bacteriophages require bacterial host to complete its life-cycle, wherein site-specific genetic recombination occurs. furthermore, homologous recombination also occur in phages in case of multiple infection of the host cell.

1. DR. VIBHA KHANNA
ASSO. PROF. (BOTANY)
S.P.C. GOVERNMENT COLLEGE
AJMER (RAJASTHAN)

2. CYTOGENETICS
• BLOCK 3: PHAGE GENETICS
• PRESENTATION 3:
GENETIC RECOMBINATION IN PHAGE

3. Genetic Recombination
• Recombination is the process in which one or
more nucleic acid molecules are rearranged or
combined to produce a new nucleotide sequence
• Usually genetic material from two parents is
combined to produce a ‘recombinant’
chromosome with the new different genotype
• Recombination results in a new arrangement of
genes or parts of genes and normally is
accompanied by a phenotypic change

4. Types Of Natural Recombination:
I. General Or Homologous Recombination
• General or homologous recombination occurs between DNA molecules of very
similar sequence, such as homologous chromosomes in diploid organisms.
• General recombination can occur throughout the genome of diploid organisms,
using one or a small number of common enzymatic pathways.
• For homologous or general recombination, each homologous chromosome is
shown as a different shade of blue and a distinctive thickness, with different alleles
for each of the three genes on each. Recombination between genes A and B leads
to a reciprocal exchange of genetic information, changing the arrangement of
alleles on the chromosomes.
• In general recombination, DNA rearrangements occur between DNA segments that
are very similar in sequence. Although these rearrangements can result in the
exchange of alleles between chromosomes, the order of the genes on the
interacting chromosomes typically remains the same.

5. Types Of Natural Recombination:
General Or Homologous Recombination
• General recombination is an integral part of the
complex process of meiosis in sexually reproducing
organisms.
• General recombination also occurs in nonsexual
organisms when two copies of a chromosome or
chromosomal segment are present. We have
encountered this as recombination during F-factor
mediated conjugal transfer of parts of chromosomes
in E. coli.
{https://www.slideshare.net/vibhakhanna1/biology-of-homologous-recombination-in-
bacteria}
Recombination between two phage during a mixed
infection of bacteria is another example.

6. Types Of Natural Recombination:
II. Site-specific Recombination
• Site-specific recombination occurs between particular short
sequences (about 12 to 24 bp) present on otherwise dissimilar
parental molecules.
• Site-specific recombination requires a special enzymatic machinery,
basically one enzyme or enzyme system for each particular site.
• Site specific recombination, can alter gene order and also add new
information to the genome.
• It can move specialized nucleotide sequences, called mobile genetic
elements , between nonhomologous sites within a genome.
• The movement can occur between two different positions in a
single chromosome, as well as between two different chromosomes.
• Some of these elements are viruses in which site-specific
recombination is used to move their genomes into and out of the
chromosomes of their host cell.

7. Types Of Natural Recombination:
Site-specific Recombination
• An example of site-specific recombination is, the
systems for integration of some bacteriophage, such as
lambda, into a bacterial chromosome.
• Site-specific recombination leads to the combination of
two different DNA molecules, illustrated here for a
bacteriophage λ integrating into the E. coli
chromosome. This reaction is catalyzed by a specific
enzyme that recognizes a short sequence present in both
the phage DNA and the target site in the bacterial
chromosome, called att.

8. Types of natural recombination:
III. Illegitimate or nonhomologous recombination
• Illegitimate or nonhomologous recombination occurs in regions where no large-scale
sequence similarity is apparent, e.g. translocations between different chromosomes
or deletions that remove several genes along a chromosome.
• Analysis of the DNA sequence at the breakpoints for such recombination, reveals that
short regions of sequence similarity are found in some cases. For example, the non-
homologous recombination between two similar genes that are several million bp
apart can lead to deletion of the intervening genes in somatic cells.
• For nonhomologous (or illegitimate) recombination, two different chromosomes
(denoted by the different colors and different genes) recombine, moving, e.g. gene C
so that it is now on the same chromosome as genes D and E. {Although the sequences
of the two chromosomes differ for most of their lengths, the segments at the sites of
recombination may be related, denoted by the yellow and orange rectangles.}

9. Types of natural recombination:
IV: Replicative Recombination
• The process of replicative recombination is used by
many transposable elements, to generate a new copy
of the transposable element at a new location.
• Specific enzymes are required for replicative
recombination
• Replicative recombination is seen, in the fig., for some
transposable elements, shown as red rectangles, again
using a specific enzyme, which in this case is encoded
by the transposable element.

10. Genetic Recombination In Phage
Site-specific Recombination
• The site specific recombination, also known as conservative
site-specific recombination, moves specialized nucleotide
sequences, called mobile genetic elements, between
nonhomologous sites within a genome.
• The movement can occur between two different positions in a
single chromosome, as well as between two different
chromosomes.
• In this pathway, breakage and joining occur at two special
sites, one on each participating DNA molecule.
• Depending on the orientation of the two recombination sites,
– DNA integration,
– DNA excision, or
– DNA inversion
can occur

11. Genetic Recombination In Phage
Site-specific Recombination
• Site-specific recombination enzymes that break and rejoin
two DNA double helices at specific sequences on each
DNA molecule often do so in a reversible way:
– the same enzyme system that joins two DNA molecules can take
them apart again, precisely restoring the sequence of the two
original DNA molecules.
• This type of recombination is therefore called
“conservative” site-specific recombination to distinguish it
from the mechanistically distinct, transpositional
recombination.
• {Transposition is the process by which genetic elements
move between different locations of the genome,
whereas site-specific recombination is a reaction in which
DNA strands are broken and exchanged at precise positions
of two target DNA loci to achieve determined biological
function.}

12. Two Types Of DNA Rearrangement Produced By
Site-specific Recombination
• The only difference between the reactions in (A) and (B) is the relative orientation
of the two DNA sites (indicated by arrows) at which a site-specific
recombination event occurs.
• (A) Through an integration reaction, a circular DNA molecule can become
incorporated into a second DNA molecule; by the reverse reaction (excision), it can
exit to reform the original DNA circle. Bacteriophage lambda and other bacterial
viruses move in and out of their host chromosomes in precisely this way.
• (B) Conservative site-specific recombination can also invert a specific segment of
DNA in a chromosome.

13. The Insertion Of A Circular Bacteriophage
Lambda DNA Chromosome Into The Bacterial
Chromosome
• In this example of site specific recombination, the lambda
integrase enzyme binds to a specific “attachment site”, DNA sequence, on
each chromosome.
• A key feature of the lambda integrase reaction is that the site of
recombination is determined by the recognition of two related but
different DNA sequences—one on the bacteriophage chromosome and
the other on the chromosome of the bacterial host.
• The integrase then catalyzes the required cutting and resealing reactions
that result in a site-specific strand exchange. Because of a short region of
sequence homology in the two joined sequences, a tiny heteroduplex
joint, which is seven nucleotide pairs long, is formed at this point of
exchange.
• A total of four strand-breaking and strand-joining reactions is required.
The lambda integrase resembles a DNA topoisomerase in forming a
reversible covalent linkage to the DNA when it breaks a chain. The energy
of the cleaved phospho-di-ester bond is stored in this transient
covalent linkage.
• Thus, this site-specific recombination event can occur in the absence of
ATP and DNA ligase, which are normally required for phosphodiester bond
formation.

14. The Insertion Of A Circular Bacteriophage Lambda
DNA Chromosome Into The Bacterial Chromosome

15. The Excision Of A Circular Bacteriophage Lambda
DNA Chromosome From The Bacterial Chromosome
• The same type of site-specific recombination mechanism
can also be used in reverse, to promote the excision of a
mobile DNA segment that is bounded by special
recombination sites present as direct repeats.
• In bacteriophage lambda, excision enables it to exit from its
integration site in the E. coli chromosome, in response to
specific signals and multiply rapidly within the bacterial
cell.
• Excision is catalyzed by a complex of integrase enzyme and
host factors with a second bacteriophage protein,
excisionase, which is produced by the virus only when its
host cell is stressed—in which case, it is in the
bacteriophage's interest to abandon the host cell and
multiply again as a virus particle.

16. Physical Consequences Of
Site-specific Recombination Events:
a. Intermolecular reaction of circle + circle = integration
b. Intramolecular reaction between two sites on circle
with direct orientation = excision or resolution.
c. Intramolecular reaction between two sites on circle
with inverted orientation = inversion ("flipping").

17. Biological Consequences Of
Site-specific Recombination Events
• a. Prophage integration/excision systems:
Temperate bacteriophage establish lysogeny by integration of
its genome (in a repressed state) into a special site on the
bacterial chromosome by recombination at the attachment (or
att) site. Bacteriophage lambda integration excision is the best-
characterized site-specific recombination system.
• b. Inversion systems:
– 1. Antigen switching/host range: Site-specific inversion
results in the expression of one of two forms. It is an
important mechanism for the generation of diversity within
a population.
– 2. FLP: The 2-micron plasmid of the yeast Saccharomyces
cerevisiae inverts a segment. This changes the relative
orientation of the replication forks, promoting
the amplification of the plasmid to high copy number.

18. Biological Consequences Of
Site-specific Recombination Events
• c. Resolution/excision systems.
– 1. Transposon cointegrate resolution: Some transposons move by a
replicative process that results in a "cointegrate" intermediate
containing two copies of the transposon. Site-specific recombination
then occurs between the two copies of the transposons.
– 2. Replicon dimer resolution: Homologous recombination between
circular chromosomes or plasmids yields dimers, trimers, tetramers,
etc. These larger structures do not segregate as well as the starting
monomer plasmid. Therefore, some plasmids encode site-specific
recombination systems to produce monomers from
multimers. Examples are the cer system of ColE1 plasmid and
the cre/lox system of bacteriophage P1 which, in the lysogenic state,
replicates as a plasmid.
– 3. Developmental excision: Bacillus subtilis undergoes a site-specific
recombination event during sporulation to assemble a transcription
factor specific for sporulation functions. The development
of Anabena, a cyanobacterium, into the nitrogen-fixing heterocyst
form involves two intramolecular site-specific recombination events
to activate nitrogen fixation genes.

19. The Phage Cross: Discovery
• Genetic analysis of any organism is accomplished by
bringing together two genetically different genomes into
the same cell and giving them an opportunity to engage
in recombination and segregation.
• Likewise two distinct phage genomes can be united, but,
because of the parasitic nature of phages, this union
must be inside a bacterial cell. In other words, a
phage cross must be made by a double infection of
bacterial cells by the two phage types.
• A single bacterium infected with several closely related
virus particles may produce a population of progeny
viruses some of which receive genetic properties
characteristic of different parental particles.
• This result was first observed by Delbriick and Bailey and
was analysed in terms of genetic recombination by
Hershey and Rotman.

20. A Phage Cross
• A phage cross can be illustrated by a cross of T2 phages
originally studied by Alfred Hershey.
• The genotypes of the two parental strains of T2 phage
in Hershey’s cross were h− r+ × h+ r−.
• The alleles are identified by the following characters:
– h− can infect two different E. coli strains (strains 1 and 2);
– h+ can infect only strain 1;
– r− rapidly lyses cells, thereby producing large plaques; and
– r+ slowly lyses cells, thus producing small plaques.

21. Recombination Due To A Phage Cross
• In the cross, E. coli strain 1 is
infected with both parental
T2 phage genotypes at a high
phage:bacteria ratio, to ensure
that a large percentage of cells
are simultaneously infected by
both phage types.
• The phage lysate (the progeny
phages) is then analyzed by
spreading it onto a
bacterial lawn composed of a
mixture of E. coli strains 1 and 2.
• Four plaque types are then
distinguishable.
• These four genotypes can be
scored easily as parental
(h− r+ and h+ r−) and recombinants
(h+ r+ and h− r−).
Phenotype of
Plaque
Inferred genotype
Clear and small h− r+
Cloudy and large h+ r−
Cloudy and small h+r+
Clear and large h−r−
Note: Clearness is produced by the h− allele,
which allows infection of both bacterial strains
in the lawn;
Cloudiness is produced by the h+ allele, which
limits infection to the cells of strain 1.

22. To Conclude….
• Recombination in phages, thus, fall under two
broad categories:
– Site Specific recombination that leads to
Integration and Excision of Phage genome and the
host chromosome, during the infection cycle.
– Homologous recombination between two phage
during a mixed infection of bacteria

23. Homologous Recombination Vs.
Site-specific Recombination:
• Homologous recombination occurs between DNA
with extensive sequence homology anywhere
within the homology.
• Site-specific recombination occurs between DNA
with no extensive homology (although very short
regions may be critical) only at special sites. The
protein machinery for the two types of
recombination differs too. Strand exchange
during site-specific recombination occurs by
precise break/join events and does not involve
any DNA loss or DNA resynthesis.