Microbiology Notes PDF

I. Introduction Chapter 16 A. Questions 1. What is meant by composite organism a) Relates to the endosymbiotic theory where relation to prokaryotic cells are drawn due to the fact that mitochondria as well a...

I. Introduction Chapter 16 A. Questions 1. What is meant by composite organism a) Relates to the endosymbiotic theory where relation to prokaryotic cells are drawn due to the fact that mitochondria as well as chloroplasts are considered organelles with their own DNA b) Plants are even more of a composite organism c) Composite organisms are able to survive on their own d) Humans cannot live without their mitochondria and mitochondria cannot be propagated on their own either 2. Initially labeled as infective heredity, what is this process now called? a) Horizontal gene transfer is what it is called now 3. What is historically inaccurate in Chapter 16 a) Described bacteria is invisible, but bacteria can be seen with with the naked eye b) Went on to credit a microbiologist responsible for an entirely different discovery (1) Dead wrong about who described bacteria growing on a potatoes surface, Robert Hooke, it was Robert Hooke 4. Why was it important to show that microbes to show that microbes has stable identities a) Under the false belief that bacteria went under immediate and unproved transformation; however, microbes were brought into microbiology as when you work with a pure culture, such as a potato slice, those microbes could be propagated and shared many similarities to plants and animals (1) Due to the fact that they were able to mainly maintain their shape and structure, they became characterized within a similar vein to plants and animals II. Lecture ~ Basic Structures and Processes in Prokaryotic Cells A. Cells (no matter prokaryotic or eukaryotic) much more complicated than it used to be thought of 1. Thought previously that cytoplasm was just an aqueous body; however even prokaryotic cells have regions not bounded by membranes as in eukaryotic cells a) But these regions of the cytoplasm differ amongst one another 2. The Bacterial Cytoplasm Has Glass-like properties and Is Fluidized by Metabolic Activity a) They grew e coli, normal lab strain, common strain e.coli b) They had these bacteria transformed (infected) with a plasmid infected with green fluorescent protein c) They propagated the bacteria that was expressing the fluorescent protein where they either had non-growing and growing populations  (1) In 18 minutes, they showed under growing conditions a fluorescence that was moving around (2) Wheres in the startup cell, the fluorescence remains in the same location across the 18 minutes (3) Representative case; however, real data must be compiled to observed a plethora of cells (4) Tracked where the fluorescence was in relation to the cell (a) Within growing populations, the fluorescence would move around (b) Within cells that have been disrupted within their respiratory abilities, the signal of the fluorescence remained unchanged (c) Been repeated for not just eukaryotic cytosol but also the eukaryotic nucleus (i) Different regions that have different physical properties (ii) Discovering how these properties are regulated and what is causing such different attributes (a) Long RNA molecules can have either intramolecular base pairing or intramolecular base pairing, there are long polymers being formed thus making the solution more viscous 3. Cytoplasm in all cells is not simple and is more complex than previously believed B. Cell Envelope and Appendages 1. Membrane Function entails the defining of the cell and acts as the MAIN PERMEABILITY BARRIER (Gram Negatives, some Gram Positives) a) Passive vs Active Transport Across Membranes (1) Passing Energy yielding Downhill (a) Diffusion and facilitated Diffusion (embodies that of a specific protein or pore that allows a specific substrate to travel down its concentration gradient (2) Active Energy requiring Uphill (a) In the form of ATP, proton gradient and a plethora of others (b) Violence factories are molecules that have to be transported against their concentration gradient 2. Where did the ‘Gram’ come from a) The decolorization step is the defining step that differentiates between Gram positive and Negative bacteria  b) The Gram Stain was developed in the late 19th century before they had any idea why the two groups of bacteria stained differently (1) Stained differently as a result of the peptidoglycan layer in Gram Positive bacteria (2) Anatomical reasoning was determined later c) Peptidoglycan (1) The function is that it provides the mechanical strength for the bacteria (a) Provides strength to the bacterial envelope in order that it withstands the internal osmotic pressure (b) In the presence of pencilion (it prevents the peptidoglycan functioning) therefore in regular high osmotic environments, e.coli for instance would experience bursting (c) Water will rush into the bacteria and thus they explode with their peptidoglycan functionality (i) Prokaryotic cells cannot do phagocytosis because they have a rigid wall (ii) If they grow they can partially break down the wall to formulate two new walls (iii) They can regulate the synthesis and degradation of the peptidoglycan layer on a long time scale (iv) Animal cells and eukaryotic microbes don't have cell walls and can adult restructure their membranes, thus engaging in phagocytosis 3. Peptidoglycans (same two carbohydrates) of Gram positive and Gram negatives are very similar a) The strength of peptidoglycan comes from the peptide portion b) The peptides cross the long molecules and the penicillin prevents the cross linkages of the peptides (1) The more crosses, the more rigid and strong the peptidoglycan membrane is (a) Gram negatives have a peptidoglycan 4. The Gram-Negative Wall and Outer Membrane (OM) a) The outer membrane is an incredible surface that is very malleable in order to support the refashioning of their membrane to interact with their environment and immune cells within the human body (1) They constantly shed LPS, also known as endotoxin, of which is a part of their outer membrane b) Periplasm is the space between the outer membrane and the cytoplasm  (1) It is like a cellular compartment, its bounded by the inner and outer membrane (2) Its signaling pathways and construction peptidoglycan to make complicated outer surface proteins (3) Think of it as its own organelle c) E. coli (1) Make pili that stick out of the outer membrane and have our proteins that interact with kidney cells (2) To make the pili, they are made up of different proteins that are coded by a separate gene (a) Gene transcription and translation occurs only within the cell (b) Individual components are transported into the periplasm and put together and transported simultaneously across the outer membrane (c) A highly synchronized process (d) Chaperones are proteins that bind to other proteins that prevent folding and misfolding where they are held in opposition to interact with other proteins they are supposed to. (e) The periplasm, if a protein goes rouge and misfolds, there are quality control measures in order to dispose of such biological error d) LPS can be rapidly remodeled in response to changing environmental conditions (1) All have to do with modification of LPS (a) The enzyme FlmK adds a glucose molecule to Lipid A, the effect of the glycosylation rates a lower affinity to toll like receptors (i) Toll receptors are there to tell if there is any LPS in the system (ii) If LPS is glycosylated, it lowers the affinity it increases the chances that the pathogen will be undetected and thus it can proliferate more before it is destroyed (b) Lipid A is the base (c) The enzyme LpxE (i) Dephosphorylates Lipid A, through a very different chemical modification, the pathogen become undetectable to an entity e) CAMP (1) (CAMP) Cationic, antimicrobial peptides  (a) Small peptides that are able to disrupt the prokaryotic cell membrane (i) They are released by human cells, especially in the intestine (ii) Gram-Negatives can become resistant to these small peptides (b) Enzyme AlmG adds a glycine to the hydroxyl group of the 3-acyloxy alkyl chain of lipid A (i) Polymyxin B is good at disrupting the small peptides f) Horizontal gene transfer puts a specific gene back into a cell (1) The locus of the gene is disrupted but the the gene is being expressed from the plasmid (2) The reading frame of gram negative bacterias can be sensitive to CAMP expression 5. The Gram-Positive Wall C. Ancestor Cell Type? 1. Gram positives have to be the ancestral type was argued countless times a) How a group looked at the genome sequences of thousands of Gram Negative bacteria to try and configure what makes more sense which bacterial group would be the ancestral cell type (1) They argued that Gram Negatives were the ancestral cell type (2) LBCA (Last bacterial common ancestor) (a) The simpler cell type is not the ancestral bacteria D. The Outliers: 1. The Acid-Fast Bacteria a) Refers to resistance to decolorization by acids and alcohols (1) There are many acid-fast species of bacteria including mycobacterium tuberculosis (a) Acid-fastness in M. tuberculosis is due primarily to mycolic acid in the mycobacterial outer membrane (2) They are biogentical Gram Positives, but they are called acid-fast due to the fact that they are acid resistant (a) Very difficult to stain 2. The Wall-less Bacteria a) Some bacteria lack peptidoglycan; the plasma membrane forms the outer boundary of the bacterium (1) Two such species, mycoplasma pneumoniae and mycoplasma, are associated with walking pneumonia E. Proton Transport 1. Transporters are important a) Listeria  (1) Is taken up by cells through a vacuole if a macrophage, and is able to breakdown the membrane of those cells and enter the cytosol (a) To be able to get into the cytosol, they have to express and secret virulence factors that enzymatically breakdown the membrane (2) Virulence factors are replaced in which they are non-covalently allowed into the peptidoglycan b) In Gram Positives (1) The pathogen can cross the inner membrane and have a lipid attached to it to remain anchored to the membrane or it can remain non covalently associated (a surface antigen) but it as enough of affinity to anchor it (2) Anchoring proteins to peptidoglycan in Gram positives (a) Amino Acids - leucine, glycine, proline tyrosine (b) LPXTG is recognized by the outside of the membrane where the enzyme is then responsible for cutting the protein at the glycine III. Lecture ~ August 26 A. Outside-in signaling 1. Signaling transduction has to be conveyed across the membrane and into the interior a) In prokaryotic cells, its less complex b) The basic paradigm of the signaling pathway has a sensor kinase (sense something outside the cell and functions as a kinase that puts a phosphate onto a histamine (1) In eukaryotic cells is usually a tyrosine c) Once a signal is detected, it gets transmitted to the kinase part in which a response regulator is activated (1) Response regulator is the substrate of the kinase and they are transcription factors themselves (a) They don't activate something else but they go directly to DNA and cause activation (2) Target gene - the enzyme that puts the lysine on the LPS modification for instance (a) Vibrio picks up the correct signals 2. Sensitive to a lot of different signals including magnesium a) Magnesium acts a repressant - the receptor kinase is in an inactive state b) Magnesium is very important, DNA and RNA polymerase are required for all life forms to keep tight control over their mechanisms (1) If magnesium levels drop, the pathway is active (derepress) c) Bacterial genetics - non italicized and not capital is a gene  (1) This signaling pathway is reversible 3. Very important pathway that Gram Negatives have a) Sensitive to misfolded proteins b) Have a sensor kinase that sits on the inner membrane c) The hydrophobic interior parts will thus be exposed if its folded incorrectly d) Target gene is protease and once made, its transported through the periplasm and utilizes the resources within the environment B. Molecular vs Anatomic based Phylogeny: Information and Storage and Expression 1. The code is universal where all life on Earth evolved from a common ancestor 2. It was not obvious that DNA was the hereditary material a) The Path to DNA (1) It was known in the early part of the 20th century that the nucleus the chromosomes were 50% chromosome and 50% nucleic acids (2) Thought proteins were structurally in the leading hypothesis (a) DNA has the genetic material and proteins contain the structure b) The Griffith’s 1928 Transformation experiment (1) In the 1920s, he worked with glycerine in mice where he injected various strains into mice (a) Rough strain (nonvirulent) (b) Smooth strain (virulent) (c) Heat killed smooth strain (d) Rough strain and heat-killed smooth strain (2) The rough strain had been converted to a smooth strain c) 1944 Transformation factor is likely DNA (1) Characterize the hypothetical transformation principle (a) Assumed it was something physical however it turned out to be chemical (2) Published in 1943, talks about transformation and who was the first to describe it within the abstract of his paper d) Took heat crude rough strain and broke them up to try and effect the virulence factors of the bacteria with specific chemicals (1) Had crude preps made of dog intestinal mucosa, rabbit bone phosphatase, swine kidney, pneumococcus normal dog and rabbit serum (2) Call Depolymerase - D-rase (3) The only crude extract able to breakdown DNA e) 1952 Phage genetic material is likely DNA (The Hershey-Chase experiment) (1) Took advantage of radioactive of sulfates and phosphates  (2) S35 (sulfur) labels protein (3) P34 labels DNA (4) Phages attach to the surface of a bacteria and inject their genetic material into the bacteria (a) The blender gets rid of the phage (b) If the bacteria was infected with P32, the bacteria would become radioactive f) By the early 50s, most biologist switch their alliance as a result of these experiments and their findings 3. DNA Structure a) Rosalind Franklin (1) She mostly used X-ray diffraction to get structural details (2) She died of breast cancer in her mid 30s (3) X-rays are very damaging to DNA b) The most important part of the model (1) Base pairing was the most fundamental aspect of the double helix and DNA structure (2) Discovery of penicillin was a great discovery but their complementary base parking model underlies all of life (a) Developed very early and the base pairing is what made life possible c) Unappreciated part of the model (1) Frank Skull talks about that G and C and A and T, not only are they complementary, the width and physical dimensions are the same (a) G and C are of the same physical width as the A and T base pair (2) DNA wouldn’t crystallize if the dimensions between AT and CG were different d) Higher effort in biology is to make organisms that can make amino acids (1) If we had a code for DNA and different types of amino acids we could make a greater variety of different proteins C. Replication and Transcription 1. Both polymerization processes mediated by enzymes (called, appropriately enough, polymerases a) Very ancient process that are essential to biology b) Involved in nucleic acid metabolism must be very well concised (1) RNA polymerase is evolutionary labeled very similarity amongst all life forms because they are all evolutionary similar c) The enzyme had to evolve 2. Possible modes of DNA replication  a) Set out to test whether or not semiconservative replication as opposed to a conservative model where a double helix comes from the preexisting double helix. 3. The perfect experiment (meselson and Stahl, 1958 a) Use radioactivity and in this case they are using a heavy nitrogen as it has an extra neutron (1) They brew a batch of e coli in the heavy nitrogen (2) Extracted the e coli's DNA and analyzed the DNA via ultracentrifugation (a) Spin it fast enough the CC-chloride will be more dense (b) DNA will migrate through the tube until the densities match one another (c) That DNA would go to a certain part of the tube b) If you changed the media to normal nitrogen for the e coli and let it grow (1) The DNA is lighter and closer to the top on the left c) If you let it grow even longer, the DNA becomes even lighter d) An experiment sets out to answer a question but this experiment answers the question right away (1) The data is only compatible IV. Lecture August 28 ~ Genomes A. Genomes and genes 1. national institute of health maintains a database of genes 2. Most proteins start with thymine a) Taa - a sense codon b) Atg - a sense codon that recognizes t RNA 3. Sequences of genes that don't contain a chromosome B. Key Numbers and Dates/Eras to Remember 1. C. Gene expression (transcription, translation, and regulation) 1. The ribosome a) Converter the linear nucleic acid polymer into a linear protein polymer b) Is itself composed of several proteins and RNAs (ribosomal RNA; rRNA) c) Is absolutely conserved among all known life forms on Earth; used to determine evolutionary relationships d) Is ‘targeted’ by several antibiotics 2. Prokaryotic Ribosome a) Large subunit: 31 proteins + 55 rRNA + 23S RNA b) Small subunit: 21 proteins + 165 rRNA (1540 nts) c) Usually squash the picture down because this shows how much intramolecular base pairing occurs (1) The same molecule and base pairing with itself (2) RNA can be intramolecular base pairing or tans molecular base pairing in order to configure a sense of regulation (a) Within the ribosome, 16s rRNA, in which stems and loops exist (i) Loops don't have base pairing because they don't have the complementary base pairing capacities (ii) In DNA intermolecular have opposite linking directions much like proteins (a) Anti polarity is evident between strands that are binding to one another in opposite directions D. PCR: Basis and analysis by electrophoresis 1. Perforin-2 (P2) knockout mouse used for infection studies a) P2 +/- female (dam) crossed to P2 +/- male should yield approximately 1 +/+; 2+/-; 1 -/- 2. Each pup has to be analyze to determine its genotype a) DNA is isolated from tail snips and analyzed by PCR using P2-specific primers 3. Essential ingredients for PCR a) Template, Primers, polymerase, Nucleotides, and Mg-containing buffer (1) Very simple as only things needed is magnesium (metal cofactor) DNA primer, polymerase 4. Steps a) First denaturing step (1) 95 degrees Celsius (2) Only need to heat for 25 seconds b) Annealing (1) Allows primers to formulate a sequence (hybridize)  (2) They are point towards each other (a) Polymerase is able to form a 3 prime linkage c) Following step (1) Raise temperature up to 72 in order to polymerize 5. The wild type and KO alleles of Perforin-2 (P2) a) PCR using primer that point towards each other (primer flank the positions of one another) (1) These alleles will be on the two different sides (2) Extract the DNA through PCR and then run on an electrophoresis gel (a) Agarose gel separates the PCR components where each one of the wells products are loaded (b) DNA is negatively charged electronegative and will migrate towards the positive end (i) The smaller fragment will migrate faster than the larger one (ii) Larger one - wild type (iii) Smaller one - regular KO V. September 4 A. Woese’s expeiremnta;l approach for comparative 16S analysis 1. Want to label RNA or DNA, you would have growing cells label them alpha phosphate and incorporate ATP and RNA a) Conventional how the fragments come together 2. Isolate ribosomes a) Discord ribosomal proteins (1) By phenol/chloroform extraction b) Digest 16S rRNA with the ribonuclease T1 c) Separate resulting digestion products (1) By 2 dimensional electrophoresis d) Results in a characteristic fingerprint 3. Exo vs endoribonuclease a) Enzyme that is an exoribonuclease chews from the ends in the 3 prime to 5 prime b) Enzyme that is endoribonuclease cuts from within c) Endonuclease (1) They can either cut in a sequence specific fashion (cut at specific bases) or be nonspecific (cut anywhere) 4. Parts of the ribosome are highly conserved or other parts that are even more variable a) Caralose turned out the be right that this molecule provides a nice plutolatry mapping b) Took RNA molecule and dropped T1 into it  (1) Would cleave at every gene (2) The T1 (particular ribonuclease) was cutting in particular spots c) Configured the sequence of each spot on RNA (1) They could eventually get the entire sequence (2) The important part of this experiment was getting specificity for the particular species 5. Was the 16S rRNA component of the ribosome Woese’s first choice to focus on a) Couldn't get the fine (gnus level) relationship b) Was making an educated guess about any connection (1) Didn't say there would be variation in ribosomal DNA c) Satisfying to see any variation as he was predicting there would be differences d) 5S didn't give enough information as it was too small so the switch to 16S was crucial B. Carl Woese 1. Could start seeing different proofs of ribosomal DNA 2. Isolated chloroplasts from algae a) Chloroplasts were hypothesized to have metabolic origin b) By isolating chloroplasts and then isolating their ribosomes and then from ribosomes he isolated their 16S and was able to configure a fingerprint c) Able to characterize and strengthen the endosymbiotic theory that chloroplasts were likely closely related to cyanobacteria 3. Using fingerprinting, he showed that plants have chloroplasts that are related to cyanobacteria 4. Take the supplement (gel) and put it up against x-rays and are able to see the film a) The autorays allow for different exposures b) Lighter and darker spots are only able to be seen at different autoluminescent C. Classification of methanogenic bacteria by 16S ribosomal RNA characterization 1. What immediately indicated the methanogens might not be a normal bacterium a) Looked at thousands of autoradiowaves b) Fingerprint produced by ribosome T1 digestion of E.coli 16S rRNA is shown (1) Each spot contains one or more individual digestion products. The spots labeled 05x and 05y contain post-transcriptionally modified nucleotides that cause them to have aberrant mobility. The absence of these highly characteristic spots on the methanogen 16s rRNA fingerprints was he first indication of the uniqueness of the Archaea 2. New Domain  a) The important thing is they didn't examine just one but 10 different species and they all lacked the two spots as well as other differences (1) Most important were the two spots cause every bacterium was found to lack those spots b) The advantages were already known to be very strange microbes (1) Their cell wall is different (a) Lack of peptidoglycan however they don't have a molecule like peptidoglycan (b) They do have a molecule that functions similar to peptidoglycan (c) The General idea and function where the same (i) Had cross-linked polymer in their cell wall that was chemically distinct from bacteria (2) Archaea membrane lipids and structure are every different (a) Known that their lipids were very different, the linkage as there are ethers in archaea and ester linkages in eukarya and bacteria (3) Nathalogens are just one group (a) At some point there was a split that some when down the acetogens avenue and other went down the methanogens (i) Methanogens do (a) CO2 + 4 H2 → CH4 + 2 H20 (b) Strict anaerobes (4) An unexpected discovery (a) Discovery of archaea - totally unexpected (b) Three domains (i) Bacteria (ii) Eukarya (iii) Archaea (a) Ribosomes were noticed to be more similar to eukaryotic organism rather than bacterial organisms (b) There is some kind of branching where eukarya are derived from archaea (c) Realized that eukaryotes are more closely related to some archaea than other archaea (i) Eukaryotes are embedded evolutionary in the domain 3. Completing hypothesi: 3 domains vs 2 domains a) Eukaryotes are derived from particular line of archaea b) Relationship between eukaryotes and archaea  (1) There are huge differences D. Why did designate the methanogens in archaebacteria 1. Regretted he inclusion of bacteria in the name 2. Ironic thing about the name is that they are most closely related at all and archaea are in fact more closely related to eukarya VI. Lecture september 9 A. There is grandeur 1. RNA it can be both an information molecule as well as an action molecule a) Serves as an information molecule and can also act as an enzyme b) Even though RNA can do those activities in a catalytic manner, not as good of an information molecule as DNA c) All cellular life switched to DNA (1) DNA is more stable and not as reactive as RNA d) RNA lost a lot of its catalytic activity (1) Proteins are better at being enzyme (a) Due to the fact that there are more chemical groups and amino acids that can do a variety of chemistry 2. Speculated that a billion or 2 billion years from now that ribosomes would be purely proteins that catalytically and evolutionary, the RNA parts of the ribosomes would get displaced by proteins B. Mitochondria found in trypanosomes 1. Some of the catalytic basis are protein based a) A ribosome where some of the RNA work has been taken over by proteins, not entirely 2. RNA follows the same kind of tends always in science a) Whn RNA was discovered to have catalytic activity b) Used by the earliest life forms c) Still learning about RNA and its biology C. Early life 1. Life arose and evolved to a sophisticated extent in the total absence of oxygen a) The game changer: splitting water (1) The reaction generate molecular oxygen so the proper reaction would be two waters (2) Life forms first started performing this reaction more than 2 billion years ago (3) Would refer to such event as a catastrophe because it was extremely bad for all life forms on the planet exposed to the atmosphere (a) No reason to make defenses against oxygen because it didn’t exist (4) Scientific controversy - can be very narrow or broad  (a) A lot of evidence to support that oxygen levels grew overtime 2. Water oxidation a) Grabbing the water off of oxygen (1) Respiratory chain - oxygen reduction (a) At the end of the respiratory chain, our mitochondria takes the low energy electron to form water (2) Oxidation of water is pulling a very low energy electron to make oxygen (a) Oxygenic phototroph - what happens in chloroplasts where sunlight is focused down and used to split water to grab the electron to energize it (b) Z diagram - useful way of thinking about the energy in a chloroplasts (3) Photosystem 2 is where the water splitting occurs (4) Photosystem 1 - additional focus of energy were the electrons are high energy enough to make a dinucleotide to create a hydrogen electron carrier (5) Reducing power is generating reducing NAD 3. Purple bacteria don't split water a) They have components such as a light machine to energize electrons where they use them in a heterotrophic manner 4. Green sulfur bacteria a) Release a high amount of energy to reduce electrons b) Some that are autotrophs because they do generate D. The OEC 1. Represent photosystem II where the oxygen actually emerges from 2. Depicting the cofactors known as mangasee (polymerase adjacent) depicts some of the pigments a) Energy is getting focused down to be used in oxidation E. Structure and Assembly mechanism of plant C2S2M2 1. Figured out how photosystem II plus the light of lamp plants 2. The structure of the cryo-electron microscopy is composed of proteins where 203 pigments, 34 lipids and 8 other cofactors exist 3. In algae a) Eukaryotes but have chloroplasts where extra structures exists b) There is an increase in complexity in photosystem II in relation to cyanobacteria F. The endosymbiotic theory of the origins of mitochondria and chloroplasts 1. The 2nd major event preceding the emergence of animals and plants 2. Sometime after the linkage diverges, one line that went further to draw relation between eukaryotic and cyanobacteria  a) Very best evidence it was a cyanobacteria were connection between chloroplasts and cyanobacteria - showed that fingerprint from a chloroplast ribosome matched the fingerprint in cyanobacteria b) In 1975, but a lot of evidence since then has supported that hypothesis (1) Chloroplasts are derived from cyanobacteria G. Life impacting Earth: Then (GOE) and now (The anthropocene) VII. Lecture - Sept 11 A. Question Review 1. Why was the addition of O2 not considered a mass extinction event a) There might have not been a sudden mass extinction - if did occur happened a couple million years ago, no fossil (1) 5 mass extinction events happened after the procreation of cellular organisms 2. How does meiosis factor into the prokaryotic/eukaryotic divide a) Huge divide between prokaryotes and eukaryotes, no prokaryotes that undergo meiosis b) Nearly all eukaryotes we know undergo meiosis 3. Why is cytoplasmic inheritance considered to be non-mendelian a) Did punnett square, you would not get the 1:2:1 ratio b) One of the battles as there was genetic material in the cytoplasm that contributed to an organism's phenotype, going against the grain (1) Though all genetic material was located in the nucleus (2) Once particular genes started to be inherited within a pattern, certain phenotypes that are paired from the parent animal became evident 4. Summarize the idea of Constantin Merezhkowsky a) He believed that chloroplasts had a bacterial origin b) The kind of evidence he had to support his hypothesis was microscopy c) Chloroplasts were noted to be involved in photosynthesis (1) Ideas put out around the 19th century and early 20th century (a) Would be confirmed with molecular data invented by Carol Woes, it was based on appearance prior 5. Summarize the ideas of Ivan Wallin a) More in the early 20th century b) One of the first, based on Merezhkovsky, noted that there was two membranes c) Recognized both of these findings as she came up with the term endosymbiotic B. MIC 323 Plant Microbe Interactions 1. Fungi a) Are eukaryotes b) Are significant pathogens of plants and cold-blooded animals  (1) Funci don't favor 37 Celsius so not a threat to humans generally and warm blooded animals c) Many amphibian species are going extinct (1) 80s, 90s and early 2000s, amphibians were mysteriously going extinct 2. Adversarial interactions a) Dead spots that are little parts of the leaf that a defense reaction has occurred (1) Hypersensitive response (much like inflammatory response) (a) It detects some sort of threat, could be physical or viral insult or fungal (b) The response is triggered right at the lesion and the surrounding tissue around the lesion (c) Starts with a microscopic lesion (i) Idea is that it is analogous to an inflammatory response (want the response to keep a potential pathogen localized rather than spread throughout the system) (d) Involves located programed cell death around lesion that blocks the ability of the bacteria to spread to neighboring cells (2) Can induce these inflammatory responses (a) Do a mechanical or use a bacterial pathogen (3) Type of response was first recognized in the 1950s (a) Flor was able to recognize that for any given plant, a microbe could trigger this response (i) Plant X and microbe Y and X is put onto the plant, a response could be triggered (ii) Noticed the plant could be genetically varied - derive some plant lines for some reason or not the response would not be triggered (iii) There is genetic component in the plant side that regulates the response (a) In many cases it was a single gene of the plant responsible (b) Also could take the original plant and take the microbe that would trigger the response and derive variants of the microbe that no longer triggered the response (i) again , the response between not being triggered still, the response is not being triggered and the disease would not contain the pathogen  (ii) Not only are there genes on the plant side controlling the reaction but also on the microbe side (a) Genetic evidence because it is just based on crosses as opposed to biochemical (b) Suggested genes both on the host and pathogen that determined whether the response would be triggered (c) Came up with gene for gene theory (i) A gene on the plant, gene on the microbe, and those gene need to be on a specific allele to have the reaction occur, some sort of matching (ii) Specific segregated genes on plants were called R genes (iii) Avr (a variant) a mutated form would not triggered the desired response in terms of the microbe b) In the 1990s (1) A system had been discovered in gram negatives that cause disease in plants possess a type three secretion system (a) Protein injection system that injects the virulence factories directly from the cytosol of the bacteria into the cytosol of the target (b) Avr proteins were substrates and secreted into the cytoplasm (c) Plant pathogens have AVR proteins (2) MAPS for microbial (a) Used to be known as PAMP (b) Plants just like us have detection systems and is activated in certain genes that are being expressed (3) Ways to block the basil immune response (a) Inject things in (effectors - used to block the receptors) (b) Plants have figured out ways to know that they are infected (c) R proteins are like censors in the cytoplasm, if something odd occurs, they are activated and then an alternative pathway is triggered (i) R protein triggered immune response (4) Gram positives have different ways of combating pathogens (a) A plant can become resistant to a pathogen if it can get these pathways to act c) Evolutionary in action (1) Plant has basil alarm system (2) Pathogens figure out to combat it (3) Plants try to know when they are infected (4) Pathogens have evolved to configure better ways of infection d) Guard hypothesis (1) Avr’s target critical components (T’s) of the plant defense response (2) R proteins monitor T activity, induces HR if e) AVR protein - how it was figured out (1) Ubiquitination system (ligases - system in eukaryotic cells involved in immune response and t -cells) f) Describing chemical activity (1) AVR PtoB (a) Suamoncis is gram negative (b) The virulence factor is injected directly into the cytosol (c) R proteins encoded by genes in plants (i) Array known that R proteins cna specifically detect the AVR protein of PtoB (ii) Plants have two different r proteins that can separate the work in detecting the activity (iii) They work independently but they both signal through common signal molecules (a) One gets activated (Rf) the other will as well as program cell death (iv) Also known that this AVR protein could suppress the signaling of one of these molecules (d) Oftentimes people can look at the sequence of the gene of which they had sequence clues to indicate what ptoB was responsible for (i) Crystallize just the portion of the protein and recovered a model (a) Model doesn't tell much but starting to compare the model they derived and all the modes in the database (b) They got hints of proteins that were structurally similar (e) Showed the alpha carbon trace protein turned doubt it was very similar (the backbone structure) (i) All the proteins AvrProG, etc… E3 ligase are only found in eukaryotic cells  (ii) They have three key residues that are key to their cellular functionality (iii) Have amino acids at those same positions (iv) Property of the bacteria (2) Nomenclature (a) F479A (i) Normally in this protein - has 50 plus amino acids, in the 479 positive has been replaced (ii) The flurobenoalaline (iii) If the substitution is made the B- is wiped out (iv) The single amino acid substitution is shown to be hypersensitive (v) Lose biological activity when disrupt that substitution (a) Biological evidence 3. Mutualistic interactions a) Frienleur plant associated bacteria also use type 3 secretion systems (1) Nodule development (a) Plant releases flavonoids in the rhizosphere (b) NodD recognizes flavonoids and return to Rhizobia to activate Nod factor production (c) Nod factors are released from Rhizobia and bind to receptors b) An example of a mutualistic plant-bacteria symbiotic relationship (and it's resembles to eukaryotic cell-mitochondria relationship (1) Incredible enzyme of nitrogenase (a) Tells that such reaction is a difficult reaction that requires a plethora of energy c) Cyanobacteria (1) Can also reduce bacteria (2) Because a reduction of nitrogen is so sensitive to oxygen, the cyanobacteria make specific cells called heterocysts (a) Nitrogen reduction occurs in these specialized cells and have very thick walls to keep the oxygen out (3) nitrogen -fixing organelle in a marine alga (a) Cyanobacteria is a prokaryotic cell (b) Cyanobacteria used to be called blue green algae (i) Before they realized it wasn't an algae but a bacterium (c) Within the same cell, you can have organelles generating oxygen and organelles containing compartments that produce nitrogen  C. Questions 1. What was the specific factor that led to the correction of identifying cyanobacterium from an algae to a bacterium? 2. How were nitrogen compartmentalized bacteria developed from an evolutionary standpoint? VIII. Lecture - Sep 16 A. The Microbiome and metabolism in the Gut 1. Concepts a) Born microbiologically pure (1) Inhabited and uninhibited zones (2) With humans very difficult to get a definitive answer as you can only look at tissue 2. Terminology a) Microbiota vs microbiome (1) No longer refers to DNA - microbiome, refers to the enteric bacteria b) Symbionts vs pathogens (1) Symbiotes are there all the time (2) Pathogens are the ones that cause harm c) Good symbionts vs bad symbionts (1) Symvions - non-pathogens (what makes a good symbiont) (2) Pathobionts - more and more sued as it refers to symbionts that are unfavorable; however they are always there d) Fermentation vs respiration (1) Fermentation - (a) Electron acceptor of non-organic molecules (2) Respiration (a) electron acceptor of organic molecules e) Homeostasis vs dysbiotic 3. Abundance in ur guts a) Comparable numbers of bacteria in gut around 3 billion bacteria and cells b) approximately equal number of humans and bacteria cells (1) Mass 0.2 bacteria c) Locations (1) Colon and mouth have about same concentration of microbes (2) However the colon is a large intestine (3) 99% of total microbiota live in the lower gut (a) Health benefits are most clear cut 4. Major phylogenetic groups are similar but species level variation between individuals a) Colon  (1) Firmicutes (a) Symbiotes (2) Actinobacteria (a) Prominent in newborns 5. Culture independent sample a) Cfu - colony forming unit (1) A colony derived from a single organism (2) Assumption is that each colony is derived from one cell (3) Ives an idea of how many bacteria were in the sample (4) Most of the microbes aren't going to grow on the plate (5) Prefer to use culture independent sampling (a) Cfu is a culture dependent assay that is depend on the culture to grow b) Culture independent sample (1) Don't give microbes anytime to grow or not grow after they are removed from their environment (2) To get an accurate view of microbes in a specific environment (3) Steps (a) Extract DNA immediately (b) Analyzing sequencing the gene (all 16S ribosomal genes) (i) Two things to find (a) What genes are present - sequence all the DNA (b) Metagenomic (all sequences) - sequencing all the genomes that are present (c) Don't care about all the species only care about the genes (d) Using massively parallel sequencing techniques c) Complexity of human Gut microflora (1) Analyzed a total of 13,355 16S RNA (a) Gene sequences from 7 different locations of 3 individuals (A, B, and C) (2) Important points (a) Complexity (i) 395 unique phylotypes (b) Novelty (i) 62% of the 395 species were unknown (at species level); 80% have not been cultivated (ii) In 2005, a majority of the human gut were not characterized  (a) Showed our level of ignorance (c) Variation between individuals (d) Published 2005 in the journal Science d) Highly abundant gut microbes and where they fit into the phylogenetic tree (1) Both groups are obligate anaerobes (they are harmed by oxygen but they don’t use oxygen in terms of their metabolism - good guys, the good symbiotes) (2) What biggest factor determining what symbionts predominate (a) Respiratory electron acceptors (O2 and NO3-) (i) In the colon, it is very anaerobic, shown to be associated with a healthy gut (ii) A healthy gut has a very low oxygen level e) Facultative anaerobes (1) These anaerobes use oxygen if available if oxygen is missing and nitrate are there it will use nitrate, however if non are there, it will fermate (a) E. coli is a facultative anaerobe (2) Electron is the best electron acceptor due to being able to accept electrons at the lowest levels (a) If oxygen is present their growth rates are the highest (3) Examine the expression of the genes for nitrate reduction, would not find it near the top of the tube (a) Would find e coli that express nitrate at the bottom of the tube (b) If e.coli for instance sense oxygen f) Difference between oxygen and nitrate (1) Energy is conserved and is used to make the proton gradient and need to transfer 6 protons (2) Nitrate starts off the same but only gets 4 protons (a) Less ATP using nitrate as a terminal electron acceptor than would come from oxygen 6. Paper a) Proteus bacteria b) Inoculated mice with an equal mixture of E. coli wildtype and mutant that lacks the gene that encodes nitroreductase (1) 1:1 ratio inoculated into the gut orally (2) 3 days later, euthanized the mic and opened the guts to discover the ratio after 3 days (a) In the untreated mice (homeostatic mice, where the gut did not get wiped out) (i) 1:1 more or less the same ratio  (b) In mice treated with strep, the range of ratios had 15 more (c) E Coli is at a compactive diags age in these conditions as it needs to be able to express nitrogendeducatse c) Genetic evidence that nitroreductase is important for a facultative anaerobe d) Take under normal conditions, measure amount of nitrate in the colon, wildtype mice that has the PPAR gene (1) What they find the the knockout mice is higher levels of nitrate (2) These genes are able to regulate electron acceptors e) Somehow, if you can get microbiota or the main products made by microbiota during the fermentation process (1) End chains are products (2) By flooding system with short chain fatty acids (a) They make it a less lastly place for facultative anaerobes like e coli. f) Intestinal cells don't express short chain fatty acids into the lumen (1) Short chain fatty acids are important because they are derived from good microbiota 7. Antonine von leunick a) Discovered microbes 8. Take nitroreductase and incubate with sufficient concentrate of tungsten a) Get replacement of mineral cofactor with tungsten b) In a nitrate reductase with tungsten, it's properties encompass different redox reaction rate where it will be more slow 9. Treat mice with sodium tungstate a) Decrease competitive advantage b) More convincing is the tissue histology (1) Normal lining of mouse gut should look like (2) Treated with DSS will be concentrated with neutrophils and be a very inflamed tissue (3) Reduce the activity of a certain nitrate (a) Restrict facultative anaerobe growth and thus limit the growth of the tissue (b) Strong correlation between facultative anaerobes and microbiome (c) E Coli is apart of the microbiome in gut 10. What is cleare a) Short chain fatty acids are generated by obligate anaerobes (1) Breaking down dietary fibers and the end products are the short chain fatty acids  b) Respiratory electron acceptors are host derived (come from humans under bad situations: tissue is destroyed microbiome is disrupted, etc.) and impact on obligate and facilitation ratio (1) Facultative anaerobes take advantage of disruption 11. What is not clear a) What is regulating hist production of respiratory electron acceptors (1) The electron acceptor come from the host but what is regulating is a point of contention (a) Models suggest one thing or another