Recombination or genetic changes
• Conjugation: DNA is transferred
between bacteria through a tube
between cells.
• Transformation: a bacterium takes
up a piece of DNA floating in its
environment
• Transduction: DNA is accidentally
moved from one bacterium to another
by a virus
Conjugation
• Discovered by Joshua Lederberg and
Edward Tatum,
• Strain A would grow on a minimal medium
only if the medium were supplemented with
methionine and biotin;
• strain B would grow on a minimal medium
only if it were supplemented with threonine,
leucine, and thiamine.
• Strains A and B are mixed together, and
some of the progeny are now wild type,
having regained the ability to grow without
added nutrients.
U tube experiment
• This possibility of “cross feeding” was ruled out by
Bernard Davis.
• He constructed a U-tube in which the two arms were
separated by a fine filter.
• The pores of the filter were too small to allow bacteria
to pass through but large enough to allow easy
passage of the fluid medium and any dissolved
substances.
• Strain A was put in one arm; strain B in the other.
• After incubation, Davis tested the content of each arm
to see if cells had become able to grow on a minimal
medium, and none were found.
• In other words, physical contact between the two
strains was needed for wild-type cells to form.
Conjugation process
• Conjugation, depends on the presence of a conjugative plasmid .
• Plasmids are small, double-stranded DNA molecules that can exist
independently of host chromosomes.
• They have their own replication origins, replicate autonomously, and are
stably inherited. Some plasmids are episomes,
• F factor plays a major role in conjugation in E. coli,
• The F factor is about 100,000 bases long and bears genes responsible for
1) cell attachment and 2) plasmid transfer
Conjugation process
• Conjugation, depends on the presence
of a conjugative plasmid .
• Plasmids are small, double-stranded
DNA molecules that can exist
independently of host chromosomes.
• They have their own replication origins,
replicate autonomously, and are stably
inherited. Some plasmids are
episomes,
• F factor plays a major role in
conjugation in E. coli,
• The F factor is about 100,000 bases
long and bears genes responsible for
1) cell attachment and 2) plasmid
transfer
• IS elements that assist plasmid
integration into the host cell's
chromosome.
F+ X F- mating.
• In 1952 William Hayes (1913-1994) demonstrated
that the gene transfer was unidirectional
• Definite donor (F+, or fertile) and recipient (F-, or
nonfertile) strains
Rolling circle replication.
• one strand of the circular DNA is nicked, and
the free 3' -hydroxyl end is extended by
replication enzymes
• The 3' end is lengthened while the growing
point rolls around the circular template and the
5' end of the strand is displaced to form an
ever-lengthening tail,
• The single-stranded tail may be converted to
the double-stranded
• form by complementary strand synthesis.
• Rolling-circle replication is initiated by a
complex of proteins called the relaxosome,
which are encoded by the F factor
F Plasmid Integration.
• The process begins with association between
plasmid and bacterial insertion sequences.
• The 0 arrowhead (white) indicates the site at which
oriented transfer of chromosome to the recipient cell
begins.
Hfr Conjugation.
• F plasmid integrated into their chromosomes.
• In this type of conjugation, the donor transfers
chromosomal genes with great efficiency but does not
change the recipient bacteria into F+ cells.
• Because of the high frequency of recombinants produced
by this mating, it is referred to as Hfr conjugation
• F factor also directs the transfer of the host chromosome.
• If the cells remain connected, the entire chromosome
with
• the rest of the integrated F factor will be transferred (100
minutes);
• However, the connection between the cells usually
breaks before this process is finished.
Hfr Conjugation.
F’ Conjugation.
• F plasmid can leave the bacterial
chromosome and resume status as an
autonomous F factor.
• Sometimes during excision an error occurs
and a portion of the chromosome is excised,
becoming part of the F plasmid.
• it is called an F' plasmid
• The recipient becomes F' and is partially
diploid because
Bacterial gene
mapping using Hfr
strain
• Interrupted mating technique: Ellie
Wollman and François Jacob
• Specifically, a culture containing a
mixture of an Hfr and an F- strain was
incubated, and samples were removed at
intervals and placed in a blender.
• The shear forces created in the blender
separated conjugating bacteria so that the
transfer of the chromosome was
terminated.
• Then the sampled cells were grown on
appropriate medium containing the
antibiotic, so that only recipient cells
would be recovered.
Bacterial gene mapping using Hfr strain
• Although genes were always
transferred linearly with time, as in
their original experiment, the order in
which genes entered the recipient
seemed to vary from Hfr strain to Hfr
• The major difference between each
strain was simply the point of the
origin (O)—the first part of the donor
chromosome to enter the recipient
Bacterial gene
mapping using Hfr
strain
• Interrupted mating technique: Ellie
Wollman and François Jacob
• Specifically, a culture containing a
mixture of an Hfr and an F- strain was
incubated, and samples were removed at
intervals and placed in a blender.
• The shear forces created in the blender
separated conjugating bacteria so that the
transfer of the chromosome was
terminated.
• Then the sampled cells were grown on
appropriate medium containing the
antibiotic, so that only recipient cells
would be recovered.
Transformation
• Transformation is the uptake by a cell of DNA, either a
plasmid or a fragment of linear DNA, from the surroundings
and maintenance of the DNA in the recipient in a heritable
form
• The transformation frequency of very competent cells is
around 10-3
Bacterial Transformation
• The mechanism of transformation has been intensively
studied in S. pneumoniae
• competent cell binds a double-stranded DNA fragment
• DNA uptake requires energy expenditure.
• One strand is hydrolyzed by an envelope-associated
exonuclease during uptake; the other strand associates with
small proteins and moves through the plasma membrane.
• The single-stranded fragment can then align with a
homologous region of the genome and be integrated into the
chromosome.
Transduction Is Virus-Mediated Bacterial DNA
Transfer
• Lederberg and Zinder mixed the Salmonella
auxotrophic strains LA-22 and LA-2 together, and
when the mixture was plated on minimal medium,
they recovered prototrophic cells.
• The LA-22 strain was unable to synthesize the
amino acids phenylalanine and tryptophan (phe-
trp-) and LA-2 could not synthesize the amino acids
methionine and histidine (met-his-).
• The two auxotrophic strains were separated by a
sintered glass filter, thus preventing contact
between the strains
• Surprisingly, when samples were removed from
both sides of the filter and plated independently on
minimal medium, prototrophs were recovered, but
only from the side of the tube containing LA-22
bacteria
Transduction Is Virus-Mediated Bacterial DNA
Transfer
• The addition of DNase, which enzymatically digests DNA, did not
render the FA ineffective.
• Therefore, the FA is not exogenous DNA, ruling out
transformation.
• The FA could not pass across the filter of the Davis U-tube when
the pore size was reduced below the size of bacteriophages.
• P22, present initially as a prophage in the chromosome of the LA-
22 Salmonella cells.
• P22 prophages sometimes enter the vegetative, or lytic, phase,
reproduce, and are released by the LA-22 cells.
• Such P22 phages, being much smaller than a bacterium, then
cross the filter of the U-tube and subsequently infect and lyse
some of the LA-2 cells.
• In the process of lysis of LA-2, the P22 phages occasionally
package a region of the LA-2 chromosome in their heads.
• If this region contains the phe+ and trp+ genes, and if the phages
subsequently pass back across the filter and infect LA-22 cells,
these newly lysogenized cells will behave as prototrophs.
Generalised Transduction
• Generalized Transduction
• Generalized transduction occurs during
the lytic cycle of virulent and some
temperate phages.
• Any part of the bacterial genome can be
transferred
In specialized transduction, only specific portions of the
bacterial genome are carried by transducing particles.
Specialized transduction is made possible by an error in
the lysogenic life cycle of phages that insert their genomes
into a specific site in the host chromosome.
The transducing particle is defective because it lacks
some viral genes and cannot reproduce without
assistance.
Transposable elements
• Mobile genetic elements were first discovered in the 1940s by
Barbara McClintock (1902-1992) during her studies on maize
genetics.
Transposable elements
Unit transposon sometimes
also referred to as non
composite transposons
Transposable elements
Two major transposition methods have
been identified: simple transposition and
replicative transposition.
Simple transposition is also called cut-
and-paste transposition. In this method,
transposase catalyzes excision of the
transposable element, followed by
cleavage of a new target site and ligation
of the element into this site
In replicative transposition, the original
transposon remains at the parental site
on the chromosome and a copy is
inserted at the target DNA site
Transposable elements insertion
mechanism
Plasmid's mobility
• Mobility is an essential part of plasmid fitness.
• two functions are deemed essential for plasmid
survival: DNA replication and horizontal spread.
• The latter may occur by conjugation if a plasmid
carries two sets of genes.
• The set of mobility (MOB) genes is essential and
allows conjugative DNA processing (the MOB genes
were also called Dtr genes, for DNA-transfer
replication).
• Besides, a membrane-associated mating pair
formation (MPF) complex, which is a form of a type 4
secretion system (T4SS), provides the mating channel.
• A plasmid that codes for its own set of MPF genes is
called self-transmissible or conjugative.
• If it uses an MPF of another genetic element present in
the cell, it is called mobilizable.
• Some plasmids are called nonmobilizable because
they are neither conjugative nor mobilizable. They
spread by natural transformation or by transduction.
