Bacterial Transformation

Question 1

what is transformation

Answer 1

ability of bacterial cells to take up free DNA from their environment

Question 2

what is transformation like

Answer 2

May be a transient phenomenon associated with growth phase

Question 3

what is the first demonstration of transformation

Answer 3

Streptococcus pneumoniae by Fred Griffiths in 1928

Question 4

what phenomenon is transformation

Answer 4

not known if transformation is a natural phenomenon in all bacteria

Question 5

when is transformation seen

Answer 5

generally only seen if it leads to a phenotypic change in the recipient cells
Perhaps widespread in nature but difficult to observe in the laboratory

Question 6

what is the transforming principle in the transformation of non-pathogenic (rough) Streptococcus pneumoniae bacteria into pathogenic (smooth) bacteria

Answer 6

DNA

Question 7

what stops the transformation process

Answer 7

DNase enzyme breaks down DNA

Question 8

if DNase was boiled/denatured how does this affect transformation

Answer 8

no effect

Question 9

bacterias natural competence - nutrition

Answer 9

Uptake of DNA would provide bacteria with a source of nucleotides. However, this seems unlikely to be an efficient method of nutrient uptake

Question 10

bacteria natural competence - repair

Answer 10

Uptake of potentially homologous DNA would allow repair. Competence induced in S. pneumoniae by DNA-damaging agents and antibiotic treatment provides strong evidence that genetic transformation is induced as a global response to stress
Little evidence that competence systems in other bacteria are induced by damage to DNA

Question 11

bacteria natural competence - diversity

Answer 11

Uptake of DNA from other strains offers the potential for increasing genetic diversity by uptake and incorporation of non-homologous DNA
S. pneumoniae are involved in pneumococcal fratricide/sobrinicide, which selectively lyses non-competent cells while protecting competent cells

Question 12

what happens in natural transformation

Process in both Gram-negative and Gram-positive bacteria is broadly similar

Answer 12

DNA first binds to the exterior of cell
DNA molecules then fragmented
- may occur concomitantly with uptake into the periplasm in Gram-negative bacteria
One DNA strand is taken up into the cytoplasm while the other is degraded
RecA mediated integration into chromosome or replication as plasmid

Question 13

what is needed in gram positive natural transformation

Answer 13

complex of 3 - 5 proteins are required for transformation, including:
a competence factor
a specific endonuclease
DNA-binding proteins
an autolysin to increase cell permeability

Question 14

what does autolysin do in gram positive natural transformation

Answer 14

degrades the cell surface and exposes the competence complex

Question 15

what happens once the competence complex is exposed

Answer 15

binds dsDNA fragments
Only dsDNA can be used to transform cells
If ssDNA is used, no transformants occur

Question 16

what happens to bound ds DNA fragments

Answer 16

digested by an endonuclease into fragments of size ~15 Kbp
An entry nuclease then degrades one of the two strands as the other enters the cell
protected by ssDNA binding proteins from further degradation

Question 17

what happens to the ssDNA

Answer 17

recombined into the host genome

Question 18

what is different in gram negative natural transformation

Answer 18

Gram-negative cells have to get DNA across 2 membranes

Question 19

what is DNA uptake associated with in gram negative natural transformation

Answer 19

DNA uptake associated with the formation of small membranous structures (transformasomes) on outside of cell

Question 20

what happens to transforming DNA

Answer 20

first taken into these vesicles then internalised into the cell
One of the two strands is degraded while the other may recombine with the host chromosome

Question 21

what does gram negative natural transformations require

Answer 21

Appears to require the recognition of specific sequences

Question 22

what is a major breakthrough in induced competence/artificial transformation

Answer 22

Stanley Cohen reported induced competence of E. coli

CaCl2 induced competency of E. coli can generate large numbers of transformants

Question 23

what is CaCl2 induced competency of E. coli can generate large numbers of transformants used for

Answer 23

Used routinely in cloning experiments

Question 24

what is the induced competence/artificial transformation method

Answer 24

E. coli log phase cells incubated with circular DNA on ice in presence of 10mM (0.01M) CaCl2
Or other cations e.g. Rb
Cells heat shocked at 42°C for 2 min
Cells transferred to broth to reinitiate growth
Plated on selective medium to identify transformed cells
Plasmids used carrying antibiotic resistance genes to identify transformed cells
Only about 1 in 1000 cells transformed

Question 25

what causes transient holes in cell membrane

Answer 25

high-voltage electric field ~12.5 kV/cm is applied briefly (5-10ms) to cells

Question 26

what must happen to cells after transient holes made in cell membrane

Answer 26

Cells must be washed in Distilled Water or salt-free buffer

reduces conductivity

Question 27

why are cells transferred into broth

Answer 27

reinitiate growth

Question 28

why are cells plated on selective medium

Answer 28

identify transformed cells