Lecture 6:

Bacterial Genetics II
I. Conjugation and genetic mapping in bacteria
II. Transformation
III. Transduction
Assigned Readings
3
rd
Ed. Chapter 15: pg. 539-548, 550-564
4
th
Ed. Chapter 14: pg. 477-482, 486-494

Assigned Problems:
3
rd
Ed. Chapter 15: 1-3, 5-6, 8, 11, 13-14, 16, 20
4
th
Ed. Chapter 14: 1-3, 5-6, 11, 16-19, 22

Adapted from F.A. Laski and J. Ribaya
High Resolution mapping in bacteria
Interrupted mating experiments give only crude
estimates of gene order and cannot distinguish the relative
position of genes within about 2 minutes of each other,
and thus give only a rough idea of gene location.

But like in eukaryotes, can use recombination
frequencies to determine gene order.
Review of Recombination in Bacteria
(F
-
cell bearing donor alleles
following conjugation)
Fine structure mapping utilizes co-integration
frequencies
Select for Leu
+
This type of analysis requires conditions under which every
marker has an equal chance to be recombined into the host
chromosome so that recombination frequencies are dependent
exclusively on the relative distances separating the marker genes.
ThereforeSince the gradient of transfer is biased toward those
markers close to the origin of transfer one must select for later
markers.
-Because of the linear transfer of genes during conjugationIf later
markers are selected then all the markers in front of it must have
entered the cell.
leu
+
arg
+
met
+

O
Hfr str
s
leu
+
met
+
arg
+
x
F
-
str
r
leu
-
met
-
arg
-
Select: MM with streptomycin
and Methione and Arginine
Order unselected genes relative to late gene
Replica plate and count numbers of colonies showing
recombination for unselected markers (arbitrarily pick 100
colonies to look at).
Leu
+
=100%
number of colonies
Met
+
Arg
+
68
Met
-
Arg
+
22
Met
+
Arg
-
0
Met
-
Arg
-
10
4 possibilities
(4 different replica plates)
Leu+ Met+ Arg+ 68
Leu+ Met- Arg+ 22
Leu+ Met+ Arg- 0
Leu+ Met- Arg- 10
Leu+, Arg-, Met-
2COs
Leu+, Arg+, Met-
2COs
Leu+, Arg+, Met+
2COs
Leu+, Arg-, Met+
4COs
Order? Map Distance?
4 COs
Raw Data
Exogenote
Order: The lowest phenotypic class is the one where a quadruple
crossover event is needed to generate the gene combination. In this case
arg differs and is therefore in the middle.

-We already know leu is last from interruption studies, therefore the
order of genes is
leu arg met
leu - met: 10 mu + 22 mu = 32 mu
Map distance:
leu - arg: (0 + 10)/100 = 0.10 = 10% Recombination = 10 mu
Leu+ Arg+ Met+ 68
Leu+ Arg+ Met- 22
Leu+ Arg- Met+ 0
Leu+ Arg- Met- 10
Order? Map Distance?
4 COs
100
arg - met: (22 + 0)/100 = 0.22 = 22% Recombination = 22 mu
O
Reordered
Data
Four Independent Hfr strains:
Strain 1: Z M U R B
Strain 2: W C N A L
Strain 3: A L B R U
Strain 4: M Z X W C
All these Hfr strains are derived from the same F
+
strain. (Each
different strain has the F factor inserted at different locations.)
M
Z
X
W
C
N
A
L
B
R
U
1
2
3
4
What is the order of these markers on the circular chromosome
of the original F+? (Include orientation of the F-factor for each
Hfr strain)
Hfr Interruption mapping
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.
-Some F strains can
carry large parts of the
bacterial chromosome
(up to 25%)

- Another transferable
plasmid found in
bacteria that is
responsible for the
spread of multiple-drug
resistance in probably
arose in this way
F elements
F elements are formed by faulty excision of Hfrs from
the bacterial chromosome
II. Transformation
Only competent cells (secrete
competent factors) are capable of
serving as recipients in transformation.
Transformation
The ssDNA taken up integrates into
the recipients chromosome
-If this DNA is of a different genotype
from the recipient, the genotype of the
recipient can become permanently
changed or transformed.
The uptake of free DNA molecules
-Discovered in 1928 by Frederick
Griffith (Streptococcus pneumoniae).
Later Avery, MacLeod, and MacCarty
identified the transforming factor
! DNA.
Competent cell
Mechanism of Transformation
Transformation and gene distance
The donor DNA molecules taken up by recipient competent cells
during transformation are small. (0.2 - 0.5 % of the complete
chromosome.)

-Therefore, unless two genes are close together, they will never be
present on the same molecule of transforming DNA.

-On the other hand, two genes close together on a chromosome will
be found on the same molecule of DNA and they will likely be
cotransformed.
b
-

b
+

b
-

b
-

b
+

Double Transformation: The transformation of two genes into a
single recipient cell.
Scenario 1 : Double transformation
for 2 genes a and b located far apart
on the chromosome

a
+
b
+
donor and a
-
b
-
recipient

a ! a
+
b ! b
+

will require two independent
transformation events.

-The probability of this occurring=
P(a
+
) x P(b
+
)
Transformation and gene distance
Scenario 2: Double transformation
for 2 genes a and b located near
one another on the chromosome

a
+
b
+
donor and a
-
b
-
recipient

a ! a
+
b ! b
+
will require just one single
transforming event.

-In this case double transformants
are formed at a high frequency.

b
+

b
+

b
-

b
-

b
-

Take home message: The frequency with with two genetic markers
(genes) are cotransformed can be used to estimate how far apart the
genes are on the chromosome.
Transformation and gene distance
III. Transduction
Bacteriophage
Greek: Eater of bacteriaparasitizes and kills bacteria
Bacteriophage "
(temperate phage)
Bacteriophage T4
(virulent phage)
Temperate phages can remain within the host cell for a period without
killing it. Their DNA either integrates into the host chromosome to
replicate with it or replicates like a plasmid, separately in the cytoplasm.
-Prophage: A phage integrated into the bacterial genome.
Virulent phages are those that immediately lyse and kill the host.
T4 Phage life cycle
Phage Plaques
After lysis, the progeny phages infect neighboring bacteria.
Repetition of this cycle and subsequent rounds of infection of
neighboring bacteria create a clear area or plaque in the opaque lawn
of bacteria.
Lawn of bacteria
Plaques
(clearing)
Transduction
Genetic material is transferred from one bacterium to
another by virus (phage).

There are two kinds of transduction:

General transduction:
Phage can transfer any segment of the bacterial genome to
another bacterium.

Specialized transduction:
Only specific segments of the bacterial genome are
transferred.
(phe
+
trp
+
tyr
+
met
-
his
-
) (phe
-
trp
-
tyr
-
met
+
his
+
)
(phe
+
trp
+
tyr
+
met
+
his
+
)
Discovery of Transduction
Lederberg and Zinder, 1951

-Used two auxotrophic strains to
study recombination in
Salmonella typhimurium
What method of recombination
does S. typhimurium use?

Transformation? Conjugation?
-Mixed the two strains together
and observed prototrophic
colonies ! recombination!

-Results appeared similar to that
for recombination in E.coli.
Pore size: Too small to allow bacteria to pass through but small
molecules (DNA, viruses, proteins, etc.) can pass through.
In other experiments one can change pore size to exclude smaller
molecules
(phe
+
trp
+
tyr
+
met
-
his
-
)
(phe
-
trp
-
tyr
-
met
+
his
+
)
U- tube experiment: Set up
(
p
h
e
+
t
r
p
+
t
y
r
+
m
e
t
-
h
i
s
-
)

(
p
h
e
-
t
r
p
-
t
y
r
-
m
e
t
+
h
i
s
+
)

U- tube experiment
1) Pore size (restricts bacteria):
Bacteria plated on MM !
growth
Recombination not due to
conjugation
They slightly increased the pore size to allow a known bacteriophage
(P22) to pass: Collected bacteria, plated on MM and observed
prototrophic colonies. (Control: Also treated medium with hydrolytic
enzymes that killed virus ! No growth on MM)
Bacteriophage P22 responsible for gene transfer
2) # Pore size (restricts virus
but DNA can pass): Bacteria
plated on MM ! no growth.
Recombination not due to
transformation
Instead of conjugation, they discovered phage-mediated
transfer of bacterial genes, or transduction.
Conclusion
Strain A: a
+
b
+
Strain B : a-
lysis
-Infrequently (1/10,000) phage accidentally incorporate bacterial chromosome
fragments carrying gene a
+
from donor strain. This DNA can be transferred to an a
-

recipient in the next round of infection.
Generalized Transduction: Faulty head stufng
Generalized Transduction: Any piece of DNA can be transferred
Generalized transduction provides a method for fine scale mapping
of genes.

- A head full of DNA is about 2 minutes of bacterial chromosome.
Linkage data from Transduction
- If two genes are close together, they have better chance to be
cotransduced by the same viral particle.
The closer two genetic markers are, the higher the cotransduction
frequency.
Linkage data from Transduction
A
B C
Markers
cotransduced
A
A
A + B
B
B + C
B + C
B + C
2 min DNA
Linkage mapping of bacterial genes by transduction
Experimental Outline:
Next, screen amp
+
colonies for the presence of lac
+
or man
+
:
Donor: amp
r
lac
+
man
+

Recipient: amp
s
lac
-
man
-
After transduction and selection by plating on MM with
ampicillin and variety of sugarsthe possible genotypes of the
recipient bacteria are:
1) amp
r
lac
-
man
-
2) amp
r
lac
+
man
-
3) amp
r
lac

man
+

4) amp
r
lac
+
man
+

Linkage mapping of bacterial genes by transduction
amp
r
colonies
(master plate)
replica plate
MM + mannose only
MM + lactose only
Assign genotypes to colonies on master plate by looking at replica
plates:
Linkage mapping of bacterial genes by transduction
MM
+
mannose only
(Select for man
+
)

MM
+
lactose only
(Select for lac
+
)
Master Plate
MM with
amp, variety
of sugars
amp
r
colonies
(master plate)
amp
r
man
-
lac
-
amp
r
man
-
lac
+
amp
r
man
+
lac
+
The higher the cotransduction frequency of those 2 genes ! The
smaller the map units between 2 genes
amp-lac > amp-man Cotransduction Frequency:
Here we are asking, of all the phage that carried amp
r
, what
percentage of them also carried man
+
?
x 100 %
Cotransduction
of amp
r
and man
+

Total colonies (amp
r

man
+
)
Total # colonies (amp
r
)

Cotransduction Frequency
amp
r
colonies
(master plate)
amp
r
man
-
lac
-
amp
r
man
-
lac
+
amp
r
man
+
lac
+
amp
r
man
+
= (1/7) x 100 = 14.3%
amp
r
lac
+
= (5/7) x 100 = 71.4%
Conclusion: amp is closer to lactose than to mannose.
You need more info to put them in some sort of order!
=
Donor bacteria:
leu
+
thr
+
arg
+
Recipient bacteria:
leu
-
thr
-
arg
-
phage infection
Transducing lysate
Centrifuge bacteria to bottom.
Phage stay in the lysate.
Using Transduction to Determine Gene Order
select transductants: (need all 3 in order to grow)
Lysate
Exp # 1: Minimal media with thr & arg

# leu
+
thr
+
= 2 %
# leu
+
arg
+
= 50 %
MM + arg
Donor: leu
+
thr
+
arg
+
Recipient: leu
-
thr
-
arg
-
or
leu arg
thr
arg leu thr
2 Possible
maps
What is the order of the leu, thr and arg genes?
Selecting for leu+
Replica plate
MM + thr
Screen
Final Map
arg leu thr
arg leu thr
leu arg
thr
or
From previous mapping, we predict 2 Possible maps
# thr
+
leu
+
= 2%
# thr
+
arg
+
= 0%
Replica plate
MM + arg MM + leu
Donor: leu
+
thr
+
arg
+
Recipient: leu
-
thr
-
arg
-
Exp # 2: MM + leu + arg

Selecting for thr+
Transduction experiment: Short Cut
Results after replica plating:
c
+
d
+
e
+
57
c
+
d
+
e
-
76
c
+
d
-
e
-
365
c
+
d
-
e
+
2
total 500
select for c+ screen for d+ and e+
replica plate
a) Determine co-transduction frequencies for c-d and c-e
b) Determine probable gene order
Donor = c+ d+ e+ Recipient = c- d- e-
Four classes of transductants:
c
+
d
+
76
c
+
e
+
2
total 78
Determining co-transduction frequencies
Donor = c+ d+ e+ Recipient = c- d- e-
c-d = c
+
d
+
/c
+
x 100 = 57 + 76/500
= 133/500 x 100 = 26.6%

c-e = c+ e+/c+ x 100 = 57 + 2/ 500
= 59/500 x 100 = 11.8%
Two possible maps
e c d
or
c d e
c+ d+ e+ 57
c+ d+ e- 76
c+ d- e- 365
c+ d- e+ 2
500
Donor = c+ d+ e+ Recipient = c- d- e-

Inferring gene order

Compare these classes.

Two possible maps
e c d
e+ c+ d+
P
4CO
c
+
d
+
e
+

c d e
Donor: Kan
R
lys+ arg+ X Recipient: Kan
S
lys- arg-
Cells are selected on a master plate with Kan Lysine and Arginine, then screened
using the following replica plates:
What genotypes will grow on each type of media?
What are the cotransduction frequencies of the three gene?
Cotrans freq Kan arg =
20
1
180
299
Master




Replica 1


Kan
r
arg+ lys+
Kan
r
arg+ lys-
Kan
r
arg- lys+
Kan
r
arg- lys-

Kan
r
arg+ lys+
Kan
r
arg+ lys+
Kan
r
arg+ lys-

Kan
r
arg+ lys+
Kan
r
arg- lys+
Replica 2


Replica 3

Calculating how many of each genotype
Co-transduction
kan-arg = (20+1)/500 x 100 = 4.2%
kan-lys = (20+180)/500 x 100 = 40%
kan lys arg or lys kan arg
X
During generalized transduction, all markers in the genome
are transferred at an equal rate, because the phage can package
any piece of DNA randomly.

Specialized transduction is characteristic of viruses that
transfer only certain genes between bacteria.
Specialized transduction
How do certain bacteriophages accomplish this?
Alternative phage life cycle- lysogeny
-Phage integrates into
host chromosome
-Phage will lay dormant,
until some signal, like
UV light, signals it to
enter lytic pathway
Specialized transduction: Phage "
-Example: Phage " (Temperate Bacteriophage)
-Specialized transducing phage
-Carries only the gal and bio genes
-Has a specific insertion site between the gal and bio genes
Specialized transducers insert into the bacterial chromosome at
one position only.
Occasional faulty excision leads to specialized
transducing phage
Just as in F plasmids, sometimes when the phage excises, it
accidentally carries along one of the flanking bacterial genes.
Normal excision Abnormal excision