Transformation

Question 1

summarize transformation

Answer 1

when a cell is competent and can take up free naked DNA which recombines with homologous regions to produce recombinant transformants

Question 2

what are recombinant transformants

Answer 2

homologous recombination between chromosomal DNA and and free naked DNA that was taken up by the cell

Question 3

what is competence

Answer 3

ability for cell to take up free naked DNA

Question 4

what experiment was used in the discovery of transformation OR transforming principles

Answer 4

Griffiths experiment with the mice, R cells, and S cells
- note: r cells avirulent mixed with dead S cells virulent produced virulent effect on mice.

Question 5

when is streptococcus pneumoniae competent

Answer 5

only in early exponential growth phase

Question 6

when is bacillus subtilis competent

Answer 6

competent when lacking nutrients

Question 7

when is haemophilus influenzae competent

Answer 7

competent when lacking nutrients

Question 8

when is Neisseria gonorrhoea

Answer 8

always competent

Question 9

what is two component system in bacillus subtilis
(hint: explain competence in reference to gm+ bacteria)

Answer 9

include comP sensor proteins which detect competence stimulating factor (pheromones)

ComP signals comA response regulator which turns on gene expression for DNA uptake (competence)

Question 10

what is ComP

Answer 10

sensor protein that detects competence stimulating factor (comX- pheromones) and auto-phosphorylates, giving P to comA

IN BACILLUS SUBTILIS

Question 11

what is comA

Answer 11

protein that receives P from comP, recruits comK (transcription) to turn on competence gene expression

IN BACILLUS SUBTILIS

Question 12

what is comK

Answer 12

transcription factor that stimulates expression of DNA uptake and machinery (RNAP)

IN BACILLUS SUBTILIS

Question 13

what is comS

Answer 13

protects comK from proteolytic degradation

IN BACILLUS SUBTILIS

Question 14

what is proteolytic degradation

Answer 14

proteases (enzymes) that break down proteins

Question 15

what is two component system in streptococcus pneumonia

Answer 15

peptide pheromone increase in density signaling comD sensor kinase which signals comE response regulator

• phosphorylated comE can signal for competence gene expression
  OR
• can signal alternative sigma factor (comX), region on RNAP which tells it what promotor to bind to for competence gene expression

Question 16

whats comD

Answer 16

sensor kinase that is signaled by density of peptide pheromone and signals comE

Question 17

Whats comE

Answer 17

response regulator that turns on gene expression in streptococcus pneumonia

Question 18

what is comG operon genes

Answer 18

gene involved in building of pseudopilus which grabs foreign DNA

Question 19

what is comGC

Answer 19

the major pilus protein with which the pilus is comprised of

Question 20

what is comGB and comGA

Answer 20

proteins involved in pilus assembly

Question 21

what is comC processing endopeptidase

Answer 21

cleaves pilus proteins involved in pilus retraction (?)

Question 22

explain Gm+ DNA uptake machinery
(bringing DNA into the cell)

Answer 22

DNA binds to pilus, comEA holds dna through electrostatic interactions
- dsDNA is cleaved by nucA
- Pilus retraction brings DNA into cell pulling one strand in with the other out -> directed by comFA
- EndA nuclease degrades outside strand while other goes through comEC channel and is held by SSB to prevent degradation
- enters 3’ to 5’

Question 23

what is comEA

Answer 23

holds DNA through electrostatic interaction

Question 24

what is NucA

Answer 24

nucleases that cleaves DNA

Question 25

what is comEC

Answer 25

channel with which ssDNA goes through

Question 26

what is comFA ATP-dependent DNA translocator

Answer 26

uses energy to direct ssDNA through comEC from 3' to 5'

Question 27

what is endA nuclease

Answer 27

nuclease that degrades other strand of ssDNA (outside cell)

Question 28

explain Gm- uptake machinery

Answer 28

uses type II secretion system - DNA goes through pilQ channel - pilE is pseudopilus that grabs DNA - comE degrades int ssDNA - comA is second channel that ssDNA goes through - pilT is motor that directs ssDNA into cell

Question 29

what is role of SSB

Answer 29

ssDNA in cell is coated with SSB to prevent ssDNA degradation - its also a substrate for recA so that it does homologous recombination

Question 30

what experiment is used to observe the efficacy of DNA uptake

Answer 30

- donor DNA is labelled with radioactivity - added to competent recipients and treated with DNase (cleaves dna) at various times - cells are precipitated and radioactivity is counted

Question 31

what type of DNA is favored for uptake

Answer 31

competence favors free DNA that is similar or the same as bacterial DNA/ plasmid DNA because foreign DNA could be lethal

Question 32

what are specific uptake sequences (USSs)

Answer 32

signals that allow for4 the uptake of bacteria/plasmids own DNA more efficiently or rapidly than other DNA

Question 33

what is the eclipse phase of transformation

Answer 33

The time elapsed between successful cell infection and the start of virus production ssDNA is in the cell but is not yet integrated into bacterial chromosome to become dsDNA

Question 34

what are arguments against competence for nutrition

Answer 34

1) some bacterial are only specific in taking up their own kind of DNA 2) competence develops in only a portion of a population

Question 35

what are arguments for competence for nutrition

Answer 35

1) uptake of DNA may keep from species uptaking harmful DNA (B.subtilis) 2) feeding on cells that are incompetent may be normal process

Question 36

what is B. Subtilis cell killing mechanism

Answer 36

process by which bacteria are programmed to kill themselves and donate their contents so that other bacterial cells can thrive (improve fitness)

Question 37

what are arguments for competence for DNA repair

Answer 37

damage in a bacterial chromosome can be replaced by foreign DNA that was taken up

Question 38

what are arguments against competence for DNA repair

Answer 38

1) recA gene is not induced during competence in all species, thus no recombination of new genes occurs in some bacterial cells 2) competence genes are not induced in response to DNA damage

Question 39

what is the evolutionary hypothesis for transformation and recombination use neisseria spp. as an example

Answer 39

hypothesis: transformation provides exchange in genetic material and increases genetic diversity an example is Neisseria spp. as it has type 2/4 secretion systems for DNA import and type 4 for DNA export - DNA export undergoes mutations and modifications continuously which lead to diversity

Question 40

what is calcium ion induction artificial transformation

Answer 40

calcium+ heat shock induces cell surface to open--> any proteins is sucked inside and induces competence

Question 41

what is electroporation artificial transformation

Answer 41

cells exposed to electric field which induce small forms which make cells permeable to molecules like DNA for competence - more efficient than calcium induction