Microbiology
BIOL 275
BACTERIAL CONJUGATION
I. OBJECTIVES
•
To demonstrate the technical procedure to monitor the conjugational transfer of
genetic material from one cell to another.
•
To learn about the various genetic elements involved in bacterial sex and
recombination events.
•
To recognize and discuss the advantages and limitations of the conjugational
procedure in genetic recombination experiments.
II. INTRODUCTION
There are three different mechanisms for genetic exchange between bacteria:
conjugation, transformation and transduction. Conjugation has been shown to occur in
many bacterial species especially the Gram negative organisms. In Enterobacteriaceae
(Escherichia, Salmonella, Shigella, Vibrio, etc.), the transfer has been shown to cross
the taxonomic lines with varying rates of efficiency. Thus, conjugation has become a tool
to study the location and function of many genes, including genes responsible for
virulence, antibiotic resistance, metabolic functions, regulatory proteins, phage
attachment sites, etc.
E. coli has been the model organism in conjugation studies. It may become
infected with an extrachromosomal genetic element (a plasmid called the F factor) which
mediates certain events such as the synthesis of a pilus (the F or sex pilus), and the
rapid infection of all susceptible bacteria with this F factor. The pilus has been shown to
facilitate attachment of the male (or F+ cells) to the female (or F- cells) which do not
contain the F factor. It was previously thought that the hollow center of the pilus
constituted a passageway for the F factor to be transferred to the female. However,
recent evidence suggests that the pilus may be used as a "hook" to bring the two cells
close together and that the DNA transfer actually occurs outside the pilus.
The genetic transfer is mediated by the F factor itself, and requires that the F
factor be semi-conservatively replicated. One of these strands passes into the female
and the female now assumes the characteristics of a male cell. Note that all these
functions are caused by genes present on the F plasmid.
Scientists found another kind of male bacteria called the Hfr strain in which the F
factor integrates into the chromosome of its host. When an Hfr mates with an F- cell, the
F factor genes mediate the transfer of the genetic material into the F- cell. Such a
conjugational event yields a recombinant F- cell because the integrated F factor is rarely
completely transferred. Hfr stands for "high frequency of recombination". The F factor
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Dr. Eby Bassiri
ebassiri@sas.upenn.edu
Microbiology
BIOL 275
appears permanently "anchored" within a specific site in the bacterial genome and is not
excised. These strains may be used for genetic studies to map the location of genes.
A bacterial chromosome is considerably longer than an F factor, thus more time
is required to replicate and transfer the complete length of the chromosome. Transfer
commences from a point within the integrated F factor and proceeds in a linear fashion.
The F factor is thus split in this process with some of the F associated genes being
transferred first and the rest at the very end of the conjugation process. Since the
conjugational juncture is relatively fragile, the conjugation may be mechanically
interrupted at different times by vigorous agitation of the mixture (male and female).
Under optimal conditions, the entire E. coli chromosome is transferred in 100 min.
The recombinants formed as a result of a mating experiment are observed by
plating the mating mixture on a medium that selects for the growth of only the
recombinants. For example, an Hfr (Leu+) is mated with and F- (Leu-) and recombinants
are selected by plating the cells on a medium that lacks leucine. The F- parent cannot
grow on this medium, but the Hfr parent and the recombinants can. We must then
arrange for some mechanism to prevent the growth of the Hfr to be able to easily
recognize the recombinants. The inhibition of the Hfr may be accomplished by a variety
of techniques such as:
•
•
•
•
Utilizing an Hfr that has a metabolic requirement and omit that essential nutrient from
the selection medium.
Lyse the donor (Hfr) cells with a virulent male specific phage.
Include antibiotics to which males are susceptible in the medium.
Incubate the selection medium at a temperature that is antagonistic to growth of the
donor (Hfr) cells.
Naturally all these methods require that the counterselection affect only the donor
and that the recipient be resistant (to the antibiotic or phage), prototrophic (for the
particular metabolic lesion), or temperature insensitive.
In the following experiment, you will receive an Hfr which is prototrophic for all
genes (i.e., Hfr is wild type) and an F- that is auxotrophic for proline, lactose, tryptophan
and histidine. (Note: This is the same E. coli strain used in the transduction exercise). In
addition, the male is sensitive to, and the female is resistant to streptomycin. So our
cross would be: F- (pro- lac- trp- his- strR) x (pro+ lac+ trp+ his+ strS). We shall include
streptomycin in our selection medium to counterselect against the Hfr, and the omission
of an essential nutrient will select against the female. In theory, only the recombinants
should grow.
III. LABORATORY SUPPLIES
F- culture (NR57.10), 2 ml
Hfr culture (NR57.8), 1 ml
Sterile dropper
Sterile empty test tube (largest size)
Sterile empty test tubes (small size)
1 tube/group
1 tube/group
2/group
10/group
7/group
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Dr. Eby Bassiri
ebassiri@sas.upenn.edu
Microbiology
BIOL 275
1.0, 2.0 and 10 ml pipets
Nutrient agar plate
MS + Strep plate
MacConkey + Strep plate
Pro- + Strep plate
Trp- + Strep plate
His- + Strep plate
MS broth
MS + Strep broth
Glass rod spreader and alcohol beaker
37°C water bath
Vortexer
as needed
6/group
1/group
1/group
4/group
4/group
7/group
100 ml/group
50 ml/group
1/group
2/lab
4/lab
IV. PROCEDURE
Note: This experiment will be performed by students at a table forming a group. Try to
keep all cultures and plates at 37°C as much as possible and use sterile techniques in
all your work.
Observation of Recombinants
1.
Obtain the Hfr and F- cultures, one MS + Strep (Mineral salts + streptomycin) and
one MAC + streptomycin plate. Label plates with your name and medium type.
2.
With a sterile dropper, place a small drop of F- culture on the left and middle of
each plate (See figure). Do not move the plates. Use a new sterile dropper to
place a small drop of Hfr culture on the right and middle of each plate. Make sure
that the F- and Hfr drops at the center of the plate mix together. The center of the
plate is the place where conjugation between F- and Hfr cells should take place.
All other drops serve as controls for natural reversions of F- cells to prototrophy
and Hfr to antibiotic resistance.
3.
Return the cultures to the 37°C water bath. Allow the drops to dry before
transferring the plates to 37°C for two days.
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Dr. Eby Bassiri
ebassiri@sas.upenn.edu
Microbiology
BIOL 275
Finding the Order of Genes
1.
Obtain a small sterile test tube and mix 0.9 ml of F- with 0.1 ml of Hfr. This is your
conjugation mixture. Note the exact time (i.e. time zero), mix gently by tapping the
tube and incubate in the water bath at 37°C. This tube should be kept at 37°C at all
times except when taking samples. You should be very quick in taking your
samples not to decrease the temperature of the mixture below 37°C.
2.
Quickly take 0.1 ml of the conjugation mixture and add to 0.9 ml of MS + Strep broth.
Vortex the tube for 90 seconds at top speed and then transfer 0.1 of this solution to 9.9
ml of MS + Strep and vortex at medium speed for 30 seconds. Place 0.1 ml of this
dilution on each of Pro-, Trp- and His- plates. Spread with alcohol flamed glass rod.
Label plates with zero time and type of plate as well as your initials.
3.
At exactly 20 min into the experiment, repeat the above step.
4.
At exactly 40 and 60 min into the experiment, take 0.1 ml of the conjugation mixture
and add 0.9 ml of MS + Strep. Vortex 90 seconds at top speed. Take 0.1 ml of this
mixture and add 0.9 ml of MS + Strep. Vortex for another 30 seconds. Place 0.1 ml
of the last dilution on the 3 different plate types, spread and label as before.
5.
Incubate the plates at 37°C for 2 days. Your T.A. will remove the plates to a
refrigerator.
Enumeration of Parental and Recombinant Cells
1.
Use your knowledge of dilution series to prepare 10-6, 10-7 and 10-8 dilutions of the
Hfr and F- cultures separately by using MS broth.
2.
Plate 0.1 ml of each dilution on a nutrient agar plate and spread the inoculum with a
sterile glass rod. Work from the lowest dilution upward so that you only use a single
pipet. Spreading should be done very rapidly not to give the inoculum any chance of
soaking in. Let the inocula dry completely after spreading before inverting the plates.
3.
Label 3 His- plates for enumeration of recombinants as 10-1, 10-2 and 10-3. About
60 minutes into the experiment, transfer 0.1 ml of the mixture from the
conjugation tube used for finding the order of genes onto the 10-1 His- plate and
use a glass rod to spread it. Dilute the mixture 10-1 and 10-2 times with MS +
Strep and plate onto the remaining His- plates. When the inocula have dried,
label plates, invert them and incubate at 37°C for 2 days.
________________________________________________________________
Use of any section of this Lab Manual without the written consent of Dr. Eby Bassiri, Dept. of
Biology, University of Pennsylvania is strictly prohibited.
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Dr. Eby Bassiri
ebassiri@sas.upenn.edu
Microbiology
BIOL 275
Results of the Bacterial Conjugation Lab Exercise
NAME ___________________________ DATE ____________ GROUP NAME ______
NAME(S) OF PARTNERS _________________________________________________
Draw the results of "Observation of Recombinants" and label well.
Time elapsed
0 min
20 min
40 min
60 min
_____________________________________________________________________
No. of colonies on Pro- plates
No. of colonies on Trp- plates
No. of colonies on His- plates
_____________________________________________________________________
What is the gene order and why?
_____________________________________________________________________
No. of colonies
______________________________
Plate
10-6
10-7
10-8
dilutions
Number of cells per
ml of original
_____________________________________________________________________
FHfr
_____________________________________________________________________
No. of colonies
______________________________
10-1
10-2
10-3
Plate
Number of cells per
dilution
ml of original
_____________________________________________________________________
Recombinants
_____________________________________________________________________
5
Dr. Eby Bassiri
ebassiri@sas.upenn.edu
Microbiology
BIOL 275
From the data you have obtained above and the fact that you used 0.9 ml of F- and 0.1
ml of Hfr in your conjugation mixture, determine the number of F- and Hfr cells that can
pair up.
From the data on the number of F-, Hfr and conjugants, derive the efficiency of
conjugation in your experiment (i.e., the % of the conjugants per parental pair).
Why do we dilute the parental mixture less and less as the conjugation time increases?
Why do we enumerate conjugants at 60 minutes and not later?
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Dr. Eby Bassiri
ebassiri@sas.upenn.edu