Genetic Recombination in bacteria

Sometimes when two pieces of DNA come into contact with each other, sections of each DNA strand
will be exchanged. This is usually done through a process called crossing over in which the DNA
breaks and is attached on the other DNA strand leading to the transfer of genes and possibly the
formation of new genes. Genetic recombination is the transfer of DNA from one organism to
another. The transferred donor DNA may then be integrated into the recipient's nucleoid by various
mechanisms. In the case of homologous recombination, homologous DNA sequences having nearly
the same nucleotide sequences are exchanged by means of breakage and reunion of paired DNA
segments. Genetic information can be transferred from organism to organism through vertical
transfer (from a parent to offspring) or through horizontal transfer methods such as conjugation,
transformation or transduction. Bacterial genes are usually transferred to members of the same
species but occasionally transfer to
other species can also occur

Horizontal gene transfer, also known as

lateral gene transfer, is a process in
which an organism transfers genetic
material to another organism that is not
its offspring. The ability of Bacteria
and Archaea to adapt to new
environments as a part of bacterial
evolution most frequently results from
the acquisition of new genes through
horizontal gene transfer rather than by
the alteration of gene functions through
mutations. (It is estimated that as
much as 20% of the genome of
Escherichia coli originated from
horizontal gene transfer.)

Horizontal gene transfer is able to cause

rather large-scale changes in a bacterial
genome. For example, certain bacteria
contain multiple virulence genes called
pathogenicity islands that are located
on large, unstable regions of the
bacterial genome. These pathogenicity
islands can be transmitted to other
bacteria by horizontal gene transfer. However, if these transferred genes provide no selective
advantage to the bacteria that acquire them, they are usually lost by deletion. In this way the size of
the bacterium's genome can remain approximately the same size over time.

There are three mechanisms of horizontal gene transfer in bacteria: transformation, transduction,
and conjugation. The most common mechanism for horizontal gene transmission among bacteria,
especially from a donor bacterial species to different recipient species, is conjugation. Although
bacteria can acquire new genes through transformation and transduction, this is usually a more rare
transfer among bacteria of the same species or closely related species.
CONJUGATION

Bacterial conjugation is the transfer of genetic material between bacterial cells by direct cell-to-
cell contact or by a bridge-like connection between two cells. Discovered in 1946 by Joshua
Lederberg and Edward Tatum, conjugation is a mechanism of horizontal gene transfer as
are transformation and transduction although these two other mechanisms do not involve cell-to-cell
contact.

Fig. 42. Genetic recombination

Lederberg and Tatum did not directly prove that physical contact of the cells was necessary for gene
transfer. This evidence was provided by Bernard Davis (1950), who constructed the U tube
consisting of two pieces of curved glass tubing fused at the base to form a U shape with a fritted
glass filter between the halves. The filter allows the passage of media and not bacteria. Davis
discovered that when two auxotrophic strains were separated by the filter, gene transfer could not
take place. Therefore direct contact is necessary for the recombination to take place as discovered
by Lederberg and Tatum.
 Fig. 43 . U-tube experiement

Bacterial conjugation is often regarded as the bacterial equivalent of sexual

reproduction or mating since it involves the exchange of genetic material. During conjugation
the donor cell provides a conjugative or mobilizable genetic element that is most often
a plasmid or transposon. Most conjugative plasmids have systems ensuring that the recipient cell
does not already contain a similar element. The genetic information transferred is often beneficial
to the recipient. Benefits may include antibiotic resistance, xenobiotic tolerance or the ability to use
new metabolites. Such beneficial plasmids may be considered bacterial endosymbionts. Other
elements, however, may be viewed as bacterial parasites and conjugation as a mechanism evolved
by them to allow for their spread. The prototypical conjugative plasmid is the F-plasmid, or F-
factor. The F-plasmid is an episome (a plasmid that can integrate itself into the
bacterial chromosome by homologous recombination) with a length of about 100 kb. It carries its
own origin of replication, the oriV , and an origin of transfer, or oriT . There can only be one copy
of the F-plasmid in a given bacterium, either free or integrated, and bacteria that possess a copy are
called F-positive or F-plus (denoted F+). Cells that lack F plasmids are called F-negative or F-
minus (F-) and as such can function as recipient cells.

Fig. 44. Cojugation

Among other genetic information the F-plasmid carries a tra and trb locus, which together are
about 33 kb long and consist of about 40 genes. The tra locus includes the pilin gene and
regulatory genes, which together form pili on the cell surface. The locus also includes the genes for
the proteins that attach themselves to the surface of F- bacteria and initiate conjugation. Though
there is some debate on the exact mechanism of conjugation it seems that the pili are not the
structures through which DNA exchange occurs. This has been shown in experiments where the
pilus are allowed to make contact, but then are denatured with SDS and yet DNA transformation still
proceeds. Several proteins coded for in the tra or trb locus seem to open a channel between the
bacteria and it is thought that the traD enzyme, located at the base of the pilus, initiates membrane
fusion.
When conjugation is initiated by a signal the relaxase enzyme creates a nick in one of the strands
of the conjugative plasmid at the oriT. Relaxase may work alone or in a complex of over a dozen
proteins known collectively as a relaxosome. In the F-plasmid system the relaxase enzyme is
called TraI and the relaxosome consists of TraI, TraY, TraM and the integrated host factor IHF. The
nicked strand, or T-strand , is then unwound from the unbroken strand and transferred to the
recipient cell in a 5'-terminus to 3'-terminus direction. The remaining strand is replicated either
independent of conjugative action (vegetative replication beginning at the oriV) or in concert with
conjugation (conjugative replication similar to the rolling circle replication of lambda phage).
Conjugative replication may require a second nick before successful transfer can occur. A recent
report claims to have inhibited conjugation with chemicals that mimic an intermediate step of this
second nicking event. If the F-plasmid that is transferred has previously been integrated into the
donor's genome some of the donor's chromosomal DNA may also be transferred with the plasmid
DNA. The amount of chromosomal DNA that is transferred depends on how long the two
conjugating bacteria remain in contact. In common laboratory strains of E. coli the transfer of the
entire bacterial chromosome takes about 100 minutes. The transferred DNA can then be integrated
into the recipient genome via homologous recombination. A cell culture that contains in its
population cells with non-integrated F-plasmids usually also contains a few cells that have
accidentally integrated their plasmids. It is these cells that are responsible for the low-frequency
chromosomal gene transfers that occur in such cultures. Some strains of bacteria with an integrated
F-plasmid can be isolated and grown in pure culture. Because such strains transfer chromosomal
genes very efficiently they are called Hfr (high f requency of r ecombination). The E.
coli genome was originally mapped by interrupted mating experiments in which various Hfr cells in
the process of conjugation were sheared from recipients after less than 100 minutes (initially using a
Waring blender). The genes that were transferred were then investigated.

Fig. 45. Conjugation – formation of an Hfr cell

TRANSDUCTION:

Transduction, a process of genetic recombination in bacteria in which genes from a host cell (a
bacterium) are incorporated into the genome of a bacterial virus (bacteriophage) and then carried
to another host cell when the bacteriophage initiates another cycle of infection. In general
transduction, any of the genes of the host cell may be involved in the process; in special
transduction, however, only a few specific genes are transduced. It has been exploited as a
remarkable molecular biological technique for altering the genetic construction of bacteria, for
locating bacterial genes, and for many other genetic experiments.

Transduction happens through either the lytic cycle or the lysogenic cycle. If the lysogenic
cycle is adopted, the phage chromosome is integrated (by covalent bonds) into the bacterial
chromosome, where it can remain dormant for thousands of generations. If the lysogen is induced
(by UV light for example), the phage genome is excised from the bacterial chromosome and initiates
the lytic cycle, which culminates in lysis of the cell and the release of phage particles. The lytic
cycle leads to the production of new phage particles which are released by lysis of the host.

Fig. 46. Lytic and lysogenic cyles.

 The packaging of bacteriophage DNA has low fidelity and small pieces of bacterial DNA, together
with the bacteriophage genome, may become packaged into the bacteriophage genome. At the
same time, some phage genes are left behind in the bacterial chromosome. There are generally
three types of recombination events that can lead to this incorporation of bacterial DNA into the viral
DNA, leading to two modes of recombination.

Generalized Transduction

Generalized transduction is the process by which any bacterial gene may be transferred to another
bacterium via a bacteriophage, and typically carries only bacterial DNA and no viral DNA. In essence,
this is the packaging of bacterial DNA into a viral envelope. This may occur in two main ways,
recombination and headful packaging.

If bacteriophages undertake the lytic cycle of infection upon entering a bacterium, the virus will
take control of the cell's machinery for use in replicating its own viral DNA. If by chance bacterial
chromosomal DNA is inserted into the viral capsid which is usually used to encapsulate the viral
DNA, the mistake will lead to generalized transduction.

If the virus replicates using 'headful packaging', it attempts to fill the nucleocapsid with genetic
material. If the viral genome results in spare capacity, viral packaging mechanisms may incorporate
bacterial genetic material into the new virion. The new virus capsule now loaded with part bacterial
DNA continues to infect another bacterial cell. This bacterial material may become recombined into
another bacterium upon infection.

When the new DNA is inserted into this recipient cell it can fall to one of three fates

1. The DNA will be absorbed by the cell and be recycled for spare parts.
2. If the DNA was originally a plasmid, it will re-circularize inside the new cell and become a plasmid
again.
3. If the new DNA matches with a homologous region of the recipient cell's chromosome, it will
exchange DNA material similar to the actions in conjugation.

This type of recombination is random and the amount recombined depends on the size of the
virus being used.
 Fig. 47. Generalized Transduction

Specialized Transduction

Specialized transduction is the process by which genes that are near the bacteriophage genome may
be transferred to another bacterium via a bacteriophage. The genes that get transferred (donor
genes) always depend on where the phage genome is located on the chromosome. This second type
of recombination event which is the result of mistakes in the transition from a virus' lysogenic to lytic
cycle is called specialized transduction , and non-viral DNA is carried as an insertion/substitution. If
a virus incorrectly removes itself from the bacterial chromosome, bacterial DNA from either end of
the phage DNA may be packaged into the viral capsid. Specialized transduction leads to three
possible outcomes:

1. DNA can be absorbed and recycled for spare parts.

2. The bacterial DNA can match up with a homologous DNA in the recipient cell and exchange it. The
recipient cell now has DNA from both itself and the other bacterial cell.
3. DNA can insert itself into the genome of the recipient cell as if still acting like a virus resulting in a
double copy of the bacterial genes.

When the partially encapsulated phage material infects another cell and becomes a "prophage" (is
covalently bonded into the infected cell's chromosome), the partially coded prophage DNA is called a
"heterogenote".
Esther Lederberg, Larry Morse, Herman Kalckar, Michael Yarmolinsky, and Yukinori Hirota
went on to do detailed studies of Galactosemia. Specialized transduction was used in these
studies for gene mapping. At about this time, Esther Lederberg, Julius Adler, and Enrico Calef
were also engaged in similar research involving Maltophilia.

Example of specialized transduction is λ phages in Escherichia coli discovered by Esther

Lederberg as well as Fertility Factor F, also discovered by Esther Lederberg.

Fig. 48. Specialized transduction

TRANSFORMATION:

Transformation involves the uptake of free or naked DNA released by donor by a recipient. It was
the first example of genetic exchange in bacteria to have been discovered. This was first
demonstrated in an experiment conducted by Griffith in 1928. The presence of a capsule around
some strains of pneumococci gives the colonies a glistening, smooth (S) appearance while
pneumococci lacking capsules have produce rough (R) colonies. Strains of pneumococci with a
capsule (type I) are virulent and can kill a mouse whereas strains lacking it (type II) are harmless.
Griffith found that mice died when they were injected with a mixture of live non capsulated (R, type
II) strains and heat killed capsulated (S, type I) strains. Neither of these two when injected alone
could kill the mice, only the mixture of two proved fatal. Live S strains with capsule were isolated
from the blood of the animal suggesting that some factor from the dead S cells converted the R
strains into S type. The factor that transformed the other strain was found to be DNA by Avery,
McLeod and McCarty in 1944.

Transformation is gene transfer resulting from the uptake by a recipient cell of naked DNA from a
donor cell. Certain bacteria (e.g. Bacillus, Haemophilus, Neisseria, Pneumococcus) can take up DNA
from the environment and the DNA that is taken up can be incorporated into the recipient's
chromosome.

By 1926 the quest to determine the mechanism for genetic inheritance had reached the molecular
level. Previous discoveries by Gregor Mendel, Walter Sutton, Thomas Hunt Morgan, and numerous
other scientists had narrowed the search to the chromosomes located in the nucleus of most cells.
But the question of what molecule was actually the genetic material had not been answered.

In 1928 Frederick Griffith, in a series of experiments with Diplococcus pneumonia (bacterium

responsible for pneumonia), witnessed a miraculous transformation. During the course of his
experiment, a living organism (bacteria) had changed in physical form.

The pneumococcus bacterium occurs naturally in two forms with distinctively different
characteristics. The virulent (S-strain) form has a smooth polysaccharide capsule that is essential for
infection. The nonvirulent (R-strain) lacks the polysaccharide capsule, giving it a rough appearance.
Mice injected with S-strain of the pneumococcus bacteria die from pneumonic infection within a few
days, while mice injected with the R-strain bacteria continue to live. Injection with heat-killed S-
strain bacteria also results in the mice surviving.

Griffith was surprised to find in his experiments that mice injected with a mixture of heat-killed S-
strain and live but nonvirulent R-strain produced lethal results. In fact, Griffith discovered living
forms of the S-strain bacteria in the infected mice. He hypothesize that the R-strain bacteria had
somehow been transformed by the heat-killed S-strain bacteria. Some "transforming principle",
transferred from the heat-killed S-strain, had enabled the R-strain to synthesize a smooth
polysaccharide coat and become virulent.

Oswald Avery, Colin McCleod, and Maclyn McCarty (1934-1944) at the Rockefeller Institute, building
on Griffith's work, showed that only DNA could cause the transformation. They isolated a cell-free
extract from the S-strain bacteria and were able to transform living R-strain into a culture containing
both S-strain and R-strain cells. The purified extract contained Griffith's "transforming principle".
Through biochemical testing, they showed it to be deoxyribonucleic acid (DNA).
Fig. 49. Evidence of transformation
Fig. 50. Transformation (a) with DNA fragments and (b) with a plasmid

Fig. 51. Mechanism of Transformation

 PLASMIDS

It is an extra chromosomal DNA molecule separate from the chromosomal DNA

which is able to replicate independently of the chromosomal DNA. Most
commonly found as small circular, double-stranded DNA molecules in bacteria,
plasmids are sometimes present in archaea and eukaryotic organisms. In nature,
plasmids carry genes that may benefit survival of the organism (e.g. antibiotic
resistance), and can frequently be transmitted from one bacterium to another
(even of another species) via horizontal gene transfer. Artificial plasmids are
widely used as vectors in molecular cloning, serving to drive the replication of
recombinant DNA sequences within host organisms.

 They vary from 1kb to 1000kb

 The term plasmid was first introduced by the American molecular

biologist Joshua Lederberg in 1952.
 The plasmid does not contribute to the genome of the bacterial cell it is present
in but often translates proteins of significance. For example
o It contains the antibiotic resistance gene that helps the bacteria survive from
that specific antibiotic.
o It can provide the bacteria with an ability to fix elemental nitrogen or to degrade
calcitrant organic compounds which provide an advantage under conditions of
nutrient deprivation.

Fig. 34 . This image shows a line drawing of a bacterium with its chromosomal
DNA and several plasmids within it.

Plasmids used in genetic engineering are called vectors. Plasmids serve as

important tools in genetics and biotechnology labs, where they are commonly
used to multiply (make many copies of) or express particular genes. Many
plasmids are commercially available for such uses. The gene to be replicated is
inserted into copies of a plasmid containing genes that make cells resistant to
particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a
short region containing several commonly used restriction sites allowing the easy
insertion of DNA fragments at this location. Next, the plasmids are inserted into
bacteria by a process called transformation . Then, the bacteria are exposed to
the particular antibiotics. Only bacteria that take up copies of the plasmid
survive, since the plasmid makes them resistant. In particular, the protecting
genes are expressed (used to make a protein) and the expressed protein breaks
down the antibiotics. In this way, the antibiotics act as a filter to select only the
modified bacteria. Now these bacteria can be grown in large amounts, harvested,
and lysed (often using the alkaline lysis method) to isolate the plasmid of
interest. Another major use of plasmids is to make large amounts of proteins. In
this case, researchers grow bacteria containing a plasmid harboring the gene of
interest. Just as the bacterium produces proteins to confer its antibiotic
resistance, it can also be induced to produce large amounts of proteins from the
inserted gene. This is a cheap and easy way of mass-producing a gene or the
protein it then codes for, for example, insulin or even antibiotics. A plasmid can
contain inserts of up to 30-40 kbp . To clone longer lengths of DNA, lambda
phage with lysogeny genes deleted, cosmids , bacterial artificial chromosomes,
or yeast artificial chromosomes are used.

Replication of plasmid

Non Integrative Replication

In this type of replication the plasmid DNA once introduced into the cell grows as
per the copy number and the multiplication of the cells

Integrative Plasmid

Episome

Under certain conditions some plasmids may integrate into the bacterial
chromosome. They are called episome or integrative plasmids. At this stage
they replicate along with the bacterial chromosome.

The plasmids in this way are classified into 2 types

Relaxed plasmids

They are the ones which are normally maintained at multiple copies per cell.

Stringent plasmids
 They are the ones which have a limited number of copies per cell.

In this case of plasmid replication, the plasmid DNA is integrated in the bacterial
chromosome and grows along with the cell. It uses the bacterial machinery for
division. A good example of this type of replication is Ti Plasmid, often used in
agricultural genetic engineering experiments. It completely uses the cell genetic
mechanism to grow.

Fig. 35. Replication of plasmid

Types of plasmid

 Fertility plasmid :

That contains the tra genes required for conjugation else known as F plasmids.
For example F plasmids of E coli.
 Fig. 36. Fertility plasmid

 Resistance Plasmid or R plasmids

Carry genes of resistance to one or more antibacterial agents such as ampicilin,

etc

 Col plasmid :

It contains genes for production of bacteriocins, proteins that kill other bacteria
example col E1.

 Virulent plasmids:
Which in turn convert bacteria into a pathogen. For example, the Ti plasmid of
agrobacterium tumefaciens induces crown gal disease on dicot of the palnts

Table 2. Size of some plasmids as vectors