RECOMBINATION IN BACTERIA

Bacteria contain a single circular chromosome and it is usually haploid. Bacteria divide by
simple fission with equal distribution of their genetic material to the progeny cells. Bacterial
cells don’t undergo mitotic or meiotic divisions, hence the genetic recombination events like
segregation,independent assortment and meiotic crossing over aren’t observed in bacteria.
Recombination is important in evolution of bacteria.in bacteria recombination ov=ccurs by three
different processes.
1. Transformation is the uptake of naled DNA molecule from donor cell by the recipient cell
2. Transduction -in bacterial DNA is carried from donor to recipient cell by bacteriophage
3. conjugation - bacterial DNA is transferred from donor cell to recipient cell through
specialized sex pili or “conjugation tubes”

Transformation
It is an active energy requiring process.Transformation occurs only in those bacterial species
possessing the enzymatic machinery involved in active uptake and recombination(eg.
Diplococcus pneumoniae,Bacillus subtils,Haemophilus influenzae).even in those species only
“competent cells” which possess the competence factor serve as recipients.the competence factor
may be a cell surface protein or enzyme(eg DNA transferase) involved in binding or uptake of
DNA.the proportion of competent cells in a culture varies with growth conditions and stage of
the growth curve(maximal in late log-phase)

Transformation occurs in 5 stages

1. reversible binding of double stranded donor DNA molecules to receptor sites on the cell
surface of recipient cells
2. “Irreversible uptake” of the donor DNA by recipient cells
3. “conversion of double stranded donor DNA to single stranded” molecule by nucleolytic
degradation of one strand
4. “Integration of” of all or part of the single strand of donor DNA into chromosome of the
recipient by replacing a segment of one strand of the recipient chromosome
5. segregation and “phenotypic expression”of the integrated donor gene
Steps 2 & 3 occur as a single process during which DNA transferase pulls one strand of donor
DNA into the cell using energy derived from the degradation of the complimentary strand.Steps
1,2&3 are not specific for homologous DNA.competent bacteria can take up any DNA in these
steps but steps 4 & 5 are specific for homologous DNA.donor DNA should atleast be partially
complimentary.
Only small fragment of the donor DNA is taken up by competent cells a DNA minimum length
of 500 bp donor DNA is required for integration.in most transformation experiments donor DNA
fragments are about 20000 bp, hence mapping experiments using transformation can be done
only if the selected genetic markers(or genes) are close together in the donor DNA.for eg.if
genes a+ & b+ are far apart in the donor DNA,they can’t be carried on a single fragment to the
recipient.therefore it will require 2 independent transformation events.since transformation of
single donor DNA fragments occurs with low freq,transformation of 2 fragments of donor DNA
or 2 independent transformation events will be extremelt rare.therefore the double transformants
of the recipient for the 2 genes a + & b+ will be rare.if the 2 genes a + & b+ are close together,
they can be carried in a single fragment of the donor DNA,hence double transformants can be
formed in one transformation event.co-transformation of the 2 genes occur with higher freq.the
frequency with which the 2 genetic markers are co-transformed can be used a s a crude estimate
of the linkage distance between them.

Specialised Transduction
This is mediated by temperate phages where chromosomes migrate at specific attachment sites
on the host chromosome.chromosomes of these phages are capable of both- autonomous
replication & integrated repliacation.these genetic elements are the examples of Episomes
During phage infection the phage DNA enters inside the donor cell.inside the bacterial cell
the phage chromosome circularize in the case of λ phage.integration of the
of λ phage chromosome involves a site specific recombination event
between the bacterial chromosome and the phage chromosome.it integrates
only at specific attachment site on the 2 chromosomes which results in the lines
insertion of the phage chromosome into the bacterial chromosome.in this integrated state the
phage chromosome is called a prophage.the lytic genes of the phage are repressed in the
prophage state.the bacterium harbouring the prophage is called a Lysogen and this phage host
relationship is called Lysogeny.these lysogens are immune to second infection by the same virus.
These temperate phages can undergo transition from lysogenic and or prophage state to the lytic
state only when induced under specific condition eg. Irradiation with UV light.in this condition
the prophage is excised from the host(donor) chromosome and replicate autonomously.this
excision and integration process are catalyzed enzymes coded by phage genes(Eg int
gene for integration and “int” + “xis” gene for excision in λ phages).the
excision is also site specific.it is usually very precise in cutting out only the
phage chromosome.occasionally, the excision event occurs at a site other
that the original attachment site.in this case a portion of the phage chromosome is left in the
host chromosome and a portion of the bacterial chromosome is excised with the phage DNA.this
mistake in prophage excision leads to the formation of “specialized transducing phages”
carrying a segment of the donor DNA.in this case only genes located with
short distance on each side in the donor cell can be transferred to the
recipient cell by these phages.eg phage λ integrates between “gal” and
“bio” genes,thus it usually transuces “gal” and “bio” markers
In generalized transduction a segment of the recipient of the chromosome is replaced during
integration.whereas during specialized transduction,a segment of the donor DNA is just added to
the recipient chromosome during integration.thus it results in the production of Partially diploid
transductant

Production of “Hft” lysate

During λ phage infection,the λ chromosome integrates between the “gal” &
“bio” genes in the chromosome of E.coli cell(donor).when lytic cycle is
induced with UV light, consider a λ phage excision from prophage carrying a
“gal” gene.then the resulting specialized transducing phage is called “λ
dg”( λ defective “gal”).it is “defective” cos genes required for growth and
maturation under lytic conditions have been left in the donor cell.the “g”
indicates that the bacterial genes (donor) transferred is “gal” gene.when this
‘λ dg’ infects a “gal” recipient E.coli cell it integrates between the “gal” and
“bio” genes.when lytic cycle is induced by UV light it doesn’t lyse the cell
because it lacks the genes required for growth and maturation.thus this “λ
dg” phage can reproduce in the recipient cell only in the presence of a wild
type λ phage called helper phage.therefore a second infection of this gal+ lysogen with
the helper phage which integrates into the recipient cell at the normal
attachment site results in the production of double lysogen(has one λ +
prophage and one λ dg).the transductant is thus gal+-/gal- partial diploids.lytic
cycle induced in this λ dg- λ + double lysogen results in the production of
50% λ dg phages and 50% λ+ phages (both prophages are excised).such lysates are called
“Hft” lysates. This Hft” lysate increases the freq of transductant events+
Conjugation
In Conjugation DNA is transferred from a donor (male) to a recipient(female) cell through a
conjugation tube.the donor cells have specialized cell surface appendages called “F pili”.the
synthesis of F pili is controlled by genes located on small circular DNA called F factor or F
plasmid.the cells having F factor is called the F+ cell(donor) and the cells without F factor is
called the F – cell(recipient).
The F factor can undergo replication in autonomous state or in integrated state which is an
example for episomes.In the F + cell the F factor is in autonomous state and during conjugation
only the F factor is transferred from the donor to the recipient which results in the recipient cell
becoming F -
In the integrated state the F factor integrates into the host chromosome .this integration is site
specific which is mediated by “IS elements”the cells carrying on integrated F factor is called an
“H fr cell”(high frequency recombination).in this integrated state the F factor mediates transfer
of host chromosomal DNA from Hfr cell to a recipient F – cell,usually only a portion of the host
DNA is transferred.only in rare cases an entire Hfr chromosome is transferred to the recipient
cell.the mechanism of transfer in both F + chromosome and in Hfr cell is by “rolling circle
replication”.transfer is first initiated by an endonucleolytic break in one strand at a specific site
on the F factor.Then the5’ end of the nicked strand is transferred through the conjugation tube
into the recipient cell.this transfer is coupled with replication with the intact inner circular strand
serving as the template.since the origin of transfer lies within the F factor only a portion of the F
factor is transferred from a Hfr cell to a recipient cell,because the conjugation tube breaks and
the cells separate before complete transfer of an entire Hfrchromosome
GENE MAPPING (interrupted mating experiment)

The most famous mapping experiment in conjugation is interrupted mating experiment .In this
experiment a specific Hfr strain called Hfr H or hfrHayens is used.in this strain the F factor
integrates near “thr” threonine and “leu” leucine loci in the host chromosome.In this experiment
the HfrH cells of genotype thr +,leu +,azi-s,TI-s,lac +,gal +,str-s was mated with F – cells of
genotype thr -,leu -,azi-r,TI-r,lac -,gal -,str-r(alleles azi,TI and str control resistance(r) or
sensitivity(s) to sodium azide,bacteriophage TI and streptomycin.while alleles lac and gal control
the ability to utilize lactose and galactose)at varying time intervals after mixing Hfrat varying
time intervals after mixing Hfr and F-cells,samples were taken and agitated vigorously in a
blender to break the conjugation tube and separate the cells.the sparated cells were tk plated in
selective medium containing streptomycin but lacking threonine and leucine where only
recombinant cells can grow .the HfrH donor cells are killed by streptomycin(str-s) and F-
recipient can’t grow in the absence of threonine and leucine (thr- & leu-).the colonies produced
by thr+,leu+ & str-r recombinants in the selective medium were then “replica plated” in series of
different selective medium to determine the donor marker(DNA or genes) transferred to the
recipient.the series of different selective medium consists of medium containing (1)sodium azide
to score azin or azi-s ,(2)bacteriophage TI to score TI-s or TI-r,(3) lactose as sole carbon source
to score lac + or lac -,and galactose as sole carbon source to score gal + or gal – a

Result :- after 8 min of mixing HfrH and F – cells no thr +,leu +, str-r recombinants were
observed.recombinants first appeared at about 8.5 mins

When the presence of other markers were scored in the recombinants after replica plating it was
observed that the donor markers were transferred in a specific sequence.the “HfrH azi-s”
recombinant first appeared after 9 mins followed by ”TI-s lac +,gal +” markers at 11,18,25
minutes of mating(mixing of Hfrh and F -cell).this result indicates that HfrH genes are transferred
to F – cell in a specific linear sequence.

Subsequent studies with F factor which intergarate at many different sites in the host (E.coli)
chromosome also indicates the specific linear sequential transfer of DNA.the transfer of a
complete chromosme from an HfrH to a F – cell takes 90-100 minutes depending on the strain
used.the rxn proceeds at a constant rate and the time interval between the transfer of any 2
markers gives the estimate of the physical distance between the 2 markers on the chromosome.a
map distance of 1 min corresponds to the length of the segment of the chromosome transferred in
1 minute during conjugation.the standard E.coli linkage map is divided into minute intervals
from 0 to 100 min on the basis of interrupted mating experiment.