Bacterial Transformation Flashcards
what is transformation
ability of bacterial cells to take up free DNA from their environment
what is transformation like
May be a transient phenomenon associated with growth phase
what is the first demonstration of transformation
Streptococcus pneumoniae by Fred Griffiths in 1928
what phenomenon is transformation
not known if transformation is a natural phenomenon in all bacteria
when is transformation seen
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
what is the transforming principle in the transformation of non-pathogenic (rough) Streptococcus pneumoniae bacteria into pathogenic (smooth) bacteria
DNA
what stops the transformation process
DNase enzyme breaks down DNA
if DNase was boiled/denatured how does this affect transformation
no effect
bacterias natural competence - nutrition
Uptake of DNA would provide bacteria with a source of nucleotides. However, this seems unlikely to be an efficient method of nutrient uptake
bacteria natural competence - repair
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
bacteria natural competence - diversity
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
what happens in natural transformation
Process in both Gram-negative and Gram-positive bacteria is broadly similar
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
what is needed in gram positive natural transformation
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
what does autolysin do in gram positive natural transformation
degrades the cell surface and exposes the competence complex
what happens once the competence complex is exposed
binds dsDNA fragments
Only dsDNA can be used to transform cells
If ssDNA is used, no transformants occur
what happens to bound ds DNA fragments
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
what happens to the ssDNA
recombined into the host genome
what is different in gram negative natural transformation
Gram-negative cells have to get DNA across 2 membranes
what is DNA uptake associated with in gram negative natural transformation
DNA uptake associated with the formation of small membranous structures (transformasomes) on outside of cell
what happens to transforming DNA
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
what does gram negative natural transformations require
Appears to require the recognition of specific sequences
what is a major breakthrough in induced competence/artificial transformation
Stanley Cohen reported induced competence of E. coli
CaCl2 induced competency of E. coli can generate large numbers of transformants
what is CaCl2 induced competency of E. coli can generate large numbers of transformants used for
Used routinely in cloning experiments
what is the induced competence/artificial transformation method
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
what causes transient holes in cell membrane
high-voltage electric field ~12.5 kV/cm is applied briefly (5-10ms) to cells
what must happen to cells after transient holes made in cell membrane
Cells must be washed in Distilled Water or salt-free buffer
reduces conductivity
why are cells transferred into broth
reinitiate growth
why are cells plated on selective medium
identify transformed cells
