MOLECULAR BIOLOGY

1. GENETIC
RECOMBINATION
IN
BACTERIA

2. INTRODUCTION:- Bacteria do not reproduce sexually like eukaryotic
organisms because their requirement of sexuality are met
through certain alternate pathway of genetic
recombination which are called
“CONJUGATION”,”TRANSFORMATION”&
“TRANSDUCTION”.
 These 3 processes mediate transfer of genetic material
(DNA) from one bacterial cell (Donor)to the other
(recipient)making possible the subsequent recombination

3. THE MOST OBVIOUS DIFFERENCE BETWEEN THESE 3 PROCESSES IS
THE MODE OF TRANSFER OF GENETIC MATERIAL (DNA) FROM DONOR
TO RECIPIENT CELL:-
 In “CONJUGATION” donor cell transfers its DNA to recipient cell
only when both the cells are in physical contact through a
specialized sex-pilus or conjugation tube.
 In “TRANSFORMATION” there is transfer to a cell-free or “naked”
DNA from donor cell to recipient cell.
 In “TRANSDUCTION” the transfer of genetic material (DNA) from
donor cell to recipient cell is mediated by a bacteriophage.

5. THE RECOMBINATION IN BACTERIA USUALLY TAKES PLACE
FORMING “PARTIAL NORMAL DIPLOIDS” ALSO CALLED
“HETEROZYGOTES” OR “MEROZYGOTES”.THUS, THE RECIPIENT
CELLS ARE CONVERTED TO HETEROZYGOTES OR MEROZYGOTES
CONTAIN FRAGMENTS OF DONOR DNA “THE EXOGENOTE” ANS A
COMPLETE RECIPIENT DNA “”THE ENDOGENOTE”.

6. 1}CONJUGATION
 Lederberg and tatum discovered conjugation in “E.coli” in the year 1946 and
detailed studies were made by Woolman and Jacobin 1956.
 “conjugation is a process by which genetic material is transferred from one
bacterial cell (Donor cell or male cell) to another (recipient cell or female cell)
through a specialized intercellular connection called “sex pilus or conjugation
tube”.
 The maleness and femaleness of bacterial cells are determined by the
presence or absence of “F-plasmid “(also called F-factor or sexfactor).

7.  F-plasmid an extrachromosomal genetic material is always present in the
cytoplasm of donor or male cells and the , latter develop specialized cell
surface appendages called “F-pilli or sex pilli “under the control of F-plasmid
recipient or female cells always lack F-plasmids and therefore , F-pilli are not
present on their surface
 F-plasmid or F-factor can exist in 2 different states:-

8. AUTONOMOUS STATE INTEGRATED STATE
• It lies free in the cytoplasm and this state is integrated into the
replicate independent of the bacterial bacterial chromosome(DNA) and replicate alongwith it.
Chromosome (DNA)
A donor or male cell containing F-factor in autonomous a donor or male cell containing F-factor in
integrated
state is called “F+cell” state is called”Hfr cells” or high frequency male
cells.
however, the recipient or female cell lacks F-factor and this is called “F – cells “.

10. 1}CONJUGATION BETWEEN A F+(DONOR)CELL AND A F-
(RECIPIENT) CELL:-
 In conjugation between a F+(donor) cell and F-(recipient) cell, it autonomous F- factor (F-
plasmid)which is transferred never the bacterial DNA.
 When the 2 cells (F+&f-) come close to each other , the F- pilus of the F+(Donor) cell attaches
with the F- (recipient)cell and acts as a conjugation tube.
 Simultaneously , the double –stranded circular F-factor DNA is nicked at a specific point and
begins to replicate producing a single –stranded copy of the F-factor DNA which migrates
through the tube into the cytoplasm of the F-(recipient )cell.
 It becomes double stranded and circulars and lies free in the cytoplasm thus , rendering the
recipient cell to become F+ donor cell.
 In this way , mixing a population of F+ (donor)cells with a population of F+ (recipient )cells
results in the conversion of virtually all the cells in the population becoming F+ (donor) cells.

12. CONJUGATION BETWEEN HFR DONOR CELLS AND RECIPIENT
(F-) CELL:-
 The Hfr donor cells are considered to be fertile because unlike F+(donor) cells , their chromosomal
segments are transferred from donor to recipient cells and the F- factor remains in sites.
 When 2 cells (Hfr and F-) come in contact , a conjugation tube develops between them .
 The circular DNA of Hfr donor cell is nicked and replication is initiated,
 The integrate F-factor always lies at the rear end of the DNA molecule.
 The replication of DNA starts towards the end near the conjugation tube & newly synthesized single
strand starts migrating through the tube into the recipient (F-) cell.
 In nature the mating of 2 cells exists for a short period and gets interrupted resulting in the migration of
only a portion of the donor DNA into the recipient cell.
 The genes of the newly entered DNA fragment may replace the homologous genes of the DNA of the
43recipient cell , resulting in a recombination genetic material .
 The newly formed recombinant genetic material now possess those male charactters that hav ebeen
transferred through recombination with the migrated DNA fragment .

14. CONJUGATION BETWEEN F’(F PRIME) MALE AND F-
(RECIPIENT)CELL(SEX-DUCTION):-
 Existence of Hfr donor cells is not absolute and the F-factor integrated into the
bacterial DNA of Hfr donor cells may dissociate and become free in the cytoplasm .
 The dissociation may be occasionally anomalous during which the dissociated F-
factor may bring with it some genes of the bacterial chromosomes.’
 When F-prime male conjugates with F- (recipient cell) the F-factor is transferred from
donor to the recipient cell and such a recipient bacterial cell becomes heterozygous
for the part of the bacterial chromosome which the F-prime factor had obtained
during its anomalous dissociation.
 Transfer of F-prime factor to recipient cell apparently occurs by the same
mechanisms as F- factor transfers in Hfr and F- cell mating .
 Genetic recombination of this type mediated by F PRIME FACTOR is called “SEX-
DUCTION OR F0-DUCTION”.

16. 2}TRANSFORMATION
 Transformation is a process of genetic recombination was first studied
by “GRIFFITH” in 1928 (an English bacteriologist ).
 He took 2 strains of the bacterium Streptococcus pneumoniae.
 One of the 2 strains was virulent or pathogenic and capsulated normal
which formed smooth colonies on culture medium .
 and
 The other strain was non-pathogenic or avirulent and non-capsulated
which formed rough colonies on culture medium.

17. HE EXPERIMENTED ON MICE AS SUMMARISED BELOW:-

18.  It is obvious from Griffiths experiment that the avirulent or non-
pathogenic strain becomes virulent or pathogenic when mixed with
heat –killed (dead) virulent strain ,thus , causing the death of mice.
 Griffith named this change of avirulent into virulent strains as
“Transformation”.
 Griffith reasoned that there was a transfer of some factor from heat-
killed (dead) virulent strain to the virulent strain and called it
“TRANSFORMING PRINCIPLE”.
 He said that the transforming principle was the polysaccharide of
capsule of heat –killed virulent strains.

19.  The idea of polysaccharide as transforming principle came to an end in 1944
when AVERY MACLOED and MCCARTY showed that it is the DNA which
works as “TRANSFORMING PRINCIPLE “ not the polysaccharide capsule
.They proved for the first time that DNA is the genetic material in organisms.
 In transformation a free naked DNA molecule is transferred from a donor to
recipient bacterial cell.
 The donor bacterium undergoes lysis to free the DNA molecule and the
recipient bacterium must be component to receive it .
 The competence of bacterial cell is not a permanent feature it has been
demonstrated in relatively few bacterial genera and depends upon the growth
of bacteria and the environmental conditions.

20.  When a donor DNA comes into contact with the competent
bacterial cell , it first binds on the cell surface and then is taken up
inside the cell.
 In some cases it is observed that the double strand DNA enters
inside the bacterial cell as such and its one strand is degraded by
“ENDONUCLEASE” enzyme there in leaving single – strand DNA
 whereas
 In others species of Bacillus and Streptococcus it appears that
only single strand DNA enters the recipient bacterial cell.

21.  An endonuclease enzyme degrades one of the strands of
double stranded DNA of recipient bacterial chromosome in
corresponding region and this gap is filled by the donor single
stranded DNA with the help of ligase enzyme which joins it
with the DNA of the recipient bacterial chromosome.
 If allelic forms of donor and recipient genes are not identical
the donor DNA forms a “HETERODUPLEX” with the recipient
bacterial DNA.
 When the bacterial cell containing “HETERODUPLEX” it
undergoes binary fission the hetroduplex replicates forming 2
“HOMODUPLEXES”.

22.  One of these is a normal duplex which is all recipient in origin
and the daughter cell containing it is like the recipient bacterial
cell .
and
The other homoduplex is a transformed duplex (hybrid genome)
different from that of either the donor or the recipient bacterial
genome .
 The daughter cell containing transformed duplex is
“TRANSFORMED CELL” and contains some of the
characteristics of the donor bacterial cell which are inherited
progeny to progeny .

24. 3}TRANSDUCTION:-
 This process of genetic recombination was discovered by “ZINDERAND
LEDERBERG”(1952) in Salmonella typhimurium during their experiments with
the objective of discovering whether E.coli type of genetic exchange also
existed in Salmonella typhimurium.
 In transformation where in DNA (naked) is transferred or fragments of DNA
are transferred from one bacterial cell to the other
but
During transduction phage mediated process of genetic material transfer in
bacteria.

25.  The bacteriophage acquires a portion of the bacterial DNA of the
bacterial DNA the host cell in which it reproduces and then ,
transfers this acquired DNA to another bacterial cell to which it
infects such bacteriophage is called “TRANSDUCING
BACTERIOPHAGE”.
 Transduction is of 2 types:-
i)Generalized transduction {non-specialised}
ii)Specialized transduction {restricted }

26. A}GENERALIZED TRANSDUCTION :-
 Transduction which results in transfer of any bacterial gene from one bacterial cell to the other is
reffered to as “GENERALIZED OR NON-SPECIALIZED TRANSDUCTION”.
 It is mediated by some virulent phages and certain temperate phages like E.coli phage P1 , Salmonella
phage P22 and Bacillus subtilis phages PBS1 and SP10 are such phages.
 In generalized transduction some of the developing progeny phages during their normal lytic cycle may
accidentally acquire pieces of bacterial DNA.
 Such phages after the lysis of the host bacterial cell and their release attach to and inject their DNA into
a new recipient cell but fail to re-establish lytic cycle.
 Once inside the recipient bacterial cell the injected DNA may be degraded by NUCLEASES in which
genetic exchange does not occur.
 The injected DNA may undergo integration resulting in homologous recombination.
 As a result , the transduced cell may possess new combination of genes .Now , the transduced bacterial
cell now undergoes,usually binary fission and produces progeny cells containing new combination of
genes.

28. B}SPECIALIZED TRANSDUCTION:-
 Specialized transduction which leads to the transfer of only
specific or restricted genes from donor to recipient cell.
 It is mediated by temperate bacteriophage {Lambda phage, mu
phage and Ø 80 phage} that usually incorporate (integrate)their
DNA into the bacterial chromosome.
 The phage DNA is called ”PROPHAGE” in its integrated state with
the bacterial chromosome , the bacterium having a prophage is
said to be “lysogenic “ and the phage –host –relationship is called
“LYSOGENY”.

29.  During transition the prophage is usually excised precisely from the specific
site of integration in its exactly originally form
 but
 It may excise imprecisely so that it takes specific portion of bacterial
chromosome which lies close to the site of prophage insertion and leaves a
portion of its own DNA remaining integrated within the bacterial chromosome .
such prophage is called “SPECIALIZED TRANSDUCING PRINCIPLE” and is
packed into developing phage particle inside the host bacterial cell.
 Phage particle so developed is called “specialized transducing phage”and is
released after the host bacterial cell undergoes lysis.

30.  When a viable specialized transducing phage infects a new bacterial cell its specialized transducing
principle that already contains specific portion of bacterial chromosome inserts into the recipient
bacterial chromosome.
 Since , the specialized transducing phage is “DEFECTIVE” phage as it has lost some genes during
excision and its functions in recipient bacterial cell only when the latter is already infected by another
phage ,i,.e , it contains missing genes and there by , complements the lost phage functions of
specialized transducing phage.
 The partial diploid contains 2 copies of the concerned genes, one from donor bacterium and other
recipient bacterium and is unstable.
 AS a result bacterial cells containing gene of donor bacterium and these containing gene of recipient
bacterium segregate at a frequency of about one in 1000 cell division.

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