Phosphorous

answer

It is a component of phospholipids and nucleic acids

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Requirements of Sulfur

answer

It is found in the amino acids cysteine and methionine.

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Sources of Nitrogen

answer

Organic molecules, ammonia, nitrate via assimilatory nitrate reduction, nitrogen gas via nitrogen fixation

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Sources of Phosphorous

answer

Most organisms use inorganic phosphorous, which is directly incorporated into their cells.

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Sources of Sulfur

answer

Most organisms use sulfate and reduce it by assimilatory sulfate reduction

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Growth Factors

answer

Organic compounds, essential cell components (of their precursors) that the cell cannot synthesize, and they must be supplied by environment if cell is to survive and reproduce

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Classes of Growth factors and their use

answer

Amino acids (needed for protein synthesis), purines & pyrimidines (needed for nucleic acid (DNA) synthesis), and vitamins (function as enzyme cofactors)

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Applications of Growth Factors

answer

Many microorganisms are used to manufacture vitamins needed for human use.

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Uptake of Nutrients by the Cell

answer

Some nutrients enter by passive diffusion. Most enter by facilitated diffusion, active transport and group translocation.

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Passive diffusion

answer

Molecules move from region of higher concentration to one of lower concentration, with rate dependent on size of the concentration gradient between the cell exterior and interior. Water, oxygen and carbon dioxide often move across membranes this way.

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Facilitated Diffusion

answer

Similar to passive diffusion, movement of molecules is not energy dependent. Direction of movement is from high concentration to low concentration. Size of concentration gradient impacts rate of uptake. Use of integral membrane proteins to recognize certain molecules. Differs from passive diffusion: uses carrier molecules (permeases) that transport closely realted solutes. Smaller concentration gradient is required for significant uptake of molecules. Effetively transports glycerol, sugars & amino acids. More prominent in eukaryotic cells than in prokaryotic cells.

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Differences

answer

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Passive V Facilitated Diffusion

answer

Rate of facilitated diffusion increases more rapidly and at a lower concentration; diffusion rate in facilitated diffusion reaches a plateau when carrier protein becomes saturated.

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Active transport

answer

Energy-dependent process (ATP or proton motive force used). Moves molecules against the concentration gradient (concentrates molcules inside the cell). Involves carrier proteins (permeases) - the carrier saturation effect is observed at high solute concentrations.

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ABC Transporters

answer

ATP-binding cassette transporters (consumes ATP). This is observed in bacteria, archaea, and eukaryotes.

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Transport using H+/Na Gradients

answer

Proton gradient powers active transport (symport/antiport)

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Symport

answer

Linked transport of two substances in the same direction

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Antiport

answer

Coupled transport of two molecules in which one molecule enters the cell as the other leaves the cell.

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Group Translocation

answer

Chemically modifies molecule as it is brought into the cell. Best known system is the phosphoenolpyruvate sugar phosphotransferafe (PTS)

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Phosphoenolpyruvate sugar phosphotransferase (PTS)

answer

Groups translocation system that transports a variety of sugars while phosphorylating them using phosphoenolpyruvate

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Iron Uptake

answer

Ferric iron is very inoluble so uptake, microorganisms use siderophores to utake, siderophore complexes with ferric ion, this complex is then transported into the cell.

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Microbial Growth

answer

Increase in cullular consituents that may result in: increase in cell number (when microorganismss reproduce by budding or binary fission), and an increase in cell size (coenocytic microorganisms have nuclear divisions that are not accompanied by cell divisions). Microbiologists usually study population growth rather than the growth of individual cells.

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Prokaryotic Cell Cycle

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Cell cycle is sequence of events from fermentation of new cell through the next cell division (most bacteria divide by binary fission); two pathways function during cycle (DNA replication and partition, and cytokinesis).

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Origin of Replication

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The site at which replication beings

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Terminus

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Site at which replication is terminated

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Replisome

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Group of proteins needed for DNA syntehsis; parent DNA spools through this as replication occurs. Multiple occur at the same time.

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Cytoskeletel Proteins

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MreB & Protein FtsZ

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Protein MreB

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Cytoskeletal protein; similar to eukaryotic actin. Plays a role in cell shape and movement of chromosomes to opposite cell poles.

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Protein FtsZ

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Cytoskeletel protein - similar to eukaryotic tubulin. Plays a role in Z ring formation which is essential for septation.

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DNA Replication in Rapidly Growing Cells

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Cell cycle completed in 20 minutes (40 minutes for DNA replication, 20 minutes for septum formation and cytokinesis); look at timing - how can this happen? Second, third, and fourth round of replication can begin before first round of replication is complete.

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Growth Curve

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Observed when microorganisms are cultivated in batch culture. Culture incubated in a closed vessel with a single bath of medium. Usually plotted as logarithm of cell number versus time. Usually has four distinct phases: log, lag, stationary, and decline.

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Possible Reasons for Entry into the Stationary Phase

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Nutrient limitation, limited oxygen availability, toxic waste accumulation, and critical population density reached.

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Lag Phase

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Occurs within the first few hours. No hard change in numbers, organisms are still getting adjusted to the environment. They metabolize new substrates and increase slightly in size.

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Log Phase

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Undergoing fission. Most cells are good and alive and active.

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Starvation Responses

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Morphological changes (endospore formation), decrease in size, protoplast shrinkage, and nucleoid condensation, production of starvation proteins, long-term survival, and increased virulence.

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Death Phase

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Two alternative hypothesis: cells are viable but not culturale (VBNC) so they are alive but dormant. Or programmed cell death (fraction of the population genetically programmed to die (commit suicide)).

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Generation Time

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AKA doubling time; time required for the population to double in size. Varies depending on species of microorganism and environmental conditions. Range is from 10 minutes for some bacteria to several days for some eukaryotic microorganisms.

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Direct Cell Counts

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Counting chambers (easy, inexpensive, and quick), electronic counters (micrboial suspension forced through small orifice; impacts electric current the flows through orifice), and membrane filters (cells stained with fluorescent dye are filtered through special membrane that provides background for observing cells).

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Viable Cell Counts

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Cell plate counting (diluted sample of bacteria is spread of solid agar surface and the numbers of organisms are determined by counting the number of colonies multiplied by the dilution factors -- results are expressed as CFU's; colon forming units)

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Measurements of Cell Mass

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Dry weight (time consuming and not very sensitive), quantity of a particular cell constituent (protein, DNA, ATP, or chlorophyll; useful if amount of substance in each cell is constant), turbidometric measures (light scattering) is a quick, easy and sensitive.

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Spectrophotometric Analysis

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More cells = more light scattered = less light detected. Measures how much light passes through an organism and can generate a growth curve.

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Open System Growth

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Continual provision of nutrients, and continual removal of wastes (continuous culture of microorganisms)

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Continuous Culture of Microorganisms

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Open system growth, maintains cells in log phase at a constant biomass concentration for extended periods, achieved using a continuous culture system.

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Continuous Culture System

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Study of microbial growth at very low nutrient concentrations, close to those present in natural environment; study of interactions of microbes under conditions resembling those in aquatic environments.

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Chemostats

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Rate of incoming medium = removal of medium from vessel. As essential nutrient is in limiting quantities. Natural type of environment controls the nutrient uptake.

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Dilution Rate

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Rate at which medium flows through vessel relative to vessel size.

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Dilution rate and microbial growth

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Cell density maintained at wide range of dilution rates and chemostate operates best at low dilution rate.

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Turbidostats

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Regulates the flow rate of media through vessel to maintain a predetermined turbidity or cell density; dilution rate varies, no limiting reagent. Operates best at high dilution rates. Certaind density can be maintained - all cells are in the same growth state.

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Extremophiles

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Archaea (eg) - grow under harsh conditions that would kill most other organisms.

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Water Activity

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Amount of water available to organisms, reduced by interaction with solute molecules (osmotic effect). A higher solute concentration has a lower water acvitiy. Reduced by adsorption to surfaces (matrix effect).

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Osmotolerant Organisms

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Grow over wide ranges of water activity. Many use compatible solutes to increase their internal osmotic concentration. Solutes that are compatible with metabolism and growth. Some have proteins and membranes that require high solute concentrtions for stability and activity.

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Halophiles

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Grow optimally at more than 0.2 M

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Extreme halophiles

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Require more than 2 M

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pH

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The negative logarithm of the hydrogen ion.

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Acidophiles

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Growth optimum between pH 0 and 5.5

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Neutrophiles

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Growth optimum between pH 5.5 and 8

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Alkalophiles

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Growth optimum between pH 8 and11.5

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pH

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Most acidophiles maintain an internal pH near neutrality. The plasma membrane is impermable to protons. Some synthesize protins that proivde protection (acid-shock proteins). Many microorganisms change pH of their habitat by producing acidic or basic waste products (most media contain buffers to prevent growth inhibition).

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Temperature

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Organisms exhibit distinct cardinal growth temperatures; minimal, maximal and optimal.

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Adaptation for Thermophiles

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Protein structure stabilized by a variety of means; heat-stable (more H bonds, more proline & chaperones). Histone-like proteins stabilize DNA, and membrane is stabilized by variety of means (more saturated, more branched and higher molecular weight lipids.

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Oxygen sensitivities

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Oxygen can easily be reduced to toxic products (superoxide radical O2, hydrogen peroxide H2O2, and hydroxyl radical OH); aerobes produce protective enzymes (superoxide dismutase SOD and catalase)

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Obligate aerobes

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Contains SOD and catalase; has an oxic zone and an anoxic zone. Dependent on atmosphere O2 for growth.

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Facultative anaerobes

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Contains SOD and catalase. They prefer to have O2 but can grow without it.

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Aerotolerant anaerobe

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Contains SOD, not catalase. They totally ignore the presence of O2 and grows regardless. Spread evenly throughout. Capnophilic species require CO2.

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Strict anaerobe

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No SOD or catalase. They cannot tolerate oxygen and will die.

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Microaerophile

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Contains SOD and low levels of catalase.They require a lower conentration of oxygen, and they grow just below the surface.

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Barotolerant

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Adversely affected by increased pressure, but not as severely as nontolerant organisms.

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Barophilic Organisms

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Require or grow more rapidly in the presence of increased pressure (400 atm of greater)

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Radiation Damage

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Ultraviolet & ionizing radiation

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Ultraviolet (UV) radiation

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Mutations cause death and formation of thymine dimers in DNA. DNA damage can be repaired by several repair mechanisms.

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Ionizing Radiation

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X-rays and gamma rays. Mutations cause death and formation of thymine dimers in DNA. DNA damage can be repaired by several repair mechanisms.

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Visible Light Damage

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At high intensities generates singlet oxygen which is a powerful oxigizing reagent. Also create carotenoid pgiments (protects many light-exposed microorganisms from photo-oxidation).

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Microbial Grwoth in Natural Environments

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Microbial environments are complex, constantly changing often contain low nutrient concentrations (oligotrophic environment) and may expose a microorganism to overlapping gradients of nutrients and environmental factors. Bacteria here have to work together to survive.

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Responses to Low Nutrient Levels (oligotrophic environments)

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Organisms become more competitive in nutrient capture and use of available resources (some can become over aggressive). Morphological changes to increases surface area and ability to absorb nutrients. Mechanisms to sequester certain nutrients (siderophores).

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Biofilms

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Ubiquitous in nature; complex, slime enclosed colonies attached to surfaces; when formed on medical devices, such as implants often lead to illness/death; can form on any conditioned surface.

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Biofilm formation

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Microbes reversibly attach to conditioned surface and release polysaccharides, proteins, and DNA; additional polymers are produced as biolfilm matures; interactions occur among the attached organisms; continuous replication and growth. The polysaccharides protect all bacteria in the biofilm.

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Acylhomoserine lactone (AHL)

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An autoinducer molecule produced by many gram-negative organisms. It's concentration present allows cells to acess population density; process called quorum sensing.

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Quorum sensing

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Cell-to-cell communication. Tell each other when to grow to produce and specific nutrients to make. AHL or other signal molecules diffuse across plasma membrane. At high concentrations, it enters the cell. Once inside the cell, it induces expression of target genes that regulate a variety of functions. Potentially contributes to bioluminescence.

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Metabolism

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The total of all chemical reactions in the cell, divided into two parts (catabolism, anabolism).

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Catabolism

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Energy-conserving reactions that also generates a ready supply of electrons (reducing power) and precursos for biosynthesis.

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Anabolism

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The synthesis of complex organic molecules from simpler ones.

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Chemical work

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Synthesis of complex molecules

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Transport work

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Take up nutrients, elimination of wastes, and maintenance of ion balances.

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Mechanical work

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Cell motility and movement of structures within cells.

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First Law of Thermodynamics

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Energy can neither be created nor destroyed and the total energy in the universe remains constant. However, energy may be redistributed either within a system or between the system and its surroundings.

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Second Law of Thermodynamics

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Physical and chemical processes proceed in such a way that the entropy of the universe increases to the maximum possible.

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Entropy

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Amount of randomness (disorder) in a system.

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Free Energy and Reactions

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Expresses the change in energy that can occur in chemical reactions and other processes. Used to indicate if a reaction will proceed spontaneously.

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Delta G

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Free energy change; amount of energy that is available to do work

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Delta H

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Change in enthalpy (heat content in calories)

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Delta S

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Change in entropy

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Relationship between free energy and entropy

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A positive delta G requires energy (endergonic reaction) and is less favorable, decreases entropy.

A negative delta G proceeds on its own (exergonic reaction) and is spontaneous, increasing entropy.

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Equilibrium

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When the rate of the forward reaction equals that of the reverse reaction. A + B > C + D

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K(eq)

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Equilibrium constant, expresses the equilibrium concentrations of products and reactants to one another. K(eq) = [C][D]/[A][B]

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Standard Free Energy Change

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Delta G naut - defined at standard conditions of concentration, pressure, temperature and pH. pH at 7

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Relationship between free energy and standard free energy

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K(eq) > 1 - exergonic (delta G is negative) & K(eq) < 1 - endergonic (delta G is positive)

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Adenosine 5'-triphosphate

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ATP - energy currency of the cell

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Role of ATP in metabolism

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Exergonic breakdown of ATP is coupled with endergonic reactions to make them more favorable.

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The cell's energy cycle

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ADP + Pi <- chemical work, transport work, and mechanial work <- ATP <- Aerobic respiration, anaerobic respiration, fermentation, and photosynthesis <- ADP + Pi

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Glycolysis

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The conversion of glucose to pyruvic acid; consists of active transport, group translocation, electron transfer, conversion, catabolism and anabolism.

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Oxidation-Reduction Reactions and Electron Carriers

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Many metabolic processes involve this and they work in conjunction with one another. Acceptor + ne- <-> donor. Electron carriers are often used to transfer electrons from an electron donor to an electron acceptor. Can result in energy release, which can be conserved and used to form ATP.

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Standard Reduction Potential

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E naut - equilibrium constant for a redox reaction. A measure of the tendency to lose electrons. More negative = better e- donoer, more positive = better e- acceptor.

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Reduction potentials

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The greater the difference between E naut of the donor and E naut of the acceptor, the more negative delta G naut.

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Electron transport chains (ETC)

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Also known was electron transport system (ETS); electron carriers organized into ETC with the first electron carrier having the most negative E naut. As a result, the potential energy stored in the first redox couple is released and used to form ATP.

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Electron carriers

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NAD (nicotinamide adenine dinucleotide) & NADP (nicotinamide adenine dinucleotide phosphate). FAD (flavin adenine dinucleotide) & FMN (flavin mononucleoide, riboflavin phosphate). Coenzyme Q (CoQ - a quinone, also called ubiquinone). Cytochromes (use iron - heme - to transfer electrons). Ferredoxin proteins (use iron to transport electrons).

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Protein cataysts

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Enzymes = Have great specificity for the reaction catalyzed and the moelcules acted on. They don't bind to just any enzyme.

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Catalyst

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Enzymes = substance that increases the rate of a reaction without being permanently altered.

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Substrates

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Enzymes = reacting molecules.

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Products

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Enzymes = subsances formed by reaction.

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Structure and classification of enzymes

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Some enzymes are composed of one or more polypeptides. Some enzymes are composed of one or more polypeptides and nonprotein components.

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Enzyme structure

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Apoenzymes, cofactors and holoenzymes.

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Apoenzyme

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Protein component of an enzyme

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Cofactor

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Nonprotein component of an enzyme (prosthetic group - firmly attached, coenzyme - loosely attached).

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Holoenzyme

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Apoenzyme + Cofactor

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Coenzymes

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Often as as carriers, transporting substance around the cell. They can report the conditions of the cell, they bind to other molecules and bring them to the enzyme

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Mechanism of Enzyme Reactions

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Typical exergonic reaction = A + B -> AB++ -> C + D.

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Transition-state complex

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Resembles both the substrates and the products

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Activation energy

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Energy required to form transition-state complex; enzyme speeds up reaction by lowering the activation energy

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How enzymes lower activation energy

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By increasing concentrations of substrates at the active site of the enzyme; by orienting substrates properly with respect to each other in order to form the transition-state complex.

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Environmental Affect on Enzyme Activity

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Enzyme activity is significantly impacted by substrate concentration, pH, and temperature

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Effect of [substrate]

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Rate increases as [substrate] increases; no fruther increase occurs after all enzyme molecules are saturated with substrate.

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Effect of pH and temperature

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Each enzyme has specific pH and temperature optima. Denaturation can occur. They are supported well @ specific temperature and pH

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Denaturation

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Loss of enzyme's sutrcture and activity when temperature and pH rise too much above optima.

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Enzyme inhibition

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Competitive inhibitor (directly competes with biding of substrate to active site) & noncompetitive inhibitor (binds enzyme at site other than active site; changes enzyme's shape so that is becomes less active -- allosteric enzymes)

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The naure & significance of metabolic regulation

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Conservation of energy and material and maintenance of metabolic balance despite changes in the environment. Three major mechanisms: metabolic channeling, regulation of the amount of synthesis of a particular enzyme, and post-transcriptional regulation.

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Metabolic Channeling

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Differential localization of enzymes and metabolites. Compartmentatio (differential distribution of enzymes and metabolites among separate cell structures and organelles). Bacteria localize around certain parts of the cell because they don't have organelles.

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Control of Enzyme Activity

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Allosteric regulation and covalent modification

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Allosteric regulation

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Noncovalent binding of allosteric effector changes activity of enzyme. Alters the shape of the active site.

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Covalent modification

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Covalent binding of a phosphoryl, methyl, or adenyl group changes activity of enzyme

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Feedback Inhibition

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Also called end product inhibition; inhibition of one or more critical enzymes in a pathway regulates entire pathway. Have pacemaker enzymes.

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Pacemaker enzymes

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Catalyzes the slowest or rate-limiting reaction in the pathway. Ensures balanced production of a pathway end product.

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End-product inhibition

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Each end product regulated its own branch of the pathway and the initial pacemaker enzymes; isoenzymes - different enzymes that catalyze the same reaction.

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Chemotaxis

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An example of a complex behavior that is regulated by altering enzyme activity; system involves a number of enzymes and other proteins that are regulated by covalent modification; a major componnt is the phosphorelay system consisting of a sensor kinase and response regulator; modulation of th activity of the phosphorelay system determines the rotational direction of the flagella and whether the cell will run or tumble.

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The Carbon Cycle

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Cycling of carbon is continuous and dependent upon nutrient availability, abiotic conditions, and microbial community present.

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The Nitrogen Cycle

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Bacteria are the major players in this cycle. There is a continuous reduction of nitrogen, which is an amino acid component.

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The Phosphorous Cycle

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Most phosphorous is not readily available. Most is inorganic (released in the soil to be taken up by the plants). Phosphorous is also extremely limited in the environment.

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The Sulfur Cycle

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Each step of this cycle is mediated by molecules to break them down. This process can also occur in soil, and not just the oceans.

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The Iron Cycle

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Iron is an insoluble solid. Specific mirobes mediate the redox or iron in the presence/absence of oxygen.

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The Mercury Cycle

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Interactions between atmosphere, oxic water and anoxic water are critical. Biomagnification - progressive accumulation of refractile compounds. This process has mainly negative effects.

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General info about Aquatic Environments

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They vary dramatically; surface areas and volumes, as well as locations (rivers, streams, lakes and oceans - human body - water-saturated soil). They also vary in pH, temperature, etc.

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Water as a Microbial Habitat - Important physical factors

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Dissolved oxygen content, temperature, pH, and light penetration.

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Dissolved oxygen content

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Oxygen solubility and diffusion rates in waters are limited. Compared to soil, waters are low oxygen diffusion rate environments. In the very deep ocean, oxygen concentration varies with depth (oxygen solubility increases with decreasing temperatures and increasing atmospheric pressures). In warm environments, oxygen may become limited a few meters below the water surface.

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pH affected by CO2

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Use of CO2 by autotrophic microorganisms increases the pH of many waters. Oceans are buffered between pH 7.6 and 8.2 by the carbonate equilibrium system.

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Light in Marine and Aquatic Ecosystems

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The penetration of light into the surface water determines the depth of the photic zone. The resultant warming of the waters can lead to the development of a thermocline.

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Ocean Photic Zone

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Photosynthetic microbes that fix approximately half the world's carbon inhabit the upper 200-300 meters of the open ocean. A constant exchange of carbon dioxide occurs at the oxygen surface (there is also limited export of carbon to the seafloor). Organic matter escapes the photic zone and falls through depths as marine snow (composed of material that is not easily degraded, much of the material in marine snow is returned to surface water in upwelling regions).

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The Microbial Loop

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Describes the transfer and recycling of nutrients between trophic levels. The ocean is the richest source of viral life.

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Viruses in the Marine Environment

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Viruses play roles in nutrient cycling; accelerate rate at which their hosts are converted to particulate organic matter (POM) and dissolved organic matter (DOM), thereby "short-circuiting" the microbial loop.

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Estuaries

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Semi-enclosed coastal region where a rivr meets the sea, defined by tidal mixing between fresh- and salt-water.

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Salt Marsh

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Unlike estuaries, lack freshwater input from a single river.

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Marine Systems: Estuaries and Salt Marshes

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Many have high levels of pulltion; can result in loss of macroscopic life; can result in harmful algal blooms such as those caused by the diatom Pseudonitzchia and dinoflagellates Alexandrium and Pfiesteria.

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Benthic Marine Environments

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The largest microbial ecosystem is under the sea. Recent studies on ocean sediments (benthos) has revealed important information on microbial communities. Previously thought to be devoid of life, we now know that total subsurface life equals that of all terrestrial marine plants. Some of the microbes are barophilic and are able to tolerate atmospheric pressures up to 1,000 atm.

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Freshwater Environments

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Recent studies indicate that active microbial communities exist in glaciers and permanently frozen lakes (Microbes have been found in the East Antarctic Ice Sheet and its underlying Lake Vostok). Streams, rivers & lakes vary in their nutrient status.

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Lakes

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Eutrophic VS Oligotrophic

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Eutrophic

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Nutrient rich, have bottom sediments rich in organic matter, may be thermally stratified.

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Oligotrophic

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Nutrient poor, remain aerobic and are oxygen-saturated, low microbial population.

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The Soil Environment

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Very complex and contains a large variety of habitats (As a result, microbial diversity in soils is greater than that found in aquatic environments). The complexity of soil as a habitat has been a challenge to our understanding of soil microbial ecology.

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Formation of Different soils

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Soil formation is the result of combined action of weathering and colonization of geologic material by microbes. Types of soil: tropical & temperate region soil, could moist soils, desert soils, geologically heated hyperthermal soils.

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Soil as an environment for microorganisms

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Dominated by inorganic geological material (modified by organisms to form soils); pores provide optimum environment for microbial growth (microbes present in thin water films on particle surfaces where oxygen is available); rainfall and irrigation alter ideal state (mini aquatic environments, water-logged soil, and carbon dioxide & carbon monoxide as well as other gases have modified concentrations).

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Microorganisms in the Soil

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Bacteria, archaea, fungi and protozoa use different functional strategies to take advantage of the complex physical matrix in soil (prokaryotes on surfaces within smaller soil pores, terrestrial filamentous fungi bridge open areas between soil particles or aggregates called peds); soil populations play roles in biogeochemical cycling and the carbon, nitrogen, sulfur, iron, and manganese cycles; only small portions have been cultured.

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Soil Microbial Loop

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Differs from that operating in the photic zone of the open ocean (plants rather than microbes account for most primary production in terrestrial environments; microbes play role in recycling the organic matter); also differs because plant-or insect-released degradative enzymes do not rapidly diffuse away.

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Associations with Vascular Plants

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Many microbes have evolved commensalism relationships with plants, the major source of terrestrial primary production (plant is neither harmed nor helped, microbe gains some advantage). Other microbe-plant interactions are mutualistic (both are benefited; some protect plant from chewing insects); in some interactions, the microbe is a plant pathogen and harms the host.

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Mycorrhizae

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Mutualistic fungus-plant associations; fungus colonize plant roots; not saprophytic; instead use photosynthetically-derived carbohydrate provided by host; provide enhanced nutrient uptake for plant. Can increase a plant's competitiveness (in wet environments they increase the availability of nutrients, especially phosphorous; in arid environments they aid in water uptake); bacteria are also associated with mycorrhizal fungi; mycorrhization helper bacteria (MHB) can also be present as symbionts.

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Fungal Activies in Soil

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Production of gases and chemicals in process of woody plant decomposition

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The Three Domains of Life

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Carl Woese and George Fox, using the nucleotide sequence of the small subunit ribosomal RNA (SSU rRNAs), determined that all living organisms belong to one of three domains: bacteria, archaea, and eukarya.

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Universal Phylogenetic Tree

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Based on SSU rRNA sequence from the three domains of life (evolutionary relationships based on rRNA sequence comparisons). The root of the tree suggests that the three domains have a single ancestor, but Archaea and Eukarya evolved independently of the Bacteria.

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Creating Phylogenetic Trees from Molecular Data

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Align sequences, determine the number of positions that are different, express difference (evolutionary distance), use measure of difference to create tree (organisms clustered on relatedness, parsimony analysis -- fewest changes from ancestor to organism in question).

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Anagenesis (microevolution)

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Small, random genetic changes over generations which slowly drive either speciation or extinction, both of which are forms of macroevolution.

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Punctuated Equilibrium

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A phenomenon caused by the slow, steady pace of evolution being periodically interrupted by rapid bursts of speciation due to adrupt environmental changes.

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Prokaryotic evolution

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Results in microbial diversity which upon selection determines the development of new species; genetic heritable changes in Archaea and Bacteria are introduced by mutation and LGT.

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Taxonomy

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Science of biological classification, consists of three separate but interrelated parts. Classifcation (arrangement of organisms into groups), nomenclature, and ID (determination of taxon to which an isolate belongs.

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Phenetic Classification

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Groups organisms together based on mutual similarity of phenotypes. Can reveal evolutionary relationships, but not dependent on phylogenetic analysis (doesn't weight attributes); best systems compare as many attributes as possible.

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Phylogenetic Classification

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Also called phyltic classification systems; phylogeny (evolutionary development of a species); usually bsaed on direct comparison of genetic material and gene products (this approach is widely accepted, large databases exist for rRNA sequences).

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Strains

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Type strain (wild-type) is usually one of the first strains of a species studied and is often most fully characterized but not necessarily most representative member of the species. They descended from a single, pure microbial culture.

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Genus

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Well-defined group of one ore more strains; clearly separate from other genera.

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Species

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Collection of strains that share many stable properties and differ significantly from other groups of strains; collection of organisms that share the same sequences in their core housekeeping genes.

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Classical characteristics

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Morphological, physiological, metabolic, ecological, and genetic.

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Molecular chracteristics

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Nuclei acid base composition, nucleic acid hybridization, nucleic acid fingerprinting, and amino acid sequencing.

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Use of DNA sequences to determine species identity

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Requires analysis of genes that evolve quickly than rRNA encoding genes; multilocus sequence typing (MLST) the sequencing and comparison of 5 to 7 housekeeping gnes is done to prevent misleading results from analysis of one gene introduced by lateral gene transfer.

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Genomic Fingerprinting

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Used because of multicopies of highly conserved and repetitive DNA sequences present in most gram-negaive and some gram-positive bacteria. Uses restriction enzymes (cleavage patterns are compared); repetitive sequences amplified by PCR (amplified fragments run on agarose gel with each lane of gel corresponding to one microbial isolate - the pattern of the bands analyzed by the computer & compared in restriction fragment length polymorphism (RFLP)); in some cases allows for ID to strain level.

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Bergey's Manual of Systematic Bacteriology

answer

Largely phylogenetic rather than phenetic; prokaryotes are divided between two domains and 25 phyla.

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Transcription

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Yields a ribonucleic acid (RNA) copy of specific genes.

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Translation

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USes informtation in mRNA to synthesize a polypeptide (also involves activities of tRNA and rRNA).

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Nucleic Acid Structure

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The nucleic acids, DNA and RNA, are polymers of nucleotidse linked together by phosphodiester bonds. RNA and DNA differ by the nitrogenous bases they contain, the sugar they contain & whether they are single or double stranded.

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DNA

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Polymer of nucleotides; contains the bases adenine, guanine, cytosine and thymine. Sugar is deoxyribose. Molecule is usually double stranded (major and minor grooces form when the 2 strands twist around each other).

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Complementary Strands of DNA

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Base pairing (adenine which is a purine and thymine which is a pyrimidine & guanine which is a purine and cytosine which is a pyrimidine). CG and connected by three hydrogen bonds while AT are connected by two.

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RNA

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Polymer of nucleotides. Contains the bases adenine, guanine, cytosine and uracil. Sugar is ribose. Most RNA molecules are single stranded.

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RNA structures

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Three different types which differ rom each other in function and in structure (mRNA, tRNA and rRNA).

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Organization of DNA in cells

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In most bacteria, DNA is a circular double helix, further twisting results in supercoiled DNA. In bacteria, the DNA is associated with basic proteins (helps to organize DNA into a coiled chromatin-like structure).

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DNA replication

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Complex process involving numerous proteins which help ensure accuracy, the two stranded separate each serving as a template for synthesis of a complementary strand, synthesis is semi-conservative (each daughter cell obtains one old and one new strand). Bidirectional from a single origin of replication.

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Replication Machinery

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DNA polymerase catalyzes synthesis of complementary strand of DNA. DNA synthesis is in the 5' to 2' direction resulting with the formation of a phosphodiester bond. These enzymes require a template (directs synthesis of complementary strand), primer (DNA or RNA strand), and dNTPS (deoxyribonucleotide triphosphates).

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Bacterial DNA Replication

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Helicases, topoisomerases and DNA polymerase III are part of the replisome. The lagging strand is synthesized in short fragments called Okazaki fragments (a new primer is needed for the synthesis of each Okazaki fragment).

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Completion of Lagging Strand Synthesis

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DNA ligase forms a phosphodiester bond between 3'-hydroxyl of the growing strands and the 5'-phosphate of an Okazaki fragment.

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Proofreading

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Carried out by DNA polymerase III. Removal of mismatched base from 3' end of growing strand by exonuclease activity of enzyme. The acitivty is not 100% efficient.

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Termination of Replication

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Replication stops when replisome reaches termination site (ter) on DNA. Catenanes form when the two circular daugher chromosomes do not separate. Topoisomerases temporarily break the DNA molecules so the strands can separate.

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Rolling Circle Replcation

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Some small circular genomes (viruses, plasmids) are replicated by this method

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Gene Structure

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It is the basic unit of genetic information; it is also defined as the nucleic acid sequence tht codes for polypeptide, tRNA, or rRNA. It is a linear sequence of nucleotides with a fixed start point and end point. Codons are found in mRNA and code for a single amino acid.

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Genes that code for Proteins

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Template strand directs RNA synthesis (read in the 3' to 5' direction); promoter is located at the start of the gene (is the recognition site or RNA polymerase; functions to orient polymerase); leader sequence is transcribed into mRNA but is not translated into amino acids (shine-dalgarno sequence important for initiation of translation).

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Coding Region

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Begins with the DNA sequences 3'-TAC-5' (produces codon AUG, codes for N-formylmethionine, a modified amino acid used to initiate protein synthesis in bacteria; coding region ends with a stop codon that is immediately folowed by the trailer sequence which contains a terminator sequence used to stop transcription).

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Importance of the Reading Frame

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Organiztion of codons such that they can be read to give rise to a gene product.

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Genes that code for tRNA and rRNA

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tRNA/rRNA genes have a promoter, leader, coding, spacer, and trailer regions. These regions are removed during the maturation process.

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Transcription

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RNA synthesis under the direction of DNA (RNA produced has complementary sequence to the template DNA); produces three types of RNA (mRNA carries message for protein synthesis, tRNA carries amino acid during protein syntehsis and rRNA molecules are components of ribosomes).

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Transcription in Bacteria

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Polycistronic (polygenic) mRNA often found in Bacteria and Archaea, and contain directions for more than one polypeptide. They are catayzed by a single RNA polymerase by ATP.

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Bacterial RNA Polymerases

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In most bacteria RNA polymeases the core enzyme is composed of 5 chains and catalyzes RNA synthesis, the sigma factor has no catalytic activity but helps the core enzyme recognize the start of the genes, and the holoenzymes = core enzyme + sigma factor). Only the holoenzyme can begin transcription.

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Transcription Process

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Initiation, elongation and termination.

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Transcription Initiation

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Only a short segment of DNA is transcribed; promoter (site where RNA polymerase binds to initiate transcription; it is not itself transcribed, has specific consensus sequence before transcription starting points and it's called the -10 (Pribnow box) and -35 sites.

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Transcription Elongation

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After binding, RNA polyermase unwinds the DNA. Transcription bubble forms and moves along with the polymerase as it transcribes mRNA from template strand (within the bubble a temporary RNA:DNA hybrid is formed).

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Transcription Termination

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Occurs when core RNA polymerase dissociates from template DNA. Two types: intrinsic and rho-dependent.

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Intrinsic termination

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They come to a U-rich sequence in the transcription bubble and a stem-loop forms that causes RNA polymerase to pause. This U-rich sequence can't hold the hybrid together so termination occurs.

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Rho-dependent Termination

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Rho is there, causes another stem loop to form and termination occurs.

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Genetic Code

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The final step is expression of protein from encoding genes. mRNA sequence is translated into amino acid sequence of polypeptide chain (process = translation).

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Organization of the Code

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Degeneracy (up to siz different codons can code for a single amino acid), sense codons (61 that specify amino acids), and stop codons (three codons used as translation termination signals - they do not code for amino acids).

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Wobble

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Loose base pairing in the 1st or 2nd position of the codon. This eliminates the need for unique RNA for each codon.

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Translation

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Synthesis of polypeptide directed by sequence of nucleotides in mRNA. Direction of synthesis - N-terminal -> C-terminal. Ribosome (site of translation, polyribosomes is a complex of mRNA with several ribosomes).

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tRNA and Amino Acid Activation

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Attachment of amino acid to tRNA. Catalyzed by aminoacyl-tRNA synthetases. There are at least 20 of these. Each is specific for a single amino acid and for all of the tRNAs to which each may be properly attached (cognate tRNAs).

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The Ribosome

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70S ribosomes = 30S + 50S subunits

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Initiation of Protein Synthesis

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Initiation codon aids to position ribosome properly at the 5' ends of mRNA.

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Elongation of the Polypeptide Chain

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Elongation Cycle (sequential addition of amino acids to growing polypeptide), consists of three phases (aminoacyl-tRNA binding, transpeptidation, and translocation); involves several elongation factors (EFs).

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Transpeptidation

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Catalyzed by peptidyl transferase.

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Translocation

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Three simultaneous events -- peptidyl-tRNA moves from A site to P site, ribosoe moves down one codon & empty tRNA leaves P site.

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Termination of Protein Synthesis

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Takes place at any one of three codons (nonsense - stop - codons UAA, UAG or UGA); release factors (RFs) air in the recognition of stop codons, 3 RFs function in prokaryotes.

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Chaperones

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Proteins that aid in the folding of nascent polypeptides. They protect the cells from thermal damage (heat-shock proteins) and aid in the transport of proteins across membranes.

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Protein Splicing

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Removal of part of polypeptide before folding. Inteins (removed) and exteins (kept)

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Bacterial Protein Secretion

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Gram-positive and gram-negative bacteria have different problems secreting proteins based on the differences between the structure of their walls; for both types of cells, the major pathway for transporting proteins across the membrane is the Sec-dependent pathway; other secretion pathways exist; all protein secretion systems require energy.

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Sec-Dependent Pathway

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Also called the general secretion pathway; translocates proteins from cytoplasm across or into plasma membrane; secreted proteins synthesized as preproteins having amino-terminal signal peptide (signal peptide delays protein folding chaperone proteins keep pre-proteins unfolded); secA translocates preprotein though the plasma membrane; when preprotein emerges from plasma mambrane a signal peptidase removes the signal peptide.

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Protein secretion in gram-negative bacteria

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5 pathways = Type II and V transport proteins across outer membrane that were translocated across plasma membrane by sec-dependent pathway. Type I and III pathways are sec-independent. Type IV usually functions independently of Sec.

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Genomics

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Study of molecular organization of genomies, their information content, and gene products they encode.

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Structural genomics

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Physical nature of genomes

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Functional genomics

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how genomic functions

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comparative genomics

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compares genomes of different organisms

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sanger method

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referred to as the chain-termination dna sequencing method; uses ddNTPS; mix single strands of DNA with primer, DNA polymerase, 4 deoxynucleotides, small amount of one ddNTP - dna synthesis occurs; random insertion of ddNTP generates DNA fragments of different lengths - four reactions carried out; each with different ddNTP - fragments in each reaction mixture separated electrophoretically - gel autoradiographed and sequence read.

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whole-genomic shotgun sequencing

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developed in 1995 by j. craig venter and hamilton smith. four stage process: (1)library construction - generates clones of portions of genome, (2) random sequencing - determines sequences of clones, (3)fragment alignment and gap closure - uses speical software to form long stretches of sequence called contigs, (4) editing

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bioinformatics

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analysis of genomie data using computers; generates data on genomic content, structure, and arrangement; also provides data on protein structure and function; uses annotation to determine location of genes on newly sequenced genome; further examination carried out using in silico analysis

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genome annotation

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process that locates genes in the genome map; identifies each open reading frame (ORF) in genome (a prokaryotic reading frame is a sequence > 100 codons that is not interrupted by a stop codon; a ribosomal binding site at the 5' end is apparent and terminator sequences at the 3' end); uses databases to assign tentative function of gene (ORF's presumed to encoe proteins are called coding sequences)

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functional genomics

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determination of how genome works; derived from alignment of gene sequences (paralogs are two or more genes found alike in the same genomic that likely arose from gene duplication; orthologs are two or more genes very similar in different organisms that are predicted to have the same function); involves analysis of translated amino acid sequnce of presumed genes (provides understanding of protein structure and function) -- a motif if a short pattern of amino aicds that may represent a functional unit within the protein, such as the active site of an enzyme

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proteomics

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the study of the proteome - the entire collection of proteins that an organism produces. provides information about genome function not available from mRNA studies. information that determines what is actually happening in the cell is referred to as functional proteomics. proteome often analyzed by 2D gel electrophoresis.

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microarray analysis

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once the identity and function of genes in a genome have been determined, it can determin which genes are expressed at a specific tim (at the RNA-level); spotted arrays (prepared by robotic application of limited number of DNA probes to a solid support like a chip); photolithography (commercially prepared microarrays, can contain probes for every ORF in the genome of an organism)

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analysis of microarray analysis

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based on hybridization between the probe DNA and the targets nucleic acids, mRNA, to be analyzed. targets are labels with fluorescent dyes and then incubated with the gene chip and the unbound target is washed off. the chip is scanned with laser beams to detect fluorescence which indicates that hybridization has occurred; a vast amount of data has been generated (often presented using hierarchial cluster analysis).

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environmental genomics

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also called metagenomics. used to learn more about diversity and metabolic potential of microbial communities. takes a census of microbial populations and can determine the presence and level of classes of genes. phylotypes (taxon characterized only by its nucleic acid sequence); finding rhodopsin-like genes and nitrogenases in marine prokaryoties is an example of an exciting discovery that requires a reassessment of oceanic/nitrogen cycling.

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different methods of cloning

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PCR, restriction enzyme digestion of DNA, gel electrophoresis, cellular transformation

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PCR

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the rapid synthesis of many copies of a specific DNA fragment from a complex mixture of DNA and other cellular components; reaction mix contains - primers, target DNA, thermostable DNA polymerase such as Taq polymerase, each of the 4 dNTPS. in the reaction, DNA is denatured, primers anneal to the target DNA, copies of the target DNA are synthesized; pieces of DNA ranging in size from 100 to several thousand bp in length can be amplified.

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reverse transciptae

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synthesizes double-stranded DNA from RNA template; used to construct complementary DNA (cDNA)

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restriction enzymes

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bind to DNA at specific sequence called the recognition site (usually 4-8 bp long); cleave DNA at this site or a defined distance from it

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gel electrophoresis

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used to separate molecules based on their charge and size; agarose or acrylamide gels can be used to separate DNA fragments; DNA is acidic, it migrates from the negative to the positive end of the gel; each fragment's migration rate is inversely proportional to the log of its molecular weight.

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cloning vectors & creating DNA

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cloning vectors are used to provide many copies of cloned DNA (via replication in a host organism); four types of cloning vectors - plasmids, phages, cosmids & artifical chromosomes.

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plasmids

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plasmids are excellent cloning vectors because they replicate autonomously and are easy to purify; some are called shuttle vectors because they can move from one host to another; often have a high copy number to facilitate production of cloned gene product

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cloning vectors & recombinant DNA

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each type of cloning vector generally has an origin of replication, a selectable marker, a unique restriction site(s) called a multi-cloning site (MCS) or polylinker

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blue-white selection of recombinant plasmids

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the lacZ gene produces B-galactosidase which forms blue colonies. a disruption of the gene prohibits B-gal function, forming white colonies

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foreign genes in a host

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cloned genes, in the new host cell, are called heterologous genes and may not be expressed unless they are modified (recombinant gene must have a promoter that host RNA polymerase recognize, introduced eukaryotic genes must be modified to have a prokaryotic leader sequence and all introns must be removed); expression vectors are used to overcome problems with expression of recombinant genes in host cells.

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genomic libraries/phenotypic rescue

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used when gene of interest is on a chromosome that has not been sequenced; the library is constructed by cleaving the genome and then gloning the fragments into vectors; the libraries are screened for the genes of interest in a variety of ways

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inserting recombinant DNA into host cells

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most common hosts (E. coli and S. cerevisiae); hosts are engineered to lack restriction enzymes and RecA; DNA introduction into microbes (transformation, electroporation

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bioengineering in plants

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plants can be transformed using the Ti (tumor inducing) plasmid of Agrobacterium tumefaciens; the Ti plasmid has been genetically engineered to improve its utility as a cloning vector; nonessential regions, including tumor-inducing genes, have been detected; selectable marker and multi-clonning site added.

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bioprospecting

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hunting for new microorganisms; most major sources of microbes for use in industrial microbiology have been natural materials; hunting for industrially useful microbes in nature continues because <1% of microbes have been cultured from nature.

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industrial microbiology

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must first find suitable microbes - genetically stable, easy to maintain and grow, well suited for extraction or separation of desired product

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industrial fermentation

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for many industrial processes, microorganisms must be grown using specifically designed media under carefully controlled conditions; to industrial microbiologists fermentation means the mass culture of microorganisms; requries precise control of agitation, temperature, pH changes, and oxygenation

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genetic manipulation and modifications

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used to produce microorganisms with ne and desirable chracteristics, such as increased yield of a product; procedures include: mutation (variety of techniques used, including site-directed and mutagenesis) - deliberately making alterations in amino acid sequence of a protein in order to generate enzymes with new or improved properties. protoplast fusion - widely used with fungi, can fuse protoplasts of different species, and an inherently mutagenuc procedure. insertion of short DNA sequences, transfer of genetic information between different organisms, modification of gene expression, and natural genetic engineering.

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protein evolution

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one of the newest approaches for creating new metabolic capabilities in a given microorganisms. employs forced evolution and adaptive mutations (involve use of specific environmental stresses to force microbes to mutate and adapt, thus creating microbes with new biological capabilities). in vitro evolution (starts with purified nucleic acids rather than whole organisms). high-throughput screening (HTS) enables the rapid selection of a single desirable mutant or molecule from tens of thousands of new constructed tains, molecules, or compounds.

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HTS (high through-put screening)

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enables the rapid selection of a single desirable mutant or molecule from tens of thousands of new constructed tains, molecules, or compounds.

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applications of genetic engineering

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in medicine (many useful proteins, gene therapy) and in agriculture (use of genetic engineers allows for the direct transfer of desirable traits to agriculturally important animals and plants).

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biopolymers/biosurfactants

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used to modify flow characteristics of liquids and to serve as getting agents. includes dextrans and other polysaccharides. used for emulsification, increasing detergency wetting and pase dispersion, and solubilization. important in bioremediation, oil spill dispersion, and enhancing oil recovery. many have antibacterial and antifungal activity; some inactivate enveloped viruses

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biofuels

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ethanol is produced by microbial fermentation of crop residues that consist of cellulose hemicellulose (these two polysaccharides are polymers of five different sugars: glucose, xylose, mannose, galactose, and arabinose.

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