Transformation Flashcards
transformation (def.)
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
transformation is both _____ and important for which two techniques?
-artificial and natural
-genetic mapping and engineering
first demonstration of transformation
Griffith’s transforming principle
-R streptococci (avirulent) acquire transforming principle from S streptococci (virulent) which allows them to infect & kill mice
bacteria must be _____ to take up naked DNA from environment
competent
competence is _____ process that ultimately leads to _______ required to take up & process DNA from environment; slightly different in Gram negative & gram positive organisms
-genetically programmed
-expression of proteins
Usually competence genes expressed under _____ conditions: Streptococcus pneumoniae? Bacillus subtilis, Haemophilus influenzae? Neisseria gonorrhoeae?
-specific
-only competent in early exponential growth phase
-only competent when nutrients limiting
-competent all the time
bacillus subtilis (gram positive) competence is regulated by _________ that senses cell density
-two-component system (ComP sensor protein & ComA response regulator)
ComP sensor protein detects ______ that builds up and indicates cell density/______. autophosphorylates on conserved ____
-secreted peptide pheromone (ComX peptide)
-starvation
-his (histidine)
ComA response regulator phosphorylated by _____ which turns on expression of genes required for DNA uptake & processing
-ComP-P
ComA when phosphorylated, changes conformation and binds DNA & recruits ______.
-RNAP to transcribe DNA
B. subtilis RNA Polymerase + ComK activator =
________
-expression of competence uptake machinery
ComA-P controls expression of two things + what they did?
ComK: transcription factor that stimulates expression of DNA uptake & processing machinery
ComS: protects ComK from proteolytic degradation
ComK degradation by ____ (2)
ClpCP, MecA
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
-peptide pheromone
-ComD
-ComE
-alternative sigma factor
-ComX
S. pneumonia RNA Polymerase + ComX alternative sigma factor = _______
expression of competence uptake machinery
DNA uptake machinery accomplished by _____, similar to other ________ machineries, slightly different in G+/G- organisms
-multi-protein machines
-pilus elaboration & secretion
Dna uptake machinery steps (1-4)
1) initiation
2) uptake
3) translocation
4) recombination
DNA uptake machinery (gram +) : bacillus, streptococcus: genes needed for transformation identified by screening for mutants that cannot take up DNA —> _____ genes
com
DNA uptake machinery (gram +): comG operon genes encode _____
pseudopilus (resemble type IV pili made by type II secretion systems, similar to some conjugation systems)
comG operon genes (3)
ComGC- major pilus protein
ComC- processing endopeptidase
ComGB, ComGA - pilus assembly
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 _____.
-pilus & ComEA cell wall protein
- 6 kb
-15 kb
-NucA endonuclease
-pilus retraction
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
-one
-NUCLEASE
- ComEC
- ComFA ATP-DEPENDENT
-ComEA & ComEC
-80 - 100
- 3’ - 5’
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
-type II
-type IV
-secretin/PilQ
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
-type II secretion/type IV pilus
-same type II secretion
-type IV
-pseudopilus
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)
-SSB
-transformation
-RecA & homologous recombination
- 8.5 to 12
-RecA
2 evidence of transformation
-efficiency of DNA uptake
specificity of DNA uptake
efficiency of DNA uptake lab technique (5)
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
specificity of DNA uptake lab technique (4)
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
specificity of DNA uptake can be measured using DNase experiments that measure _______
transformation efficiency
specificity of DNA uptake experiments show that some bacterial species have specific _______ (USSs). Has USS= take up DNA containing these sequence much more ______
-uptake signal sequences
-efficiently
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 ________
-dsDNA
-ssDNA
-isolating it at various times & using it to transform a fresh recipient.
transformation eclipse phase
DNA goes through a phase where it cannot be reisolated & be active for transformation (ssDNA)
transforming DNA undergoes 3 transitions during transformation
dsDNA -> ssDNA (eclipse) –>dsDNA
3 roles of natural transformation
1) nutrition
2) DNA repair
3) Recombination
arguments against nutrition role of natural transformation
-some bacteria only take up own DNA
-competence develops in only portion of population
arguments for nutrition role of natural transformation
-this may be to avoid uptake of “dangerous” parasitic DNAs
-cell death & cannibalism may be part of normal colony development
cell death & cannibalism in B. subtilis; S. pneumoniae; E.coli
- 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
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
-homologous recombination to repair DNA
- recA gene
-competence genes
role of natural transformation: recombination hypothesis
transformation provides genetic exchange that increase diversity -> speeds up evolution
Neisseria spp. have 2 different secretion systems: ______ system for DNA import
- ______ secretion system (e.g. conjugation system) for DNA export
-type II secretion / type IV pili
-type IV
Mutation of the DNA export ____ leads to less Neisseria genetic diversity
-T4SS
reason for artificial transformation
many microbes are not naturally transformable; have engineered ways to make bacteria take up DNA in the lab
calcium ion induction- artificial transformation
-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
calcium ion induction steps
-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
electroporation- artificial transformation
-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
electroporation steps
-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
