Learning Objectives Final Microbio PDF

lOMoARcPSD|31356638 Learning Objectives Final Microbio General Microbiology (University of Ottawa) Scan to open on Studocu Studocu is not sponsored or endorsed by any college or university Downloaded by amelia safi ([email protected]) ...

lOMoARcPSD|31356638 Learning Objectives Final Microbio General Microbiology (University of Ottawa) Scan to open on Studocu Studocu is not sponsored or endorsed by any college or university Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 LEARNING OBJECTIVES FINAL Here are the learning objectives for the 昀椀nal exam. Use these to guide your studying. Mastering these learning objectives will help you answer questions that are more applied, but you still need to synthesize and contextualize this knowledge in the best way you can. BACTERIA Describe common cell morphologies and cellular arrangements typical of bacterial cells o Identify and name the main shapes and structures of bacteria. A typical bacterial cell contains: a cell membrane, chromosomal DNA that is concentrated in a nucleoid and extrachromosomal DNA called plasmids ribosomes, and a cell wall. Some prokaryotic cells may also possess flagella, pili, fimbriae, and capsules. Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 Compare and contrast the cell walls of gram-positive, gram-negative, and acid-fast bacteria. § The cell wall (or envelope) confers shape and rigidity to the cell and helps it withstand turgor pressure. § The bacterial cell wall (or envelope), consists of a single interlinked macromolecule.  Like a flexible mesh bag or scaffold  This mesh is made of peptidoglycan.  In prokaryotic cells, the cell wall provides some protection against changes in osmotic pressure, allowing it to maintain its shape longer.  The cell membrane is typically attached to the cell wall. - Rigid sugar protein coat - o Describe the structure, function, and biochemistry of the bacterial plasma (cytoplasmic) membrane. Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 Lipid bilayer Separatein/out Fluidity Membrane proteins Integralor peripheral  Transport  Osmosis  Energy(cell respiration)  Sensing  Secretion  Polar and charged molecules must be transported.  Transport proteins accumulate solutes against the concentra琀椀on gradient Holds transport proteins in place Genera琀椀on of proton mo琀椀ve force Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 o Describe the structure, biochemistry, and function of the bacterial cytoskeleton in comparison to the eukaryotic cytoskeleton and explain how cells maintain their morphology. Cytoskeleton: - Implicated in cell division - Shapes the bacterium - Transport (Par system) Diamter: FtsZ, tubulin like protein Length: MreB, actin-like protein Curve: Crescentin, filament like protein o Describe the function of the nucleoid and ribosomes Nucleosome: region where the genetic material and extrachromosomal plasmids are found - no membrane Origin of replication: area attached to the envelope where DNA is attached to Ribosomes: always running - in prokaryotes, translation is tightly coupled to transcription - Ribosomes are translating before transcription of mRNA is even complete o Describe the structure and function of endospores. Endospores: - Structures: o Exosporium  outermost o Spore coat o Core wall o cortex Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 o DNA - contains dipicolinic acid and is enriched in Ca2+, both form the calcium- dipicolinic acid (DPA) complex o Helps cope with dehydration and stabilze DNA - SASPS  bind and protect DNA and function as carbon and energy source for outgrowth o “Small acid-soluble spore proteins” - Core contains cytoplasmic membrane, cytoplasm, ribosomes and other cellular essentials for germination Function: highly differentiated cells resistant to heat, harsh chemicals, and radiation - Endure unfavourable growth conditions - “Dormant” stage of life cycle - Present in only some gram positive bacteria (bacillus and clostridium) o None in Archaea o Di昀昀erentiate between 昀氀agella, 昀椀mbriae, and pili. Fimbriae: stick to surfaces or form pellicles (thin sheets of cells on a liquid surface) Pili: Longer than fimbriae, fewer found per cell than fimbriae - Conjugative sex pili  facilitate genetic exchange between cells Flagella: Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 Identify structures unique to bacteria. Thylakoids  extensively folded intracellular membranes found in photosynthetic bacteria Carboxysomes  polyhedral bodies packed with the enzyme rubisco for CO2 昀椀xation Gas vesicles  Protein bound gas 昀椀lled structures that increase buoyancy (storage) Pili  attachment and/or sex conjugation for exchange of DNA Stalks and holdfasts  membrane embedded extensions of the cytoplasm that attach bacteria to a surface o Tips secrete adhesion factors called holdfasts o Asymmetric o Mix bacteria in place  Fimbriae is a rough attachment (do not confuse) Inclusions: Energy reserves, carbon reservoirs, or have special functions - Enclosed by a thin membrane - Reduces osmotic stress - Carbon storage polymers o Glycogen o poly-β-hydroxybutyric acid (PHB): lipid polymer, stored as lipid droplets. Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 Rotary Flagella Endospores Nanotubes ARCHAEA  Explain why archaea are considered a separate domain of life Phylogenetic tree was originally based on prokaryotes and eukaryotes Carl Woese - Sequenced 16s ribosomal RNA and transcriptional machinery of a halophile DNA to compare to eukaryea and bacteria o Enzymes for replicating the genetic code of archaea are like humans and dissimilar to bacteria. Conclusion  they are like microbes but the enzymes they use for replication/transcription translation are more similar to humans - We are the direct descendants of archaea - Only archaea are methanogens  3rd domain of life o Many differences between bacteria and archaea = different tree of life  Describe internal and external structures of archaeal cells in terms of their physical/chemical structure and function o General shapes and characteristics of archaea - Size 0-5 – 5um (similar to bacteria) - No nucleus (similar to bacteria) - Similar shapes to Bacteria/Eukarya - o Cell wall structure - Psuedomurein (NO peptidoglycan layer) o NAG and NAT o NAG and NAT are linked via Beta 1-3 glycosidic bonds  Lysozymes in our tears would not affect these archaea Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638  Affects Beta1-4 link only  Makes them resistant to certain antibiotics that target only the cell wall structure of bacteria o L-Stereo isomers only in peptide chains (L and D isomers in bacteria) o S layer (glycoprotein) present  Resist changes in osmotic pressure  In addition to the pseudomurin Note*** Cell walls of some archaea lack pseuomurein and use a different polysaccharide polymer OR have only an S layer o Plasma (cytoplasmic) membrane structure Archaeal cytoplasmic membranes have different lipid constituents and chemistry but are structurally similar Structure of Plasma Bacteria Archaea membrane Phospholipid Head Fatty acid Isoprene chains - Form linkages Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 between chains to form a monolayer Linkages Ester linkages (and in Ether linkages Eukaryotes) o Archaeal cytoskeleton: comprised of eukaryote and bacteria homologs - Tubulin (Eukarya) and FtsZ (bacteria) are homologous to TubZ in archaea - Crenatin (actin like filament unique to archaea) - ESCRT no homologous, completely different system that exists in archaea and is important for cell division Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 o Archaella and hami Hami: Archaeal “grappling hooks” assisting in surface attachment, forming biofilms Archaella: - Simpler than bacterial or eukaryotic flagella - Uses ATP (see phototrophic mechanism) rather then the proton motive force as an energy source - Rotating flagella o Eukaryotes have whip-like flagella  Identify and describe structures, metabolic pathways unique to archaeal cells Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 Similarities: - DNA/RNA machinery (DNAP/RNAP) is shared with eukaryotes - Gene regulation shared with bacteria Unique differences: 1. Plasma membrane  Ether-linked membrane lipids (covered above) 2. Methanogens 3. Retinal associated light driven membrane pumps for H+ or Na+ 4. Nucleic acid structure and function Methanogens: - Only known organisms to produce methane - Chemolithotrophy  using chemicals to create ATP - Chemoorganotrophy  uses simple organic molecules to create ATP Retinal-Based Photoheterotrophy: - Most haloarchaea are photoheterotrophs - Supplement their utilization of organic substrate energy by using light-driven ion pumps - Do not fix carbon (no photosynthesis) - Do not produce O 2 o Respire with O2 or anaerobically with nitrate Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 Terms: Bacteriorhodopsin  H+ in (archaea only) o Contains retinal  captures light  Ionized retinal, trans  cis Halorhodopsin  Cl- in to cell (archaea only, halo) o Allowing Cl- in creates negative potential inside which powers ATP synthase to create ATP o Creates a proton motive force: negative potential draws protons inside, increasing ATP synthesis Sensory Rhodopsin 1  Red light (halo) o Hrt1 signal transducer to histidine-kinases o Rotate and swim ahead Sensory Rhodopsin 2  Blue light (halo) o Hrt2 signal transducer to histidine-kinases o Tumble and change direction Goal: Phosphorylate a protein that regulates the direction of rotation of the flagellar motor o Why move? To orient toward light that will activate the rhodopsins (bacterio and halorhod) to produce ATP, while staying away from harsh UV light that may kill it Nucleic Acid structure and function: unique histones and reverse gyrase - Singular, circular chromosomes (like bacteria) - Gene size and density similar to bacteria - Complexed with histones (like eukaryotes), bacteria do not have histones in their DNA - DNA replication enzymes look like eukarya - tRNA genes are interrupted by introns (similar to eukarya, bacteria do not have introns) Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 Reverse Gyrase in Archaea similar to topoisomerase in eukaryotes, unique to archaea - Creates positive supercoils in chromosomal DNA which helps protect it against high temperatures Histones in Archaea  form tetramers histones - Eukarya form octamers - Histone tails can be modified for epigenetic purposes - Nucleosome (stacking of histones) structure allow the formation of superhelices Genome of Archaea - Homolgy to both eukarya genes (transcription/translation) and bacteria (central metabolic pathways and cell division) - 40% of genome with no homology to any living organism - ARCHAEOSINE = guanosine analog, only found in archaea Gene structure of Archaea: more similar to eukarya - Contain similar promotors o TATA box o BRE = B- recognition element o TFB  archaeal transcription factor B o TBP  TATA bidning protein - Require a pre-transcription initiation complex Gene regulation: similar to bacteria - Regulatory units are closer to the DNA vs far away in eukaryotes - RNA pol II structure similar to that of eukaryotes - Multiple regulatory elements (binding sites) direct gene expression Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 - Activators and repressors resemble = bacterial DNA binding proteins than eukaryotic transcription factors  Compare the distinguishing characteristics of bacterial, archaeal, and eukaryotic cells.  Inclusion bodies (like bacteria) gas vacuoles for carbon storage  Protein content (similar to bacteria, ribosomes, enyzymes)  Cytoskeleton (similar to eukarya and prokaryotes)  Do not form spores  No nucleus  nucleoid VIRUSES  Describe the structures and composition of viruses  Common structures among all:  Genome  Protein shell (Capsid)  Lack metabolism/replicating themselves; nonliving Size:  from 0.02 to 0.3 µm  Range between very small to large Structure:  Capsid  protein shell that surrounds the genome of a virus particle  Naked virus  no other layers  Nucleocapsid only  Enveloped viruses (e.g., many animal viruses) have an outer layer consisting of a phospholipid bilayer (from host cell membrane) and viral proteins. Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638  Genomes: vary  DNA or RNA  ssDNA or dsDNA  Linear, circular or segmented  Di昀昀erentiate among bacteriophages and animal viruses  Bacteriophages  infect bacteria  Prophage  integrates into bacterial host’s genome  Animal virus (humans)   RNA and retrotransposons  Human genome  old viral DNA  Provirus  integrated viral genome into host human cell  Describe the general characteristics of viral life cycles and its di昀昀erent phases   Attachment  what proteins recognize each other between the irus and the host cell  Penetration: injection of nucleic acids  Virus itself never enters the cell, just the nucleic acids Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638  Synthesis  of virus nucleic acid and protein by host cell metabolism as redirected by virus  assembly of capsids and packaging of viral genomes into new virions  release of mature virions from host cell  Or lsysogenic (hiding)  Intracellular replication complex: Within a host cell, the viral gene products direct the cell’s enzymes to assemble progeny virions at “virus factories” called replication complexes.  Viral genome integrated within host DNA  Some types of viral genomes may integrate within a host chromosome as a prophage or provirus. This may be a permanent condition.  Describe the mechanism of lytic and lysogenic life cycles of bacteriophages  Lytic cycle   Lysogenic cycle Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638   Bacteriophages exhibit two di昀昀erent types of replication cycles: 1. Lytic cycle 2. Lysogenic cycle (HIDING)  Can become lytic depending on the environmental cues (stress, starvation) may cause the prophage to excise out of the bacterial genome and start the lytic cycle  Describe how phages, using T4 as an example, deliver DNA into its host Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 1. Virions attach to cells via tail 昀椀bers that interact with polysaccharides on E. coli LPS layer. 2. Tail 昀椀bers retract, and tail pins contact cell wall. 3. T4 lysozyme forms small pore in peptidoglycan. 4. Tail sheath contracts, and viral DNA passes into cytoplasm. 5. Capsid stays outside.  Explain the di昀昀erence between a virulent and temperate virus Virulent: Viruses always lyse and kill host after infection. Temperate: Viruses replicate their genomes in tandem with host genome and without killing host, establishing long- term, stable relationship. can be lytic/virulent can enter lysogeny: most viral genes are not transcribed, viral genome is replicated with host chromosome and passed to daughter cells lysogen: host cell that harbors temperate virus can result in lysogenic conversion with new properties (e.g., virulence in pathogens) Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 Viral DNA can be integrated into host DNA  Stay dormant instead a population of cells and they replicate YOUR DNA  Describe the several mechanisms of defense bacteria have against viral infections o Genetic resistance o Restriction endonucleases o CRISPR o Genetic resistance  variants as they’re being replicated in a host, mutate randomly creating slight di昀昀erences in their bacterial cell biology - Bacteria changes cell surface recognized by phage - Phage adapts to recognize new surface molecule - Bacteria can produce a molecule that masks the phage target o Restriction endonucleases  recognition of a palindromic sequence, bacteria use them to cleave DNA lacking methylation - Phage can change is nucleic acids to prevent enzymes that would otherwise degrade it (endonucleases) o Bacteria and phage go back and forth Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 o CRISPR  adaptive immune system o Clustered Regularly Interspaced Short Palindromic Repeats o Collect pieces of DNA from a virus and have a way to recognize them if the bacteria are infected again  Genetic record of past infections by a virus  Cuts piece of viral DNA piece (PAM sequence) and inserts it into the bacteria genome  “spacer” Evolution of Viruses to avoid CRISPR: mutation of PAM regions production of Cas inhibitors  Describe the 3 mechanisms of entry utilized by animal viruses Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 1. Host attachment 2. Viron entry (di昀昀erent, depends if naked/envelope acquired) 3. Uncoating (unique to animal viruses) 4. Gene expression (di昀昀erent, depends on genome type) a. Replication takes plae in the nucleus since animals are eukaryotes 5. Virion Assembly a. Migrate toward cell surface 6. Viron Release a. Some may kill the cell (lytic) or be released and NOT kill the cell Tropism  ability to infect a particular tissue type within a host - Some viruses can only e昀昀ect epithelial cells o Ex/ For example, Ebola virus exhibits broad tropism by infecting many kinds of host tissues, whereas papillomavirus shows tropism for only epithelial tissues. Entry mechanisms: (as compared to phages that only inject DNA) A. Endocytosis of non-enveloped virus B. Membrane fusion of an enveloped virus C. Endocytosis of an enveloped virus Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 Lytic cycle Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 - Uncoating  DOES NOT HAPPEN IN PHAGES (DNA injection only for phages) - Release  exocytosis/budding - Di昀昀erent types of genome of a virus change replication cycle (utilize di昀昀erent host machinery/enzymes) o DNA viruses o RNA viruses o Retroviruses Lysogenic cycle in Animal cells” Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 - Provirus o Make tons of virus or stay in genome o Dormant, do not release any viruses (still have HIV)  Describe the replication process of animal viruses (what is particular to animal infections) Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638  Explain what viral tropism is.  Describe unique characteristics of retroviruses and latent viruses. - Reterovirus  carry an enzyme needed to copy their genome into complimentary DNA o Reverse transcriptase + mRNA (- strand)  Can start copying mRNA into DNA right away  T cells only that carry the CD4 proteins 1. The adhesion of the virus gp120 envelop protein to the cell surface marker CD4 (only present on T helper cells) and its coreceptor is mandatory for infec琀椀on. 2. The viral envelop fuses with the lipid bilayer of the host cell, the viral capsid is delivered in the cytoplasm. 3. The capsid is digested by proteases and the ssRNA genome delivered into to the nucleus with the reverse transcriptase enzyme necessary to convert ssRNA in cDNA which can then be converted to dsDNA by the host cell. 4. An integrase, also delivered with the virus, helps the transduc琀椀on of the virus into the host genome. 5. New viral mRNA and proteins are produced by the host cells, virions assemble within the cytoplasm and envelop proteins are integrated into the host plasma membrane. 6. Encapsulated new viral par琀椀cles exocytose, the vesicle with which they exocytose forms the new envelop, the virions are mature and can infect new cells. MICROBIAL METABOLISM  De昀椀ne what is metabolism and how it operates on a planetary level (birds eye view)  The local, temporary gain of energy enables the cell to grow (if you have energy, you can do work/processes to grow)  Growth requires continual gain of energy and continual radiation of heat – this is true for the entire biosphere  On Earth, the total metabolism of all life-forms must ultimately dissipate most energy as heat Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 EARTH ITSELF IS A HEAT REACTOR BECAUSE ALL ORGANISMS CONTINUOUSLY GENERATE AND RELEASE HEAT (growing)  The primary source for energy on the planet is the sun  Energy from the sun is taken by photosynthetic organisms.  Create complex molecules that heterotrophs can use to power metabolism  Decomposers can decompose the complex molecules that heterotrophs and photosynthetic organisms create  All energy forms on earth release heat (obeys the law of thermodynamics)  Di昀昀erentiate between catabolism and anabolism  Catabolism: breakdown of complex molecules into smaller ones  Provides energy for anabolism  Some energy is released as heat  Anabolism: reactions that build molecules  Put energy into a system to assemble smaller molecule into larger ones  Against thermodynamic law to create a larger molecule without energy  Describe the roles of ATP, NAD+, and other electron carriers in metabolism and explain why they are so important in a cell  Many of the cell’s energy transfer reactions involve energy carriers:  Molecules that gain or release small amounts of energy in reversible reactions  NADH, ATP, PEP Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638  These energy carriers also transfer electrons  Electron donor: a reducing agent  Electron acceptor: an oxidizing agent  Compare and contrast autotrophs and heterotrophs, chemotroph, phototroph, organotroph, lithotroph and organotroph (and combinations of). Distinguish these terms regarding energy source, carbon source, electron source. Energy source: - Photo  light absorption captures energy - Chemo  chemical reactions yield energy without absorbing light Electron Source/Electron acceptors: - Litho  inorganic molecules donate electrons o Ex/ using a non-organic compound to use its electrons to power its metabolism - Organo  organic molecules donate electrons Carbon source for biomass: - Auto  make your own: CO2 is fixed and assembled into organic molecules - Hetero  outsourced: Outsourced organic molecules are acquired and assembled into new organic molecules - - Chemo = using chemical reactions as an ENERGY source - Litho = using INORGANIC molecules as an electron donor - Autotroph = carbon source is fixed into organic molecules - Photoheterotrophy vs photoautotrophy Source of Source of Source of Electron Carbon energy electrons acceptor PhotoHETEROtrophy Organic Sun Organic H2 Compounds (outsourced) PhotoAUTOtrophy Fixing CO2 Sun Inorganic H2 - Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 - ***Photoautotrophs need to fix CO2/decompose it before entering a central metabolism pathway. They need a different source of electrons as compared to photoHETEROtrophs who have their carbon source and electron source all-in-one ready to go (no fixation) -  Name sources of carbon, energy, and electrons (2 each)  Sources of carbon: organic compounds (polysaccharides, lipids, carbohydrates), CO2  Sources of energy: light, chemical bonds  Sources of electrons: organic compounds; inorganic compounds like thiosulfate; Fe+2  Compare and contrast the main steps of cell respiration in a prokaryotic cell and a eukaryotic cell o from glycolysis to pyruvate, pyruvate to acetyl-coA, Krebs cycle, ETC and ATP production Eukaryotic cell respiration: Prokaryotic Cell respiration: Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 Cell respiration = glycolysis  pyruvate  acetyl-coA  Kreb’s cycle  ETC  ATP production - Glycolysis : o In cytoplasm for both prokaryotic and eukaryotic cells - Conversion of pyruvate to acetyl-coa: o In cytoplasm for prokaryotes o In mitochondria for eukaryotic cells - Kreb’s Cycle (Citric Acid Cycle) o in cytoplasm for prokaryotes o in mitochondria for eukaryotes - ETC and ATP production: o On cell membrane (plasma membrane) for prokaryotes o In mitochondria for eukaryotes - Heterotrophy  Energy via catabolism of chemical bonds of an organic molecule  ETC  proton motive force  ATP - Phototroph  Energy via the sun  ETC  Proton motive force  ATP  Explain what the proton-motive force is - Generated as protons are pumped across the inner mitochondrial membrane during the ETC; important for ATP synthesis, active transport, flagellar rotation  Describe the function and location of ATP synthase in a prokaryotic versus eukaryotic cell  Function = synthesis of ATP by using the energy generated by the proton motive force  Location:  cell membrane (plasma membrane) in prokaryotes  mitochondria (inner mitochondrial membrane) in eukaryotes  Compare and contrast aerobic and anaerobic respiration (in principle, not metabolic details) - Aerobic respiration: o Final electron acceptor in ETC = O2 o Can only occur in presence of oxygen o Produces a bunch of ATP - Anaerobic respiration: o Final electron acceptor = NO3-, SO4 -2, etc. o Doesn’t require oxygen  Explain what fermentation is and why it does not require oxygen - Glycolysis  breaks down glucose into two molecules of pyruvate and forming NADH Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 - Then NAD+ regeneration - No ETC, no terminal electron acceptor - It does not require oxygen bc it does not rely on the presence of external electron acceptor  it uses internal molecules like pyruvate (byproduct of glycolysis) as electron acceptors Fermentation: is the completion of catabolism without the electron transport system and an exogenous terminal electron acceptor. The hydrogens from NADH + H+ are transferred back onto the products of pyruvate, forming partly oxidized fermentation products.  Most fermentations do not generate ATP beyond that produced by substrate-level phosphorylation. Microbes compensate for the low efficiency of fermentation by consuming large quantities of substrate and excreting large quantities of products  Compare and contrast fermentation and anaerobic respiration (in principle, not metabolic details) - both occur in the absence of oxygen as final electron acceptor; NAD+ regeneration in both - fermentation: Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 o final electron acceptor: internal, typically organic molecule like pyruvate (byproduct of glycolysis)  allows NADH to be oxidized back to NAD+ - anaerobic respiration: o final electron acceptor = SO4 -2, NO3-, S0 o uses specific electron acceptors o uses ETC  proton motive force  ATP production - fermentation produces less ATP than anaerobic respiration MICROBIAL GROWTH  Explain what binary 昀椀ssion is.  Cell division following enlargement of a cell to twice its minimum size  Describe how nutrients are transported into cells o Simple transporters, ABC system 1) Simple diffusion – no channels necessary 2) Passive transport: channel-mediated or carrier-mediated 3) Active transport: against concentration gradient, energy required for movement (ATP, PMF, or PEP), carrier protein used a. ABC transporters: i. Gram – use periplasmic binding proteins ii. Gram + and archea use substrate-binding proteins on external surface of cytoplasmic membrane iii. Uptake ABC transporters = transport nutrients iv. Efflux ABC transporters = multidrug efflux pumps (pumping solutes out of the cell)  Describe the di昀昀erent growth phases in a batch culture  1) Lag phase = no increase in # of living bacterial cells  2) log phase = exponential increase in # of cells  3) stationary phase = plateau; rate of cell division = death rate  4) death/decline phase = exponential decrease in # of cells Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638  Di昀昀erentiate between sessile growth, planktonic growth, bio昀椀lms, and cell mats  Planktonic growth = growth as suspension  Sessile growth = attached to surface; can develop into biofilms  Biofilms = attached polysaccharide matrix that contains embedded bacteria  Stages: planktonic cells attach together  sticky matrix forms  Microbial mats = multilayered sheets with different organisms in each layer (e.g., hot springs)  Describe di昀昀erent categories of microbes with temperature requirements for growth: psychrophile, mesophile, thermophile, hyperthermophile.  Psychrophile: low T, found in cold environments  Mesophile: midrange, most commonly studied  Thermophile: high, found in hot environments  Hyperthermophile: very high, found in extremely hot habitats like hot springs and deep-sea hydrothermal vents Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638  Describe the di昀昀erent categories of microbes with pH requirements for growth: acidophiles, neutrophiles, and alkaliphiles.  Acidophiles:  grow at pH 0-5  chemoautotrophs  Neutrophiles:  Grow at pH 5-8  pathogens  Alkaliphiles:  Grow at pH 9-11  Found in soda lakes  Describe di昀昀erent categories of microbes with requirements for growth with or without oxygen: obligate aerobe, obligate anaerobe, facultative anaerobe, aerotolerant anaerobe, microaerophile, and what is the di昀케culty encountered when growing in these environments.  Strict aerobe = can only grow in oxygen  Microaerophile = grow only at low O2 levels  Strict anaerobe = die in the least bit of oxygen  Aerotolerant anaerobes = grow in oxygen while retaining a fermentation-based metabolism  Facultative anaerobe = can live with or without oxygen  Can do fermentative metabolism and respiration (anaerobic and aerobic) Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638  Aerobes use O2 as terminal electron acceptor to extract energy from nutrients  Difficulty = ROS (breakdown products of O2) which damage DNA, RNA, proteins and lipids  need to have the proper enzymes to destroy ROS  Explain how oxygen can be toxic and how oxygenic organisms cope with ROS.  Toxic to anaerobes and to all cells that do not have the enzymes capable of efficiently destroying the breakdown products of oxygen  ROS  seriously damage DNA, RNA, proteins and lipids by stripping them of electrons  Anaerobes avoid oxygen altogether to prevent this  Describe di昀昀erent categories of microbes with speci昀椀c growth requirements such as altered salinity (halophiles), osmotic pressure (osmophiles) or dry environments (xerophiles).  Osmophiles: organisms that live in environments high in sugar as solute  Xerophiles: organisms able to grow in very dry environments  Lowest water activity = 0.61; physiochemical constraints on obtaining water at lower water activity  Halophiles: organisms that grow best at water activity of 0.98 (seawater); have a specific requirement for NaCl  Halotolerant: can tolerate some additional dissolved solutes but grow best without the added solute  Extreme halophiles: require very high levels (15-30%) of NaCl; unable to grow at lower concentrations Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 Biofilms  Explain what a bio昀椀lm is  Specialized, surface-attached communities; one or many species; can form on organic/inorganic surfaces  Are hardy (difficult to dislodge)  Protect bacteria (eg. Prevent chemicals from penetrating)  Describe the formation, biochemistry, and characteristics of bio昀椀lms in general.  Form when nutrients are plentiful (once scarce  individuals detach from community to look for new sources)  Quorum sensing = chemical signals that enable the bacteria to communicate and form biofilms  Biofilm development: adherence of cells to substrate  formation of microcolonies  formation of complex channeled communities that generate new planktonic cells Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638  Bacteria growing in biofilms have cell differentiation bc of physiological conditions developing in different layers of biofilm  Cells can perform different functions (like greater motility while others sporulate)  Changes in genetic expression  changing from planktonic state to sessile state for instance  Molecular constitution (can vary even in one species)  EPS  Polysaccharides in Gram+  Cellulose in Gram-  Proteins (type IV pili)  Extracellular DNA (can be released extracellularly; - charge  protects from antimicrobials that are +; sticky so helps structure and coherence; release favours exchange of genetic material within biofilm communities; source of nutrients in low levels of nutrients conditions)  Describe the mechanism of bio昀椀lm formation for P.aeruginosa  P. aeruginosa:  Quorum sensing  c-di-GMP expression  Accumulation of c-di-GMP  binds proteins that reduce activity of flagellar motor, regulates attachment proteins, mediates biosynthesis of extracellular matrix polysaccharides  Bacterial population is dense  increase in c-di-GMP  increase in biofilm formation  Accumulation of AHLs indicates population is growing  c-di-GMP synthesis  extracellular polysaccharide production; decreased flagellar function  Identify health and environmental risks associated with bio昀椀lms, what causes these risks and how they can be addressed. Biofilms  chronic infections; difficult to treat; due to biofilm presence on mucosa or biofilm formation on medical devices (urinary catheter eg) Environmental risks: form on ship hulls, pipelines  damage to infrastructure and increased energy consumption bc don’t work as well Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 Also corrosion of metals/concrete  degradation of infrastructure Water contamination bc of pathogenic organisms  What can we do?  Design resistant materials to biofilm formation or coat them with molecules that prevent formation  Quorum quenching (inhibit the signals that stimulate formation); inhibit receptors of quorum sensing molecules MICROBIAL REGULATORY SYSTEMS Quorum sensing  Explain what quorum sensing is.  Cell-cell communica琀椀on; allows bacteria to assess the popula琀椀on density; allows them to sense and respond to changes in their local popula琀椀on density by detec琀椀ng the concentra琀椀on of autoinducers (signaling molecules)  facilitates coordinated group behaviours; regulates genes that are useful only when bacteria is in a community/group  Explain the roles and advantages in cell-to-cell communication and coordination of cellular activities through quorum sensing (see above)  Explain what is an autoinducer  membrane-permeable second messenger that allows cells to regulate gene expression based on the density of cells in the environment.  Describe molecular mechanisms of quorum sensing in Gram – and Gram + bacteria (use one example for each).  Explain what quorum quenching is and why it could bene昀椀t us clinically (3 examples)  Discuss what could be drawbacks (if any) in using quorum quenching techniques for clinical bene昀椀ts. Spatial organization  Explain the role of each of these structural proteins: MreB, FtsZ, Crescentin and FilP.  Describe molecular mechanisms that bacterial cells use to maintain their morphology Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 (MreB, FtsZ, Crescentin and FilP)  Explain what cell polarity is  Describe two functional examples of bacterial cell polarity or spatial patterning: one example must be symmetric and the other asymmetric. Regulatory systems  Describe the di昀昀erence between negative and positive gene regulation in prokaryotes  Nega琀椀ve regula琀椀on: repression in which the repressor (form of regulator) binds to the operator site in front of the promoter site  Polymerase binds to promoter site  When we remove repressor, transcrip琀椀on proceeds as usual  Ac琀椀vator binds to ac琀椀vator binding site; behind the promoter  Compare and contrast di昀昀erences and similarities in the transcriptional control mechanisms between bacteria, archaea and eukarya.  Archaea and Bacteria use ac琀椀vator and repressor proteins, whereas Eukarya uses chroma琀椀n modi昀椀ers and transcrip琀椀on factors  Bacterial RNA polymerase is a holoenzyme (formed of mul琀椀ple proteins) o One of these proteins = sigma factor  helps RNA polymerase bind to certain DNA sequences and adds speci昀椀city to the RNA polymerase Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 o Housekeeping (principle) sigma factors = responsible to direct RNA polymerase to genes that are always expressed (tRNA, cytoskeletal proteins, ETC, etc.) o Alterna琀椀ve sigma factors: direct RNA polymerase to transcribe other genes  depends on sigma factor   Ground state in prokaryotes is on bc genes are generally expressed  O昀昀 in eukaryotes bc genes not expressed generally  Compare and contrast di昀昀erences in the genetic and genomic structures between bacterial, archaeal and eukarial cells.    Describe the structure of an operon and the general mechanisms by which operons are regulated (keep it general) Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638  Repressor binds to operator to stop transcrip琀椀on  The structural genes here code for 3 di昀昀erent proteins  Ac琀椀vator binds to ac琀椀vator binding site  Polymerase binds to promoter  Inducible operon: environment with lots of glucose  normal state is o昀昀 bc no need to produce lac enzyme which digests lactose  Induced state: lots of lactose in environment, not much glucose  can induce enzyme if needed  produces enzyme that digests lactose  Repressible operon: normal state is ON (eg. Always on to make arginine; turned OFF when there is a lot of Arg in environment)  Repressed state: usually turned on, but can be repressed if plenty of Arg in environment  Environment  gene琀椀c control  phenotype  Describe what is a two-component system.  Changes in extracellular condi琀椀ons are detected with plasma membrane-embedded signaling molecules that tell the cell what is happening outside  Two component signal transduc琀椀on systems: each system regulates a di昀昀erent set of genes or proteins  Two-component systems are based on protein phosphoryla琀椀on:  Sensor kinase = membrane-bound enzyme  binds environmental signal  causes it to transfer a phosphate group from ATP to itself or to another target protein Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638  Response regulator = cytosolic protein  S琀椀mulated by ac琀椀vated sensor kinase  binds operator and alters gene expression  Controlled by covalent modi昀椀ca琀椀ons instead of ligands   Explain how a two-component system functions (mechanism) using an example  Sensor kinase detects condi琀椀on outside the cell (environmental signal)  signal triggers autophosphoryla琀椀on  phosphate is transferred to a response regulator in the cytoplasm  response regulator binds DNA (the operator sequence)  s琀椀mulates or represses target genes.  Describe how chemotaxis is regulated in E. coli with Che proteins when presented with the name of each protein. (If asked to describe the regulatory process, I would provide at minimum the name of the proteins in play or most likely, a schematic representation of the signal transduction that is NOT the one you have seen in your slides)  Apply your understanding of regulatory mechanisms in bacteria. For example, this means I could present you with a completely new Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 regulatory pathway and ask you questions on that pathway that would demonstrate you can transfer knowledge from a general conceptual understanding of regulatory mechanisms to a new situation.  Explain what the stringent response is and what is its purpose. Important prokaryo琀椀c second messengers:  ppGpp = 2nd messenger that allows cells to handle abrupt changes in nutrient availability by switching from rapid growth to slower growth  stringent response: nutrient scarcity leaves many ribosomes idle.  These ribosomes:  Trigger synthesis of ppGpp  binds to RNA polymerase  lowers its ability to transcribe rRNA (ribosomal RNA) genes  decrease in rRNA transcripts made for ribosome assembly  fewer ribosomes and overall decrease in growth  Stringent response shuts down macromolecule synthesis and ac琀椀vates stress survival pathways; controls adapta琀椀on to nutrient depriva琀椀on  Reduc琀椀on of amino acids in nutrients  stringent response launched  slows down growth un琀椀l bacteria can adapt to their new condi琀椀ons  Alarmones are produced (pppGpp and ppGpp)  slow growth, inhibit cell division and reduce the energy (↓GTP/ATP) available for biosynthesis. Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638   Shi昀琀 down = reduc琀椀on of amino acids in nutrients  If certain or all amino acids become limited, tRNA stay empty and eventually, uncharged tRNAs block protein synthesis by stalling the ribosome.  RelA = protein associated to the ribosome.  When ribosomes stall (a signal of stress)  RelA starts producing alarmones.  Alarmones = ppGpp and pppGpp:  Decrease rRNA, tRNA synthesis by directly inhibi琀椀ng RNA pol transcrip琀椀on  Ac琀椀va琀椀on of biosynthe琀椀c pathways to produce missing amino acids  Cell division arrest  Ac琀椀va琀椀on of stress survival pathway  If condi琀椀ons get be琀琀er, tRNA and rRNA synthesis and ribosome produc琀椀on slowly restart  Side note: Most microorganisms produce alarmones (pppGpp or ppGpp) as second messenger, but the produc琀椀on of alarmones may be s琀椀mulated by di昀昀erent signals (like SpoT) and produce di昀昀erent stress responses (oxida琀椀ve stress survival, oxida琀椀ve stress survival, osmo琀椀c stress survival, etc.)  Describe one example of a stringent responses due to environmental conditions.  E.coli transi琀椀on from nutrient-rich mammalian colon to nutrient-poor soil or aqua琀椀c environments  In colon: low (p)ppGpp levels because enough nutrients (also, cells are large and contain mul琀椀ple replica琀椀on forks) Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638  Outside colon: high (p)ppGpp levels because not enough nutrients (also, cells are smaller and contain either one or zero replica琀椀on forks)  Describe what is a general global network response  Global control systems: regulate the expression of many di昀昀erent genes simultaneously (e.g., lactose operon and maltose regulon).  Catabolite repression is an example of global control:  controls use of carbon sources if more than one present  Synthesis of unrelated catabolic enzymes (e.g., lactose operon and maltose regulon) is repressed if glucose is present in growth medium.  ensures that the "best" carbon and energy source is used 昀椀rst  RpoS is a sigma factor that control a stress response.  Act on mul琀椀ple operons and genes  Explain what a regulon is.  Some regulatory proteins can control the transcrip琀椀on of mul琀椀ple operons  Regulatory protein = ac琀椀vators, repressors, alterna琀椀ve sigma factors  Regulon = collec琀椀on of coregulated operons (controlled by the same regulatory protein) that are sca琀琀ered around the bacterial chromosome  Coordina琀椀ng the regula琀椀on of mul琀椀ple operons of di昀昀erent func琀椀ons allows the cell to adjust gene expression according to a par琀椀cular environmental condi琀椀on.  Explain what a stress-response is and what is its purpose.  Purpose = transcrip琀椀on/ac琀椀va琀椀on of stress genes - An琀椀-sigma factor is normally bound to sigma factor and inhibits it - During stress, an琀椀-sigma factor binds more strongly to ac琀椀ve PhyR and releases sigma factor Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 - Sigma factor can now bind to RNA polymerase holoenzyme and direct transcrip琀椀on of stress genes. - Also possible: an琀椀-sigma factor inhibits sigma factor from binding to RNA polymerase - Unstressed cells: PhyR is inac琀椀ve, sigma factor inhibited by an琀椀-sigma factor - Stress  PhyR is phosphorylated and interacts with an琀椀 sigma factor  releases sigma factor  sigma factor binds to RNA pol. to transcribe stress genes  Explain the concepts of persistence and dormancy in bacteria  Persistence: popula琀椀on of an琀椀bio琀椀c- sensi琀椀ve bacteria produces rare cells that are transiently tolerant tomul琀椀ple an琀椀bio琀椀cs.  Persisters = gene琀椀cally iden琀椀cal but dormant (viable but do not grow)  Dormancy prevents an琀椀bio琀椀c from killing cell  When an琀椀bio琀椀c treatment stopped  cells emerge from dormancy and grow  Cause of recurring M. tuberculosis and P. aeruginosa infec琀椀ons  Bacterial persisters defy an琀椀bio琀椀c treatments  Persister cells = phenotypic variants of regularly growing bacteria; survive lethal an琀椀bio琀椀c treatment in a nongrowing, dormant state  Termina琀椀on of treatment  rescuscita琀椀on of persister cells can replenish popula琀椀on  Describe the mechanism of persistence using the Toxin-Antitoxin module system - Toxin-An琀椀toxin module: o Gene琀椀c loci encodes a toxin whose produc琀椀on inhibits cell growth and an an琀椀toxin Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 o Toxic ac琀椀vity promotes cellular adapta琀椀on by slowing cell growth  ensures survival during stress  hipAB genes encode TA module that triggers persistence in E. coli  HipA: toxin that inhibits transla琀椀on  HipB: an琀椀toxin  Normally HipA and HipB form a stable complex.  Polyphosphate (polyP) are produced at di昀昀erent concentra琀椀ons in bacteria (phenotypic heterogeneity).  Lon is a protease ac琀椀vated by polyphosphate (polyP). A small popula琀椀on of bacteria producing higher concentra琀椀ons of polyP will ac琀椀vate Lon.  If Lon is ac琀椀vated, HipB is degraded, HipA is then freed to do its work.  HipA phosphorylates glutamyl-tRNA synthetase (GltX), GltX is unable to charge its a.a., leading to ribosome stalling and ac琀椀va琀椀on of RelA.  Stalling inhibits transla琀椀on and s琀椀mulates produc琀椀on of the alarmone (p)ppGpp by RelA = induc琀椀on of stringent response pathway.  Cells that have triggered stringent response become dormant. - polyP produc琀椀on  ac琀椀vates Lon protease  HipB (an琀椀toxin) degraded  HipA freed to do its work  phosphorylates GltX  GltX unable to charge its amino acid  ribosome stalling (which inhibits transla琀椀on)  ac琀椀va琀椀on of RelA  produc琀椀on of alarmones  stringent response pathway  dormancy and persistence Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 MICROBIAL GENETICS AND GENOMICS  Explain how prokaryotic organism acquire genetic diversity  Asexual reproduction: one organism needed to make one  Horizontal gene transfer (HGT) between closely or sometimes more distantly related species (only small pieces of DNA at a time)  Vertical gene transfer  between mother and daughter cells.   Discuss the bene昀椀ts and trade-o昀昀 of sexual vs asexual reproduction  Sexual reproduction:  o Slower to produce o昀昀springs  o Generates diversity  Material from 2 parents  Crossover  Independent assortment of chromosomes during meiosis  Chromosomal segregation not always correct Asexual reproduction: Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 Generates more o昀昀springs more quickly Generates less diversity Identical genome passed down to daughter cells Mutations could give rise to a selective advantage Mutations are random and rare(ish) Both = vertical transmission of genetic material  Explain in detail each of these processes (mechanism): transformation, transduction, conjugation (ie, no need for any more details than what we have seen)  transformation:  with vesicles carrying DNA  With naked eDNA  Many bacteria are naturally “competent” and can bind DNA in the environment, transport across the plasma membrane and make it single stranded.  Double stranded DNA would normally be digested by endo or exonucleases in the cytoplasm, but these nucleases are mostly ine昀昀ective against ssDNA.  This ssDNA can recombine with the genome. The DNA that was added to the wildtype genome is called “recombinant DNA”.  Natural vs arti昀椀cial competence: Natural competence is regulated in bacteria, for example, quorum sensing regulates competence in Streptococcus pneumoniae. Arti昀椀cial competence has been genetically engineered in lab strains of E.coli to be chemically or heat induced (or via electroporation).  Transduction:  Genes are transferred between cells in a virus  Specialized or generalized - Generalized: - - Generalized transduc琀椀on by phage vectors can move any segment of donor chromosome to a recipient cell. The number of genes transferred Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 in any one phage capsid is limited, however, to what can 昀椀t in the phage head. - Specialized transduc琀椀on is restricted to moving host genes 昀氀anking the phage a琀琀achment site. The number of genes transferred is limited by the size of the phage head. The resul琀椀ng recipient, or transductant, chromosome becomes a par琀椀al diploid, in this case, for the gal gene. (names not important, only mechanism) -  Conjugation:  Pili, type 4 secretion system (T4SS) or nanotubes are all synonymous to a degree  Use of a hollow tube called a conjugation pilus to transfer genes between cells  DNA transferred from one cell to another by a needle-like structure (conjugation pilus)  links the 2 cells together  DNA can be transferred through cell contact to closely or even distantly related cells (di昀昀erent genus)  Conjugation pilus = F pilus; component of T4SS  In E.coli, capacity to form F pilus encoded on plasmid  F+ cells have the plasmid and can make a pilus  DONOR cells Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638  F- cells do not have the plasmid  RECIPIENT cells  F plasmid = episome – can integrate into host chromosome  Cells possessing a non-integrated F plasmid = F+  Cells possessing integrated F plasmid = Hfr (high frequency of recombination)  High rates of genetic recombination between genes on donor (Hfr) and recipient (F-) chromosomes - A bacteria may attempt to transfer the whole chromosome to an F- cell (rare bc contact bw cells is transient and short-lived)  can be su昀케cient to transfer operons -  Vesicles and nanotubes:  Transport of eDNA by vesicles or through nanotubes (di昀昀erent than pilus) Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 Whatever was coded on this recombinant DNA that is now incorporated into the bacterial genome becomes an integral part of the bacterial genome and can change the phenotype (new toxin, enzyme, structural protein...) However, there is relatively little environmental DNA, it is rarely a whole coding piece of DNA and recombination requires that the “recombinant DNA” be at least in part similar to the genomic DNA for a length of nucleotides for recombination to occur. Also, although less e昀케cient, nucleases still digest the ssDNA fragments. - Vesiduction = transformation through the incorporation of DNA from vesicles released by microbes -  Distinguish between generalized and specialized transduction.  - Generalized transduction: any piece of DNA can be transferred from one infected cell to another if it is erroneously (by chance) incorporated into the capsid when the virions are assembling.  Specialized transduction: a speci昀椀c piece of DNA is lifted from the infected cells from either side of where the lysogenic phage had integrated into the host cells. These sequences are then encapsulated and transported to other cells and will integrate in the newly infected cells at the viral integration site.  Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638  Discuss the consequences for bacterial genetic diversity for each of these processes: transformation, transduction, conjugation, and transposition.  Transduction is a major contributor to genetic hypermobility and both phage and bacterial evolution.  So transformation is not very e昀케cient at introducing diversity. Regardless, it is still an important mechanisms for the acquisition of tolerance to Abx or virulence.  Explain what vertical transmission and horizontal (or lateral) transmission of genetic material are  In summary, vertical transmission is the transfer of genetic material from parent to o昀昀spring, ensuring the continuity of traits within a lineage, while horizontal transmission involves the transfer of genetic material between individuals of the same generation or di昀昀erent lineages, potentially contributing to genetic diversity and adaptation.  Describe and contrast what are pan genomes and core genomes Core genome: shared by all strains of the same species Pan genome: core genome plus genes that are not shared by all the strain in a same species Chromosomal (genetic) islands code for genes not essential for survival, but ones that can increase 昀椀tness for a speci昀椀c stress or environment:  Pathogenicity island  Antibiotic resistance islands  Metabolic pathway islands  Symbiosis islands Describe what chromosomal islands are and explain why we think they are of foreign origin - Chromosomal island = cluster of genes for specialized functions not essential for survival o Certain strains of the species will possess this island, some will not  more genetic diversity within a single species o Chromosomal islands are considered to be of foreign origin for 3 main reasons: o The extra genes are often "flanked" by inverted repeats (indicating they might have been inserted there by transposition). o The base composition and codon bias of the island may differ substantially to that of the rest of the genome. o Chromosomal island are found only in certain strains of the species but not all of them, again suggesting their presence was not via evolution of vertically transmitted DNA, but by an insertion event potentially through lateral transfer (HGT). Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 MICROBIAL SYMBIOSIS WITH HUMANS  Discuss, in a general way, what we know of the diversity and dynamics of microbial populations in humans so far.  Most are harmless, many are bene昀椀cial to the host  Our bodies carry about as many bacterial cells as human cells (1013 to 1014 microbes ), and about 100 times more nonredundant bacterial genes than human genes.   Explain what symbiosis is.  Explain what dysbiosis is.  caused by pathogenic microorganisms  consequences: localized gut in昀氀ammation, systemic in昀氀ammation, increased oxidative stress, increased production of endotoxins and other biotoxins, intestinal permeability, etc.  Describe the di昀昀erent microbial microhabitat in the oral cavity and airways, the characteristic of the microbiota present and what mechanisms the oral cavity and airways have to clear or contain the microorganisms present. (no bacteria names needed) - oral cavity = complex, heterogeneous microbial habitat - saliva contains antimicrobial enzymes - high concentrations of nutrients near - surfaces in the mouth promote localized microbial growth. - The oral microbiota are normally harmless, but they can cause disease. - Dental procedures, for instance, will often cause these organisms to enter the bloodstream, producing what is called bacteremia. - This can ultimately lead to subacute bacterial endocarditis. - Microbes thrive in the upper respiratory tract. - Bacteria continually enter the upper respiratory tract from the air during breathing. - Most are trapped in the mucus of the nasal and oral passages and expelled with nasal secretions or swallowed and then killed in the stomach. - The lower respiratory tract has a limited microbiota in healthy adults. - Ciliated mucosal cells move particles up and out of the lungs. - Lungs and trachea harbor normal microbiota - Ciliated mucous lining of trachea, bronchi, and bronchioles = mucociliary escalator  sweeps foreign particles up and out of the lungs - Epithelial cells (lungs) defend themselves: Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 o Secretion of mucus o Secretion of surfactants  decrease surface tension but can also be microbicides o Can detect pathogens and secrete microbicidal polypeptides, or cytokines such as o IL-25 and IL-33, following their activation o A large population of resident alveolar macrophage constantly clear debris and o intruders by phagocytosis. o Dendritic cells (DCs), present in the respiratory epithelium, also seem to set the tone for the immune response with their ability to present Ag to cells of adaptive immunity.  Describe the di昀昀erent microbial microhabitat in the urogenital tract, the characteristic of the microbiota present and what mechanisms it has to clear or contain the microorganisms present. You should know Lactobacilli is the dominant species in the vagina. - Kidney and bladder normally sterile. - Altered conditions can cause potential pathogens in the urethra (such as Escherichia coli and - Proteus mirabilis) to multiply and cause disease. - E. coli and P. mirabilis frequently cause urinary tract infections in biological women, for example, when the pH changes. - The vagina of the adult female is weakly acidic and contains signi昀椀cant amounts of glycogen. - Lactobacillus acidophilus, a resident organism in the vagina, ferments the glycogen, producing lactic acid. - Lactic acid maintains a local acidic environment (pH~5.0). - L. acidophilus only colonizes the vagina at puberty and disappears during menopause. Before puberty and after menopause, pH is neutral (no lactic acid being produced), so the microbiota changes as biological women become fertile or lose their fertility. - Male genitalia: circumcision = largest in昀氀uence on composition of penis microbiome  less anaerobic bacteria - Age and sexual activity a昀昀ect composition - Species resemble that of the gut - Female and male partners seem to share genital microbiota. - Anaerobes associated with bacterial vaginosis also found on uncircumcised penises.  Describe the di昀昀erent microbial microhabitat in the skin, the characteristic of the microbiota present. (no bacteria names needed)  Dry skin, moist skin, sebaceous skin  Two distinct layers:  Epidermis:  o Keratinocytes  o Squames (enuclated keratinocytes crosslinked to fortify the skin barrier)  Dermis:  o Sub-cutaneous tissues, include sebaceous  and sweat glands, hair follicles and immune cells.  Mostly Gram-positive bacteria  More resistant to salt and dryness  Staphylococcus epidermidis  Cutibacterium acnes (formerly  Propionibacterium acnes)  - Degrades skin oil  - In昀氀ames sebaceous glands  - Causes acne  Describe how the microbiota of any ecological niche on the human body can both protect against pathogenesis and at times participate in diseases. What needs to happen for an infectious or opportunistic agent to cause disease (especially in the gut).   The acidity of the stomach and the duodenum of the small intestine (~pH 2) prevent many organisms from colonizing the GI tract.  Few microbes survive.  Helicobacter pylori  Survives stomach at pH 1 Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638  ― Burrows into protective mucous lining  ― Occasionally may causes gastric ulcers  Decreased stomach acidity = Hypochlorydia  Caused by malnourishment  Vibrio cholerae survives stomach passage.  ― Establishes infection in less acidic intestine  Large intestine: ratio of 1000 anaerobes: 1 facultative organism  The small amount of oxygen that di昀昀uses from the intestinal wall into the lumen is immediately consumed by facultative bacteria  By constantly monitoring intestinal antigens, the immune cells of the gastric mucosa are always on the alert and eliminate threats locally, without our knowledge.  For an immune response to occur:  There must be stimulation of the innate cells:  Physical barriers must be broken  balance between pathogenic and commensal must be broken (ecological niche favorable to pathogens)  Or a deregulation of the innate system favorable to pathogens  Severity of the pathology is a consequence of the host/pathogen relationship  Virulence of microΦ in昀氀uence immune response  Aggressiveness, opportunistic microΦ, toxin, quantity of pathogens ingested?  Quality and number of commensals  Presence of immune de昀椀ciency (over or under activation)  Break in tolerance (overreactive immune response)  Dysbiosis and/or virulence = pathogenesis   Explain the role and utility of the commensal bacteria in our gut.  Microbes in gut a昀昀ect early development, health and predisposition to disease  Colonization of gut begins at birth  Responsible for digestion of food, absorption of nutrients, and production of nutrients by the indigenous microbial 昀氀ora  Intestinal microorganisms carry out a variety of essential metabolic reactions that produce various compounds.  vitamin production  modi昀椀cation of steroids  amino acid biosynthesis Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638   Describe the microbial microhabitat in the gastro-intestinal tract and the interactions between the commensal microbiota and the GI tract (GI tract tissues and immune system underlying the GI tract) (no species names needed, but know the main phyla)   Main phyla: 昀椀rmicutes, Bacteroidetes, proteobacteria  Individuals may have mostly Firmicutes, or mostly Bacteriodetes, or a mix of the two.  In contrast to the limited phylum-level diversity, the species diversity in the mammalian gut is  enormous (3,500 to 35,000 species but no more then ~200 in one individual).  Archaea, yeasts, fungi and protists are either absent or form a very little part of the gut community in humans.  The colon is essentially an in vivo fermentation vessel (anoxic so no respiration using O2  as 昀椀nal electron acceptor), with the microbiota using nutrients derived from the  digestion of food.  Most organisms are restricted to the lumen of the large intestine, while others are in  the mucosal layers.   Describe how the composition of the commensal 昀氀ora in the GI tract may change through time or under various selective pressures (bottle fed infant vs breast-fed infant, meat diet vs complex 昀椀ber diet, etc....) - Microbiome begins to develop before birth; source may be placenta - Once outside of embryonic membrane  bunch of microbes in birth canal and outside world - Early colonizing microbes are a source of vitamins and tend to be facultative rather than obligate anaerobes. - Indeed, young babies have microbiomes that are more diverse than those of adults. - By the time babies are 3 years old, diversity assumes adult composition. Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 - Vaginally born infants have a microbiome more like that of their biological mother than those born via C-section; colonized by lactose digesting bacteria at 4 months old - Breastfed infants have more commensal bacteria bc breast milk has variety of complex oligosaccharides that promote their colonization - breast-fed microbiota including taxa commonly associated with probiotics and gut lining protection and immune system education, particularly Bi昀椀dobacterium longum, whilst bottle-fed infants have a greater abundance of - C. di昀케cile and Citrobacter and Enterobacter. - Hygiene hypothesis: our use of soaps, antibiotics, disinfectants  severely restricted our access to microbes  microbiota les diverse  immune system not as trained; could contribute to in昀氀ammatory and autoimmune diseases - Targeted hygiene hypothesis: lack of microbial diversity (rather than clean environment) that understimulates our immune system  less e昀昀ective at discerning what is dangerous and what is not - Farmers, pet owners, and what we would call underdeveloped countries, seem less likely to develop immune disorders. - Individual adult microbiota is stable; 昀椀rmicutes and proteobacteria not as stable - Early experiences determine gut microbiome  parental transmission of microbiota early in life determinant for rest of adult life - Geographical location and age are stronger determinants than physiological or demographic variables - Aging  decreased microbial diversity (increased proportion of Bacteroidetes)  Describe in昀氀ammatory disorders in the GI tract (IBD) and what using mouse models have shown about the capacity of the microbiota to impact our health.   - chronic in昀氀ammation of the gut and disruption of homeostasis (dysbiosis)  Epidemiology: 4X more frequent then in the 50's, genetic risk cannot explain this increase.  Emergence greater in countries that are actively "westernizing". Diagnosed at anytime  during life. Incidences rather high in Canada compared to other countries.  Potential causes:  Failure to develop a tolerance to commensal bacteria in the gut early in life  (breastfeeding vs not, vaginal birth vs C-section, hygiene hypothesis)? Leaky gut?  Antibiotic use increases the risk of developing IBD  Links with Western diet (rich in animal proteins which can produce harmful  metabolites also associated with colon cancer, food additives), high 昀椀ber diet seems to be bene昀椀cial  Once developed, IBD may be transmissible between family members.  Presence of an adaptive T cell response against commensal gut bacteria is detected when it should not (break in tolerance).  Individuals with IBD seem to have lower gut microbiome diversity. Data inconsistent with recent meta- analysis suggesting more studies need to be done to associate microbial diversity to IBD Mouse models: - Normal mice have 40% more fat than germ-free mice with same diet - Germ-free mice given normal mouse microbiota  started gaining weight - Mice that are genetically obese have di昀昀erent microbiota than normal mice - Obese mice have more 昀椀rmicutes Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638  Like the mouse model, obese humans have more Firmicutes than non-obese humans.1  The nature and transferability of gut microbiota is dependent on diet as well as genetics.  Transplanting fecal material from the gut contents of a paired identical human twin study group (one twin was obese and the other lean) to germ-free mice showed that the obese twin microbiota made the mouse obese. Conversely, transfer of gut contents from the lean twin did not contribute to an obese phenotype.   Microbiota seems to in昀氀uence obesity in two major ways.  Harvesting of energy from ingested foods  Triggering of intestinal in昀氀ammation  The metabolic dexterity of intestinal microbes allows them to digest many foods that we cannot.  In the process, these microbes produce short-chain fatty acids (SCFAs) that our cells use in many ways.  Numerous investigations suggest that the amounts and ratios of these SCFAs in昀氀uence obesity.  Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638   Archaea in the gut might also tilt metabolic balance toward obesity. About 50% of humans have signi昀椀cant numbers of methane- producing archaea  In昀氀ammation induced by gut microbes also may in昀氀uence the development of obesity and diabetes.  Lipopolysaccharide (LPS, or endotoxin) produced by Gram- negative species promotes in昀氀ammation.  A high-fat diet can promote absorption of endotoxin across the intestinal epithelium.  Oral antibiotics decrease most microbes in the human gut (both target and non-target).  Use of antibiotics during the 昀椀rst few months of life increases the risk of developing IBD and other disorders related to dysbiosis.  Distinguish between probiotics and prebiotics.  Probiotics are living microbes that are ingested to restore the natural microbial balance. Bi昀椀dobacterium and lactobacillus (yogurt and probiotic drinks)  Live organisms that confer a health bene昀椀t to host  Most destroyed by acid pH conditions in stomach and ileum; enzymes or bile acids; they are obligate anaerobes (oxygen toxic); don’t colonize the gut and disappear in feces when u stop eating them  Prebiotics are typically carbohydrates that are indigestible by human hosts, but provide nutrition for fermentative gut bacteria, presumably to promote the growth of “good” bacteria. HOST-PATHOGEN RESPONSES (Weapons of mass infection)  Describe what conditions need to be met for an infection and/or disease to occur (in humans or animals)  Describe the cell structures or biochemical armoury that microorganisms can deploy to infect us (virulence factors) and explain the mechanisms with which they can cause disease: o For adhering to cells and tissues (adhesins, capsule, 昀椀mbriae, pili, and 昀氀agella) o For invading cells and tissues (enzymes, cytolytic exotoxins) Downloaded by amelia safi ([email protected])  lOMoARcPSD|31356638 o For helping their growth and toxicity (virulence plasmid, exotoxins, endotoxins, anti-phagocytic proteins, immune inhibitors, T3SS and e昀昀ector proteins,...)  Distinguish between the terms infection, bacteremia, and septicemia.  Explain what virulence means.  Explain what LD50 and ID50 are.  Distinguish between the terms cytotoxin, endotoxin, exotoxin, hemotoxin.  Describe the virulence mechanisms of the following toxins and their e昀昀ect at the cellular, tissue or systemic levels when appropriate: o Diphtheria toxin o Botulinum and Tetanus toxins o Cholera toxin o Cytolytic toxins (hemolysins and Staph alpha-toxin o Endotoxins Describe how bacteria can deploy toxins and e昀昀ectors using various secretion systems (in particular the T3SS). Describe what are e昀昀ector proteins. Explain how e昀昀ector proteins can in昀氀uence pathogenicity (what role can they play during infections) Downloaded by amelia safi ([email protected])