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The basis of diversity in bacteria
Recombination
DNA replication
Mutation
Repair
Homologous
Non-homologous
Rearrangements/transpositions
Deletions
Plasmid
conjugation
DNA
transformation
Phage
transduction
Transformation
DNA extracted
from trp+ strain
Plating on
selective
medium
(absence of Trp)
trp-
strain
Transformed
cells
Auxotrophy = inability of cells to grow unless the medium is
supplemented with a particular substance
Prototrophy = ability of cells to grow when a particular
substance is absent from the medium
The average fragment size of DNA is 32 Kb
and transforming activity decreases with size
The minimum required is 0,5 Kb.
Synthesis of competence factor.
DNA associates with the membrane. Both Gram-positive and Gramnegative bacteria exhibit DNA binding, respectively.
Double stranded breaks have been introduced into the entering DNA by
the endogenous membrane-bound endonuclease. In addition DNA
duplex is converted to single stranded DNA which recombines with
chromosome. The region of mismatch is repaired by the DNA repair
enzymes. In the example, lac- cells are transformed in lac+ cells.
In most system
saturation is achieved by
adding 1mg to 10 mg of
DNA to 1 ml of culture.
Transformation is a rare event in nature
Competence, defined as the ability of a bacterium to take up DNA from
the medium, can be naturally or artificially induced (divalent cation
method for E. coli and related gram-negative bacteria, wall-degrading
enzymes plus PEG for gram-positive bacteria, electroporation for
eukaryotes and prokaryotes).
Cells
(N°/ml)
Bacillus subtilis
2
1
Frequency of
transformation
(%)
0
time
Natural competence  B. subtilis, S. pneumoniae, H. influenzae, N. gonorrhoeae
Mediante trasformazione è possibile determinare la
concatenazione di geni, la distanza e il loro ordine
La probabilità di co-trasformazione è data dal prodotto
delle probabilità di trasformazione dei singoli geni.
Se due geni sono vicini è
probabile trovarli sullo
stesso frammento di DNA.
La frequenza di cotrasformazione è
inversamente proporzionale
alla distanza tra i due geni.
Quando 2 geni sono molto vicini,
la loro frequenza di cotrasformazione è simile alla
frequenza di trasformazione di
ciascun gene
Esempio:
p+ q o
= 2.5x10-3
p q+ o
= 2.8x10-3
p q o+
= 2.6x10-3
p+ q o +
= 1.5x10-7
p+ q+ o
= 1.5x10-3
p q+ o+
= 5x10-6
p
•
q
•
o
•
Conjugation (the F factor)
A)
Lederberg and Tatum demonstrated bacterial recombination (1946)
This experiment was designed to determine whether mutation or
recombination explained the appearance of new bacterial strains.
B)
Davis’s U-tube experiments (1950) The pores in the filter were too small for
bacteria to pass through but large enough to allow DNA molecules to pass freely.
Lederberg
experiment
Davis
experiment
Conclusions: bacterial cell contact (coniugation) is required for DNA
transfer and transformation could not explain recombination in E.coli.
thr, treonina; bio, biotina; leu, leucina; thi, tiamina; met, metionina
Il trasferimento di DNA da
un una cellula batterica ad
un altra avviene in una sola
direzione.
F+  FHayes, nel 1953 propose
l’esistenza di un fattore di
fertilità (Fattore F).
Il fattore F contiene i geni
per la sintesi dei pili F o
pili sessuali.
Un ponte citoplasmatico
mette in contatto la cellula
donatrice con quella
ricevente.
La cellula F- è priva del
fattore F.
Coniugazione in E. coli
Nell’ incrocio F+ x F- la cellula
F- diventa F+.
Cellula FCellula F+
1) An F+ cell may conjugate with an 2) Hfr (high frequency recombination)
F- cell and transfer the F factor to the
Luca Cavalli
F cell
Sforza discovered
Hfr strains
The F plasmid
is an episome
1) Recombination fr = 10-6
2) Recombination fr = 10-3
Time-of-entry gene mapping (interrupted mating)
Wollman & Jacob started the experiment with two E. coli strains
(1956):
Hfr strain (donor) genotype
thr+ : Can synthesize threonine
leu+ : Can synthesize leucine
aziR : Resistant to azide
tonR : Resistant to the infection by T1 phage
lac+ : Can metabolize lactose
gal+ : Can metabolize galactose
strS : Killed by streptomycin
F– strain (recipient) genotype
thr– leu– aziS tonS lac– gal – strr
thr, leu and str were genes allowing selection of recombinants
while azi, ton, lac and gal were genes to be mapped
Recombinant cells were thr+, leu+ and strr.
Recombinant colonies could be selected by culturing on medium containing
streptomycin blocking the growth of Hfr strain and lacked threonine and
leucine blocking the growth of F-strain.
The researchers knew that the thr and leu genes were close to origin and that str
gene was farther from the origin than the 4 genes to map.
Interpreting the Data
Minutes that
Bacterial
Cells were
Allowed to
Mate Before
Blender
Treatment
There were no surviving colonies
after 5 minutes of mating
Percent of Surviving Bacterial Colonies
with the Following Genotypes
thr+ leu+
aziR
tonR
lac+
gal+
5
––
––
––
––
––
10
100
12
3
0
0
15
100
70
31
0
0
20
100
88
71
12
0
25
100
92
80
28
0.6
30
100
90
75
36
5
40
100
90
75
38
20
50
100
91
78
42
27
60
100
91
78
42
27
1) After 10 minutes,
the thr+ leu+ genotype
was obtained
4) The lac+ gene
enters between 15 &
20 minutes
2) The aziR gene
is transferred
first
3) It is followed
by the tonR gene
5) The gal+ gene
enters between 20
& 25 minutes
The map of the four genes of interest (minutes)
Complete transfer of the entire chromosomal DNA
from Hfr cells requires 100 minutes in E. coli
The F-factor does not have a
predetermined site on the
chromosome for integration
The F-factor orientation and map
location of 25 Hfr strains of E. coli
Detailed E. coli maps have been constructed using different F- and Hfr
strains by means of time-of-entry mapping
F- x Hfr
Arbitrarily assigned the starting point
minutes
Combining the results of mating experiments with different Hfr
strains Campbell proposed that the chromosome of E. coli
was circular.
The F plasmid is a large circular DNA, ~100 kb in length
A large (~33 kb)
region of the F
plasmid, called the
transfer region (~40
genes), is required
for conjugation.
1) traJ is a positive regulator that turns on
both traM and the traY-I operon. On the
opposite strand, finP is a regulator that codes
for a small antisense RNA that turns off traJ.
2) The genes traS and traT code for
"surface exclusion" proteins.
3) The gene traA codes for the single
subunit protein, pilin, that is polymerized
into the pilus.
4) Transfer of the F factor is initiated at a site
oriT while in its free form F utilizes its own
replication origin oriV and is maintained at a
level of one copy per bacterial chromosome.
5) There must be a channel through
which DNA is transferred, but the pilus
itself does not appear to provide it.
TraD is an inner membrane protein in
F+ bacteria that is necessary for
transport of DNA and it may provide
or be part of the channel.
F’ factors (sexduction)
F’ factor is formed when different IS
are used in excision and integration
process.
Once an F’ factor enters a recipient cell,
the recipient cell has 2 copy of the piece
of chromosomal DNA introduced on the
F’ factor - merozygote or partial
diploid.
Complementation Analysis
Experiments of Jacob and Monod using F’lac (1950-’60)
Complementation tests are valuable in determining whether
or not two mutations belong to the same gene.
Transduction
Virulent phage enters the lytic
cycle soon after infection.
Temperate phage may enter the
lytic cycle soon after infection or it
may enter the lysogenic cycle,
where it integrates (prophage) into
the host cell chromosomal DNA
and replicates as part of the
chromosomal DNA.
Plaques of T2
The discovery of transduction (Zinder and Lederberg, 1952)
Salmonella typhimurium
LA2 strain (phe+, trp+, met-,
his-)
Salmonella typhimurium
LA22 strain (phe-, trp-, met+,
his+)
Presumably a substance passed across the filter and transmitted DNA (FA).
FA (filterable agent) was not affected by DNase treatment neither
transformation or conjugation could occur. A series of additional
experiments, showed a consistent association between P22 and the
observation of prototrophy, leading Zinder and Lederberg to conclude that
the FA was the P22 phage. Salmonella LA22 strain was lysogenic for P22
phage while LA2 was lytic.
Specialized transduction  phage particles contain mostly phage
DNA but carry a specific fragment of chromosomal DNA ()
Generalized transduction  phages particles contain DNA from any
part of bacterial chromosome (P22, P1)
Generalized
Transduction
Specialized Transduction
To enter the lysogenic condition, free lambda DNA must be integrated into the
host DNA. To be released from lysogeny into the lytic cycle, prophage DNA
must be excised from the chromosome.
attachment
sites
Integration occurs through the attachment
sites.
The sequence O (core) is common to attB
and attP. The flanking regions B, B’ and P, P’
are referred to as the arms; each is distinct in
sequence. The products of the recombination
(prophage) are called attL and attR.
The reaction proceeds through a Holliday
junction. Exchanges of single strands take
place sequentially. The Integrase protein
(Int), in the presence of IHF (Host
Integration Factor), can resolve Holliday
junctions, and is responsible for the cutting
and ligation reactions.
Together, Int, Xis, and IHF cover virtually all of attP
23 bp
The function of attP requires a
stretch of 240 bp, but the function
of attB can be exercised by the
23 bp fragment extending from
– 11 to +11, in which there are
only 4 bp on either side of the
core.
The disparity in their sizes
suggests that attP is providing
additional information necessary
to distinguish it from attB.
Int and IHF bind cooperatively to attP, and their affinity for the site is
enhanced by supercoiling. The high stoichiometry suggests that the proteins do
not function catalytically, but form a structure that supports only a single
recombination event.
When Int and IHF bind to attP, they
generate a complex (Intasome) in
which all the binding sites are pulled
together on the surface of a protein.
Supercoiling of attP is needed for the
formation of this intasome.
Int does not bind directly to attB in
the form of free DNA. The intasome
is the intermediate that "captures"
attB.
Int can form a similar complex
with attR only if Xis is added. Such
complex results in the excision
process.
Formation of dbio and dgal transducing phages
Fr = 10-6
Fr = 10-6
Cell lysis
LFT (Low Frequency of Transduction)
This event
occurs at low
frequency
producing
lambda
defective
phages
LFT is used to infect a lysogenic E.coli strain (gal-)
In addition the gal or bio genes in
phage may recombine with its
homologous counterpart in the
chromosome producing a gal+ or
bio+ strains.
HFT
(High Frequency of Transduction by U.V. induction)