Transformation Flashcards
(46 cards)
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
