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NADIA is used in metabolic pathways like glycoside and fatty acid synthesis. LEO says EGGER. Lose electrons oxidation, Gain electron reduction. Know the general time line for biology and evolution on the Earth; What major events altered evolution especially in terms of microbial driven changes 4. 5 billion years- formation of Earth 4 billion yr- life begins 3. 5 billion yr- photosynthetic prokaryote (non oxygen); synthesis of amino acids originated 2. 8 billion yr- photosynthetic contractible (oxygen) 2. 4 billion yr- aerobic bacteria 2 billion yr- unicellular eukaryote . Billion yr- multicultural eukaryote . 5 billion yr- plants and animals Carl Woes – what did he contribute? What kind of biological molecules are best to enervate phylogeny trees, and why? How does this compare to enzymes? In 1950-ass’s: Carl Woes in Illinois was studying arches bacteria, methanol’s from cow. He started working on RNA, and sequencing. He noticed that there are clusters of microbes. He was doing crude sequencings of methanol’s. He had a prediction that there are 2 clusters of prokaryote: architecture & phylogeny tree.
He generated the 1 AS/ASS RNA tree. Using different enzymes would generate different phylogeny trees because genes and proteins evolved Independently from ribosome. The RNA coding regions are giggly conserved because they code ribosomal RNA which Is essential for protein synthesis. Even a single nucleotide polymorphism In this region would code for Inefficient and ‘sick” ribosome, so these coding regions are essentially Identical across species. Conventional Definition: -DNA to DNA habitation between total genomic DNA of 2 isolates of 70% or greater. ASS RNA similarity has to be greater than 97%: if they are, they are in the same genus and species. Natural Species Concept: -relates species with function, gene diversity exceed that of ASS RNA -role of organism in the environment did notes: * Bacteria and arches: ASS RNA *Eukaryote: ASS RNA What are open genomes? What are closed genomes? What is meant by the ‘core’ genome? Open genomes are able to incorporate genetic material that is foreign to the species’ own genetic material. Genes are fluid and may be found in separate (or related) genomes e. G. C. Difficult genes being found in the E. Coli genome.
An open genome is determined by comparing an isolates genome to a genomic library of that same species – if 10% or more (general guideline) of the genome has never been observed in that species, it has an open genome. The differences between genomes are outside the core region, in the highly variable regions. Closed genomes are the polar opposite of open genomes, and these genomes are conserved across all isolates of that species. In this model, the genome is not open to foreign genes and will evolve independently of outside genetic influence. The only differences you see in these genomes are the Snaps. An example of a closed genome is B. Anthrax’s (Anthrax) Core genomes are the essential genes in all genomes and is conserved across all pathogenic species; these genes are very often involved in metabolism and growth. These genomes hardly change because they are central to the microbe and slight changes may kill the organism. A pan-genome is the entire gene pool for that pathogen species, and includes genes that are not shared by all strains. Pan- genomes may be open or closed depending on whether comparative analysis of multiple strains reveals no new genes (closed) or many new genes (open) compared to the core genome for that pathogen species.
Short stuff: Open genome = C. Difficult = many different isolates and strains. There are large variations between same genus, species (new genes + core genome). Closed genome = B. Anthracic = very, very little variation when different isolated have been sequenced. Phenomena = all the genes that have ever been sequenced from E. Coli. Also known as microbial Pangaea. All the genes in that genome. Arches are usually found in what kind of environments? How does this support Woe’s hypothesis for three domains of life? What does this likely say about early evolution on Earth?
They are found in extreme environments exhibiting extreme (often toxic to other forms of life). This supports Carl Woes in that he posited that if you are competitive in extreme environments, evolution favors those who can persist n those environments. Earth’s early environment was very volatile and inhospitable to the lifeworks that we know today, but architecture regularly thrive in conditions that are analogous to early Earth’s environment. This means that architecture are good candidates for the progeny of the originators of life on Earth, essentially evolved with Earth.
Arches – no established pathogens? How does this support Woe’s hypothesis as well? Arches are not pathogenic for plants or animals, supporting Woe’s hypothesis in that they are found only in extreme environments, arches have evolved and developed their cellular metabolism independent of bacteria and eukaryote; therefore, interaction between arches and eukaryote (particularly humans) is minimal especially when in regards to pathogenic. Not antibiotic sensitive, enzymes resemble eukaryote, extreme thermopiles and methane producers. Ass RNA.
It could potentially be a big problem for humans if some archival organism went pathogenic as we would have little to quickly combat it. 8. ) Know and understand the terms: minimal medium; defined medium; undefined medium; rich medium; protractor; Minimal medium: minimal medium given for an organism to grow. -slow growth, more work. -oxidized glucose + 02 to produce 50% CO 50% biomass. -could be defined medium -protractor: don’t require a specific nutrient to grow, converts any carbon source to what it needs (I. E. Amino acids). Defined Medium – a medium that is made up of chemicals that are pure.
Exact contents have been measured out. There are no or “unknowns”. This tells us little about the requirements for growth. Undefined Media – any time you have an extract that is used as a medium for bacterial growth, usually used because it is cheap and all that is needed is to grow he bacteria, almost always a rich medium because yeast extract (most commonly used) contains more nutrients than already present in minimal media. We don’t know the exact components Just that it has a lot of organic nutrients. Brain-Heart infusions and Lurid Broth are another example of undefined media.
Rich medium = contains plenty of nutrients, usually resulting in a large amount of growth. ”20 minute doubling times. Defined minimal medium = the simplest composition of a defined medium for the microbe to grow. It forces the microbe to makes all its amino acids. Selective medium = takes advantage of resistance to an agent and competitive use of a carbon or nitrogen source. Differential medium = use colony morphology to help identify genus or even species level identification. Protractor = organism capable of synthesizing all of its own metabolites What defines growth? Are changes in the number of cells in a test tube also defining growth?
Why or why not? Growth is strictly defined as a change in mass of a culture. Cell number may not change during this time (e. G. Smaller cells becoming larger, Turbidity (Optical Density):Measure of the light that is scattered in a phosphorescent as a representative measure of the number of cells Assumptions: All cells are of equal size Linear over a range of concentrations In Practice: A standard curve is required to determine if linear relationship is true, and to relate ODD to the number of cells Pretty common method (if not the most common) * High cell densities can lower the accuracy of the turbidity reading!
Total Cell Counts: Uses a counting chamber (glass slide with defined area and depth) which is divided like a grid. You microscopically count one grid and they multiply by the total. Electronic Cell counting can also be used, which operates by passing the cells through a narrow pore with an electrical field.
The bacterium passes the conductivity changes and a voltage difference is triggered Limitations: Cannot distinguish between live and dead cells (Because dead cells also have a resistance) Cannot be used to count very low cell density cultures Viable Cell Counts: Cells are serial diluted and plated on a growth medium Each colony represents a viable cell in culture tested Limitations: Clumps of cells will be represented by a single colony, and most bacteria grow in luster Some cells plate with a poor efficiency (do not survive the manipulation or simply grow better in a liquid medium) Dry Weight/Protein Cells are harvested by centrifugation, dried and carefully weighed Dried cells can also be assayed for protein content, which also increases with growth These techniques are not typically used in today’s lab as they are not amenable to assaying many cultures at several time points, etc. (since the cells are “killed” when assayed) – But when this is found it is the best marker for measurement since protein increases tit mass Growth is strictly defined as a change in mass of a culture. Cell number may not change during this time (e. G. Mailer cells becoming larger, larger cells dividing to smaller cells when growth is not occurring, etc) Understand the phases of growth, and also the physiological changes that are occurring during each phase, and especially between each phase of the typical batch growth curve (see graph) Fig 2. 3 -Lag is prolonged when it is taken from rich media and placed into minimal media – When we discuss E. Coli, we are talking about E. Coli KEG, it has about 4400 genes -In he order of 1000 genes need to be turned on to start making everything from scratch making everything, slowly start using carbon to generate TAP and biomass, then slowly increase in size -As they accumulate biomass, they become larger, and start dividing during log phase). In exponential phase, cells are growing as fast as they can with the nutrients available -Fish protein active because it turns on ribosome synthesis (more ribosome= better growth) -When the growth curve flattens, cells run out of an essential nutrient or a toxic product (metabolite) builds up, or there are too many cells and they are crowded At that moment, they stop increasing in biomass. They still divide in stationary phase, mass remains flat, number of cells can change, no growth -As the cells divide they become smaller in size but the overall mass of the population is unchanged. The reason that they continue to divide is to spread out their numbers in hopes that a higher number of cells can find the nutrients more quickly.
H-INS protein will be active here: binds to Fish promoter region and ribosome synthesis stops -In the transition from exponential to stationary phase, the number of cells increases (large cells divide) without a change of mass, no Roth Know the roles for Fish and H-INS in regulation of ribosomal synthesis; also note the physiological state of ribosome (abundance) during log phase growth and what happens upon starvation for nutrients (pig – stringent response) Fish and H-INS DNA binding proteins (Fish – specific; H-INS – non-specific) Fish activates RNA gene transcription – inhibited by H-INS H-INS levels increase during stationary phase – binds to Fish’ promoter region results in inhibition of ribosome synthesis Stationary phase sees cells transition from a lot of ribosome to a few ribosome Fish is high in actively growing cells (in log phase) until starvation is hit (at the beginning point of stationary phase), Fish levels drop and H-INS levels increase and bind to promoter to stop transcription (p)pig “Magic Spot” – slang term guanidine tetra/Pentecostal derivative shift from rich to minimal medium results in scum. F uncharged tarn (= stringent response) causes ribosome to stall –When there aren’t enough amino acids to make (because not enough nutrients/ elements available) the tarn loses an efficient charge which causes the ribosome tailing -When a ribosome cannot read through a cod, it means that the tarn is not charged. Real sits with the ribosome to sense the stalling and turn on pig Real (p)pig syntheses I in complex with stalled ribosome is activated by uncharged tarn pig synthesized & slows tarn/RNA transcription Triggers a slow down in ribosomal synthesis, do not know mechanism Recently reported that it triggers the arrest of the replication fork 16. ) Understand the timing of DNA replication during either fast growing cells (e. G. 20 minute doubling time) versus slow growing cells (e. G. Hour doubling time); when do replication forks start? ; how can you divide in 20 minutes overall? Etc…. Cells reach critical mass (sensed internally) DNA duplex Other replication proteins bind to form a complex Chromosome replication and partition to opposite poles (Par and Musk proteins assist) Septum forms (often in center of cell) by inward growth of CM, PIG, & MM (Cell Membrane, Pedagogical, Outer membrane) (LIPS also in gram negative) Separation & cell separation occur in parallel The overall synthesis of the DNA in a rapidly growing E. Coli cell (generation time of sees than one hour) takes about 40 minutes. The actual partitioning of the cell and final separation requires another 20 minutes Thus, the overall process from the initiation of replication of the chromosome to the complete separation is roughly one hour. For fast growing cells, the number of ribosome is also increased. This allows protein synthesis to take place in a more efficient way because ribosome operate at a single speed. Synthesis of DNA may also begin in previous generations.
Know the changes in the relative amount of DNA, RNA and protein during fast vs… Slow growth rates for E. Oil Protein synthesis is ‘one speed’ – to make more cells faster you have to make more ribosome. More hands working on building = faster building. The relative level of protein and RNA is significantly affected by growth rate 0. 6 represents slower growth (minimal medium) while 2. 5 doubling per hour represents rich medium –0. 5 doubling per hours=2 hour replication time/l doubling per hour”I hour replication time -DNA increases as a result of the multiple replication forks in fast growing conditions Understand the definition of growth yield Y, for carbon sources. Also know what the Y alee is for E. Oil growing in glucose minimal medium with oxygen as an electron acceptor (and why this is true) Grams of biomass over the grams of carbon source (biomass/carbon source) For glucose, growing under aerobic conditions the Y is about 0. 5 – which translates into 50% biomass, 50 % carbon dioxide (what media is this? I would say rich probably – this is for minimal media, rich is better; this is only applicable to a single carbon source; I. E. Glucose by itself). If growing in optimal conditions with Glucose yields 50% CO and 50% biomass, 0. 5 is the best Y because glucose is the best carbon source. Growing in another C source will be less. Private for example, is more oxidized than carbon and some of that source will be have to converted to make glucose anyway. ‘says.
If 10 g private given, and 1 g of E. Coli bio mass is produced, the Y is 0. 1 Growth in rich media–OHIO% CO production because you don’t need to utilize that carbon to make other amino acids Grow e coli with glucose as a carbon source, and it using nitrate as an electron donor, growth yield = 0. 4 ‘Expect that 0. 4 Goff biomass produced. He said this in class. Why isn’t it true? When growing in minimal medium, oxygen is the most efficient electron acceptor, meaning the Y would be 0. 5, meaning half of the glucose would be used to make energy and half would be used for biomass. Other electron acceptors, like nitrate, aren’t as efficient, so they will always have a Y of less than 0. Because oxygen is the most efficient. Under those conditions, the value will never be more Anyone remember the question about this on Test 1? More Oxidized = Lower Y value? Private is an example of a more oxidized carbon source and it would have a lower Y value because it takes more work for the cell to change private into glucose. Now the differences between substrate level phosphorescently and membrane gradients AZ(I. E. Oxidative phosphorescently); know the basic chemical reaction behind SLP chemistry 1 . ) Substrate level phosphorescently (Chapter 8 in revised textbook) flow of e- from donors (E’ O) coupled to TAP synthesis in cytology 2. Oxidative phosphorescently or electron transport flow of e- from donors (E’ O) coupled to TAP synthesis in membrane systems Substrate level phosphorescently was the first studied synthesis of TAP Substrate level phosphorescently harnesses energy through membrane gradients, always in the cytology, not in the membrane Oxidative phosphorescently is in the membrane Cells do work to push protons out, harness energy from cytology into the gradient, called proton pumping Protons build up around the cell and enter through other proteins NADIA (primary energy input) can act as a battery to push the protons Protons fall back through proton channels (back inside the cell) to generate TAP Peter Mitchell – what did he propose and what were the three driving principles of his proposal Mitchell was the first to propose a mechanism other than substrate bevel phosphorescently (which dated to around 1953) Mitchell was the first to propose the proton gradient Semiotics Theory: Driving principles of the theory: 1 . The cell membrane must be essentially impermeable to H+ & OH- permeable to water, but not to differently charged molecules It is This causes it to act as a capacitor, separating charge and generating energy 2. ) Enzymes (e. G. Respiratory, photosynthetic, TAP hydroplaning) that translate H+ outside the cell to form an electrochemical gradient are located in the membrane, and use coercing reactions to drive proton translation 3. An electrochemical gradient develops by this action that is composed of: PAP or pH gradient (acidic out & basic in)–More positive charge outside VA (SSI) or membrane potential (+ out & – in) Talking about E. Coli systolic membrane, innermost membrane The E.
Coli cell membrane acts as a capacitor that gets charge Enzymes in the membrane do redo chemistry that translate protons, deposit them on one side of the membrane The change in available protons contributes to the capacitance of the cell membrane, it is more positive on the outside of the membrane (more acidic pH) now the MAP equation and understand the difference between delta SSI and delta pH Ap is proton motive force Ap = – APIPA where Ap is the potential energy stored in the electrochemical proton gradient as Ap becomes more negative the potential to do work becomes greater VA = -60 log [X+ negative; VA (membrane potential gradient) is limited by the membrane capacity (Max ” 40,000 H+ or -200 NV) PAP = pH in – pH out as the pH drops outside the cell (more H+ ions, increase in acidity), Ap becomes more negative Understand the concepts behind the potential energy to do work with an electrochemical gradient, and how this ties into the core physiology of the cell (central metabolism) overall 1 . ) The cell membrane must be essentially impermeable to H+ & OH- 2. ) Enzymes (e. G. Respiratory, photosynthetic, TAP hydroplaning) that translate H+ outside the cell to form an electrochemical gradient are located in the membrane, and use coercing reactions to drive proton translation 3. ) An electrochemical gradient develops by this action that is composed of: PAP or pH gradient (acidic out & basic in) VA (SSI) or membrane potential (+ out & – in)
Ap = VA – APIPA where Ap is the potential energy stored in the electrochemical proton gradient As Ap becomes more negative, the potential to do work becomes greater -60 log [X+ in]/[X+out] as positive ions accumulate outside the cell the VA and thus Ap become more negative; VA is limited by the membrane capacity (Max ” 40,000 H+ or -200 NV) PAP = pH in – pH out as the pH drops outside the cell, Ap becomes more negative -rest Understand the nature of the electron donors, H-carriers, and electron carriers (what molecules are responsible for these roles in the cell) Electron Donors AND+/NADIA, negative redo potential (H2O, HAS, Fad Spinach) H- Carriers- Flavorings, Quinine’s Electron Carriers Flavorings, Quinine’s, Fee-S protein, Stockrooms. The electrons are not carried in the protein, but in a nonprofit molecule bound to the protein called prosthetic group. Iron-sulfur protein: Fees clusters n: flavor (FIN/FAD) Stockrooms: hem molecule bound or used by a protein, so quinine’s do not have prosthetic groups.
Flavorings- e/H, catalyst oxidation reduction reactions in cytoplasm (not only e- transport in the membrane) , Flavor FAD and FIN are from Riboflavin ebb. Villains reduce carry H, Villains give up 1 e- at a time because Fees cluster can only carry one e-. NADIA (which carry 2 e-) must interact with Florentine (gives up 1 e- at a time but holds 2) Ubiquitous- found in Mitochondria , mobile in membrane carries e- from complex 2 to 3. (Eukaryotic complexes not on test, but ubiquitous is also found in bacteria) Most y can accept 2 e-) Quinine’s- mobile in the lipid phase of cell membrane, carry e/H to and from complexes of protein electron carriers that are not mobile not proteins abundant of quinine’s. Has suppression side chains. Shuttles AH+ to and from non- mobile complexes
Uneasiness-from vitamin K , investigations, because they have lower electrode potentials than US, they are used during anaerobic respiration when the e- acceptor has low potential (ex fumigate) Platitudinous- chloroplasts and contractible found in microbes Fees- Uneasiness are only e. Catalyst oxidation reduction reactions in cytoplasm and membranes Non-hem Feb.. Acid labile S meaning when pH is 1 HAS is released from the protein Fee is bound in protein by 4 Sulfurs (2 acid labile sulfurs, 2 sulfurs from cytosine Carries 1 E- Wide range potential -move to +move so it can carry oxidation-reduction sins at low and high potential. Stockroom- After translation sulfur atom is added. , send electrons to final electron acceptor -e donor: flavor FAD, Fees, Quinine, Fees, stockroom: e acceptor Hem is the prosthetic group Hem (4 payrolls) called tetra-payroll (porphyry’s are substituted payroll) Active site is the center of hem which is Fee and is Stockroom B carrier, and modifies redo potential (has varying redo potentials) Ferric Fee+; Reduced-Ferrous Fee+ Fee is the e- Oxidized- Reduced state alpha bands Beta is lower in wavelength, and Gamma is blue region of spectra deco potential (standard electrode potential), and how is the energy derived from molecules with different redo potentials used to drive production of MAP Electrons flow from a negative potential electron donor to a less negative (or positive) electron potential acceptor. The transfer of electrons from donor to acceptor is so favorable that it provides the driving force for the protons to be pumped against their gradient. What are the role of quinine’s in the electron transport chain? Quinine’s are lipid soluble hydrogen carriers. They contain suppression side chains (that contribute to heir lipid solubility) and are mobile in the membrane. Because of their membrane, carrying hydrogen and electrons to and from the complexes of protein electron carriers that are not mobile. They are part of the e- transport chain and the p+ translation.
What is the role of Fees clusters, and why are they so critical in the coupling of redo potential to the development of MAP (what do they carry? – what are their redo potentials? ) Fees clusters are 1 electron carriers and have a wide range of redo potentials from -400 NV to +350 NV. They therefore can carry out oxidation/reduction sections at both the low-potential end and the high-potential end of the electron transport chain and are found in several locations. Stockrooms – what molecule is the redo active site – what are the oxidation states? How does the environment around this molecule affect the redo potential of the hem/stockroom? Hem is the prosthetic group. The iron is the electron carrier. O Oxidized – ferric (Fee+) o Reduced – ferrous (Fee+) Bacterial stockrooms include stockrooms bad and boo d and o Prokaryote- The groups around the ring modify the redo potential of the iron atom (electron withdrawing or donating) What is the general scheme for an electron transport chain? NADIA (Florentine) ”+ Fees ”+ quinine ”+ Fees ”+ stockroom ”+ 02 What makes bacterial electron transport chains unique (as opposed to mitochondrial) – why do bacteria have these type of modular electron transport chains? How does this affect the bacterial cell’s ability to survive? Similar to mitochondrial ETC, bacterial chains are organized into dehydrogenate and oxides complexes connected by quinine’s.
The quinine’s accept electrons from dehydrogenates and transfer them to oxides complexes that reduce the terminal electron acceptor. Bacteria re capable of using electron acceptors other than oxygen during anaerobic respiration (can use nitrate and fumigate). During anaerobic respiration, the enzyme complexes that reduce electron acceptors other than oxygen are called reeducates. Bacterial ETC is unique in that the pathways are branched. The modular chains are to balance efficiency since every electron that passes through the chain has a different efficiency (in the mitochondria, this is constant… Which is why mitochondria have linear instead of branched chains).
The two major differences between mitochondrial Test’s and bacterial is that many bacteria can alter their electron transport chains depending on growth conditions and bacteria have branched routes to oxygen (the branch point being a quinine or stockroom). The ability to have branch pathway affect the MAP and allows the bacteria to select terminal oxides with high affinity for oxygen. Switching to an oxides with higher affinity for oxygen allows the cells to continue to respire even when oxygen tensions fall to low values. This ability is important to ensure the restoration of the reduced quinine’s and NADIA so that cellular oxidations such as oxidation of glucose to private or the oxidation of private to CO can continue.