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1: GENET 270 > Transformation > Flashcards

Transformation Flashcards

1
Q

transformation (def.)

A

recipient bacterium acquires genes from free DNA in environment derived from a donor bacterium; DNA can recombine with homologous regions in chr. to yield recombinant transformants

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2
Q

transformation is both _____ and important for which two techniques?

A

-artificial and natural
-genetic mapping and engineering

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3
Q

first demonstration of transformation

A

Griffith’s transforming principle
-R streptococci (avirulent) acquire transforming principle from S streptococci (virulent) which allows them to infect & kill mice

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4
Q

bacteria must be _____ to take up naked DNA from environment

A

competent

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5
Q

competence is _____ process that ultimately leads to _______ required to take up & process DNA from environment; slightly different in Gram negative & gram positive organisms

A

-genetically programmed
-expression of proteins

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6
Q

Usually competence genes expressed under _____ conditions: Streptococcus pneumoniae? Bacillus subtilis, Haemophilus influenzae? Neisseria gonorrhoeae?

A

-specific
-only competent in early exponential growth phase
-only competent when nutrients limiting
-competent all the time

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7
Q

bacillus subtilis (gram positive) competence is regulated by _________ that senses cell density

A

-two-component system (ComP sensor protein & ComA response regulator)

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8
Q

ComP sensor protein detects ______ that builds up and indicates cell density/______. autophosphorylates on conserved ____

A

-secreted peptide pheromone (ComX peptide)
-starvation
-his (histidine)

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9
Q

ComA response regulator phosphorylated by _____ which turns on expression of genes required for DNA uptake & processing

A

-ComP-P

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10
Q

ComA when phosphorylated, changes conformation and binds DNA & recruits ______.

A

-RNAP to transcribe DNA

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11
Q

B. subtilis RNA Polymerase + ComK activator =
________

A

-expression of competence uptake machinery

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12
Q

ComA-P controls expression of two things + what they did?

A

ComK: transcription factor that stimulates expression of DNA uptake & processing machinery
ComS: protects ComK from proteolytic degradation

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13
Q

ComK degradation by ____ (2)

A

ClpCP, MecA

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14
Q

Streptococcus pneumonia (gram positive) competence is regulated similar to B. subtilis with two-component system. It has _____ to indicate cell density. ____ is sensor kinase and ____ is response regulator. Difference is ComE-P regulates expression of _______ ComX (not a protein activator like ComK). ____ associates with RNAP to transcribe DNA uptake & processing machinery

A

-peptide pheromone
-ComD
-ComE
-alternative sigma factor
-ComX

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15
Q

S. pneumonia RNA Polymerase + ComX alternative sigma factor = _______

A

expression of competence uptake machinery

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16
Q

DNA uptake machinery accomplished by _____, similar to other ________ machineries, slightly different in G+/G- organisms

A

-multi-protein machines
-pilus elaboration & secretion

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17
Q

Dna uptake machinery steps (1-4)

A

1) initiation
2) uptake
3) translocation
4) recombination

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18
Q

DNA uptake machinery (gram +) : bacillus, streptococcus: genes needed for transformation identified by screening for mutants that cannot take up DNA —> _____ genes

A

com

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19
Q

DNA uptake machinery (gram +): comG operon genes encode _____

A

pseudopilus (resemble type IV pili made by type II secretion systems, similar to some conjugation systems)

20
Q

comG operon genes (3)

A

ComGC- major pilus protein
ComC- processing endopeptidase
ComGB, ComGA - pilus assembly

21
Q

DNA UPTAKE MACHINERY (Gram +): DNA binds ____ directly via electrostatic interactions. dsDNA is cleaved into ____ (S. pneumoniae) or ___ (B. subtilis) pieces by ______. pieces of DNA brought through cell wall through _____.

A

-pilus & ComEA cell wall protein
- 6 kb
-15 kb
-NucA endonuclease
-pilus retraction

22
Q

DNA uptake machinary (Gram +): ____ strand of DNA degraded by EndA _____. other strand transported across cytoplasmic membrane through _____ channel. Facilitated by _______ DNA translocator. ; requires _____ ; ____ nucleotides/sec! (10 kb in 2 minutes); ____ polarity

A

-one
-NUCLEASE
- ComEC
- ComFA ATP-DEPENDENT
-ComEA & ComEC
-80 - 100
- 3’ - 5’

23
Q

DNA uptake machinary (Gram -): ____ secretion system/_____ pili based; very much like in Gram positive organisms except use pore called ____ to get DNA across outer membrane

A

-type II
-type IV
-secretin/PilQ

24
Q

some microbes use dedicated ______ system for DNA uptake = B. subtilis (G+). others use ______ system for both DNA uptake and secretion of other substrates. Neisseria spp. have ____ pilus modified with minor pilin proteins to convert it to a _____ for DNA uptake

A

-type II secretion/type IV pilus
-same type II secretion
-type IV
-pseudopilus

25
Q

DNA processing after uptske: DNA is coated with _____, may be a special SSB dedicated to______. It becomes a substrate for _______ if DNA is homologous. ______ kb segments can be incorporated into chromosome via homologous recombination (using _____ protein)

A

-SSB
-transformation
-RecA & homologous recombination
- 8.5 to 12
-RecA

26
Q

2 evidence of transformation

A

-efficiency of DNA uptake
specificity of DNA uptake

27
Q

efficiency of DNA uptake lab technique (5)

A

1) label donor DNA with radioactivity
2) add to competent recipients
3) Treat with DNase at various times (cannot enter cells)
4)Precipitate cells & count radioactivity
5) Radioactivity in cells/radioactivity added = efficiency of DNA uptake

28
Q

specificity of DNA uptake lab technique (4)

A

1) label donor DNA from different sources
2) add to competent recipient
3) precipitate cells at different times
4) measure amount of radioactivity incorporated into recipient cells

29
Q

specificity of DNA uptake can be measured using DNase experiments that measure _______

A

transformation efficiency

30
Q

specificity of DNA uptake experiments show that some bacterial species have specific _______ (USSs). Has USS= take up DNA containing these sequence much more ______

A

-uptake signal sequences
-efficiently

31
Q

Genetic evidence for conversion of transforming DNA between ss and ds states during transformation based on the knowledge that ______ not _____ can bind cell surface receptors to initiate transformation. Therefore we can study what happens to DNA during transformation by ________

A

-dsDNA
-ssDNA
-isolating it at various times & using it to transform a fresh recipient.

32
Q

transformation eclipse phase

A

DNA goes through a phase where it cannot be reisolated & be active for transformation (ssDNA)

33
Q

transforming DNA undergoes 3 transitions during transformation

A

dsDNA -> ssDNA (eclipse) –>dsDNA

34
Q

3 roles of natural transformation

A

1) nutrition
2) DNA repair
3) Recombination

35
Q

arguments against nutrition role of natural transformation

A

-some bacteria only take up own DNA
-competence develops in only portion of population

36
Q

arguments for nutrition role of natural transformation

A

-this may be to avoid uptake of “dangerous” parasitic DNAs
-cell death & cannibalism may be part of normal colony development

37
Q

cell death & cannibalism in B. subtilis; S. pneumoniae; E.coli

A
  • stationary phase cell-killing mechanisms in B. subtilis
    -S. pneumoniae cell wall hydrolase that kills only non-competent cells
    -competence genes in nontransformable E. coli
38
Q

role of natural transformation: DNA repair -> uptake of DNA from dying siblings in response to DNA damage could provide substrates for ______. But _____ (needed for recombination) not induced during competence in all species and ______ not induced in response to DNA damage

A

-homologous recombination to repair DNA
- recA gene
-competence genes

39
Q

role of natural transformation: recombination hypothesis

A

transformation provides genetic exchange that increase diversity -> speeds up evolution

40
Q

Neisseria spp. have 2 different secretion systems: ______ system for DNA import
- ______ secretion system (e.g. conjugation system) for DNA export

A

-type II secretion / type IV pili
-type IV

41
Q

Mutation of the DNA export ____ leads to less Neisseria genetic diversity

A

-T4SS

42
Q

reason for artificial transformation

A

many microbes are not naturally transformable; have engineered ways to make bacteria take up DNA in the lab

43
Q

calcium ion induction- artificial transformation

A

-cells treated with calcium ions will take up (CaCl2 solution) both ssDNA and dsDNA
- DNA is added and forms calcium-DNA complexes that bind to cell
-heating causes transport of DNA into cell
- if DNA can replicate, it becomes permanently incorporated into cell
- requires selection in order to detect

44
Q

calcium ion induction steps

A

-log phase culture
-resuspend bacterial pellet in CaCl2 solution
-chill on ice
-aliquot competent cells (80 C)
-add plasmid DNA (has selectable marker) & chill on ice
-heat shock (42C H20 water bath)
-plate on selective plate

45
Q

electroporation- artificial transformation

A

­-cells are exposed to an electric field
­- small pores transiently form in membrane, making cell permeable to exogenous molecules, like DNA
-much more efficient than calcium transformation

46
Q

electroporation steps

A

-log phase culture
-resuspend bacterial pellet in H20 solution
-chill on ice + centrifuge + resuspend pellet in H20
-use competent cells
-add plasmid DNA (has selectable marker) & electroshock
-add saline buffer
-plate on selective plate

1: GENET 270 (15 decks)

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