Download Chap 5 – Gene Transfer

GENES TRANSFER
Izzati Adilah bt. Azmir
02-039
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
Mechanisms 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 Frecipient cell.
• Occurs in Gram -ve enteric bacteria like E.coli
• Plasmids carry genes that are nonessential for the
life of bacteria.
• Uses pili (sex pilus).
• E.g. plasmid replication enzymes.
• Causes medical Problem: R-Factor = antibiotic
resistance!
Discovery…
• Discovered in 1946 in bacteria by Joshua Lederberg and
Edward Tatum
• They were studying strains of E. 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
Plasmid
• Small, circular pieces of DNA that are separated
and replicated 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 (rdeterminant)
Episome VS Plasmid
• Plasmid DNA is an extra-chromosomal DNA molecule, it
cannot link up with chromosomal DNA, and it contains the
genetic informations that are necessary for its own
replication.
• Episomes is any kind of extra-chromosomal DNA that can
link up with chromosomal DNA. That is the main difference
between them two.
• Episomes are usually larger than other extra-chromosomal
DNA. An example of episome are the viruses, because they
intergrate their genetic material into the host's
chromosomal DNA.
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.
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
•
•
•
•
•
•
•
•
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)
strs : Sensitive to killing by streptomycin (an antibiotic)
strr : Resistant to killing by streptomycin (an antibiotic)
lac+ : Able to metabolize lactose and use it for growth
gal + : Able to metabolize galactose and use it for growth
• 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
Summary
Conjugation Mechanism and its
Product
Basic conjugation (F+ and F-)
• F+ and F• The difference F+ has a Fertility factor (F factor)
• The F factor is an episome that contains 19 genes and
confers the ability to conjugate upon its host cell.
• From F+ cell to an F- cell, and the genetic material
transferred is the F factor itself.
• The F+ cell initiates conjugation by extending an F pilus
toward the F- cell. Among the genes present on the F
factor are the genes encoding the proteins required for
pilus construction.
Basic conjugation
1
3
2
4
Basic conjugation
• When finished, F pilus temporarily connects
the two cells.
• F factor strand is nicked, and unwinding
begins from the other strand. The nicked
strand begins to transfer through the F pilus to
the F- cell.
• As it does so, this strand begins to be
replicated, as does circular strand remaining
behind in the F+ cell.
Basic conjugation
1
3
2
4
Basic conjugation cont’d
• Eventually, the nicked strand completely passes
through to the recipient cell, and is completely
replicated.
• Produces a new F factor in the recipient cell.
• Pilus is broken, severing the connection between
the two cells.
• Both cells now contain an F factor, become F+.
• New F+ cell (which was the F- cell), can now
initiate conjugation with another F- cell.
Conjugation Involving Hfr Bacteria
• Occasionally, the F factor integrates into a
random position in the bacterial chromosome.
When this happens, the bacterial cell is called
Hfr instead of F+. Hfr bacteria are still able to
initiate conjugation with F- cells, but the
outcome is completely different from
conjugation involving F+ bacteria:
Hfr cells
1.
2.
3.
4.
5.
When F factor integrates into the bacterial chromosome at a
random location.
When DNA transfer begins, the Hfr cell tries to transfer the entire
bacterial chromosome to the F- cell. The first DNA to be
transferred is chromosomal DNA, and the last DNA to be
transferred will be the F factor DNA.
Transfer of the bacterial chromosome is almost never complete.
Pili tend to break, disrupting DNA transfer before the entire
chromosome can be transferred. So, F factor almost never
transferred to the recipient cell & remains F-.
But this cell will receive new DNA from the Hfr and new DNA can
undergo recombination at a high frequency with the host
chromosome.
F' Conjugation to F• F factors can occasionally integrate into the bacterial
chromosome (producing an Hfr cell from an F+ cell).
• Integrated F factors can occasionally excise themselves
from the bacterial chromosome.
• If this excision occurs properly, the Hfr cell becomes an
F+ again. But the excision is sometimes poor.
• F factor takes a small segment of the bacterial
chromosome with it. So some of the chromosomal
DNA become associated with the episome.
• When this happens, the cell is called an F'.
Conjugation involving F' cells allows for the
possibility of recombination, as shown below:
F' cells are able to
initiate conjugation
with F- cells because
of the presence of
the F factor.
When F factor begins
to transfer its DNA to
the recipient cell, it will
transfer the small
segment of
chromosomal DNA as
well.
Just as in the F+/Fmating, both cells wind up
with a copy of the
episome. The cell that was
F- now has the F factor
and is now F'.
F' Conjugation to FThe F' cell has a full complement of chromosomal genes; however, some of those genes
are now on the episome. F' cells are able to initiate conjugation with F- cells because of
the presence of the F factor.
In the transfer, it will transfer the small segment of chromosomal DNA as well.
• Just as in the F+/F- mating, both cells wind up with a copy of the episome.
• The cell that was F- now has the F factor (along with the piece of chromosomal DNA)
and is therefore now F'.
• This cell, however, also has a complete chromosome, so it will be diploid for the
segment of chromosomal DNA on the episome.
• Such a partially diploid bacterial cell is called a merozygote.
•The chromosomal DNA on the episome can undergo recombination at high frequency
with its homologous sequence on the chromosome.
Let's summarize
• F+ mating with F- produces 2 F+
• Hfr mating with F- produces Hfr and F• F' mating with F- produces F' and F'
merozygote (an incomplete bacterial zygote having only a
fragment of the genome from one of the two parent cells)
Quiz
• What happens when:
– F+ meets F– Hfr meets F– F’ meets F– What is F’ by the way?
– What is Hfr and how did it occur?
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
Virulent phages only
undergo a lytic cycle
Temperate phages can
follow both cycles
Prophage can
exist in a
dormant
state for a
long
time
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.
It will switch to
the lytic cycle
Virulent phages only
undergo a lytic cycle
Temperate phages can
follow both cycles
Prophage can
exist in a
dormant
state for a
long
time
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 non-encapsulated 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 heat-killed
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
•
•
•
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!