Transcript Genetic Transfer

Genetic Transfer
Siti Sarah Jumali
06-4832123
[email protected]
Overview on Bacterial Gene Transfer
• Bacteria are usually haploid
– Makes it easy to identify loss-of-function mutations in bacteria
than in eukaryotes
• These usual recessive mutations are not masked by dominant genes in
haploid species
• Bacteria reproduce asexually
– Therefore crosses are not used in the genetic analysis of bacterial
species
• Rather, researchers rely on a similar phenomenon called
genetic transfer
– In this process, a segment of bacterial DNA is transferred from
one bacterium to another
Genetic transfer
• A process to transfer genetic material from a
bacterium to another bacterium
• Enhances genetic diversity
– Confer resistance to antibiotic when one a
antibiotic resistant bacterium transfer the gene to
another bacterial cell
Mechanism of Gene Transfer
• Conjugation
– Direct physical interaction between Donor and
recipient cell
• Transduction
– When virus infects a bacterium and transfer
genetic material
• Transformation
– Information is taken from a dead bacterium which
releases it to the environment
Mechanism of Gene Transfer
CONJUGATION
CONJUGATION
• Direct physical interaction
between Donor and
recipient cell
• E.g plasmid is transferred
to a recipient cell from a
donor
• Requires the presence of a
special plasmid called the
F plasmid.
Conjugation cont’d
• A “mating” process between a donor F+ (bacteria
with fertility factor =plasmid) and an F- recipient
cell.
• Occurs in Gram - enteric bacteria like E.coli
• Plasmids carry genes that are nonessential for the
life of bacteria.
• Uses pili (sex pilus).
• Eg. plasmid replication enzymes.
• Causes medical Problem: R-Factor = antibiotic
resistance!
Conjugation
• Discovered in 1946 in bacteria by Joshua
Lederberg and Edward Tatum
• They were studying strains of Escherichia
coli that had different nutritional growth
requirements
• Auxotrophs cannot synthesize a needed
nutrient
• Prototrophs make all their nutrients from
basic components
• One auxotroph strain was designated bio–
met– phe+ thr+
– It required one vitamin (biotin) and one amino
acid (methionine)
– It could produce the amino acids phenylalanine
and threonine
• The other strain was designated bio+ met+
phe– thr–
• The genotype of the bacterial cells that grew on
the plates has to be bio+ met+ phe+ thr+
• Lederberg and Tatum reasoned that some genetic
material was transferred between the two strains
– Either the bio– met– phe+ thr+ strain got the ability to
synthesize biotin and methionine (bio+ met+)
– Or the bio+ met+ phe– thr– strain got the ability to
synthesize phenylalanine and threonine (phe+ thr+)
– The results of this experiment cannot distinguish
between the two possibilities
The need for physical contact
• Bernard Davis later showed that the bacterial strains
must make physical contact for transfer to occur
• He used an apparatus known as U-tube
– It contains at the bottom a filter which has pores that were
– Large enough to allow the passage of the genetic material
– But small enough to prevent the passage of bacterial cells
• Davis placed the two strains in question on opposite
sides of the filter
• Application of pressure or suction promoted the
movement of liquid through the filter
• The term conjugation now refers to the transfer of DNA
from one bacterium to another following direct cell-to
cell contact
• Many species of bacteria can conjugate
• Only certain strains of a bacterium can act as donor
cells
– Those strains contains a small circular piece of DNA termed
the F factor (for Fertility factor)
• Strains containing the F factor are designated F+
• Those lacking it are F–
– Plasmid is the general term used to describe extrachromosomal DNA
• Plasmids, such as F factors, which are transmitted via
conjugation are termed conjugative plasmids
– These plasmids carry genes required for conjugation
Some info on plasmid
• Small, circular pieces of DNA that are separate and
replicate independently from the bacterial chromosome.
• Contains only a few genes that are usually not needed
for growth and reproduction of the cell.
• But important in stressful situations
• F plasmid, facilitates conjugation
– Can give a bacterium new genes that may help for survival
in changing environment.
• Some plasmids can integrate reversibly into the
bacterial chromosome.
– An integrated plasmid is called an episome.
Plasmid
There are several types of plasmids:
a. Conjugative plasmids – genes for sex pili and conjugation
b. Dissimulation plasmids – genes for enzymes that catabolize unusual organic
molecules (Pseudomonas species – toluene, camphor, petroleum products)
c. Plasmids carrying genes for toxins or bacteriocins
d. Plasmids carrying genes for resistance (R) factors
i. Consist of two sets of genes – RTF (resistance transfer factor) and specific resistance
genes (r-determinant)
Mechanism of Conjugation
• The first step in conjugation is the contact between
donor and recipient cells
• This is mediated by sex pili (or F pili) which are made
only by F+ strains
• These pili act as attachment sites for the F– bacteria
• Once contact is made, the pili shorten
• Donor and recipient cell are drawn closer together
• A conjugation bridge is formed between the two cells
• The successful contact stimulates the donor cells to
begin the transfer process
• The result of conjugation is that the recipient cell
has acquired an F factor
– Thus, it is converted from an F– to an F+ cell
– The F+ cell remains unchanged
• In some cases, the F factor may carry genes that
were once found on the bacterial chromosome
– These types of F factors are called F’ factors
• F’ factors can be transferred through conjugation
– This may introduce new genes into the recipient and
thereby alter its genotype
Hfr Strains
• In the 1950s, Luca Cavalli-Sforza discovered a
strain of E. coli that was very efficient at
transferring chromosomal genes
– He designated this strain as Hfr (for High
frequency of recombination)
• Hfr strains are derived from F+ strains
Mechanism in Hfr Strains
• William Hayes demonstrated that conjugation
between an Hfr and an F– strain involves the
transfer of a portion of the Hfr bacterial
chromosome
• The origin of transfer of the integrated F factor
determines the starting point and direction of
the transfer process
– The cut, or nicked site is the starting point that will
enter the F– cell
– Then, a strand of bacterial DNA begins to enter in
a linear manner
• It generally takes about 1.5-2 hours for the entire
Hfr chromosome to be passed into the F– cell
– Most matings do not last that long
• Only a portion of the Hfr chromosome gets into the F– cell
• Since the nick is internal to the integrated F factor, only part
of the plasmid is transferred and the F– cells does not
become F+
• The F– cell does pick up chromosomal DNA
– This DNA can recombine with the homologous region
on the chromosome of the recipient cell
– This may provide the recipient cell with new
combination of alleles
•
Hfr (High Frequency Recombination)
• Hfr- bacterial plasmid integrates into the
chromosome.
• Medical Problem: Hfr antibiotic resistance genes
are passed during binary fission (every time the
cell divides). Therefore, antibiotic resistance
spreads very rapidly!
• When Hfr mate with F – bacteria, only the
bacterial genes cross NOT plasmid genes.
• Genetic diversity results in this case due to
recombination.
Hfr (High Frequency Recombination)
Interrupted Mating Technique
• Developed by Elie Wollman and François Jacob in the
1950s
• The rationale behind this mapping strategy
– The time it takes genes to enter the recipient cell is directly
related to their order along the bacterial chromosome
– The Hfr chromosome is transferred linearly to the F–
recipient cell
• Therefore, interrupted mating at different times would lead to
various lengths being transferred
– The order of genes along the chromosome can be deduced
by determining the genes transferred during short matings
vs. those transferred during long matings
• Wollman and Jacob started the experiment with two E.
coli strains
– The donor (Hfr) strain had the following genetic
composition
•
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•
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thr+ : Able to synthesize the essential amino acid threonine
leu+ : Able to synthesize the essential amino acid leucine
azis : Sensitive to killing by azide (a toxic chemical)
tons : Sensitive to infection by T1 (a bacterial virus)
lac+ : Able to metabolize lactose and use it for growth
gal+ : Able to metabolize galactose and use it for growth
strs : Sensitive to killing by streptomycin (an antibiotic)
• The recipient (F–) strain had the opposite genotype
– thr– leu– azir tonr lac – gal – strr
– r = resistant
• Wollman and Jacob already knew that
– The thr+ and leu+ genes were transferred first, in
that order
– Both were transferred within 5-10 minutes of
mating
• Therefore their main goal was to determine the
times at which genes azis, tons, lac+, and gal+
were transferred
– The transfer of the strs was not examined
• Streptomycin was used to kill the donor (Hfr) cell
following conjugation
• The recipient (F– cell) is streptomycin resistant
• From these data, Wollman and Jacob constructed
the following genetic map:
• They also identified various Hfr strains in which
the origin of transfer had been integrated at
different places in the chromosome
– Comparison of the order of genes among these strains,
demonstrated that the E. coli chromosome is circular
TRANSDUCTION
TRANSDUCTION
• The transfer of genetic material from donor
bacteria to recipient bacteria via transducing
agent (bacterial viruses called bacteriophage).
– Discovered in 1952 by Zinder &
Lederberg.
– Two kinds of transduction:
• generalized and
• specialized.
Transduction
• A bacteriophage is a virus that specifically
attacks bacterial cells
– It is composed of genetic material surrounded by a
protein coat
– It can undergo two types of cycles
• Lytic
• Lysogenic
It will switch to
the lytic cycle
Prophage can
exist in a dormant
state for a long
time
Virulent phages only
undergo a lytic cycle
Temperate phages can
follow both cycles
Transduction
• Phages that can transfer bacterial DNA include
– P22, which infects Salmonella typhimurium
– P1, which infects Escherichia coli
– Both are temperate phages
Generalized transduction
• Starts with the LYTIC CYCLE where a T- even
phage infects E. coli killing the host cell, and
synthesizing 2,000 copies of itself.
• The T-even phage randomly packages bacterial
DNA in a few defective phages.
• Once a T –even phage infects another E. coli, this
genetic information can be recombined into the
host cell without causing the lytic cycle.
• New genetic information is thereby transduced
from one bacteria to another.
Generalized Transduction
Generalized
Transduction
Specialized Transduction
• Lambda phage infects E.coli but does not lyse the cell
immediately. Instead it integrates into chromosome
of the bacteria as a prophage and remains dormant.
– This is called the LYSOGENIC CYCLE. Phage genes are
replicated and passed to all daughter cells until the bacteria
is under environmental stress, from lack of nutrients, etc.
– Then phage gene will excise from the nucleoid and enter
the LYTIC CYLE taking one adjacent gene for galactose
metabolism.
Specialized Transduction cont’d
• The gal transducing phage (lambda) makes
~ 2,000 copies of itself with the gal gene,
and infects other E.coli.
• When gal integrates into the nucleoid of
other E. coli, it may provide these bacteria
with a new capacity to metabolize
galactose.
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Comparison of Bacteriophage
• Comparison of bacteriophage transduction in
E.coli.
Generalized
T even phage
lytic cycle
random packaging
Specialized
lambda phage
lysogenic
specific gal gene
TRANSFORMATION
TRANSFORMATION
• The passage of homologous DNA from a dead
donor cell to a living recipient cell.
• Occurs in Streptococcus pneumoniae.
• When S. pneumo dies the DNA can be absorbed
by a living S. pneumo and recombined into the
chromosome.
• The gene for capsule formation is obtained in this
way, as is a gene for penicillin resistance.
• Discovered in 1929 by Fredrick Griffith.
Griffith’s Transformation Experiment
Griffith’s experiment
(a) Inject living encapsulated bacteria into
mice, mice die, encapsulated bacteria isolated
from dead mice.
(b) Inject living nonencapsulated bacteria into
mice, mice remain healthy, a few nonencapsulated bacteria can be isolated from the
living mice – most phagocytized by
leukocytes.
(c) Inject heat-killed encapsulated bacteria
into mice, mice remain healthy, no bacteria
isolated from the living mice.
(d) Inject living non-encapsulated and heatkilled encapsulated bacteria into mice, mice
die, isolated encapsulated bacteria from dead
mice.
The Experiments of Avery, MacLeod and
McCarty
• Avery, MacLeod and McCarty realized that Griffith’s
observations could be used to identify the genetic
material
• They carried out their experiments in the 1940s
– At that time, it was known that DNA, RNA, proteins and
carbohydrates are major constituents of living cells
• They prepared cell extracts from type IIIS cells
containing each of these macromolecules
– Only the extract that contained purified DNA was able to
convert type IIR into type IIIS
Hershey and Chase Experiment with
Bacteriophage T2
• In 1952, Alfred Hershey and Marsha Chase provided
further evidence that DNA is the genetic material

They studied the
bacteriophage T2

It is relatively simple
since its composed of
only two
macromolecules

DNA and protein
Inside the
capsid
Made up
of protein
Life cycle of the
T2 bacteriophage
• The Hershey and Chase experiment can be
summarized as follows:
– Used radioisotopes to distinguish DNA from proteins
• 32P labels DNA specifically
• 35S labels protein specifically
– Radioactively-labeled phages were used to infect
nonradioactive Escherichia coli cells
– After allowing sufficient time for infection to proceed,
the residual phage particles were sheared off the cells
• => Phage ghosts and E. coli cells were separated
– Radioactivity was monitored using a scintillation
counter
Transformation
• The process by which a bacterium will take up
extracellular DNA released by a dead bacterium
• It was discovered by Frederick Griffith in 1928
while working with strains of Streptococcus
pneumoniae
• There are two types
– Natural transformation
• DNA uptake occurs without outside help
– Artificial transformation
• DNA uptake occurs with the help of special techniques
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TRANSPOSITION
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Transposons (jumping genes) are big
chunks of DNA that randomly excise and
relocate on the chromosome.
Transposons were discovered in 1950 by
Barbara McLintock in corn.
Causes antibiotic resistance in Staph. aureus,
the famous methicillin resistant
Staphlococcus aureus (MRSA) strain!
End of Slides