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294 Cards in this Set
- Front
- Back
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photosynthesis
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biological conversion of light energy into chemical bond energy
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photosynthesis occurring where due to who?
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half by bacteria, half by plants and algae
half in oceans and lakes, half on land |
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eukarotic and prokaryotic
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eukaryotic are plants and algae (photo-autotrophs
prokaryotic are bacteria (photo-autotrophs and photo-heterotrophs) |
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who doesn't do photosynthesis and what do they lack?
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archaea, lack chloroplast-type oxygen producing mechanisms.
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what 2 things do you need for oxygenic photosynthesis?
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2 photosystems with distinct PRC's:
Photosystem I: cyclic or non-cyclic Photosystem II: non-cyclic only (that splits H2O) |
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light reactions occur in what with what?
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chloroplast with thylakoids in grana, stroma
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photosynthetic microbes differ in (5)?
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1. wavelength of light used
2. source of electrons (DH) 3. pH range for growth 4. temperature for growth 5. salinity for growth |
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explain microbial mats
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different organisms living in different layers of mud: cyanobacteria, purple bacteria, green bacteria. with aerobic on surface, anaerobic subsurface.
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photo pigments differ in the 3 organisms. explain. which likes which wavelengths?
also, what is the chemical reaction on surface and subsurface? |
reflects ability to harvest different wavelengths of light! different niches, can use almost entire visible and infrared spectrum.
cyanobacteria like short wavelength. purple like longest, then green like middle. surface: O2 production -> 100% O2 subsurface: sulfide oxidation (H2S) |
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cyano light harvesting antenna
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phycobili-proteins: rods with blue light-harvesting pigment phycocyanin. core with allophycocyanins (A and B) which are bluish in color, linked via polypeptides to photosystems in thylakoid membrane.
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structures of chlorophylls (4 parts)
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porphryn ring
with magnesium in the center. different side groups: related chlorophylls. phytyl side chain provides hydrophobic tail that anchors the chlorophyll to membrane protein complexes, the site of light reactions. |
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carotenoids
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tons of double bonds, very nonpolar, with rings on ends.
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cyanobacteria really close to..? (connection with evolution)
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chloroplast 16s.
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how did chlorophyll get passed and from where to where?
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lateral gene transfer from B->E.
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bacterial phototrophs come in 5 flavors, what are they photo-?
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purple nonsulfur bacteria: photo-autotroph and organotroth
purple sulfur bacteria: photo-autotroph green sulfur bacteria: photo-autotroph cyanobacteria: photo-autotroph heliobacteria: photo-organotroph |
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photosynthesis factory: 3 general divisions
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1. photoreceptor apparatus: to gather and harvest light energy
2. an electron transfer pathway: to generate pmf and generate NADH 3. an ATP synthase: to make ATP. |
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photoreceptor apparatus in more detail. mention analogy. mention 4 excited states
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(like pinball machine)
1. photon strikes 2. excited state Then it's a pathway by which light energy is converted to chemical energy, the essence of photosynthesis. breaks into... 4 excited states: 1. thermal dissipation (heat comes out) 2. fluorescence (photon of fluorescence comes out) 3. exciton transfer, to neighboring molecule (physical resonance) 4. electron transfer (oxidized M to M+) |
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more about outcome III
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photon hits light-harvesting antenna pigment-protein complexes, absorb light energy, transfer it from one antenna molecule to another until it reaches the specialized chlorophylls of the reaction center. ground state to excited state, and physical resonance. pass to another molecule.
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outcome IV
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eject e-, put to ETC. light from sun, pass it thru, PMF, ATP.
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step of photosystem (6)
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-photoreceptor reaches light capturing molecule
-electron flows from photosynthetic reaction center to a pair of bacteriochlorophyll b molecules (Bchl 870)... -energized electron leaves the PRC -transferred to an electron transfer pathway, and the electron is passed down the chain. -passed to ubiquinone -then passes thru a series of quinone carriers in cytochrome bc1 complex. -cyclic electron flow in the cytochrome bc1 complex drives protons out of the cell and then proton motive force is made (or NADH) (cytochrome c brings e- back to photosynthetic center. cyclic. -protons reenter the cell thru ATP synthase, ATP made. |
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pmf is formed by...?
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the Q-cycle via the bc1 complex.
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cells need to make NADPH for biosynthesis. why?
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many anabolic reactions require NADPH (and ATP)
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also need NADPH and NADH for what?
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fix CO2.
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so what is active in cyclic but not in acyclic?
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bc1
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when non-cyclic, what is missing and what happens? requires what ""?
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bc1 complex not used. after electron is passed to Q pool, electron goes to NAD+ to make NADH, and electron comes from lactate to make pyruvate (not H2O) and goes to cyt c2 to go back to BChl 870.
DH+A -> D+AH where DH is lactate and A is NAD+ "reversed electron transfer" |
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what enzyme is necessary in non-cyclic? why do we need this electron anyway? other examples
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dehydrogenase (lactate dehydrogenase) is made to use the electron donor.
electron provides the missing electron to restore PRC, to the "ground state" other cells use different dehydrogenase and utilize an alternative donor molecule: ethanol dehydrogenase to use ethanol, sulfide oxidase to use H2S. |
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what to energy currencies are necessary for cells to perform anabolic metabolism?
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ATP, NADPH.
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cyclic vs non-cyclic electron transport analogy
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factory that makes different products: uses common tools to make both products, but not all tools needed for each product.
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what is the essential role of bc1 complex that doesn't occur when it's non-cyclic?
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forming proton motive force.
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green sulfur bacteria don't get much light. so?
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a lot of antenna molecules per chlorosome to catch a lot of photons.
live in low light, so grow slow. use long wavelength of light, specialist. |
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green sulfur bacteria use what for electrons?
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inorganic electron donors.
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of the organisms in the mud.. photo pigments reflect what?
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populations in the microbial mat from surface down.
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all photosynthetic reactions need membranes (6 facts)
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1. many diff arrangements (tubes, vesicles, stacks, bundled tubes, lamellae)
2. one of these was origin of chloroplasts 3. again, diff photo-pigments 4. diff donor types 5. diff habitats 6. diff ecological roles |
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invagination of cytoplasmic membrane why?
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higher surface area, maximize photosynthesis, these membrane folds look like organelles
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"halo-archaea" (6)
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-(halo bacteria) high salt.
-photon comes in, excited in periplasm -proton goes to to N+ - H, pumped out while making N without H -pmf made -ATP synthase. -they get NADH from breakdown of organic materials (other microbes, dead flies, birds, plants) -no photosystem II -no "non-cyclic ability" -can't fix CO2 |
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archaea rhodopsin differs from?
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cyclic/non-cyclic modes -> further molecular support for separate domains of life.
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microbes are critical for CO2, how do they fix this?
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photosynthesis and chemosynthesis
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what are microbes critical for?
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recycling organic carbon back to CO2
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where is most of the carbon in earth and in what form?
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rocks and sediments, in inorganic form (99.5%)
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what's the next small fraction of carbon and where is it?
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bicarbonates in oceans, atmosphere, streams, rivers, bogs, etc (0.5%)
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smallest fraction of carbon where and in what?
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organic, in form of either living or decaying life forms
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what carbon is not counted in this?
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hydrocarbons (coal, oil, natural gas)
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who fixes CO2?
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autotroph
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autotropic routes to organic carbon. how many out there??
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6 microbial pathways
2 newly discovered archaea pathways. |
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what branched together concerning photosynthesis? (3 things)
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chloroplast 16S branch with bacteria, as does calvin cycle.
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what about archaea relating to CO2?
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they lack chloroplast-type CO2 fixing mechanisms, has it's own unique pathways!
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how do we know the oxygen content of earth in history?
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stromatolites and other fossils record the events, the change in earth's oxygen levels was early.
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start of earth, start of life, lots of O2??
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4.6 BYR
4 BYR 2.4 BYR |
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how did earth go from dry, anaerobic to aerobic? also alternative?
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started out with tons of iron dissolved in acidic seas
photosynthetic organisms generated oxygen iron in earth's oceans was precipitated out as iron oxides or maybe microbes who oxidized FE2. |
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when did photosystem I and II come up?
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middle earth.
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stromatolites are communities of photosynthetic bacteria (4 facts)
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-total oxygen locked up in banded iron beds is perhaps 20 times volume of oxygen present in modern atmosphere.
-banded iron ore important commercial source of iron ore (steel). -carbonates were deposited by bacteria. -chemoautotropic iron oxidizing bacteria. |
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how much is atmospheric CO2 changing? numbers!
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36% in 190 years.
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carbon dioxide change measured in hawaii...
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1. today CO2 is at 390ppm-ish
2. 1832 antarctic ice core was 284ppm |
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what is the annual fluctuation of carbon dioxide and why is it there?
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3-9ppm, follows northern hemisphere growing season
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for every naturally occurring organic compound...?
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some microbe can degrade it! as a source of cell energy or carbon!
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CO2 rise because of what for sure and what potentially?
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humans
natural causes |
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microbes can form micro-colonies on soil particles, but where? (4)
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1. inside quartz
2. in air b/w particles 3. in water b/w particles 4. in anaerobic part b/w particles |
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what do microbes do in these habitats? (3)
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1. respire with O2
2. respire without O2 3. ferment |
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what two cycles interface?
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carbon and nitrogen
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overall nitrogen cycle on earth (8)
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1. N2 goes thru nitrogen fixation by bacteria and archaea
2. -NH2 (organic nitrogen or amino group, this is cellular nitrogen compounds) goes thru ammonification 3. NH3. -can go thru ammonia assimilation back to -NH2. -can be made into N2O -goes to anammox, a process carried out only by members of plantocycetes (back to N2) 4. N2O goes thru nitrification, by select bacteria and archaea and goes to NO2- 5. NO2- to NO3- (which can go thru nitrate assimilation back to -NH2 or go thru nitrate ammonification back to NH3) 6. NO3- goes to NO2- 7. NO2- goes thru denitrification by bacteria under anoxic conditions to make N2) 8. N2O goes back to make N2. |
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what's awesome about NH2?
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can be electron donor/acceptor
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cells need N for what else?
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proteins, purines, pyrimidines
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what is the favorite way of uptake? equation?
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ammonia. NO3- + 8H+ --> NH4+
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laziness of cells.. so?
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uptake is preferred to synthesis.
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nitrogen fixation... what's the importance of this step?
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plants can't do, so has bacterial partner to do it for them. plant symbiotic nitrogen fixation needs bacteria.
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cells need a nitrogen supply. how prioritize? (4)
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1. look for what's available
2. amount of energy needed? 3. what enzymes to make? 4. regulate them as needed! |
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for example, how get nitrogen supply? (4)
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1. induce uptake systems
2. induce breakdown enzymes 3. induce biosynthesis reactions 4. induce fixation reactions |
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priority of what cells want to take in (5)
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1. organic nitrogen
2. amino acids 3. ammonia asimilation 4. nitrate assimilation 5. nitrogen fixation |
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4th. nitrate assimilation (6 facts)
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-assimilatory nitrate reduction
-assimilate N from NO3- into cell material -NO3- + 8H+ -> NH4+ -> protein -catalyzed by cytoplasmic enzymes these reactions do not produce energy -rather they require energy input -enzymes are nearly universally found in bacteria, plants, archaea, and fungi |
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who can fix gaseous nitrogen and where??
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some bacteria and archaea: obligate aerobes, facultative and strict anaerobes. usually found in low "N" environments (freshwater, oceans, soils, deserts, lakes)
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equation for fixing nitrogen
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N2 + 10e- + 10H+ -> 2NH4+ + H2
requires a lot of energy (endergonic) deltaG=+680 |
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nitrogenase (3 facts, and general pathway)
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remarkably conserved enzyme
2 components: FeS (nitrogenase reductase) and FeS-Mo-Co (Nitrogenase) electrons provided by a Fd. Electron from NADH to reduced ferredoxin (Fd) and electron is used to catalyze the reduction of N2 to 2NH4+ and 2H+ to H2. |
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vast amounts of N2 fixed on earth (% done by??)
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85% fixed biologically
3% lightning 12% by fertilizer plants |
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because only prokaryotes can fix N2 (2)
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1. plants and animals must acquire fixed "N" by uptake of ammonia, nitrate, or organic "N"
2. or interact symbiotically with bacteria. |
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fate of ammonia (2) ? consider...? (3)
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1. acquired by cells for new growth (biologically recycled)
2. enters geological cycle (non-biological fates) 1. soils 2. streams, rivers, lakes 3. atmosphere |
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aerobic nitrification occurs in two steps
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1. ammonia oxidation to nitrite
2. nitrite oxidadation to nitrate. 1. each is a respiratory process to obtain energy 2. each occurs only under aerobic conditions 3. electron donors are either NH4+ or NO2- 4. electron acceptor is always O2 5. electron transport generates pmf and thus ATP |
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example of nitrification
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feedlots in central valley, animal wastes converted to ammonia (ammoniafication) and then oxidized to nitrite and nitrate in the soil zone.
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nitrate forms what??
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plume.
numerous nitrate groundwater plumes exist in california. most sites are associated with dairies, feedlots, industrial waste, septic systems, waste water holding ponds, leaking sewer lines, fertilizers, and manufacture of chemicals. may contaminate groundwater as it flows "downstream" 52% of community water wells 57% of domestic water wells up to 15% of wells in agricultural and urban areas have nitrate levels exceeding US EPA limit for drinking water (10mg/liter) |
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effects of nitrate on human and animal health
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nitrate can be toxic, especially to infants.
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what about wells should be considered?
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location and depth
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nitrate travel distances depend on?
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type of soil and bedrock present
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contamination of aquifer
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can take years, where reversal takes thousands of years.
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nitrification occurs in two steps (2 equations)
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NH4+ + O2 -> NO2- + H2O
NO2- + O2 -> NO3- + H2O First step: nitrosomonas species 1. oxidizes ammonia to nitrite by gram negative chemo-autotrophs. 2. ammonia oxidizing enzymes make nitrite. 3. cytochrome oxidase reduces oxygen to make pmf 4. ATP synthase uses pmf to make ATP 5. all enzymes are membrane bound 6. a lot of membranes for ammonia to nitrate (electron transport). Second step: nitrobacter species 1. oxidizes nitrite to nitrate 2. gram negative "chemo-autotrophs" 3. internal membrane assays. 4. nitrite oxidase generates nitrate cytochrom oxidase reduces oxygen to make pmf ATP synthase uses pmf to make ATP |
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these microbes that do nitrification are...?
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1. obligate aerobes
2. must acquire all energy from inorganic compounds 3. have pretty good energy yields: ammonia oxidation to nitrite -275, nitrite oxidation to nitrate -76. 4. nitrate accumulates geologically, unless some other organisms metabolize it. 5. they can't use any organic carbon for growth 6. they must fix CO2 into cell carbon. |
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anaerobic denitrification in 2 flavors
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1. nitrate is reduced to N2 by denitrifiers
2. nitrate is reduced to ammonia by nitrate ammonifiers (more energy) 1. serves "dissimilatory" role (vs an assimilatory role) 2. respiratory process for energy harvesting 3. occurs only under anaerobic conditions 4. enzymes are membrane bound 5. the electron donor is often NADH 6. but can be formate, or other reduced compounds. |
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denitrification to N2 pathway and enzymes.
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NO3- -> NO2- -> NO -> N2O -> N2, with NADH feeding 2 e- inbetween. the arrows are
1. nitrate reductase 2. nitrite reductase 3. nitric oxide reductase 4. nitrous oxide reductase |
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nitrate ammonification (pathway, enzymes)
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NO3- -> NO2- -> NH4+ with 2e-'s going inbetween.
1. nitrate reductase 2. nitrite reductase (diff from enyme in denitrifiers) protons pumped by NADH dehydrogenase, protons consumed by Nar nitrate reductase. E.Coli has second nitrite reductase type enzyme. |
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microbes that do nitrate ammonification (2)
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often facultative (aerobes/anaerobes): many types of gram - and + soil bacteria, some purple photosynthetic bacteria, some bacteria
use organic and inorganic compounds to acquire energy (very good energy yields) |
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sulfur oxidation
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oxygen is electron acceptor and is reduced. H2S -> S0 -> SO4 2-
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sulfur oxidizing bacteria (8)
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1. chemo-autotrophs
2. must fix CO2 3. electron donors are reduced to S 4. electron acceptor always O2 5. sulfuric acid is formed 6. electron transport generates pmf 7. ATP synthase generates ATP 8. considerable energy obtained. |
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acid mine drainage (4 facts)
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oxygen+water+sulfuric acid -> heavy metals -> fish mortality.
extraction decreases groundwater depth and natural filtration and increases groundwater contamination. |
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sulfate reducing bacteria (10)
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1. respiratory option when no O2 is present
2. chemo-heterotrophic group of microbes. 3. electon acceptor is SO42-, SO32-, S0, etc. 4. electron donor: organics (NADH) 5. end product is hydrogen sulfide: H2S or HS- 6. sulfate is first reduced to sulfite 7. sulfite is further reduced with electrons from NADH 8. likewise, for thio-sulfate, and elemental sulfur 9. RLP generates pmf, which gives ATP 10. considerable energy obtained from bacteria. |
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passive motility (2)
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air currents or ocean currents
aerosolized (cough or sneeze) |
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active motility
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microbes use cellular structures to move in their environment
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why motility? (3)
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1. find nutrients
2. avoid hostile environments 3. pathogens use motility to invade host or disseminate within host. |
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cost to motility
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requires energy
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flagellar arrangements (4)
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monotrichous (polar): one hair
amphitrichous: two hairs on either rod ends lophotrichous: tuft of hair peritrichous: around equally, bundle for forward motion. |
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flagella size, see how?
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3-20 micrometers long
20nm thick see by staining (not light microscope) darkfield works |
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spirochetes flagellar arrangements (3)
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1. spirochetes use endoflagella for movement (b/w outer and inner membrane in gram -), protected by membranes
2. anchored at one pole, extends about 2/3 length of cell 3. cork-screw type of movement, 1-2 micrometer/second. |
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experiment to test if flagella required for motility
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1. mutagenize wild-type with ENU: ethylnitrosourea -> does transitions and transversions: purine to purine, purine to pyrimidine. kill half. all kinds of mutations
2. plate on low nutrient soft agar so cells wiggle and make halo 3. select non-motile mutants 4. control = ??? run thru negative control and see all have halo. know mutation is due to metagen, is caused by this chemical. add ENU, 1 in 10,000 mutant is good. positive control. put on mutant that is already flagella -, put it on there and see it gave you this kind of phenotype. no flagella or mutant paralyzed flagella, so know it's required for motility. screen! make pool of mutants, mutenize heck out of them, lay down on plates, we're gonna look at them all, and take out mutant from the population. have to look at them all in a screen. it's like 1/10000. find individual mutant, which is manual labor intensive. |
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what is selection?
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only get out your mutants. like for instance, you're only looking for drug resistant mutants. like select for resistance to an antibiotic, we'll kill everyone else, so living is mutant.
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how is motility driven? (3 steps, 4 findings)
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1. two models: whip model and propeller model
2. silverman and simon 1973: isolated "polyhook" mutants, shot one, no tangle, just had a hook, had antibody for the hook protein 3. so they could mount antibody on slide, tether and adhere bacteria to glass slide via an antibody. predict: if whip: windshied wiper, if propeller, propeller. spin around like airplane 1. propeller!!! 2. rotation is CCW 3. slows to stop 4. CW. |
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structure of the bacterial flagellum
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1. filament: composed almost entirely out of protein called flagellin (outside hook)
2. hook: primarily 1 protein: bend, imp like motorboat. 3. basal body series of rings in membranes. LPS layer has L ring, P ring in peptidoglycan, MS and C ring in cytoplasmic membrane. series of rings and they all rotate. 4. flanking MS and C rings are these mot proteins or motor proteins. driving force. 5. Fli proteins are motor switch, from CCW->CW. do this w/ proton motive force, energy. 6. "proton turbine" model by PMF. particular charges in polypeptide of MS ring and C ring -> repulsion, attraction that gets proper spinning. with mot protein with protons going thru. |
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structure of gram - flagellum
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filament, hook, outer rings at outer membrane and peptidoglycan, rod, then inner rings at cell membrane.
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structure of gram + bacteria flagellum
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filament, hook, shaft, outer protein ring on inside of peptidoglycan, and inner protein ring in cytoplasm.
MS and C ring for sure, but no P ring. |
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flagellar biosynthesis (4)
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1. 50 genes needed for assembly and regulation
2. sequential assembly 3. flagellin filament assembles at the tip 4. cap required for guiding flagellin molecules to the tip. |
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flagellar biosynthesis order! (8)
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1. MS ring
2. motor proteins 3. P ring 4. L ring 5. early hook 6. cap and late hook, organize hook proteins, then another cap to organize 7. filament hollow tube, so made in cytosol, pumped up the hole and attaches. add on happens at end, organized by cap. polypeptide unfolded to be linear to be pumped up, then refolded to attach with help of organization of cap |
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flagellar movement in polar-flagellated bacteria: 2 kinds
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reversible: CCW rotation, CW rotation
unidirectional: CW rotation. cell stops or floats, reorients. CW rotation in another direction. |
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run and tumble movement in peritrichous
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1. bundled flagella in CCW rotation (1 sec)
2. tumble: flagella pushed apart (CW rotation), spazzy (0.1 sec), change direction 3. flagella bundled (CCW rotation) |
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how many genes involved in flagellar motion?
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50
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can propel cells how fast?
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60 cell lengths/sec
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movement dependent on?
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flagellar arrangement
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energy from proton motive force
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1000 H+ per rotation
360 rpm |
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pfeffer 1883
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capillary tube with diff liquid, put in a pool of bacteria. if attractant, more cells per tube, if repellant, less cells per tube.
chemotaxis! -> responding to different chemoreceptors, bacteria can go find food and go get it. |
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5 kinds of taxes
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1. phototaxis: light
2. thermotaxis: heat 3. aerotaxis: oxygen 4. osmotaxis: osmotic strength 5. magnetotaxis: magnetic field -> has magnetostome, sequesters something called magnitite, deposits in bacteria, upholded by inner folding of inner membrane. depoits organize in magnetic field. |
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requirements for taxes (4)
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sense external signal
transmit the signal intracellularly respond to signal scalable: adapt to a range of stimuli |
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if attractant...?
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CCW increases. some CW increases too, but higher run tumble frequency. if concentration get higher, continue to run longer. but still tumble once in a while. if repellant, shorter run tumble frequency. down concentration, frequency changes opposite.
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run and tumble in chemotaxis (4 facts)
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1. bacteria make temporal comparisons
2. respond to spatial gradients 3. compare environment of present with environment of past 4. respond by changing run/tumble frequency. |
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non-flagellar based motility (4)
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1. pilus-based motility
2. gliding motility 3. slime motility 4. actin-based motility (host cell) |
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pilus (3 things, and what model and motility?)
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1. pilus extension from polar end, fro base, not tip like flagella
2. attachment or grab 3. retraction from base 4. movement of bacteria on solid surfaces "grappling-hook model" or "twitching motility" |
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archaeal flagella (5)
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1. analogous to flagellum of bacteria
2. extends from base instead of the tip, ATP driven. 3. proteins evolved related to pilus. 4. rotary, moves like flagella. 5. thinner, smaller hole, cant fit monomers to send up and add on, so have to add on at base, like pilus add on at base. |
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gliding motility
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1. movement of bacteria on solid surfaces
2. typically rod or filamentous-shaped bacteria 3. move at 0.2-10 micrometers/second 4. gliding racetrack model: proton goes from periplasm to cytoplasm, then movement of outer membrane proteins and cell moves the other way. outer membrane touching substrate, peptidoglycan, and cytoplasm. have transmembrane proteins, 1 side grabbing onto substrate (outer membrane), reaches out thru peptidoglycan, contacts another transmembrane proteins into cytoplasmic membrane. interact, energy is PMF. |
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cyanobacterial slime excretion
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pores in membrane, kick out slime. release slime out from substrate, then contracts, filaments inside, contracts itself forward. because of slime, move its butt up, like inchworm.
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gas vesicles
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1. inclusion of body in aquatic prokaryotes confers buoyancy on the cell
2. gas vesicles are impermeable to water, but permeable to gases. 3. phototropic organisms - vertically adjust position in the water column to a light intensity optimal for photosynthesis. 4. 2 proteins: GvpA (beta sheet) and GvpC (alpha helix that connects sheets) -> watertight structure |
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actin-based motility (host actin)
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1. found in some intracellular pathogens: Listeria, Shingella, etc. zoom around cytosol.
2. rapid intracellular and cell-cell movement, cel to cell spread. cell next door, shove into next cell. 3. bacteria polymerize host actin - Act A protein |
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motility in eukaryotic pathogens: pathogens use motility to...?
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1. infect/invade host (food)
2. disseminate within host (niche) |
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which hallmark of eukaryotic cell is essential for eukaryotic microbe motility?
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cytoskeletal filaments
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microtubules (2)
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polymer of alpha, beta-tubulin
forms a hollow tube structure |
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actin (2)
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long, thin filaments
polymer of actin when polymerize, globular? |
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polymerization
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assembly of cytoskeletal protein molecules into filaments
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cytoskeletal filament importance
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form scaffold, cage-like that is important for movement liek super highway, regulated by a large number of accessory proteins for 1. linking to each other and other cellular components
2. assembly/disassembly. |
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flagella and cilia
|
flagella: long (5-200um), thick (200nm), 1 or a few/cell
cilia: short (<5um), 100-1000's. 1. very similar structures 2. highly conserved from protozoa to mammals 3. both composed of microtubules 4. total number of proteins unknown. |
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assembly of microtubules
|
polymerization of alpha,beta-tubulin into filaments. dimers
polymerization of tubulin occurs at the + end: nucleation stage with individual subunits, oligomers, elongation with growing microtubule, then steady state with microtubule with subunits coming on and off. |
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chlamydomonas
|
2 flagella, haploid, a lot longer, 10x as thick. has asexual and sexual reproduction.
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microscopy to study flagellar structure and found..? (2 general)
|
1. plasma membrane surrounds the flagellum
2. basal body is located in the cytoplasm. |
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cross-section of flagellum reveals microtubules...
|
1. 9+2 arrangement.
2. highly organized structure 3. 9 pairs of outer microtubules 4. 2 central "=central pair 5. 9+2 known as axoneme 6. flagella extend from "basal body" which is composed of centrioles (as in mitotic spindle) 7. basal bodies: 9 outer triplets with no central pair. orientation as in mitotic spindle. |
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euk flagella picture shows...?
|
microtubules at bottom and above extending.
basal body with kinetosome flagellum extending cross section shows plasma membrane outside, protein spoke and dynein motor. |
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flagellar microtubule connections (3)
|
1. outer pair microtubules are connected by dyneins
2. dyneins are ATP-dependent motor proteins 3. radial spokes connect the outer doublets to the central pair |
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flagellar motility
|
1. observation: isolated flagella can beat on their own (all ommponents necessary for motion present in isolated flagella)
2. isolate flagella to test motion in vitro 3. add detergent to remove plasma membrane 4. limited proteolysis to digest cross-links 5. add ATP, buffer: outer doublet microtubules slide apart (ATP dependent) |
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how is sliding motion converted to flagellar beat? experiment (3)
|
1. isolate flagella to test motion in vitro
2. fix to solid support, add ATP, buffer (not proton) 3. whip-like beating motion of flagella observed in vitro: ATP dependent action of dynein motors on microtubules converts sliding into beating motion of eukaryotic flagella. (not propeller) |
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cilia in your lungs
|
grab microbes and digest them. smoke burn off: microbes stay in lungs.
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eukaryote flagella number of genes and regulation
|
unknown.
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flagella and cilia motility (2 similarities, 3 differences)
|
1. flagella/cilia use sliding microtubules for motility
2. different from propeller-based movement in prok 1. flagella move in an undulating whip-like fashion driving the microbe forwards 2. cilia use a "power stroke" and a "recovery stroke" for movement 3. cilia often cover the entire organism and work in a coordinated fashion for movement. |
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genetic study of motility: isolate cell motility mutants. explain. (6 facts) screen or selection?
|
1. WT chlamydomonas stay dispersed in liquid (they swim)
2. mutagenize (chemical or uv), invert to mix (Mot+ and Mot-). kill 1/2. aspirator and suck up all except mutants. do few times b/c maybe lazy swimmers on bottom. 3. perform selection(s), clone. 4. examine Mot- mutants: mut1: shortened flagella. mut2: paralyzed flagella. have multiple types 5. 3 kinds of mutants: dynein-, spokes-, cp- 6. motile flagella are required for motility. |
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genetic approach is (1 sentence)
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robust method for determining key proteins.
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how to determine which flagellar proteins are affected? (6 steps)
|
1. isolate intact flagella from wt/mut
2. solubilize proteins 3. SDS-PAGE, stain coomassie. 4. ID bands absent in mutant 5. ID corresponding gene by mass spec because diff sizes. 6. complement mutant with wild-type gene to ensure that this gene is responsibly for the phenotype. if swimming, bingo! |
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protein ID mass spec details (4)
|
excise protein band from gel
digest with trypsin fractionate peptides by chromatography determine masses of peptides and compare to predicted masses for a match, from all predicted proteins from genome down to theoretical trypsin digest. |
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toxoplasma gondii (4 general facts)
|
1. protozoan parasite: obligate intracellular parasite
2. cause of toxoplasmosis in neonates (pregnancy) and immunocompromised (AIDS) 3. infection from undercooked meat or cat feces 4. gliding motility linked to host cell invasion |
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more on ultrastructure of toxoplasma gondii (4)
|
1. toxoplasma contains a membrane system under the plasma membrane named inner membrane complex.
2. toxoplasma is an Apicomplexan - characterized by rhoptries, dense granunules, and micronemes 3. rhoptries, dense granules and micronemes are secretory organelles 4. micronemes release a series of molecular adhesins for attachment to surfaces/host cells |
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more on microneme proteins 5 steps)
|
1. fusion of micronemes with plasma membrane releases microneme proteins onto parasite's surface, triggered by Ca2+
2. MIC's are attached by membrane spanning domains. 3. aldolase connects MICs to the actin-myosin motor 4. myosin A tail is anchored in the inner membrane complex 5. MIC proteins are released by a parasite protease. |
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amoeboid gliding motility
|
1. driven by directed assembly - disassembly of actin filaments
2. actin polymerization drives leading edge (Pseudopod) forward 3. actin de-polymerization allows lagging edge to move forward 4. entire cell changes shape 5. ATP dependent. |
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what is a genome?
|
total DNA complement of a given organism
chromosomes plasmids viral organellar |
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how does genome information benefit us?
|
1. metabolic capabilities of an organism
2. leads to the development of new therapeutic agents 3. relevant for industrial and medical applications 4. allows comparisons of different organisms 5. furthers our understanding of how life has evolved. |
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gene organization in microbial cells
|
So 1. composition, 2. chromosome, 3. size (nt/genes)
Viruses: 1. RNA/DNA (ss and ds) 2. linear/circular 3. 10^3+, <25 Bacteria 1. dsDNA 2. circular (1) (haploid) + plasmids 3. 10^6, ~10^3 eukarya 1. dsDNA 2. linear (multiple) (haploid/diploid) + organelle + plasmids (rare) 3. 10^7-10^8, 10^3-10^4 |
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viral gene organization (4)
|
naked virus has DNA and capsomeres, enveloped virus has nucleic acid, capsid (bilayer membrane), and envelope.
-very diverse (5000-200,000nt) -very few genes (<25, often overlapping) -require host machinery for replication and expression. |
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prokaryotic gene organization (3 parts)
|
Chromosome:
-large, single copy, typically circular -nucleoid -housekeeping genes non-chromosomal - Plasmid -small, circular (<10^4 bp) -replicate independent of chromosome -multicopy -"specialty genes": can transport genes, like drug resistance. transposable elements |
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compaction of the genome
|
-in a relaxed state, the E.coli genome would occupy 10x its volume
-utilizes ATP-dependent topoisomerases to compact the genome = "supercoiling" -proteins are involved in further formation of supercoiled domains |
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supercoiling via topoisomerase (5)
|
1. relaxed circle
2. one part of the circle is laid over the other: result is contact between the helix in two places, note that no twisting has as yet been introduced 3. topoisomerase II makes double-strand breaks 4. twisting (a negative supercoil) is introduced. 5. supercoiled DNA |
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gene organization in prokaryotes: operons (4)
|
1. genes with related functions are often grouped together
2. transcribed as a single mRNA=polycistronic 3. single promoter, several genes in mRNA = operon 4. transcription and translation coupled: starts translation before transcription done. |
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eukaryotic gene organization (3)
|
-DNA enclosed in nucleus
-compaction occurs via topoisomerase and nucleosome -linear chromosomes (centromeres/telomeres) |
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eukaryotic gene regulation
|
1. typically monocistronic
2. pre-mRNA contains introns 3. processing generates mature mRNA |
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where is promotor?
|
left of exon 1
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what do you do to mature mRNA and why?
|
cap onto mature mRNA at polyA tail, to get out of cytosol, bind to ribosome to make proteins
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2 genome sequencing methods
|
1. chromosome walking: template DNA in plasmid (libraries and sequence that, a bunch), make copies, tons, a lot of time
2. shotgun sequencing |
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whole-genome shotgun sequencing part 1 (5)
|
1. shear DNA into fragments (random, no restriction enzymes, some long, some short)
2. insert fragments into plasmids and produce library (small library and large library) 3. sequence DNA fragments. random. (oligonucleotides act as primers) 4. transform into e. coli, each colony will have diff sequences. expand. 5. culture, prep plasmid DNA. each well separate plasmid with different sequences. use vector primers (common to all inserts). chromosome -> new primer, make new one every 100 or so bases. |
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computer analysis of sequenced DNA fragments (5)
|
1. search for overlaps (small overlaps. just long enough to get a little overlap to be able to put together)
2. assemble fragments to continuous larger DNA fragments (contigs) 3. close gaps between contigs 4. chromosome 5. identify genes. |
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whole-genome shotgun sequencing part 2
|
1. assemble overlapping fragments
2. sequence DNA by PCR amplification |
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why are there gaps when you do shotgun sequencing?
|
1. toxic DNA: resolve by PCR, sequencing -> piece of DNA stubborn (ex: cryptic promotor). if plasmid, E.Coli expresses and dies (not supposed to express). fix by using PCR to jump gaps, make primer on both ends and sequence properly. not in vivo, only use PCR, not bacteria.
2. repeats (chromosome walk large library): can't tell what goes where, so kicks out. so go back to large library and can see repeated parts so do old fashioned way with primer, etc. |
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review on PCR (3)
|
melting/denature
anneal. amplify with tac polymerase. |
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DNA sequencing (6)
|
1. 4 different tubes
2. chain termination 3. different chains with different colors 4. mix 5. run in one well 6. different fragment of DNA |
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whole-genome shotgun sequencing (part 3)
|
1. annotation:
2. identify genes and functions 3. 1 genome equivalent sequenced, some parts not sequenced 4. computer can tell which ones are errors, kick out. good thing about shotgun. |
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|
both conventional and large insert genomic DNA libraries should be constructed (tell purposes of each, and an important factor of both)
|
small insert library will be used for bulk of the sequencing in order to generate suitable coverage of the complete genome
large insert library (BAC, cosmid etc.) will be used as a scaffold during the sequence closure phase. it is crucial to ensure that both libraries are as random as possible - mechanical shearing is often used to generate small DNA fragments. |
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DNA sequences are generated using...?
|
vector primers for both ends of inserts
|
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|
how much sequence needs to be generated?
|
by sequencing random clones one can expect to sequence the same region many times.
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|
poisson distribution for sequencing
|
P=e^-m
P=probability that a base is not sequenced e=constant (2.718) m=sequence coverage: number of bases sequenced / genome size. |
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|
what fold coverage is standard?
|
10.
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|
annotation of a genomic sequence (4 parts in sequence)
|
1. chromosomal DNA
[6 reading frames, start and stop codon, Shine-Dalgarno sequence] 2. gene (open reading frame, Orf) [translate (computer)] 3. protein (rRNA, tRNA) [search database for homologous proteins] 4. example: nitrogenase |
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start codon
|
ATG
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stop codon (3)
|
TGA, TAG, TGA
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how do you know which is real ORF?
|
longer, more likely to be gene because higher frequency of bogus frequency smaller than 350. 98% chance at 500 of it being a real ORF.
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EST databases stands for?
|
expressed sequence tags
|
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procedure of EST databases (4)
|
1. isolate mRNA (because it's a way to sort out if expressed or not. if mRNA, expressed!)
2. reverse transcribe cDNA 3. clone into plasmid 4. sequence with vector primers |
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utility (4)
|
1. reflects genes that are expressed
2. identify coding regions 3. alternative splicing (eukarya) 4. gross expression levels |
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what's not good about EST? (2)
|
nothing about order. don't tell anything about control at upstream because use mRNA.
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BLAST stands for? what is it generally?
|
-basic local alignment sequence tool.
-a way to look at proteins after translation. -most of the time, comparison between protein vs protein. -BLASTp (protein query, protein database) scans protein database for matches. -BLASTp will ID protein domains prior to full searching of the databases. |
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5 different methods of BLAST
|
1. BLASTP: protein query - protein database
2. BLASTX: translated nucleotide query - protein database 3. TBLASTN: protein query - translated database. 4. BLASTN: nucleotide query - nucleotide database 5. TBLASTX - translated query - translated database |
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raw score
|
a high score indicates a likely relationship
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E value
|
probability that this match would occur by chance - the lower the better
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what do you do after you get raw score and E value?
|
label the genes
make a map look at transporters see relationships. |
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E.coli genes and divisions of their functions
|
88%: coding (ORF)
10% regulatory 1% tRNA and rRNA 0.5% non-coding repetitive sequences |
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special facts about E.coli genome? (2)
|
1. very compact: average distance b/w genes 118bp
2. average ORF 317 aa |
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|
how are the genes organized in E.coli?
|
-one chromosome (haploid)
-only one copy of each gene, some genomes contain several copies -genes may be on either strand |
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|
M. genitalium genome:
|
1. circular chromosome, 580,000 bp
2. only 470 predicted genes for DNA replication (because acquire a lot from host), transcription and translation, DNA repair, cellular transport and energy metabolism 3. coding regions comprise 88% of genome (similar to H. influenzae (85%) and suggests that genome reduction has been due to loss of genes and not reduction in gene size or increase in gene density (found out from BLAST, pathways eliminated because can get proteins from host) |
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|
methanococcus jannaschii
|
1. first archaea sequenced
2. grows at 95%, 2600m below sea level (in hydrothermal "smokers") 3. anaerobe, makes methane gas 4. one chromosome 5. 1,692 genes |
|
|
distribution of M. jannaschii genes in 3 domains of life (5)
|
universal: 359 genes
archaeal/bacterial: 110 genes archaeal/eukaryotic: 610 genes archaeal: 381 genes 332 are M. jannaschii specific |
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|
what's the surprise buried in M. jannaschii genom?
|
1. introns
2. inteins |
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|
prokaryotes usually don't have...?
|
introns
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|
what in an intein? (2)
|
-protein equivalent of intron
-internal and spliced out. -take mRNA, translate, and has intein within polypeptide, get spliced out to get mature protein. -some have more than one intein. |
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|
dnaE gene of synechocystis: (4)
|
-dnaE very conserved.
-when sequenced, 1/2 (ORF1) present in genome, other half (ORF2) far away and pointing to the other direction. -do alpha and beta subunits comt together and do its thing? NO, 2 pieces pieces made independently, transcribed. -had little extra sequence at one end on both, spliced out to remove inteins -> trans-trans-splicing (compared to before cis-splicing from 1 polypeptide). this is 2 independent polypeptides! |
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|
surprises discovered in whole genome research (3)
|
1. some bacteria have 2 chromosomes
2. some contain one circular and one linear chromosome 3. some contain 2 linear plasmids |
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|
example of horizontal gene transfer : E.coli and E.coliO157 (foodborne illness, kills people) (5)
|
-identical backbone sequence, but interspersed sequence
-O157 has 1,387 more orfs -consist of pathogenicity islands: include virulence factor) -includes virulence factors: adhesins, toxins, invasins -acquired by horizontal gene transfer. |
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|
horizontal gene transfer in general (4)
|
-when sequence genes and sequence others, and use BLASt, etc., some parts look like bacteria, some other parts look like archaea
-acquired! -extent varies on how well they can pick up DNA, how much is around, etc. -more common in prok |
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|
metagenomics: what is it? examples? another name? how many current projects?
|
-study of genomes recovered from environmental samples
example: sargasso sea, acid mine, etc. -"community metabolism -123 current projects |
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|
next generation sequencing technologies compared to shotgun sequencing (4 kinds)
|
-454 (pyrosequencing)
-SOLiD -illumina (solexa) -heliscope |
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|
shotgun sequencing (4 parts)
|
1. have DNA fragmentation
2. in vivo cloning and amplification with colonies, miniprep, isolate plasmids 3. cycle sequencing with template and primer (polymerase, dNTPs, labeled ddNTPs) 4. electrophoresis (1 read/capillary) (run all 4 in a single capillary tube and shine laser, see different chain links come off, get standard gene sequencing results |
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|
next generation sequencing (4 parts)
|
1. DNA fragmentation
2. in vitro adaptor ligation (no cloning into plasmid) add linkers on it, array on glass slide (like microscope) on just right dilution, just close together but not too close, stick to slide. 3. generation of polony array. can't sequence single fragment of DNA, need more, so make more copies by generating polomy array -> polymerase colony. so put on glass slide, amplification with tac polymerase off primers that you added to make copies. trap right on spt -> polymerase colony or polomy. so balls of DNA on slide. can make 1 million on single slide. 4. cyclic array sequencing (10^6 reads/array). add 1 nucleotide at a time, so get all colors, add 1 nucleotide. scan it. add next nucleotide, keep cycling thru. |
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|
what's good about next generation sequencing technologies? (2 general, 4 more specific important)
|
1. cheaper
2. really fast, powerful, massive amount sequencing. 1. toxic DNA not problem because no in vivo, just PCR on glass slide 2. not 1 sequencing reaction, 1 million. so much cheaper, but a lot shorter read. so get million of these reactions and look for overlap. 3. can do De Novo (unknown bug and do it) 4. easy with reference organism (overlay millions of sequences). |
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|
experiment: how do we know bacteria can take up DNA? (Frederick Griffith 1928, Streptococcus pneumoniae, he died in air raid in WWII) (4 steps, proposal)
|
1. have typeS (smooth, capsule) bacteria: virulent and typeR (rough, no capsule): avirulent. clear infection. can't get anything back or recover bacteria.
2. if both types put into mice, typeS bacteria recovered and killed mice. typeR had no live bacteria recovered and mouse lived. 3. heat-killed typeS -> no live bacteria recovered so mouse lived 4. living typeR + heat-killed typeS (transformation) -> living typeS bacteria recovered. Concluded that: killed virulent bacteria had passed some material to the avirulent one to make it virulent. Proposed an inheritance molecule and called the process of passing = transformation |
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|
follow-up experiments (Avery&McCarthy, 1941) for transformation (6)
|
to determine the cellular constituent that was being passed.
treat cellular lysates with proteases, RNases, or DNases to determine which is responsible for transformation. 1. type S cells 2. divide up into DNA, RNA and protein 3. added the degrading enzymes for DNA, RNA, and protein. 4. found that both typeR and typeS ells present when RNA and protein were degraded. 5. only typeR were present when used DNA-degrading enzyme. 6. transformation still happens when used RNase and protease because DNA still present, so DNA is the transforming molecule. DNA convert R-S strain. |
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|
transformation in gram positive bacteria (3 facts)
|
-generally 10kb max
-DNase-sensitive process -mechanism varies throughout bacteria and archaea. |
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|
transformation in gram positive bacteria (processes 6)
|
-dead bug release DNA
-transforming DNA sticks to DNA binding protein in membrane, grag -cell surface nuclease cuts from double strand to single strand and import into cytoplasm. -single strand binding DNA binding protein is competence-specific (same as replication). -integration via homologous recombination, so has to have homologous buddy to recomine. -RecA dependent (enzyme): if homology present, this protein incorporates into bacterial chromosome |
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|
another way of transformation (change at end, still gram positive)
|
heterologous recombination, stick in anywhere, no double crossover event.
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|
ReCA important to function of bacteria?
|
not essential, so can be knocked out. ose transformation ability, so could be good thing.
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|
gram - transformation (6 processes)
|
1. take double strand
2. import into periplasm 3. start clipping with nuclease 4. import into cytosol 5. ss binding protein binds 6 homologous recombination. |
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|
1 sentence for transformation
|
donor-free uptake of DNA in the environment
|
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|
competence: definition and special case
|
ability to take up DNA
some bacteria are only naturally competent during a particular stage of growth |
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|
2 kinds of competence and details about the second
|
1. naturally competent
2. artificially competent (for chemically artificially competent): -calcium chloride: bacteria becomes weak with calcium chloride, then do heat shock -electroporation: electric shock. cuvettes with 2 metal plates on either side. DNA and cells inside, huge charge -> some cells shear open, DNA in. Some cells will die, some may recover. -gene gun (inefficient): DNA on micromicron projectile, put in gun, fire into target cells. usually do in plants because can't do otherwise. rips open cell, very few will get a little DNA in and few may live. |
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|
importance of transformation (2)
|
1. how bacteria can do horizontal gene transfer by getting DNA from environment
2. we can exploit this. how to do experiments -> use transfer and homologous recombination to do gene knockouts. |
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|
knockouts. say gene x assumed to be for spore formation (6)
|
1. take gene x with internal EcoRI restriction site, put it into plasmid.
2. cut x with EcoRI 3. insert drug marker called kanacycin cassette is inserted into plasmit and interrupt x, so now inactivated gene x. 3. take plasmid and linearlize with BamHI. 4. transform into cell, so use chemically competent cells, take linearlized DNA, get it in. 5. recombination 6. select with kanamycin cassette so only get plasmid ones (can't be replicated so will only get recombined ones) |
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|
so say knockout of spore formation gene is successful. so can you say no more phenotype just because of disruption of gene? how do we make sure?
|
no, because might have affected stuff upstream/downstream, called polar effects.
complement recombination: gene x, on plasmid. transform into mutant. leave on plasmid or put in another, if rescue then yes! |
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|
can DNA be transferred directly from one cell to another? (Dederburg and Tatum 1946)
|
used E.coli nutritional mutants to examine direct transfer: auxotrophic mutants - mutants that have lost the ability to synthesize a compound necessary for growth.
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|
isolation of nutritional mutants using method called..? and 4 steps
|
replica plating method
1. mutagenize 2. grow on master plate 3. imprint 4. plate on complete and minimal 5. if don't grow on minimal, take that colony from complete and you know they're nutritional mutant. |
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|
2 different strains missing ability to synthesize 2 diff compounds each necessary for growth. what happens when put on plates in diff conditions?
|
neither can grow by themselves on minimal
incubation of a mixture of the two strains produces wild type recombinants, which grow on minimal medium. |
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|
"incubation of a mixture of the two strains produces wild type recombinants, which grow on minimal medium. " -> is this simply transformation?
|
1. far less efficient
2. boiling of cells = no transfer occurred (so no transformation) 3. E.coli not naturally competent 4. appears distinct from transformation |
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|
cell-cell contact required? (Davis 1950)
|
1. used U-shaped tube with a glass filter: filter permeable to liquid, DNA, filter impermeable to bacterial cells.
2. when strains requiring methionine are incubated with strains requiring threonine, leucine, and thymine, WT recombinant colonies are produced 3. when mutant strains are separated by a filter Yes, cell-cell contact required, sex in bacteria. conjugation!!!! |
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|
why use double mutants and not single?
|
because chemically mutagenized and can get revertants. if single, mutate back, not doubles.
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|
|
mechanism of conjugation
|
-efforts into the mechanism led to the discovery of "plasmids"
-key observations: 1. cell-cell contact is required 2. not every cell is a donor 3. transfer is one-way (donor -> recipient) 4. recipient becomes donor (donors remain donor) 5. chromosomal genes rarely transferred. -led to the hypothesis that donors carry non-chromosomal DNA elements called "fertility factors" |
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|
what cell part do conjugation use?
|
pilus, genes for pilus in plasmid
|
|
|
mechanism of conjugation (5)
|
1. extrachromosomal "F plasmid"
2. pilus attachment to recipient, relaxase (TraI) nicks at OriT, DNA in plasmid of F+ donor. 3. transfer via coupling protein at mating bridge 4. rolling circle replication during transfer 5. both cells result in F+ |
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|
F plasmid
|
1. genes for replication and segregation in cells
2. transfer - tra genes: *oriT specifies the start and direction of transfer, *traI encodes the nicking protein, *pili formation (eg TraA), surface exclusion (TraS, TraT) so donors don't hit other donors. 3. transposable elements (3 IS's, 1 transposon in original F) 4. cells can have multiple types of plasmids, but exclude similar. |
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|
plasmids can be 2 things in conjugation
|
1. self-transmissible: contain all of the genes for transfer
2. not self-transmissible: require helper for some function |
|
|
some traits transferred by plasmids
|
fertility, drug resistance, toxins, virulence, metabolic pathways.
|
|
|
is RecA needed in conjugation?
|
no, because no recombination. just shove in, use DNA polymerase and primer.
|
|
|
if cells have incompatibility groups...?
|
multiple plasmids. if different in compatibility group, can have 2 plasmids instead of 1. so if no TraI (nicking) to get trasfer, need second plasmid with functional TraI, called help plasmid.
|
|
|
importance of conjugation
|
R plasmids can get drug resistance by transposition events. multiple drug resistance genes moved to one giant plasmid: multi-drug resistance. so 1 R+, transfer to all R-.
|
|
|
general knowledge of antibiotic resistance: 3 ways to get rid of antibiotic by bacteria.
|
1. cleave antibiotic
2. modify antibiotic (post translational modification) 3. pump out antibiotic |
|
|
integration of F+ plasmid into the chromosome (7)
|
can do in chromosome at IS sequences that are transposable elements. so single crossover form Hfr cell. can get homologous recombination between F plasmid and your genome.
-Hfr is high frequency of recombination. -"mobilizes" chromosome. -transfer still initiates at OriT. -Hfr transfer initiates at OriT -Hfr transfer rarely transfers the whole chromosome -thus, F- typically remain F-. |
|
|
detection of conjugation with mobilized Hfr plasmids (4)
|
-Hfr donor: Thr, Leu, Lac+'s, StrS.
-F- recipient: Thr, Leu, Lac -'s, StrR -mating to allow conjugation, followed by plating onto agar media -can select for certain recombinants. |
|
|
interrupted mating (Francis Jacob) (6)
|
-Donor, Hfr+ (StrS) (Thr+Leu+Gal+Trp+)
-recipient F- (StrR) (Thr-Leu-Gal-Trp-) -carry out time-course, blend: add strep, plate, count -markers always transfer in the same order/time from 1 Hfr strain, so tells you order, how close to Hfr, and direction. -determine relative gene position by time taken to transfer. -need RecA because integrade this piece into genome, if dragged from another cell, need cross over event to make all positive. not full plasmid because never takes in the whole chromosome. |
|
|
do you need RecA for conjugation?
|
no because it's plasmid.
|
|
|
perfect excision
|
makes F+, because F plasmid just pops back out.
|
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|
F' plasmid (5)
|
-when you get imperfect excision from the host chromosome, F' transfer, becaus F plasmid carries genes from chromosome.
-if most of F intact, recipient becomes donor -F' plasmid passes to recipient cell, and recipient cell will have genes A and B -may form merodiploid (partial diploid) with genes that are passed on in F' -don't need homologous recombination because it's a plasmid. |
|
|
transposable elements
|
mobile DNA elements that can change position in the genome. movement is called transposition.
|
|
|
3 basic types of transposable elements
|
1. insertion sequences (IS)
2. transposons (Tn) (how you get all this drug resistance) 3. retrotransposons (eukarya, requires an RNA intermediate) |
|
|
insertion sequences (4)
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-most simple.
-it just has gene transposase in middle -2 terminal repeats of inverted orientation. -can be found in chromosomal, plasmid, phage DNA -make regions of homology in plasmid and gene. -can find in genome, plasmid, phage, because jump around. |
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transposons
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-larger IS's that carry other genes (like drug markers)
-composite transposons: genes between 2 full IS sequences at both ends. identical IS sequences, inverted. transposate is knocked out on IS50L, only need 1 copy. IS on both ends, boodies in middle. |
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transposition results in duplication of the..?
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target sequence. so transposable element with IR's on outside will squeeze between duplicated target sequence, essentially random target site.
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conservative transposition (6)
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-transposase cuts transposon (blunt, inverted at plasmid) and target (sticky), transposon is excised from one location
-ligation of transposon (Tn), recipient leaving overhang, ligase in transposon 5' of E.Coli to 3' of plasmid. tethered transposon. -fill-in of 3'OH with polymerase -no duplication of the transposon DNA eg Tn5, 1 copy. -degrade free plasmid. -way to get multi-drug resistance |
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look for transposons? (3)
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1. tranposase
2. inverted repeats (IR) 3. direct repeats. |
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replicative transposition (7)
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1. transposase cuts (sticky both)
2. ligation of the Tn 3. replication across Tn because have 3'OHs. 4. resolution of co-integrate 5. 2 copies of the transposon. get second copy made in plasmid replicated. so 1 ID to source, 1 present at the target. still duplication at the target site. 6. looks like original transposon + plasmid. second copy made in chromosome with tandem duplicated repeats. 7. way to get multi-drug resistance) |
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integrons (6)
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-capture and express genes (like drug resistance, one by one)
-integrase: catalyzes site-specific recombination: attl: site-specific integration, Pout=promoter drives expression of captured gene. -integrase ia always being made. -so, recombination event, attc an attI integrade drug gene, then promoter turns on, starts making product. sample DNA in, if good, keep, bad, toss. can do multiple times. keep integrating more drug resistance at same promotor. -capture genes turns on with own promotor. -know integrase is constitutively expressed, captures genes, and turns it on via own promotor. |
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why do we care about integrons? (2)
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because 1. drug resistance and 2. transposon in lab.
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transposon mutagenesis (utility of integrons) (4)
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1. engineer transposon on a suicide plasmid. specialized Ori (coli, not cholerae), transosase outside IS.
2. transform or transfect it into another cell (by interrupting) into a specific gene in the chromosome 3. select with antibiotic 4. analyze for mutants: perform selection on entire pool or clone and screen individuals. |
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what is suicide plasmid?
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origin of replication can replicate E.Coli, grow up in E.Coli but can't replicate in cholera. so has oriC for E.Coli.
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how do you determine which gene was interrupted? (5)
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inverse PCR.
1. isolate genomic DNA 2. digest with restriction enzyme (cut every 4kb outside the restriction site) 3. circularize (ligase to itself) 4. PCR outward (because known sequence of transposon)to identify flank and amplify product 5. clone and sequence |
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why do transposon mutagenesis when good with chemical mutagenesis?
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because in chemical, don't know which gene we interrupted very easily. have to have whole genome library, transform it to rescue it, so very hard! in transposon, we know sequence of transposon: use this info to know which gene you interrupted. inverse PCR.
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transduction: 2 varieties
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1. simple
2. specialized |
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simple (6)
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1. use phage, inject DNA into E.Coli and genome goes in.
2. virus makes phage heads or proteins heads and packages them 3. lyse the cells 4. sometimes the phage accidentally packages host chromosome (not viral) and stuff fully effective in putting in (transducing particle) but not effective in getting viral gene. 5. DNA insensitive because has phage head. can transfer any part of host DNA 6. RecA positive: shoving in random genomic DNA from another bug. |
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specialized
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lambda -> integrates in 1 part of host DNA into the circularization (like F') by taking neighboring chromosome and transforming it, only get genes close to insertion site.
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e.coli has what kind of replication?
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-bidirectional, theta structure.
-2 replications going on at once to produce 2 circular DNA. -so fast, 20 min! (DNA polymerase requires 40 min to replicate chromosome -not 1 replication fork under good growth conditions, initiate 1 and make 2 more replication forks. another round of division. higher order replication, divide divide... |
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replication
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1. protein unwind DNA
2. single stranded binding protein jumps on. 3. RNA primer laid down by primase 4. DNA polymerase add on 1 strand continuously, and okazaki 5. clean up and duplicated, RNA primers are replaced with DNA. |
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problem with replication of linear DNA
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shortening chromosome! use protein to put 3' OH onto 5' terminus. anchor protein, put on end of linear chromosome so when addition by DNA polymerase occurs, not shorter piece of DNA at each replication cycle.
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replication in eukaryotic microbes (4)
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1. eukaryotic chromosomes are typically much longer
2. linear chromosomes 3. replication requires multiple origins 4. autonomous replicating sequences: ARS |
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gene organization in prokaryotes: Operons
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1. genes with related functions are often grouped together
2. transcribed as a single mRNA = polycistronic 3. operons are genes transcribed together. |
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transcription in bacteria (4 things, 4th one long)
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1. mRNA starts upstream to ATG, regulated by promoter.
2. so you have UTR, start at +1. 3. pribnow box: AT rich at -10, and -35 box. 4. driven by bacteria RNA polymerase: 4 subunits that can be seen on gel. alpha, sigma, beta, and beta' called holoenzyme. can separate sigma with ion-exchange chromatography, if sigma taken out, poor activity of transcription. so sigma is essential, "add-back" -> restores activity. this is for tight binding to promotor. |
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what is sigma factor used for? (4)
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-tight binding of the RNA polymerase to the promotor sequence: to the -35 and pribnow box so transcription can happen.
-competition experiment using filter-binding assay: filter doesn't stick to DNA, sticks to protein and protein + DNA. holoenzyme sticks because has sigma, but core doesn't because no sigma. -bind radiolabelled DNA with core or holoenzyme -compete with unlabelled DNA |
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what happens to sigma after transcription starts?
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kicked off because doesn't need it.
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termination of transcription
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-DNA with inverted repeats
-transcription of one strand (get RNA) -and it folds to form secondary structure: stem-loop in RNA immediately upstream from a run of uracils leads to termination of transcription. base pairing of mRNA. transcription will step there (crash) and stop. |
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alternative sigma factors to control gene expression (6)
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-sigma 70 controls most of genes, most of growth related genes.
-by swapping in sigma factor into core polymerase (polypeptide) control core to drive transcription. -RPOD if doing a lot -RPOS (stationary) -appropriate sigma for appropriate time and function -regulon: multiple operons controlled by response to a common signal. |
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sigma factors and flagellar biosynthesis (general and then 3 sequences)
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have to make sequentially all the rings, flagellum protein with cap, flagellan filaments would be toxic if built too fast (building monomers). late flagellan genes don't want to be made before other stuff already built.
1. transcription of sigma28-dependent late genes is prevented by the association of sigma28 with FlgM (anti-sigma factor). 2. the formation of the basal body and hook complex creates a secretion channel for FlgM, decreases its intracellular levels 3. sigma28 now binds RNA polymerase core and proceeds to transcribe genes for flagellin and proteins needed during the flagellar assembly or for its function in motility. |
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transcription and translation in prokaryotes
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coupled.
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16s, 23s, and 5s
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all transcribed together, handy because need same concentration.
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eukaryotic microbe transcription (4)
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1. monocistronic
2. need 5'-cap and 3'-polyadenylation 3. introns excised out (spliced) 4. after binding to ribosome, translation! |
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splicing of introns (3)
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1. assembly of spliceosome
2. cutting of 5' splice site, formation of lariat 3. cutting of 3' splice site, joining of exons. |
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gene regulation (2)
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1. microbes must respond rapidly to changing conditions: some processes no longer needed, other pathways must be initiated.
2. microbes efficiently direct activities by maintaining tight control over gene products. |
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fastest way to block
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regulation of activity typically faster than regulation of enzyme synthesis because if you block at synthesis, already some product existent.
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transcriptional control (3)
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1. proteins binding to regulatory sequences
2. DNA binding proteins that regulate transcription are called transcription factors 3. transcription factors bind DNA in a sequence-specific fashion. |
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structure of transcription factor (2)
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-Tx factors often dimers: domain containing protein-protein contacts, holding protein dimer together
-bind to IR's: DNA binding domain fits in major grooves and along phosphate backbone. |
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common structures of transcription factors (3)
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1. helix-turn-helix, lac and trp repressors: stabilizing helix, turn, recognition helix.
2. zinc finger 3. leucine zipper |
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regulation by repression
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1. enzymes involved in tryptophan biosynthesis (tryp a-e) made in the absence of tryp
2. +tryp (which is the corepressor itself), so corepressor binds to inactive repressor to make active repressor, and tryp repressor binds operator and blocks tx (RNA polymerase) |
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regulation by derepression (lac operon)
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lactose absent, then active repressor binds to operator and blocks transcription. if lactose present, lactose becomes allolactose (inducer) and bind to the active repressor to become inactivated, and transcription may proceed.
-lacZ encodes B-gal -lac repressor encoded by lacl -inducer allolactose |
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lac operon is also regulated positively
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-diauxic growth on glucose and lactose: cells use best carbon source first, which is glucose because growth on lactose is slower.
-high glucose, low cAMP -low glucose, high cAMP: when glucose level is low, synthesis of cAMP is simulated with ATP input. cAMP forms a complex with catabolite activator protein (CAP). CAP-cAMP complex binds next to the promoter site and enhances transcription. |
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regulation by induction: the maltose regulon
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malT gene produces inactive protein MalTi. MalTi is activated to MalTa by binding of maltose. MalTa binds to the promoters of at least four operos, and transcription proceeds.
-positive regulation of the maltose regulon by induction -activator = maltose activator protein (MalTi) -inducer: maltose -binding of maltose allows binding to the promoter, recruits sigma. |
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activators/binding can be far from RNA polymerase
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the gene has activator binding site and promoter. with activator protein going onto activator binding site and RNA polymerase going on to promoter.
If activator binding site is far, it bends so that promotor is above activator binding site, and so when activator protein sticks on to the activator binding site, it is in contact with RNA polymerase and so transcription can begin. |
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attenuation of the trp operon
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-negative regulation at the operator (weak repression)
-promoter, operator, an leader regulatory elements -a second level of regulation exists called attenuation -leader encodes a short peptide with 2 Trp residues: high Trp, leader peptide synthesized, low Trp, ribosome stalls, leader peptide not synthesized. |
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attenuation of the trp operon (9)
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-negative regulation at the operator (weak repression)
-promoter, operator, and leader regulatory elements -a second level of regulation exists called attenuation -leader encodes a short polypeptide with 2 Trp residues (at 1+2): *high Trp, leader peptide synthesized *low Trp, ribosome stalls, leader peptide not synthesized. -transcription And translation coupled. -THESE 2 TRP TRANSLATED LEADER ALLOW FOR HAIRPIN TO BE FORMED (3+4) while mRNA synthesized. so stop transcription. -little E to make this protein. -without trp, hang right there, bind with 2+3 and these don't make terminator hairpin. Read thru and make all mRNAa and protein. -other amino acids have same thing (6-7 residues at leader) and don't transcribe. similar leader control. |
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two component regulatory systems
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component 1: sensor kinase
component 2: response regulator |
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sensor kinase (3)
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-detects environmental signal
-autophosphorylates in response to signal -transfers phosphate group to response regulator, activating the regulator |
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response regulator (2)
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many: DNA-binding proteins: upon activation, regulate initiation of transcription of certain genes
some: upon activation, regulate activity of other proteins |
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how two-component regulatory systems work (5)
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1. environmental signal comes in from outside cell
2. binds to sensor kinase 3. in the cell, uses ATP to phosphorylate the sensor kinase/signal duo. 4. phosphate from sensor kinase goes to response regulator, and the duo attaches to operator and blocks transcription. 5. then phosphatase dephosphorylate to turn off signal. |
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osmoregulation (4)
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1. unphosphorylated OmpR is the transcriptional activator of the ompF gene at low osmolarity. (OmpF in low, OmpC in high)
2. in environment of high osmolarity, the membrane protein EnvZ (sensor kinase) undergoes autophosphorylation using ATP 3. phosphorylated EnvZ transfers its phosphate to OmpR (response regulator). 4. which then binds near the promoters of the porin genes. it blocks the transcription of the ompF gene and activates transcription of the ompC gene. |
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two component systems: flagellar motility
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1. signals are transduced via methyl-accepting chemotaxis protein (transmembrane), all MCP proteins.
2. CheA is the sensor kinase 3. CheY is a response regulator 4. CheY-P interacts with the flagellar motor (FliM), causing CW (tumble) 5. CheZ dephosphorylates CheY 6. CheR continually adds methyl groups to the MCP tranducer at a slow rate 7. CheB-P is a demethylase removing methyl groups from MCP (the more methyl residues on receptor, less sensitive the receptor. so as signal from receptor induces demethylation of receptor in feedback loop, system is continuously adjusted to environmental chemical levels, remaining sensitive for small changes even under extreme chemical concentrations. 8. level of methylation of the MCPs affects their conformation and controls adaptation to a sensory signal. |
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what happens in attractant?
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less phosphoA, less phosphoY, less CheB. so more methyl. so longer run. when maximally methylated, they'll release their attractant (control amount of attractant). so always a quick tumble every once in a while.
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what happens in repellant?
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more PCheA, more PCheY, more CheB, more tumbles.
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