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MICROBIAL GENETICS: RECOMBINATION AND PLASMIDS
I.
II.
GENETIC RECOMBINATION:
A.
Genetic material from two individuals combined in single individual:
New genotypes arise in absence of mutation
B.
Genetic recombination in bacteria:
One-way gene transfer from donor to recipient:
C.
Movement of DNA occurs in three ways:
1.
Conjugation:
Transfer of DNA via cell-to-cell contact
2.
Transformation:
Free DNA inserted directly into recipient cell
3.
Transduction:
Transfer of DNA from one bacterium to another via a virus
Conjugation:
A.
Genetic material transferred from one living cell directly to another cell via cell to
cell contact:
Depends on a Plasmid:
Small circular DNA molecule:
Can exist independently of host chromosome:
Present in:
Bacteria
Yeast
Fungi
Have their own Replication Origin:
Autonomously replicating
Stably inherited
Have 30 or fewer genes
Genetic information is not essential for host survival
One (single copy plasmids) to 40 (multi-copy) plasmids per cell
Can be eliminated by curing:
Spontaneously
B.
Induced by treatments that inhibit plasmid replication but do no affect host cell
reproduction:
Ultra violet and ionizing radiation
Acridine dyes (mutagens)
Thymine starvation
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Growth above optimal temperatures
C.
Classification:
Episome:
Can exist either integrated into the host's chromosome or separate from
it
Conjugative plasmid:
Have genes for pili:
Can transfer themselves to other bacteria during conjugation
Fertility Plasmid:
Maleness in bacteria determined by a genetic factor:
FERTILITY FACTOR or F FACTOR:
Carries genes responsible for cell attachment and plasmid
transfer during conjugation:
Determines the presence of sex pili:
Aids in DNA transfer
Contains insertion sequences:
Assist plasmid integration into host chromosome.
D. Conjugation:
Cells must be in contact with each other
Cells must be closely related
Cells must be of the opposite mating type:
One partner is the donor (F+, male)
Male cells with the F+ factor referred to as F+ cells
Other partner is the recipient (F-, female).
Genetic material always transferred from donor to recipient:
Bacterial chromosomal genes are not transferred:
Plasmid genes are transferred.
E.
Hfr conjugation:
Certain strains do transfer chromosomal genes with great efficiency:
Do not change recipient to donor.
Fertility factor is an episome:
May integrate into main bacterial chromosome at several different
locations:
1/100,000 cells
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Hfr cells:
Cells with Fertility Factor attached to the main chromosome
G.
Integrated plasmid:
Directs synthesis of sex pili
Transfers genetic material to F- cells:
During conjugation circular chromosome containing F+ factor
breaks at the F+ and is transferred to recipient cell:
After donor chromosome enters recipient cell:
May be degraded
Incorporated into F- genome by recombination
H.
Transfer of genetic material:
Always starts at same place:
Always sequential:
Gene A always enters the recipient cell before gene B:
Gene B always enters the recipient cell before gene C etc.
Conjugation can be interrupted at any time:
Wearing blender
Allows location of genes along the DNA molecule.
III.
Bacterial transformation:
A.
First process discovered that indicated that DNA is the genetic material:
Fred Griffith (1929):
Cell-free extracts of dead encapsulated Staphylococcus pneumoniae Type
III transform Staphylococcus pneumoniae Type II into encapsulated
Staphylococcus pneumoniae Type III
Transforming substance:
Cell-free extract which transformed type II
B.
Avery, McCleod and McCarty (1941):
Transforming substance is DNA:
Bacterial macromolecules purified:
Protein
Polysaccharide
RNA
DNA
Added separately to recipient bacteria:
Only DNA caused transformation:
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After treatment with DNAase:
DNA no longer causes transformation
Only certain cells able to be transformed:
Recipient cells must be competent:
Able to take up a molecule of DNA and be transformed.
Competence is inherited property of cell:
Only certain strains of bacteria are competent:
Competent cells:
Do not have slime layer or capsule
Do not secrete DNase
Produce a protein factor which allows attachment of DNA to surface
of cell
Are at the end of log phase
C.
During transformation:
DNA extracted from donor cells:
Fragmented:
Broken into pieces:
Mixed with living recipient cells:
DNA binds to outside of competent cell:
Cell may bind several thousand pieces of DNA
Single stranded DNA taken into cell:
Inserted into DNA of recipient cell by H bonding:
Results in incorporation of genetic material from donor into
chromosome of recipient cell:
Produces hybrid DNA molecule:
IV.
Transduction:
A.
Transfer of DNA from one bacterium to another via a virus
B.
Two types of transduction:
1.
Generalized transduction:
Any portion of the bacterial chromosome is transferred from one
host to another by a virus
2.
C.
Specialized transduction:
Specific portion of donor's chromosome incorporated into virus
chromosome:
Transferred during lysogeny:
Requires a temperate virus:
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Temperate virus always:
Incorporated at the same place in host chromosome:
Lambda:
An E. coli phage
Always incorporated between:
Genes that control galactose utilization and
Genes that control biotin synthesis
D.
Lysogenic bacterium:
Contains temperate virus in its chromosome:
Host viral chromosome and host chromosome:
Replicate together for many cell generations:
Viral DNA leaves host chromosome after many cell generations:
Starts a new lytic cycle:
New viruses made
Host cell lysis
E.
Sometimes (1/100,000) when virus DNA leaves the host chromosome:
Takes small part of host chromosome with it:
Piece of host chromosome replicated along with viral DNA:
Incorporated into new virus particles:
Virus particles infect new host and become incorporated into new
host's chromosome:
DNA has been transferred from one bacterium to another by the
virus
F.
SPECIALIZED TRANSDUCTION same genes are always transferred because
virus always inserts its chromosome at the same place in the bacterial
chromosome
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