Overview of Conjugation
F+ x F-
F+, F+ products
Generation and
transfer of Hfr
chromosome
Insertion of the F factor
into the E. coli chromosome
• Multiple positions
• Two orientations
The order of gene
transfer depends the
position and the
orientation of the
inserted F facter in
the Hfr chromosome
Transfer of the integrated F factor
results in transfer of genomic markers
Hfr a+ x F- a-
Hfr a+ + F- a+
Two types of conjugation.
F+ x F- crosses
107 F+ cells x 107 F- cells
104 different Hfrs
All markers transferred equally at
about 1/107 frequency
Hfr x F- crosses
107 Hfr cells x 107 F- cells
all 107 same Hfr
Markers transferred in specific order
at about 1/104-1/103 frequency
Hfr Strain1 = MZXWC
2 = LANCW
3 = ALBRU
4 = ZMURB
MZXWC
WCNAL ALBRU
BRUMZ M
Z
U
R
X
B
W
L
C
A
N
The Genetic Map of Ecoli
minutes
How to do fine scale mapping.
Interrupted mating experiments cannot distinguish
the relative position of genes within about 2
minutes of each other, and thus give only a rough
idea of gene location.
Do 1. Three point crosses.
2. Reciprocal crosses.
Both use recombination frequencies to determine
gene order.
Lets look more closely at what happens in the Fcell (the exconjugant). Recombination (crossover) in F- cell.
incomplete genome from the donor
Hfr chromosome.
complete genome from F- cells
Interrupted mating says:
mal
arg/met
10 minutes 6 minutes
Use a 3 point cross to figure out order of arg and met.
Hfr strsarg+met+mal+ X F- strRarg-met-mal-
How to do the
experiment????
StrS
(book says mal+,met+)
StrR
1. Select for
mal+, the last
marker, ensures
that all genes
transfer. That
way you only
look at
recombination.
2. Replica plate
onto both
arginine or
methionine
minus plates..
metarg
mal
Crossovers?
2
Most frequent
2
Pretty frequent
2
Less frequent, smaller interval
least frequent
4
Reciprocal Crosses
Another way for ordering markers that are
close to one another, also takes advantage of
the lower frequency of quadruple crossovers.
a
bc
?
a
cb
?
or
Cross I
Hfr strs a+ b+ c- x F- strr a- b- c+
select strr a+ b+ c+
Cross II
Hfr strs a+ b- c+ x F- strr a- b+ c-
select strr a+ b+ c+
• If the order is abc, then will get many
more a+b+c+ exconjugants from cross I than
cross II.
• If the order is acb, then will get many
more a+b+c+ exconjugants from cross II
than cross I.
Cross 2
Cross 1
a+
b+ c-
a+
b- c+
a-
b+ c-
a+
c+ b-
Hfr
Hfr
If abc
a-
b- c+
a+
c- b+
F-
Hfr
Hfr
If acb
a-
c+ b-
F-
Therefore If abc cross I > cross II,
F-
a-
c- b+
If acb cross II > cross I.
F-
F’ elements (F prime elements)
•  F factors which carry a part of the
chromosome.
•  Allows very high frequency transfer of F’
markers.
•  Allows one to make partial diploids.
•  Can use for complementation tests.
Bacteria tend to cluster genes
of similar function.
F’ plasmids have
every thing they need to transfer.
Bacterial chromosome
Bacterial chromosome
F+
F’
metA
metB
metC
metA
metB
metC
Hfr
F’
Bacterial chromosome
Bacterial chromosome
F’
F’ male
metA
metB
metC
X
F- female
metA
metB
metC
Bacterial chromosome
F’
metA
metB
metC
F’ partially diploid male
metA
metB
metC
Complementation test, an example.
Bacterial chromosome
F’
metA-
metB+
metC+
F’ met- m1
male
Your met- mutant
Bacterial chromosome
X
metA
metB
met??-
metC
F- met- m2
female
Your friends met- mutant
F’
metA-
metB+
metC+
Bacterial chromosome
metA
metB
met??-
metC
F’ partial diploid Test for complementation by growing on met- plates.
If it grows on met- plates =
If no growth =
Different genes = complement.
Same gene = fail to complement = metA-.
Lederberg and Zinder, 1951
•  Question: Is there conjugation in Salmonella
(closely related bacteria)?
–  phe+trp+met-his- x phe-trp-met+his+
–  Found prototrophs
•  Experiment: U-tube to separate two
auxotrophic strains, then check for growth on
minimal media
U- tube experiment
(phe+trp+met-his-)
minimal media
no prototrophs
(phe-trp-met+his+)
<< the size of bacteria
~ the size of P22 Phage
minimal media
Prototrophs!!
Bacterial
Phage
Conclusion: Instead of
conjugation, they
discovered phagemediated transfer of
bacterial genes, or
TRANSDUCTION.