Microbio Plasmids & Transposons (7/8/9)

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Microbio Plasmids & Transposons (7/8/9)
2013-11-03 21:01:06

Exam 4
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  1. microbio 7
  2. What are the 3 mechanisms of transfer of DNA into bacterial cell?
    • 1. transformation
    • 2. transduction
    • 3. conjugation
    • such processes are of great biomedical importance as they are the basis for virulence & drug resistance gene transfer among bacteria
  3. competent bacteria
    • bacteria with the ability to import DNA
    • aka they have the ability to make specific proteins that import DNA into the cell
  4. What can limit the ability of DNA from one species to be acquired by a different species?
    restriction enzymes --> cleave foreign DNA that enters the cell
  5. How may bacteria be induced to take up DNA from the environment?
    by changing their cell envelope structure
  6. Transformation
    • introduction of free DNA from the environment into bacteria
    • confers a special property to transformation: if you add DNAse, DNA will be consumed & no transformation will occur
    • (DNAse = nucelase that destroys DNA)
  7. Transduction (2)
    • the transfer of DNA from one bacterium to another by a bacteriophage vector
    • generalized transduction: when a transducing virus packages random fragments of host DNA
    • specialized transduction: when viruses pick up genes that lie near the site of prophage integration
  8. Stages of Generalized Transduction
    • 1. Adsorption/Attachment
    • 2. Injection of Bacterial DNA
    • 3. Transduction of Bacterial DNA
    • able to interact with new host chromosome via homologous recombination
  9. Conjugation
    • allows the direct transfer of DNA from one bacterium to another
    • two cells, the donor cell and the recipient cell, join via sex pilus, a special attachment structure, & a single strand of DNA moves from the donor to the recipient cell
  10. What is the one thing that conjugation requires that neither transformation nor transduction require?
    • conjugation requires cell to cell contact
    • donor cells physically attach to recipient cells
  11. Recombination
    • the generation of new versions of DNA from precursor DNA molecules by enzymes that can break and ligate DNA
    • is a COVALENT association (bond-wise)
  12. conjugative plasmids
    • plasmids that carry special conjugation genes
    • donor DNA that's transferred may be plasmid or portions of chromosomal DNA
  13. Steps of Conjugation
    • 1. donor bacterial cell makes sex pilus using genes found on it's chromosome's “transferable element” or a “plasmid” it has
    • 2. donor’s sex pilus attaches to the recipient cell --> brings the donor and recipient cells together
    • 3. a single-stranded copy of the plasmid (transferable element) is transferred into the recipient cell
    • 4. the single-stranded copy of plasmid in the recipient cell circularizes & replication completes the complementary strand --> new sex pilus can be made
  14. Where are the genes that enable a cell to make a special sex pilus?
    the genes may be found on the transferable element in the bacterial chromosome or its extrachromosomal plasmid
  15. What happens if the recipient cell gets only a piece of the chromosome that's transferred?
    • it's integrated into the recipient cell’s chromosome by homologous recombination
    • if the recipient gets the entire plasmid, a new donor cell is created
  16. RecA
    • enzyme that aligns homologous DNA regions & functions in the recombination process
    • cuts & pastes incoming DNA into a bacterial chromosome
    • a gene might be immediately selected for & integrated because it can confer antibiotic resistance or utilize nutrient in environment (i.e. sugar)
  17. What are all the different ways bacteria can undergo genetic changes?
    • 1. mutations
    • 2. acquisition of new DNA (transformation, transduction, conjugation)
  18. What are the DNA elements in bacterial cells?
    • Chromosome
    • Plasmids
    • Transposons
  19. plasmid
    • non-essential, extra-chromosomal, self-replicating DNA (replicon)
    • usually circular
    • non-essential = not required for normal cellular survival
    • can carry genes necessary for pathogenesis (eg. anthrax)
    • can confer drug resistances upon their hosts
    • can be spread among bacteria or other species
  20. replicon
    • any molecule of DNA that can be autonomously replicated (capable of self-replicating)
    • eg. the chromosome, ALL plasmids & some autonomous viruses
  21. origin of replication (oriR)
    • special site in/on plasmids where replication initiates
    • is a specific, short DNA sequence DIFFERENT from the chromosomal origin of replication
  22. rep protein
    encoded by plasmid itself binds to oriR & recruits host DNA pol to initiate replication
  23. Is plasmid segregation highly regulated?
    • YES, plasmid segregation is highly regulated to ensure that each daughter cell receives at least one copy of the plasmid
    • plasmids DO NOT partition randomly to the daughter cells
  24. What controls the plasmid copy number?
    plasmid genes
  25. incompatible plasmids
    • if you try to introduce into a bacterium a plasmid that has the SAME oriR-rep system as a plasmid that was ALREADY PRESENT in the bacterium, only ONE of the two plasmids will be maintained
    • plasmids of the same family type are incompatible within the same cell
  26. What are 3 intrinsic properties of plasmids?
    • 1. replication: they control their own (oriR & rep)
    • 2. segregation: they themselves make sure one copy is given to a daughter bacterium
    • 3. how many of themselves should be present in each bacterium
  27. transferable/conjugative plasmid
    a plasmid which contains an oriR sequence, a rep gene, & something like the tra operon, a series of genes that code for transfer proteins
  28. tra operon
    a series of genes that code for transfer proteins that allow the host bacterial cell to act as a donor of genetic material by conjugation
  29. oriT (origin of transfer)
    sequence of DNA found in an F plasmid's tra operon where transfer replication initiates
  30. Non-Transferable Plasmids
    • plasmids that aren't self-transferable, aka they do not contain an oriT sequence or encode the tra proteins that mediate conjugation
    • they could also lack both an oriT sequence & the tra operon
  31. Mobilizable Plasmids
    • a plasmid which itself lacks a tra operon BUT has an oriT sequence recognizable by a separate transferable plasmid's Tra proteins
    • if these two exist in the same cell the non-transferable plasmid may be mobilized by the transferable plasmid & passed to a recipient cell
  32. R factors/plasmids
    • plasmids that carry drug resistance genes rendering the host cell resistant to certain antibiotics
    • can be transferable
  33. transposable elements
    • units of DNA that can move, within the cell, from one site to another on the same DNA molecule or from one DNA molecule to another
    • often carry within them drug resistance genes
    • "jumping genes"
  34. microbio 8
  35. bacterial gene exchange
    • 1. transDUCtion: DNA transfer between donor and recipient is mediated by a virus
    • 2. transFORmation: donor DNA in solution is taken up by recipient cells
    • 3. Conjugation: direct contact between a donor and recipient cell occurs via F plasmid for DNA transfer to occur
  36. Steps of Conjugation
    • 1. donor cell contain genes for sex pili synthesis encoded in a plasmid (F plasmid) & therefore expresses sex pili on its surface
    • 2. an extended sex pilus makes initial contact with a recipient cell
    • 3. the pilus is retracted bringing the two cells in contact w/ each other
    • 4. a copy of the F plasmid is nicked at oriT
    • 5. F plasmid DNA is transferred as a linear single strand into recipient bacterium
    • 6. the newly transferred F plasmid is circularized & converted to double-stranded DNA in recipient cell while the F plasmid of donor cell is converted back to dsDNA
    • (non-conjugal plasmids WILL NOT transfer)
  37. Will a non-conjugative plasmid with an oriT site in the donor cell transfer if there's a conjugative plasmid being transferred?
    • Yes, it can be transferred individually or by using the proteins made by the conjugative plasmid
    • (if the OriR is the same between 2 plasmids, only ONE will surivive, NOT OriT)
  38. oriT (origin of transfer)
    • sequence on the F plasmid nicked by an endonuclease (encoded in the tra operon)
    • cutting the plasmid DNA at oriT is necessary to initiate transfer of the plasmid to the recipient cell
    • the first segment of DNA to be transferred to a recipient cell is adjacent to the nick
  39. tra operon
    • region of the F plasmid which encodes genes involved in conjugation
    • said genes encode many proteins involved in the synthesis of the sex pili & DNA transfer proteins, including the endonuclease that nicks the DNA at oriT
    • other proteins encoded by the tra operon include pilin, the major component of the pilus, the proteins of the structural base of the pilus, pilus assembly proteins, & proteins that modify the surface of the donor cell
  40. surface exclusion
    • the phenomenon that conjugative plasmids encode proteins which modify the bacterial cell surface of the cell in which they reside
    • these modifications BLOCK the ability of the sex pilus of a second cell carrying a related plasmid to attach to the cell surface
    • this prevents a cell that carries a conjugative plasmid from inheriting additional, related conjugative plasmids [rendering the original one useless]
  41. tra repressor gene
    • inactivates the synthesis of traJ (positive regulator of tra operon)
    • found in the R factor - a drug resistance plasmid similar to the F factor plasmid
    • most conjugative plasmids regulate their fertility
  42. Why are tra repressor genes seen in most conjugative plasmids?
    • because the pili plasmids encode can easily be bound by phages, resulting in the bacterium's death
    • it is advantageous to be able to control pili expression
  43. Why is the tra operon of F expressed constitutively?
    because the repressor is nonfunctional - long ago an insertion mutation occurred in the repressor gene
  44. What are some regions of homology shared between the F factor & the bacterial chromosome (+ other plasmids)?
    • IS3 gene (2 copies)
    • IS2 gene (1 copy)
    • Tn1000 (1 copy of transposon)
    • *in the presence of RecA, these regions of homology can serve as RECOMBINATION SITES
  45. How can a transferable (conjugative) plasmids mediate the transfer of chromosomal genes?
    • chromosomal genes may only be transferred if the conjugative plasmid becomes INTEGRATED into the bacterial chromosome
    • this is a rare event (occurs in 1 out of every 106 cells) BUT in a culture of a billion cells there would be about 1000 cells in which independent integrations would occur
  46. Where in the bacterial genome can transferable plasmids integrate?
    • transferable plasmids can integrate at a variety of sites (unlike temperate phages like lambda which integrate at a specific site in the chromosome)
    • therefore a population of cells carrying a transferable plasmid is heterogeneous: some cells contain free plasmid DNA, some have a plasmid integrated in their chromosome, and those cells will have the plasmid integrated in different sites of their chromosome
  47. Hfr Strains
    • a new strain of bacteria that have an F factor integrated into a chromosome - it can transfer genes proximal to the integrated conjugative plasmid at high frequency
  48. F' factor
    • an F factor that has excised itself from a bacterial Hfr chromosome taking with it a piece of bacterial DNA
    • Type I excision: F factor has some bacterial DNA & is itself incompletely excised
    • Type II excision: F factor is completely excised & takes with it some bacterial DNA
    • these excisions are an important mechanism by which plasmids acquire additional genes, notably genes for pathogenesis factors which they can then pass on to future generations and to other cells
  49. microbio 9
  50. R100
    • an R factor (a plasmid) that conveys drug resistance against antibiotics chloramphenicol, streptomycin, tetracycline, & sulfonamide to bacteria such as Shigella dysenteriae (cause dysentery)
    • contains a tra operon, so is a transfer plasmid
    • fertility is repressed in established cultures but not in cells that have NEWLY acquired R100 because the R100 sex pilus is s receptor for many phages
  51. What happens if a cell contains both R100 (R factor) and an F factor?
    transfer of either plasmid occurs at LOW frequency
  52. R factor
    • plasmids which encode resistance to at least one antibiotic
    • they can be conjugative plasmids or have no conjugative capability at all
    • some R factors have very limited host ranges (can only be maintained in one or few related species), while others have very broad host ranges
  53. Which R factor is the most 'promiscuous'/transferable?
    • RP4
    • not only does it transfer to almost ALL bacterial species, it can even transfer DNA to yeast cells and to plants
  54. Which R factor genes generally contain antibiotic and chemical resistances?
    genes that are parts of transposable elements: DNA sequences that can move as a unit to new sites on the same DNA molecule OR to other DNA molecules within the same cell
  55. What are the 4 classes of transposable elements?
    • 1. Insertion Sequences (IS) [700-1500 bp]
    • 2. Simple or Noncomposite Transposons
    • 3. Composite Transposons
    • 4. Itegrating Conjugal Elements (ICE)
  56. Insertion Sequences (IS)
    • mobile, short (~0.7-1.8 kb) DNA elements that ONLY encode a single protein, transposase, involved in their transposition
    • they are found ubiquitously (in the F factor, bacterial chromosome, R factors, phage genomes…)
    • can transpose between any DNA molecule present in the SAME cell, aka their transposable properties are limited to only functioning intracellularly
  57. What do all IS elements tend to have at their ends?
    short (<100 bp), inverted repeat sequences at their 5' & 3' ends
  58. Why are chromosomes not riddled extensively with transposable elements?
    • despite the fact that an IS can hop randomly, transposable elements have evolved so that the transposase protein both mediates transposition AND represses its own transcription
    • too much transposition would inactivate genes & be deleterious
  59. What happens at the target site of transposition where the transposon or IS element is incorporated into the piece of DNA?
    • after the target site is cut & the transposon or IS element is ligated in, DNA polymerase fills in gaps that resulted from cutting of the target site, effectively DUPLICATING it
  60. Transposon
    • a mobile genetic element containing additional genes unrelated to transposition (eg. a gene encoding drug resistance, toxins, conjugative properties, etc.)
  61. Composite Transposons
    • typically consists of a drug resistance gene sandwiched between two matching IS sequences
    • the whole transposon can either "hop" as a unit or the ends can transpose independently
    • two matching IS sequences can transpose themselves & everything in between
  62. composite vs. simple transposon
    • composite: a drug resistance gene between by two matching IS sequences
    • simple: a single drug resistance gene, a transposase gene, & a resolvase gene between short terminal repeats
  63. Simple (Noncomposite) Transposons
    • are structurally similar to IS elements, but carry a SINGLE drug resistance gene, a transposase gene, & a resolvase gene between the short terminal repeats (STRs)
    • simple transposons MUST transpose as a unit
    • unlike how composite transposons, there is no independent IS sequence at either end of a simple transposon that can 'hop'
  64. resolvase
    resolves the cointegrate: a transposition intermediate
  65. Tn3
    • classic examples of a simple transposon
    • contains an It has an AMP resistance gene between the short terminal repeats
    • its transposase functions are provided by two genes
    • doesn't use a cut & paste mechanism like ISs use; instead it inserts itself into new genetic material via replicative transposition
  66. replicative transposition
    • the mechanism by which transposons move between & within genetic material
    • 1. trasposon is copied/duplicated
    • 2. form a cointegrate: intermediate consisting of the donor and receptor DNA sequences in a characteristic configuration ('theta')
    • 3. one copy of transposon remains where it existed originally & the copy is transferred
  67. What proteins are capable of resolving the cointegrate transposition intermediate resulting in the original genetic material and the new, slightly altered genetic material?
    • resolvase (TnpR in Tn3): resolves quickly because it's dedicated to this specific function
    • RecA: resolves more slowly
  68. What is another way a nonconjugal plasmid can be transferred from a donor to a recipient cell?
    • by forming a cointegrate with a transfer factor & the complex is transferred in it's cointegrate form
    • when resolved in recipient cell, nonconjugal plasmid has now been TRANSFERRED
  69. Integrating Conjugal Elements (ICE)
    combines properties (genes) of transposons, temperate phages, AND conjugal elements all in the same unit
  70. STX element
    an integrating conjugal element from Vibrio cholera that integrates at a specific site in the V.cholerae genome, promotes DNA transfer to recipient cells, & contains a transposon that includes a gene for drug resistance
  71. Conservative (Cut & Paste) Transposition
    • some transposons are transmitted by a cut-and-paste mechanism where the transposon is actually excised from the site at which it resides and is inserted into a new site
    • there is NO duplication of target site
    • NO cointegrate formation
    • the transposon LEAVES the donor site & MOVES to the target site