intro MCB4414

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intro MCB4414
2013-09-09 23:13:32
mcb4414 woese

exam 1
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  1. The elements of life are:
    • C,H,N,O,P,S
    • Trace minerals: Ca, Mg, Mn, Mo, Ni, etc.
  2. How the reduction and oxidative of elements of live drive bioenergetics?
  3. Referring the general time line for biology and evolution on Earth. What major events altered evolution especially in terms of microbially driven changes?
    • Microbes have inhabited Earth as much as 80% of the time, (animals) humans<<<less than 1%.
    • Most metabolic pathways and niches were likely evolved in this time, far in advance of higher plants and animals.
    • Only a few microorganisms are capable of existing without help from other organisms (all rely on the sun's energy in some way).
  4. What did Carl Woese contributed?
    • generated 16S/18S rRNA tree
    • line length represents evolutionary differences
    • single progenitor or universal ancestor assumed when rooting the tree.
    • genes use to root the tree assumed to have duplicated before divergence of three domains.
  5. What kind of biological molecules are best to generate phylogenetic trees? Why? how does this compares to enzymes?
  6. What defines a species?
  7. What are open genomes?
  8. What are closed genomes?
  9. What is meant by the "core" genome?
  10. Archaea are usually found in what kind of environments?
    Ruminants and waste water treatment plants.
  11. How does this supports Woese's hypothesis for three domains of life?
  12. What does this likely say about early evolution on Earth?
  13. Archaea-No stablish patogens? How does this support Woese's hypothesis as well?
    No Archaea has been determined to act as a pathogen on either plants or animals. Archaea and Bacteria are easily classified by rRNA sequence, however they also have quite unique structural characteristics.
  14. chapter 2
    growth and cell division
  15. Measuring cell growth:
    • Turbidity
    • Viable Cell Counts
    • Total Cell Counts
    • Dry weight
    • Protein Determination
  16. Total cell counts
    uses a counting chamber (glass slide with defined area and depth)
  17. Electronic cell counting
    operates by passing the cells through a narrow pore with an electrical field. The bacterium passes, the conductivity changes and a voltage difference is triggered.
  18. limitations of electronic cell count
    • cannot distinguish between live and dead cells
    • cannot be used to count very low cell density cultures
  19. What defines growth?
    • growth is strictly defined as a change in mass of a culture.
    • in bacteriology, growth is the most informative parameter to study and most widely used.
    • growth serves as a measurable assessment of how bacteria is dealing with the environment (e.g. in the presence of an antibiotic, low phosphorus, with or without oxygen)
  20. are changes in the number of cells in a test tube also defining growth? why or why not?
    Cell number may not change during this time (e.g. smaller cells becoming larger, larger cells dividing to smaller cells when growth is not occurring, ect)
  21. Optical density
    a common method to measure growth
  22. Turbidity
    measure of the light that is scattered in a spectrophotometer as a representative measure of the number of cells.
  23. Viable cell counts
    • cells are serial diluted and plated on a growth medium
    • each colony represents a viable cell in culture tested
  24. limitations of viable cell count
    • clumps of cells will be represented by a single colony, and most bacteria grow in clusters.
    • some cells plate with a poor efficiency (do no survive the manipulation or simply grow better in a liquid medium).
  25. Dry weight/protein
    • cells are harvested by centrifugation, dried and carefully weighed
    • dried cells can also be assayed for protein content, which also increases with growth
  26. limitations dry weight/protein
    these techniques are not typically used in today's lab as they are not amenable to assaying many cultures at several time points, etc.
  27. growth curve-batch
  28. starvation
    • molecular mechanisms (bacteria):
    • RpoS sigma factor
    • Fis and H-NS
    • PolyPi and Lon
    • Dps
    • ppGpp
    • cAMP/CRP complex
  29. RpoS sigma factor (σS)
    • global regulator
    • required for synthesis of approx. 30 proteins: e.g. catalase, exonuclease, acid phosphatase
    • rpoS mutants display attenuated virulence, reduced resistance to stress
  30. RpoS levels increase during starvation mediated by:
    • increased transcription and translation of rpoS
    • stabilization of RpoS protein.
  31. Starvation: Fis and H-NS
    • DNA binding proteins (Fis-specific; H-NS-non-specific)
    • Fis activates rRNA gene transcription-inhibited by H-NS
    • H-NS levels increase during stationary phase- results in inhibition of ribosome synthesis.
  32. Starvation: (p)ppGpp
    • guanosine tetra/pentaphosphate
    • shift from rich to minimal medium results in accum. of uncharged tRNA (=stringent response)
    • causes ribosomes to stall
  33. Starvation: (p)ppGpp
    • RelA (p)ppGpp synthetase I in complex with stalled ribosomes is activated by uncharged tRNA
    • ppGpp synthetized and slows tRNA/rRNA transcription
  34. Diauxic growth of E. Coli
    glucose and lactose
  35. Mechanism behind Diauxic Growth: cAMP/CRP complex
  36. Mechanism behind Diauxic Growth: cAMP/CRP complex
    • Global transcriptional regulator
    • cAMP levels increase during C starvation (e.g. E. Coli=glucose depletion
    • induces alternative metabolic pathways resulting in diauxic growth
    • mediated through Adenylate cyclase enzyme that is activated by PTS system
  37. cell division (E. Coli as model)
    • cells reach critical mass (sensed internally)
    • DNA sysnteshis initiated by DnaA binding to replication origin (oriC) which opens DNA duplex
    • other replication proteins bind to form complex
  38. cell division (E. Coli as model)
    • chromosome replication and partition to opposite poles (Par and MukB proteins assist)
    • septum forms (often in center of cell) by inward growth of CM, PG and OM
    • septation and cell separation occur in parallel
  39. DNA replication/cell separation
    the overall synthesis of the DNA in a rapidly growing E.coli (generation time of less than one hour) takes about 40 minutes.
  40. DNA replication/cell separation
    the actual partitioning of the cell and final separation requires another 20 minutes.
  41. DNA replication/cell separation
    Thus, the overall process from the initiation of replication of the chromosome to the complete separation is roughly one hour
  42. how can an E. coli cell divide in roughly 20 minutes?
  43. Growth rate as it relates to physiology
    • increasing growth rates leads to:
    • - number of doublings per hr increases (m)
    • - higher proportion of cells is ribosome
    • - DNA replication begins in previous cell cycle w/several replication origins
    • - greater cell mass consistent with minimum cell mass per replic. origin
  44. Cell size and composition
    protein synthesis is one speed- to make more cells faster you have to make more protein synthesis machines (ribosomal RNA and protein).
  45. Cell size and composition
    the relative level of protein and RNA is significantly affected by growth rate
  46. Cell size and composition
    0.6 represents slower growth (minimal medium) while 2.5 doublings per hour represents rich medium
  47. Growth Yield-Y
    If E. coli is using a single carbon source as a source of energy and carbon (such as glucose), then you can determine a growth yield constant for a given condition (Y)
  48. Growth Yield-Y
    this is the weight of cells made divided by the weight of carbon used (weight cancels-no units)