Transcript second of four for Chapter 9

Chapter 9
Genetics of
Bacteria and
Their Viruses:
Transformation
Conjugation
Jones and Bartlett Publishers © 2005
Types of genetic transfer in bacteria
• Transformation – does not require contact;
one cell lyses, another takes up the DNA
from the lysed cell.
• Conjugation – requires actual physical
contact between the donor cell and the
recipient.
• Transduction – involves a bacteriophage.
Phenology of cell competence (for
transformation) in bacterial colonies
Overview of
Bacterial Transformation
Mechanism of Bacterial
Transformation
Donor DNA is
trimmed and
joined to host
(recipient) DNA
by DNA Ligase
Cotransformation
• Cotransformation can occur for two genes near
each other.
• Cotransformation is the probability of
simultaneous transformation of two genes.
• If the rate of cotransformation is much higher
than the product of the individual frequencies,
then this implies that the two genes are close to
each other in the bacterial genome.
Genetic transformation can be used to
create genetic maps in bacteria
Lederberg and Tatum experiment
The sex factor of E. coli (F-plasmid) can recombine
into the circular bacterial chromosome
The fusion creates a
high frequency
recombination (Hfr)
strain.
Because the F factor
can exist alone, or in
the chromosome, it is
called an episome.
Conjugation in E. coli between a
Hfr (male) and a F- (female) cell
Differences between F factor
transfer and Hfr transfer
• Transfer of F factor takes 2 minutes; the
entire chromosome takes 100 minutes.
• F factor is 100 kb; E. coli chromosome is
4600kb.
• Matings are usually interrupted before
complete transfer of chromosomal genes
(but hundreds of genes can be transferred).
Differences between F factor
transfer and Hfr transfer
• Hfr cells do not convert F- recipient cells,
because the final segment of F is not
tranferred.
• In Hfr transfer, some of the transferred
DNA fragment gets incorporated into the
recipient chromosome.
• This will result in the F- cell becoming
recombinant, but the Hfr cell stays the
same.
Selectability
• How can recombinants be identified?
• The F- cell can have an allele that can be
selected, such as antibiotic resistance.
• Recombinants for nutrient requirement
(leu+) from Hfr cells can be screened.
• Selected marker (leu+)
• Counterselected marker (str-s).
• Recombination is not very efficient.
Interrupted-mating technique
The time of entry of genes into the F- cells
can be used to make genetic maps
Different Hfr strains
transfer genes starting at
different locations and in
one of 2 different
orientations (clockwise
or counter-clockwise).
The relative location of
the genes (genetic map)
remains the same
The circular genetic map of E. coli
showing some of the about 4,000 total genes
Note that the map is
in minutes rather
than in map units.
It takes 100 minutes
to transfer the
entire E. coli
chromosome to a Fcell.
Details of 2 minutes (2%) of the
E. coli genetic map
The single origin
of DNA
replication (oriC)
in the
chromosome is
located at about
84.4 minutes.
F’ plasmids
Abnormal excision of the F-factor from the Hfr chromosome can transfer
chromosomal genes to the excised F-plasmid
(now named F’-factor; F’(lac) in the example shown)
Formation of a F’-Plasmid
Partial diploids or merodiploids – when F’ plasmids spread.