MMG 2010 Learning Objectives Exam 2

Chapters 9-15

Define and describe metabolism

The sum of all chemical reactions in an organism to maintain life

Includes catabolism (exergonic pathways) and anabolism (endergonic pathways)

Compare and contrast autotrophs and heterotrophs

Autotrophs - organisms that convert inorganic CO2 into organic carbon compounds (ex. plants and cyanobacteria)

Heterotrophs - rely on more complex organic carbon compounds as nutrients, typically provided initially by autotrophs (ex. e.coli, humans)

Phototrophs, chemotrophs, organotrophs, lithotrophs

Phototrophs - obtain energy from light

Chemotrophs - obtain energy by breaking chemical bonds

• organotrophs - obtain from organic compounds (human, fungi, etc.)

• Lithotrophs - obtain from inorganic compounds

Importance of redox reactions in metabolism

Reactions that move electrons from one molecule to another

Oxidation = loses electrons (donor)

Reduction = gains electrons (acceptor)

Most of the energy stored in atoms and used to fuel cell functions is in form of high energy electrons. This transfer allows cell to transfer and use energy in small amounts at a time rather than all at once

Describe why ATP, FAD, NAD+, and NADP+ are important in a cell

These molecules are energy carriers that bind and shuttle high-energy electrons between compounds in pathways. Easily reduced or oxidized

NAD+ (oxidized form)/NADH (reduced form) - most common e- carrier in metabolism, used a lot in energy extraction from sugars during catabolism in chemotrophs

NADP+ (oxidized form)/NADPH (reduced form) - plays important role in anabolic reactions and photosynthesis

FAD (oxidized form) / FADH2 (reduced form) - used a lot in energy extraction from sugars during catabolism in chemotrophs

NADH/NADPH/FADH2 all have reducing power (willing to lose/donate electrons, can be a reducing agent)

Describe ATP, ADP, AMD and why it is a good energy source

ATP is the energy currency of a cell.

AMP is the most reduced form? (adenosine + ribose sugar + 1 phosphate group) → ADP (2 phosphate groups) → ATP (3 phosphate groups)

Adding a phosphate group requires energy, Pi groups negatively charged and repel each other when arranged like in ADP and ATP, making the molecules inherently unstable - high-energy phosphate bonds

Processes that generate ATP (3)

-Substrate level phosphorylation: direct transfer of phosphate from organic compound to ADP (image) → anaerobic, aerobic respiration, fermentation, phototrophy and chemolithotropy all use the ATP/ADP + Pi cycle in which ADP + Pi is phosphorylated to generate ATP. This ATP is used to do chemical work, mechanical work, or transport work.

-Oxidative phosphorylation: indirect, proton motive force (electrochemical gradient)

-Photophosphorylation: light used to form proton motive force

Describe key points of glycolysis (very simplified)

Catabolism of glucose, substrate level phosphorylation

Glucose + 2NAD+ 2ADP + 2 Pi → 2 pyruvate + 2 NADH + 2H+ + 2ATP +2H20

Electron donor: glucose

Electron acceptor: NAD+

Followed by Respiration or Fermentation

Includes EMP, ED, and PPP pathways (diferent organisms may use different routes)

Key points of fermentation

Fermentation = anaerobic catabolism (no O2 required), performed after glycolysis in the absence of O2

• Conserves energy - produce energy containing high-energy bonds for ATP synthesis

• Redox balance (oxidized NADH back to NAD+ so glycolysis can continue)

• e- donor: organic compound, e- acceptor: organic compound

Common waste products of fermentation include: CO2, propionic acid, lactic acid, ethanol, acetone, isopropanol, acetic acid

Homolactic (LA only fermentation product) vs Heterolactic (Mixture of LA, ethanol/acetic acid, CO2 products)

Glycolysis followed by aerobic respiration: ETC/ Oxidative phosphorylation

Aerobic catabolism

e- donor: organic compound, Terminal e- acceptor: O2

ATP produced by: electron transport to generate prton motive force (ETC), oxidative phosphorylation

Glycolysis followed by anaerobic respiration

Follows the same general pathway as Aerobic respiration: 2 pyruvate in cytosol transported into mitochondria where citric acid cycle takes place, NADH and FADH2 zip over to the inner mitochondrial membrane where the ETC and chemiosmosis occurs in order to produce the majority of ATP. The only difference is that rather than O2 being consumed in complex 4 acting as a terminal electron acceptor, sulfur or nitrogen containing compounds take that role.

Electron carriers in respiration

NADH and FADH2

Produced in glycolysis and citric acid cycle and must be reoxidized for redox balance for glycolysis to occur

-In respiration, reoxidation occurs during electron transport in the cytoplasmic membrane, used to form electrochemical gradient that conserves energy through ATP synthesis

Identify the structure and structural components of an enzyme, difference between aponenzyme and holoenzyme

Enzymes are proteins, have active sites, lower activation energy

Apoenzyme - lacks necessary cofactor or coenzyme (inactive)

Holoenzyme - has necessary cofactor or coenzyme (active)

Describe the differences between competitive and noncompetitive enzyme inhibitors

Competitive - binds at active site and blocks substrate binding (ex. sulfa drugs)

Noncompetitive/Allosteric inhibitors - Bind at allosteric site, causes conformational change in enzyme that no longer allows substrate binding

Describe why glycolysis is not oxygen dependent

Because it doesn’t require oxygen; is an ancient universal metabolic process every living organism carries out some form of it

Define and describe the net yield of three-carbon molecules, ATP, and NADH from glycolysis

2 main stages:

-Energy investment phase (Glucose → 2 GAP): 2 ATP consumed → ADP +Pi

-Energy payoff phase (2 GAP → 2 pyruvate): NAD+ converted to NADH and 2 ADP + Pi phosphorylated into 2 ATP (per GAP molecule, so net of 4 ATP produced in 2nd stage, cancelling 2 of those out with the 2 consumed in the 1st stage)

Chemiosmosis, PMF, ATP Synthase

Chemiosmosis - flow of hydrogen ions across the membrane (diffusion down the gradient, energetically favorable)

PMF (proton motive force) - electrochemical gradient formed by the accumulation of H+ on one side of the membrane compared to the other (also caused pH change). ETCs pumping H+ across a membrane forms this energy source

ATP synthase - membrane-bound complex in which chemiosmosis occurs and uses to convert ADP + Pi → ATP

Compare and contrast the electron transport system location and function in a prokaryotic cell and a eukaryotic cell

Prokaryotic ETS - occurs in the inner part of the cell membrane

Eukaryotic ETS - specialized protein complexes in inner mitochondrial matrix

Describe the function and location of ATP synthase in a prokaryotic versus eukaryotic cell

ATP synthase - membrane-bound enzyme complex that uses the force of H+ diffusing down the proton gradient to convert ADP + Pi → ATP

Prokaryotes - located in the cell membrane

Eukaryotes - Located inner mitochondrial membrane. Also found in the thylakoid membranes of chloroplasts where it produces ATP during photosynthesis

Compare and contrast aerobic and anaerobic respiration

Aerobic respiration:

• e- donor: inorganic compound

• Terminal acceptor: O2

• ATP produced by ETC and oxidative phosphorylation

Anaerobic Respiration:

• e- donor: inorganic compound

• Terminal e- acceptor: sulfur or nitrogen-containing compounds

• ATP produced by ETC and oxidative phosphorylation

Define and describe the importance of microorganisms in the biogeochemical cycles of carbon, nitrogen, and sulfur

Carbon Cycle: Carbons exchanged between heterotrophs (produce CO2 as a result of respiration or fermentation and autotrophs (use CO2 for fixation). Eukaryotes participate in aerobic respiration, fermentation, and oxygenic photosynthesis.

Nitrogen Cycle: Prokaryotes play essential roles by transforming nitrogen between various forms from own needs, indirectly benefitting other organisms who cant incorporate it from the atmosphere.

Sulfur cycle: Part of AA Cys and Met, found in several vitamins, and necessary for synthesis of important biomolecules like coenzyme A. Many bacteria and plants can use sulfate as sulfur source, decomposition dead organisms by fungi and bacteria remove sulfur groups from AA producing H2SO4, returning inorganic sulfur to environment

Define bioremediation

Microbial bioremediation uses microbial metabolism to remove xenobiotics or other pollutants (bacteria, fungi, plants) to clean up environmental pollution by eating or catabolizing toxic chemicals

Describe binary fission

1 cell → 2 genetically similar daughter cells. Most bacteria reproduce by binary fission

Consequences: short generation time, progeny are genetically identical, exponential growth

Steps:

1. Cell elongates, DNA replicated

2. Plasma membrane begins to constrict, new wall made

3. Cross wall forms, completely separating 2 DNA copies

4. Cells separate

Define the generation time for growth based on binary fission

Time required for a population to double in size (bacteria do not all grow at the same rate)

Every generation doubles the population

Identify and describe the activities of microorganisms undergoing typical phases of binary fission (simple cell division) in a growth curve

1. Lag phase - intense activity prepping for pop. growth, but no actual growth yet

2. Log phase - exponential increase in population

3. Stationary phase - microbial deaths = production of new cells. No net growth

4. Death phase - population logarithmically declines due to lack of nutrients, secondary metabolites?, no space, quorum sensing

Describe examples of cell division not involving binary fission, such as budding or fragmentation

Binary fission most common in prokaryotes, but not only one

Budding/Asymmetrical division - formation of a long, narrow extension that swells and forms a smaller cell that eventually detaches from parent (most common in yeast)

Fragmentation - Many nucleoids accumulate in an enlarged round cell or along the filament leading to many new cells at once, split from parent and float away (commonly obs in actinomyces)

Spore production in aerial filaments - aerial mycelia (bacteria) grow upward using filaments. Filamentous bacteria (ex. streptomyces) tip of aerial hyphae undergo synchronous cell division and septation to form chains of uninucleoid spores

Describe the formation and characteristics of biofilms

Biofilms are complex and dynamic ecosystems that form on many dif. environmental surfaces (usually attached to surface) and embedded in an extracellular matrix. Highly structured communities that provide a selective advantage to constituent microorganisms

Polymicrobial, Extracellular polymeric substances (EPS) secreted by organisms in biofilm, matrix interspersed with open water channels

1. Attachment - flagella/pili/surface molecules, reversible

2. Colonization - quorum-sensing

3. Growth and cell division

4. Maturation - EPS production and formation of water channels

5. Dispersal - Attachment of secondary colonizers and release planktonic cells to new sites

Biofilm matrix can: hold cells together, retain nutrients, maintain moisture, protect from environmental stress, limit penetration of some antimicrobials, help communicate

Identify health risks associated with biofilms and how they are addressed

Microbes growing in biofilm can behave differently from the same microbes growing as free-floating cells

Dental plaque → dental caries/periodontal disease

Catheters → healthcare-associated infections

Chronic wounds → persistent infection

Pseudomonas → chronic respiratory infections (main cause of cystic fibrosis)

Describe quorum sensing and its role in cell-to-cell communication and coordination of cellular activities

Quorum sensing is the ability to detect and respond to cell population density by gene regulation

Discovered 1970 with bacterial luminescent system in Vibrio fischeri (GN bacteria)

Bacterial cell produces and emits and autoinducer (AI), at low cell densities the AI will diffuse away

At high densities the AI enters bacterial cell, binds to specific DNA regions and activated gene transcription resulting in a positive-feedback loop

Can be intraspecies or interspecies communication - In bacteria they produce signaling molecules, release into environment, detect the concentration of those molecules, and respond when enough cells are present

Allows cells to coordinate biofilm formation, virulence factors, enzyme production, bioluminescence, and other population-level behaviors

Identify and describe different categories of microbes with requirements for growth with or without oxygen: obligate aerobe, obligate anaerobe, facultative anaerobe, aerotolerant anaerobe, microaerophile, and capnophile

Obligate (strict) aerobes - cannot growth without an abundant supply of O2

Obligate Anaerobes - killed by oxygen

Facultative anaerobe - Organisms that thrive in presence of oxygen but also grow in absence by relying on femerntation or anaerobic respiration

Aerotolerant anaerobes - indifferent to the presence of oxygen, dont use but not harmed by it

Microaerophiles - require goldilocks oxygen concentration, not too much and not too little. O2 must be just right for growth

Illustrate and briefly describe minimum, optimum, and maximum temperature requirements for growth

Minimum growth temp - lowest temperature at which organism can survive and replicate

Optimum growth temp - temperature at which growth rates are the highest

Maximum growth temp - highest temperature at which growth can occur

Define: psychrophile, mesophile, thermophile, alkaliphile, neutrophile,
acidophile, halophile, halotolerant

Psychrophile - Grow at extreme low temperatures (<0 C)

Psycrotrophs - Prefer cooler temperatures (~4 - 25 C)

Mesophile - Grow and reproduce at moderate temperatures (around human body temp); e. coli., salmonella

Thermophile - Grow at hotter temps (>50 C), do not multiply are room temps; Thermus aquaticus, Geobacillus

Hyperthermophile - Grow at extreme hot temperatures ( >80 C); Pyrobolus, pyrodictium

Acidophiles - grow optimally at pH < 5.5

Neutrophiles - Grow optimally at ± 1-2 from pH 7

Alkaliphiles - Grow optimally pH between pH 8-10.5

Halophile - require high salt concentrations for growth (often found in marine environments)

Halotolerant - do not need high concentrations of salt for growth, but will survive and divide in presence of high salt.

Define virulence and pathogenicity

Pathogenicity - ability of a microorganism to cause disease in a host (can cause or not)

Virulence - the degree/severity of disease caused by a pathogen: essentially how harmful a pathogen is

Differences between vertical and horizontal gene transfer in bacteria

Vertical gene transfer - transmission of genetic information from generation to generation (main mode)

Horizontal gene transfer (HGT) - in prokaryotes, introduction of genetic material from one organism to another organism within the same generation (even distantly related species to share genes and phenotypes)

• Transformation, transduction, conjugation

Explain the two functions of the genome

• DNA is the genetic material responsible for inheritance and passed from parents to offspring - accurate replication with full complementation and organization into genes arranged on chromosomes and plasmids

• Direct and regulate the construction of proteins necessary for growth and reproduction in a particular cellular environment

Explain the meaning of the central dogma of molecular biology

Flow of genetic information: DNA → RNA → Protein

Replication → transcription → translation

Differentiate between genotype and phenotype and explain how environmental factors influence phenotype

Genotype - cell’s full collection of genes

Phenotypes - set of observable chracteristics that result from those genes. Product of the array of proteins being produced by cell at a given time which is a product of the cells genotype and the environment

Compare the processes of transformation, transduction, and conjugation including the source of transferred DNA and how DNA is transferred between bacterial cells.

Transformation - naked DNA is taken up from the environment typically the result of cells that lysed upon death and released their contents. Competent cell = actively bind to environmental DNA, take it into cytoplasm and make it single stranded. Natural transformation vs laboratory transformation.

• Transformation can contribute to acquisition of AMR, new metabolic traits, and virulence associated traits

Transduction - Genes are transferred between cells in a virus. Bacteriophages move short pieces of chromosomal DNA from one bacteria to another.

• Generalized transduction - Random bacterial DNA is packaged inside a phage and
  transferred to a recipient cell

• Specialized transduction - Specific bacterial genes are packaged inside a phage and transferred to a recipient cell

Conjugation - Genes transferred from one bacterium to another through cell-cell contact (sex pilus or mating bridge). Cells must be opposite mating types (Donor cells has conjugative plasmid = F plasmid, recipient lacks it)

• F+ cells are donor cells, carry the F plasmid

• F- cells do not have F plasmid

• If conjugation successful, F- cell turns into F+ cell

• If the F plasmid integrates into donor chromosomes = Hfr cell (High frequency of recombination)

Explain the role of plasmids and other mobile genetic elements in the acquisition and dissemination of bacterial traits, including antibiotic resistance.

Extrachromosomal elements are genetic elements that exist outside chromosomes, occurring in prokaryotic and eukaryotic organisms. Includes:

• Plasmids - self-replicating circular pieces of DNA. Small, found primarily in bacteria and yeast, can carry AMR, toxins, metabolic enzymes, virulence factors, etc.

• Mobile genetic elements (transposons) - move from one chromosome to another or from one cell to another. Allows disruption of genes, and acquisition/spread of new traits through HGT.

Explain how horizontal gene transfer can contribute to the acquisition of bacterial virulence traits

HGT allows even distantly related species to share genetic materials (transduction, transformation, conjugation). Allows bacteria to acquire genes that help produce toxins, adhere to host cells, evade immune system, invade host tissues

Describe the characteristics of pathogenicity islands and explain their role in bacterial pathogenicity

PAIs are pathogenic genes clustered in one are of chromosome (builds on idea of operons). Cluster of genes that provides capabilities associated with disease

Regions containing genes that contributed virulence, host invasion, adhesion, toxin production, secretion systems, avoiding host defense

PGIs may have been acquired through HGT

Acquisition of a PAI can produce new phenotype, new interaction with host, potentially increase in virulence

Explain how bacterial pathogens regulate the expression of virulence factors in response to environmental and host-associated signals

Virulence genes are not always on, a pathogen doesn’t express every virulence factor continuously. Can respond to temp, pH, nutrients, O2, cell density, host signals, stress, location in host.

Virulence genes are tightly regulated:

Genetic info → gene → expression → protein function/product → phenotype → interaction with host → disease

Compare the mechanisms by which bacteria, fungi, protozoa, helminths, and viruses acquire, regulate, or express genetic information

Fungi - eukaryotes, DNA in nucleus, multiple linear chromosomes. Genetic variation can influence antifungal resistance, virulence, host adaptation, environmental adaptation

• Candida can undergo genetic and phenotypic changes that affect its ability to survive different host environments yeast infections

Protozoa - eukaryotes with complex life cycles. Genetic variation can influence host adaptation, antigenic variation, drug resistance

• Protozoan genetics important for understanding why parasitic infections can be difficult to control

• Plasmodium: genetic variation contributes to differences in drug susceptibility, surface antigens, parasite fitness

Helminths - multicellular eukaryotes with complex life cycles. Genetic variation can influence development, host specificity, reproduction, drug specificity, immune invasion

• Schistosoma mansonoi - blood fluke. Genetically distinct populations can differ in worm reproduction/fertility, egg production, tissue egg burden, liver and spleen pathology

Virus - Acellular with DNA/RNA. Viral genomes can change through mutation, recombination, reassortment, other.

Viruses can acquire genetic changes that affect host range, tissue tropism, antigenicity, drug susceptibility, virulence

• Influenza - segmented genome = genetic reassortment can occur when dif. influenze viruses infect same cell

Apply principles of microbial genetics to explain how genetic variation and gene regulation can influence microbial adaptation, antibiotic resistance, and disease

no.

Explain how asexual gene transfer results in prokaryotic genetic diversity

Through HGT (transformation, transduction, conjugation)

Explain the structure and consequences for bacterial genetic diversity of transposons

Transposons move within a bacterial chromosome or between a chromosome and a plasmid (allowing transfer through cells)

May carry additional genes (ex. antibiotic resistance) or disrupt a gene

Describe why regulation of operons is important

Operon regulation allows bacteria to turn on/off

• Conserves energy and resources

• Respond to envrionmental changes (temp, pH, stress, etc.)

• Maintain metabolism

Identify tools of molecular genetics that are derived from microorganisms

Restriction enzymes - cuts DNA → recombinant DNA

DNA ligase - joins DNA → recombinant DNA

Plasmids - Carry cloned DNA → genetic engineering

Transformation - Introduce new DNA into cells

DNA polymerase - Sanger sequencing

Taq. Polymerase - PCR

CRISPR - Genome editing

Describe the methods used to create recombinant DNA molecules

Recombinant DNA technology - process by which DNA sequence is manipulated in vitro, recombinant DNA then introduced into host organism making host transgenic

RE’s and Ligases:

1. Restriction enzymes cut double-stranded DNA at recognition sites forming sticky ends

2. DNA from another source is cut with the same restriction enzyme

3. Join by base pairing, fragments form a linear or circular molecules

4. DNA ligase unites the fragments producing rDNA

Plasmid Vectors: small circular DNA inserted into bacteria through environmental natural uptake or bacteriophage transduction

• Typically have a polylinker site/ multiple cloner site (MCS)

PCR: amplifies DNA sequence, primers added to amplify and join in vector, DNA fragments for recombinants DNA techniques

Describe methods used to introduce DNA into prokaryotic cells

Transformation in conjugate bacteria - take up free DNA from surroundings. Can induce this using heat shock or electroporation

Transduction - bacterial cells infected with bacteriophages to the recombinant phagemids can be introduced. May be integrated into bacterial genome (lysogeny) or exist as a plasmid in hosts cytoplasm. DNA fragments engineered into phagemids

Protoplast fusion - removing cell walls from 2 bacteria allow them to fuse; recombination may then occur between DNA derived from parent cells

• Conjugation?? - F plasmids transferred between bacterial cells through pilus/mating bridge

Electroporation - brief electrical pulses to temporarily increase permeability of cell membrane, allowing DNA to enter.

Describe the methods used to introduce DNA into eukaryotic cells

Transfection - introduction of recombinant DNA molecules into eukaryotic hosts

Electroporation - brief electrical pulses temporarily decrease membrane permeability, allowing DNA to enter

Microinjection - DNA fragments can be directly injected into cytoplasm using a glass micropipette

Gene Guns (plants) - used because plants hack thicker cell wals. Gene gun used to shoot gold or tungsten particles coated with recombinant DNA molecules into plant protoplast at high speeds.

Shuttle vectors (plants) - plasmids that can move between bacterial and eukaryotic cells

• Tumor-inducing plasmids (T_i)

Viral vectors - method often used in gene therapy to introduce healthy genes into human pts suffering from disease that result in genetic mutations.

Explain the principle of restriction fragment length polymorphism (RFLP) analysis and its uses

Compares DNA banding patterns of different DNA samples after restriction digestion

• Used to track/identigy microorganisms implicated in food poioning outbreaks, determine inheritance patterns of chromosomes with variant genes (paternity/heritable diseases), DNA fingerprinting

Explain the principles and uses of microarray analysis

Technique that capitalized on hybridization between complementary nucleic acid sequences

Useful for comparison of gene expression patterns between different cell types. Study how gene expression is affected in organisms that are infected by bacteria or viruses or subjected to certain chemical treatments

1. Isolate nucleic acid samples

2. Label with fluorescent dyes

3. Hybridize them to immobilized probed on a chip

4. Scan with laser

5. Process digital data to measure gene expression or genetic variants

Explain the method and uses of polymerase chain reaction and DNA sequencing

Permits rapid amplification in the number of copies of specific DNA sequences

Uses: determine sequence of nucleotides in specific region of DNA, amplify target region of DNA for cloning a plasmid vector, identify source of DNA sample left at crime scene, paternity test, compare samples of ancient DNA with modern organisms, determine presence of difficult to culture microorganisms in humans or environmental samples

1. Denaturing - strands separate

2. Annealing - DNA Primers bind to template

3. Extension - synthesis of new strand by Taq. polymerase

Summarize the advantages of genetically engineered pharmaceutical products

Genetic engineering allows creation of new pharmaceutical products called recombinant DNA pharmaceuticals

Includes antibiotic drugs, vaccines, hormones, etc.

Has been important in developing therapeutic proteins like insulin, human growth hormone, interferons, etc.

Allows pharmaceutical products to be manufactured more efficiently, safely, and consistently while expanding the range of medicines available to treat human disease

Summarize the mechanisms, risks, and potential benefits of gene therapy

Gene therapy attempts to correct genetic abnormalities by introducing a nonmutated, functional gene into a patients genome.

• Gene addition - introduce functional copy of a gene to replace/compensate for a faulty gene

• Gene editing - Use tols like CRISPR-Cas9 to modify specific DNA sequences

• Gene silencing - Turns off or reduces expression of a harmful gene

• Gene replacement or modification - Alters genetic instructions to cells produce a functional protein or stop producing a harmful one

Benefits: treat genetic disorders, long-lasting effects, targets underlying cause (rather than treating symptoms), personalized medicine

Risks/Limitations: Immune reactions, off-target effects, unintended genetic changes, variable effectiveness, expensive, ethical concerns

Identify ethical issues involving gene therapy and the regulatory agencies that provide oversight for clinical trials

While current research is focused on gene therapy for genetic diseases, scientists might one day apply these methods to manipulate other genetic traits not perceived as desirable.

• Which genetic traits worthy of being “corrected”

• Should gene therapy be used for cosmetic reasons or to enhance human abilities

• Should genetic manipulation be used to impart desirable traits to the unborn?

• Is everyone entitled to gene therapy, or could the cost create new forms of social inequality

• Who should be responsible for regulating and policing inappropriate use of gene therapies

Current oversights on the federal level: FDA office of human research protection (OHRP), and recombinant DNA advisory committee (RAC) at the NIH.

Compare somatic-cell and germ-line gene therapy.

Somatic cells - non-reproductive cells within the body. This is what current gene therapies typically target

Germ-line - sperm or egg, resulting traits could be passed on to offspring. Could be potentially used to combat heritable diseases, but also could lead to unintended consequences for future generations. Not currently funded by federal institutions

Explain how microbial enzymes became tools for manipulating DNA

Microbe useful biological mechanism → Identify mechanism → isolate or modify mechanism → becomes laboratory tool → tool enables new applications

Describe the role of restriction enzymes and DNA ligase in recombinant DNA technology

RE’s are endonucleases that cut DNA at specific sequences. Bacterial DNA is protected by methylation pattens, but incoming foreign DNA is cut and degraded by cell.

RE’s have a specific recognition sequence and cut at a specific place within that (ex. EcoRI). Can produce either sticky ends (ssDNA overhands) or blunt ends (no overhang)

DNA ligase - enzyme that joins breaks in DNA backbone by forming phosphodiester bond between adjacent nucleotides

• Involved in DNA replication, DNA repair, recombination, DNA maintenance

Describe how CRISPR-Cas systems can be adapted for genome editing

CRISPR systems provide a form of adaptive defense against foreign nucleic acids (particularly bacteriophages)

• Can recognize foreign genetic material, use stored sequence information, target matching nucleic acid

CRISPR-based editing can provide targeted recognition of a specific DNA sequence, making genome manipulation much more programmable

• Cas9 & gRNA produced

• Target search

• Target DNA cut

• Outcome (DNA destroyed in prokaryotes, DNA edited in adapted method)

Compare disinfectants, antiseptics, and sterilants

Sterilization (objects/equipment): Destorys/removes all microbial life, including endospores

Disinfection (nonliving surfaces): Eliminates many or all pathogens, but not necessaryily endospores

Antispetics (skin): Reduces/eliminates pathogens on living tissue

Sanitization (food/contact surfaces): Reduces microbial numbers to public health standards

Describe the principles of controlling the presence of microorganisms through sterilization and disinfection

Physical vs Chemical control methods. All of these methods have different uses in controlling microorganism.

Understand and compare various physical methods of controlling microbial growth with heating

Heat - consists of moist or dry heat

Moist heat methods: Boiling, steam, autoclave (steam and pressure), Pasteurization, High-temp hot shot (HTST). water transfers heat more efficients and is generally more effective than dry heat at the same temperature

• Boiling does not sterilize, it kills many vegetative cells but doesn’t reliably destroy endospores

• Autoclaving = sterilization, high temperature steam + pressure can destroy endospores

• Pasteurization = controlled heating to reduce harmful microorganisms and reduce spoilage while preserving food quality. Does not sterilize

• Kills by denaturing proteins, disrupting membranes, damaging nucleic acids

Dry heat methods: Flaming, incineration, Hot air sterilization

• Requires higher temperatures, longer exposures. Kills by oxidizing cellular components and damaging proteins and membranes

Understand and compare various physical methods of controlling microbial growth through cold temperatures, desiccation, lyophilization, osmotic pressure

Decreased temperature has a bacteriostatic effect

Refrigeration slows growth

Freezing shows no significant growth

Lyophilization - Freeze drying, snap frozen and placed under vacuum. A method of desiccation.

Dessication - dehydration. controls microbial growth, but doesn’t kill all microbes or endospores.

Osmotic pressure - high concentrations of salts and sugars to create hypertonic environment → causes plasmolysis

Understand and compare various physical methods of controlling microbial growth through Radiation and Filtration

Radiation: Ionizing vs Non-ionizing

• Ionizing radiation = sterilization. Includes x-rays, gamma grays, electron beams. Penetrates packaging. Causes lethal DNA damage (ds breaks).

Non-ionizing radiation = disinfection. Less energy than ionizing radiation, does not penetrate packaging. Introduces thymine dimers, leading to mutations

• UV light causes DNA damage

• Blue light produces 0 radicals, acne treatments

Filtration - Passage of liquids/gases through screenlike material. Different membrane filter sizes. HEPA filters use this. Useful for heat-sensitive materials

Understand and compare various chemicals used to control microbial growth, including their uses, advantages and disadvantages, chemical structure, and mode of action

Uses: Disinfection, antisepsis, sanitization, sometimes chemical sterilization

• Inanimate objects: Detergents, alcohols, aldehydes, phenols, halogens, Peroxygens, ethylene oxide, Chlorine dioxide

• Living tissue - Detergents, alcohols, phenols (diluted), halogens (diluted), Peroxygens (dilutes)

Alcohols ethanol, isopropanol, disrupt membranes and denature proteins. Skin antisepsis, surface disinfection. Not reliable sterilization agent, doesn’t effectively kill endospores

Aldehyde - formaldehyde, ethylene oxide. alkylating agents, disinfectant and sterilant at low temperatures.

• Ethylene oxide highly penetrating, can sterilize items within plastic bags. Useful for heat-sensitive agents

Halogens Chloride, Iodine, damage cellular components through oxidation. Disinfection

Peroxygens Hydrogen peroxide, disinfectants or antiseptics. Strong oxidizing agent that produces free radicals that cause extensive cellular damage to proteins, DNA, membranes

Bisbiguanides chlorhexidine, antiseptic. Disrupts cell membranes (low), causes cell cytoplasmic contents to congeal. Bacteriostatic at low conc., bacteriocidel at high conc.

Surfactants - Lower surface tension of water, have polar and nonpolar parts allowing to bind to dirt and wash away

Preservatives - SO2 prevents wine spoilage. Organic acids inhibit metabolism. Nitrites and nitrates prevent endospore germination, primarily used in meat products

Compare and contrast the disk-diffusion, use-dilution, and in-use methods for testing the effectiveness of antiseptics, disinfectants, and sterilants.

Disk-diffusion - applying different chemicals to separate sterile filter paper disks, then placed on bacterial lawn and zone of inhibition is measured

Use-dilution - used to determine a chemicals disinfection effectiveness on an inanimate surface. Cultures of targeted microorganisms exposed at various concentrations of disinfectant for different amounts of time.

In-use - determines whether an actively used solution is contaminated. Used disinfectant sample + sterile broth with disinfectant inactivator → plated and allowed to grow

Explain why some microorganisms are more resistant to control methods than others

Different control methods work for different microbes.

General pattern: Endospores → mycobacteria → non-enveloped viruses → fungi → vegetative bacteria → enveloped viruses

Describe how endospore, virus, protozoan, and prion levels are each controlled

Endospores are dormant structures that survive drying, radiation, boiling, chemicals, and heat. Sterilization methods = autoclaving, hydrogen peroxide vapor, ethylene oxide, chlorine dioxide, sporocides.

Protozoa - different stages of a protozoans life cycle can resist certain control methods. Treatments = filtration, CO2, UV, ozone

Viral - Viruses can be resistant to some measures, lipids in envelope sensitive to heat, drying and detergents. naked viruses are usually inactivated by chlorine-based agents

Prions - infectious proteins, eliminated through a combination of chemical treatments and autoclaving

Describe the chemotherapeutic approaches of ancient societies

Humans used substances to treat infections long before modern antimicrobial drugs

• Plant extracts and herbal preps.

• Honey and other naturally antimicrobial substances

• Moldy materials applied to wounds

• Mercury and other chemicals

• Salvarsan and other early chemical treatments

Describe the historically important individuals and events that led to the development of antimicrobial drugs

Golden age of antibiotics = 19402-1960s

Paul Ehrich (1910) = Developed Salvarsan to treat syphilis: Magic Bullet concept

• Sahachiro Hata found compound 606 targeted Treponema pallidum

Alexander Fleming (1928) = Developed penicillin from Penicillium mold. First natural antibiotic

Chain and Florey (1940s) = Purified and mass-produced penicillin during WWII

Selman Waksman = Discovered streptomycin → first antibiotic effective against TB

• Actinomycetes source of more than half of all natural antibiotics

Natural vs Semisynthetic vs synthetic antimicrobial drug

Natural - produced by microbes

Semisynthetic - chemically modified natural product. Modifications can improve stability, absorption, spectrum of activity, resistance to bacterial enzymes

Synthetic - completely manufactured (ex. sulfonamides)

Contrast bacteriostatic versus bactericidal antibacterial activities

Bacteriostatic: inhibits bacterial growth and reproduction, doesnt necessarily kill bacteria

• Bacteria → Growth → Immune system removes bacteria

Bactericial: kills bacterial cells

• Bacteria → cellular damage → bacteria cells die

Distinction is based on a drugs effect on bacteria, not whether patient gets better. Both types are effective

Contrast broad-spectrum drugs versus narrow-spectrum drugs

Broad-spectrum - Active against many types (ex. tetracyclines)

Narrow-spectrum - Active against a specific group (ex. Penicillin G)

Explain the significance of superinfections

Superinfection - a secondary infection in a patient having a preexisting infection. Develops when antibacterial intended for preexisting infection kills protective microbiota allowing another pathogen resistant to the antibacterial to proliferate and cause a secondary infection.

Ex. C. dif developed after taking antibiotics for a sinus infection

Discuss the significance of dosage and the route of administration of a drug

Dosage - amount of drug administered

• Too little may not reach effective concentration, infection may persist, can contribute to selection of resistant microbes

• Too much can cause increased risk of toxicity and side effects

Route - how drug enters body

• Oral: must survive GI tract, convenient, absorption can vary

• IV: directly into bloodstream, rapid and predictable

• Topical: Directly to skin or mucous membrane, useful for localized infections

• IM, inhaled, intrethecal

Identify factors and variables that can influence the side effects of a drug

Drug-related factors: Dose, route, duration of treatment, drug interactions

Pt-related factors: age, body size, kidney/liver function, genetics, pregnancy, other medical conditions

Microbiome: Drugs may affect beneficial microorganisms as well as pathogens

Describe the significance of positive and negative interactions between drugs

Synergistic - Drug A + Drug B = Greater effect than either drug alone

Antagonistic - Drug A + Drug B = Reduced effectiveness

Drug combinations must be selected carefully

Define antimicrobial drugs, empiric therapy, and prophylaxis

Antimicrobial drugs - therapeutic compounds that kill microbes or inhibit their growth

Empiric therapy - medical treatment begin on the basis of a clinical “educated guess” before specific causative agent is known (ex. kid with mneumonia start on antibiotics before lab results back)

Prophylaxis - process that prevents infection or disease in a person at risk (ex. antimalarials)

How are antibiotic dugs classified

By cellular targets: Cell wall, membranes, ribosomes, DNA synthesis, metabolic pathways

Major MOAs: Cell wall synthesis, protein synthesis, nucleic acid synthesis, plasma membrane integrity, metabolic pathways

For each antibiotic inhibitors of cell wall synthesis, identify: cellular target (mode of action) and whether they are bacteriostatic or bactericidal

Major classes are β-lactams, vancomycin, bacitracin (meh)

• β-lactams include penicillins, cephalosporins, carbapenems. Bactericidal, cause cell lysis. They bind transpeptidase (PBP), blocking transpeptidation step in PG cross linking. Broad range spectrum

• Vancomycin binds the terminal amino acid of pentapeptide (PG momomer D-Ala-D-ala), inhibiting the assembly of PG. Bactericidal, causes cell lysis. Exclusively gram positive. Used especially for MRSA

For each antibiotic inhibitors of bacterial protein synthesis, identify: cellular target (mode of action) and whether they are bacteriostatic or bactericidal

Bacteriostatic

Helpful because bacteria have different ribosomes than eukaryotes, allowing to specifically target bacterial ribosomes

• Aminoglycosides bind the 30S subunit, cause misreading of mRNA

  • Streptomycin

• Tetracylines bind the 30S subunit, block tRNA binding at A-site

• Macrolides bind the 50S subunit, black ribosome translocation, no peptide bond formation

  • erythromycin

For each antibiotic inhibitors of nucleic acid synthesis, identify: cellular target (mode of action) and whether they are bacteriostatic or bactericidal

Bactericidal

• Quinolones/Fluoroquinolones (Ciproflaxcin) target DNA synthesis by inhibiting bacterial DNA gyrase preventing DNA replication

• Rifamycins (Rifampin) targets RNA synthesis by binding to RNA polymerase and blocking transcription

• Metronidazole targets DNA, is activated under anaerobic conditions and damages DNA

For each antibiotic inhibitors of metabolic pathways, identify: cellular target (mode of action) and whether they are bacteriostatic or bactericidal

Broad-spectrum, bacteriostatic

• Sulfonamides and Trimethoprim inhibit steps in folic acid synthesis. Humans obtain folate from diet (dont produce) → selective toxicity

  • Synergistic effect: Combination blocks sequential steps = greater efficacy

  • Blacks folate precursors, inhibition of folic acid synthesis inhibits bacterial cell growth, folic acid needed for RNA/DNA and protein synthesis

For each antibiotic disruptors of plasma membrane, identify: cellular target (mode of action) and whether they are bacteriostatic or bactericidal

Bactericidal

Human and microbial membranes are similar → selective toxicity

• Polymyxins target cell membranes, interact with LPS in gram-negative bacteria and disrupt membrane. toxic to kidneys so mainly topical use

• Daptomycin targets cell membranes, inserts into gram positive membranes and depolarizes the cell

Explain the differences between modes of action of drugs that target fungi, protozoa, helminths, and viruses

Antifungal agents - fungi are eukaryotic so fewer selective targets. Most antifungals target fungal cell walls, plasma membranes, nucleic acid synthesis, chitin biosynthesis, microtubules, and ergosterol synthesis

• Predominant sterol in humans is cholesterol, whereas in fungi it is ergosterol, making it a good target for antifungal drug development

Antiviral drugs - treat infections but dont usually cure them. Target steps inteh viral replication pathway in order to limit tham rather than cure. Fewer options than antibiotics

Antiparasistic agents - effective ones are difficult to develop because pathogen/host are both eukaryotes and parasties have complex life cycles, so targeting one stage may not be effective during another.

• Antimalarial drugs (Quinine/Chloroquine/Primaquine increases vacuole pH disrupts membrane stability), antihelminthic drugs (Biltricide/ Praziquantel alters membrane permeability in worms; Mebendazole inhibits microtubule function in helminths), antiprotozoan drugs (metronidazole: flagyl treats intestinal/vaginal protozoa)

Explain the concept of drug resistance

Drug resistance is the reduction or loss of a medications ability to perform its function because the target organism or cell has adapted to survive it.

It is acquired either intrinsically (natural) or acquired through mutations or HGT

Describe how microorganisms develop or acquire drug resistance

4 main mechanisms:

• Efflux pumps - Microbe acquires gene product that pumps drug out of cell → drug spends little/no time in cell → resistance

• Blocked penetration → microbe acquires a gene product that prevents cell uptake of drug → drug no longer enters cell → resistance

• Inactivation by enzymes - microbe acquires an enzyme that inactivates drug → drug no longer effective → resistance

• Target modification - Microbe acquires a gene product that produces a modified drug target → drug no longer binds to target → resistance

Mechanism of Drug resistance: Inactivation by enzymes

Gene: β-lactamases → β-lactam resistance (penicillin, cephalosporins, carbapenems)

Gene: acetyltransferases → chloramphenicol resistance (Aminoglycosides, macrolides, rifamycins)

Hydrolysis of β-lactam drugs (penicillin, cephalosporins, carbapenems) by β-lactamases

Acetylation of drugs by Chloramphenicol acetyltransferase (CAT)

Mechanism of drug resistance: minimize drug concentration in cell

Blocked penetration: Carbapenem resistance in Pseudomonas aeruginosa

Efflux pumps tend to remove diverse classes of drugs, contribute to multidrug resistance

• Pseudomonas aeruginosa; azole resistance in Candida spp.

Mechanism of drug resistance: Target modification

Every antibiotic has a specific target in the bacterial cell: if the target is altered then the drug will no longer interact with target and it will have no effect.

• β-lactam drugs target transpeptidases (PBP), in resistant bacterial cells, a resistance gene mecA encodes for a different version of PBP called PBP2a. Strains that express PBP2a are resistant to all β-lactam drugs → MRSA

  • Change the target with same functionality → drug dont work

• Altered pentapeptide of PG: van genes encode an alternative version of pentapeptide that vancomycin does not recognize (D-Ala-D-Lac). Strains that express van genes are resistant to vancomycin

  • Vancomycin-resistant enterococcus (VRE) contains a terminal D-Lactate or D-serine rather than D-ala

Describe how the Kirby-Bauer disk diffusion test determines the susceptibility of a microbe to an antibacterial drug.

Kirby-Bauer disk diffusion test - zone of inhibition correlates with susceptibility

Cannot distinguish between bacteriostatic and bactericidal, differences in zone sizes cannot be used to compare drug potencies or efficacies. Only provides information on the antibacterial to which a bacterial pathogen is susceptible or resistant

Zone of inhibition determines susceptibility or resistance of the bacterial pathogen to the drug

Explain the significance of the minimal inhibitory concentration and the minimal bactericidal concentration relative to the effectiveness of an antimicrobial drug

MIC - minimum inhibitory concentration is the lowest concentration of antibiotic preventing visible growth

MBC - minimal bactericidal concentration - lowest drug concentration that kills >99.9% of the starting inoculum.

Quantitative, observe the turbidity of broth to determine.

Describe how broth dilution and Etest determine antimicrobial susceptibility

Both give quantitative values of MIC to determine antimicrobial susceptibility.

Broth dilutions observe the lowest dilution that inhibits turbidity, this dilution is the MIC (often done in ~96 well plates)

Etest is a combination of the Kirby-Bauer disk diffusion and dilution methods. A Etest strip shows a gradient of antibacterial on it, and is placed on bacterial lawn where antibiotic diffuses directly related to drug concentration (essentially adds a quantitative measure to it) and can determine MIC.

MBC cannot be determined with Etest method

Describe the methods and strategies used for discovery of new antimicrobial agents.

Not a lot of progress for developing new antimicrobial agents within the past decades (steady decline since the 80s)

iChip technology to test and grow microbes in soils, checking for anitmicrobial properties.

Bacteriophage therapy is also a new technique beign developed

Define intrinsic and acquired resistance

Intrinsic resistance is a natural resistance to antimicrobial drugs. Microbe lacks the structural target or are impermeable to the drug. Is generally not transferable

• Mycoplasma pneumoniae - lacks cell wall; intrinsic resistance to drugs that target cell wall

• C. diff - Forms endospores, dormant intrinsic protection

• GN bacteria - outer membrane is impermeable to many compounds, intrinsically resistant to daptomycin and vancomycin

Acquired resistance is the acquisition of new/altered gene that makes microbe resistant to drug. form from spontaneous mutation or HGT (transduction, transformation, conjugation)

Describe β-lactams, β-lactamase, β-lactamase inhibitor

β-lactam is a type of antibiotic including penicillin, cephalosporin, and carbapenem. β-lactam drugs target transpeptidases (PBPs) that block the joining of PG layers, disrupting synthesis of the cell wall.

β-lactamases are enzymes that a bacteria produces that hydrolyzes β-lactam drugs, therefore making them harmless to the cell

β-lactamase inhibitors are drugs that inhibit the function of β-lactamases, so they cannot hydrolyze the β-lactam drugs, and allowing the β-lactam drugs to destroy the bacterial cell membrane.

Describe the role of PBP2A, CAT, porins and efflux pumps in the development of antibiotic resistance

PBP2A is a type of transpeptidase encoded by gene mecA that bacteria may produce that β-lactam drugs do not recognize, therefore do not bind and cannot break the PG layer. Strains that express PBP2a are resistant to all β-lactam drugs.

CAT is a bacterial enzyme that acetylates drugs, leading to chloramphenicol resistance (chloramphenicol are the RNA synthesis inhibitors: Aminoglycosides, macrolides, rifamycins)

Porins with different structures (mutated) wont allow the drug in the cell, so it cant serve its function

Efflux pumps quickly pump the drug right back out, again so it cant function in the cell.

Describe the mechanisms of antibiotic resistance that are seen in biofilms

Resistance at the biofilm surface - slow diffusion of the drug through the biofilm due to the ECM: exopolysaccharide, DNA, and protein

Resistance within biofilm microenvironments - diverse microenvironments relative to metabolic byproducts, waste nutrients and low O2 levels

Resistance of bacteria persister cells - Persisters exist in a dormant state and do not divide.