Nonchromosomal Antibiotic Resistance in Bacteria: Genetic Transformation

Of Escherichia coli by R-Factor DNA.

Sidra Zafar

Introduction Methods Results 1 Results Conclusion

The rapid spread of antibiotic resistance in microbes is
a growing biological concern around the world. David
and Barry Zimmerman, in their book, the Killer Germs,
state that microbes are finding ways to counteract or
neutralize the effects of antibiotics, making them
useless. This led to the question: Can bacteria be
genetically transformed to become antibiotic resistant
in the laboratory? if yes, how can this help us in the
future? Cohen, Chang and Hsu answered this
question in their experiment on Non- chromosomal
antibiotic resistance in bacteria: the Genetic
transformation of Escherichia coli by R- Factor DNA.
The experiment shows the transformation of
Escherichia coli cells treated with Calcium chloride to
multiple antibiotic resistance by purified R-factor*. The
scientists report that treatment of Escherichia coli cells
with calcium chloride makes the Escherichia coli
capable of taking up molecules of R-factor (drug
resistant) DNA. Drug resistance is seen in a small
fraction of the bacterial population immediately after
uptake of DNA. They also find that the introduced R-
factor DNA can persist in these cells as an
independently replicating plasmid, and has the same
reproduction and antibiotic resistance functions as that
of the parent R-factor.
*Genetic component of bacteria that provides
resistance to antibiotics and can be transferred from
one bacterium to another by conjugation.
TABLE 2. Expression of different drug resistance markers in
transformed bacteria.
Bacterial Strains and R Factors:
• I-like - R factor( R64-11) which specifies resistant to
tetracycline ( Tc) and Streptomycin ( Sm) were used and
F-like R factor that carries resistance to kanamycin
( Km), neomycin ( Nm), Choloramphenical ( Cm) and
Sulphonomide ( Su) was used.
DNA prep:
• Covalently closed R factor DNA was isolated and
purified from Escherichia Coli.
Transformation Reaction mixture:
• Escherichia coli was grown at 37° with OD of 0.85 at
590 nm. Cells, chilled and centrifuged. After
centrifugation, bacteria were re-suspended in 1/2 original
volume of calcium chloride and kept at 0° for 20 mins.
• 0.2 ml of cells treated with Calcium were added to 0.1
ml of DNA solution and incubated for 60 mins at 0°.
• Bacteria then heat pulsed for 2 mins allowing uptake of
R factor DNA, chilled and platted onto agar containing
appropriate antibiotics or diluted 10 times in L broth.
• Cell survival was greater then 50% after CaCl2 treatment
and heat pulse.
• Drug resistance was assayed on nutrient agar plates
with the antibiotics indicated.
Results 2
Number resistance to
Fig 1. Kinetics of expression of Kanamycin resistance in transformed
Escherichia coli . After incubation of CaCl2 treated cells with R6 DNA,
bacteria diluted 10 –folds into antibiotic free broth. At times shown,
samples of bacteria spread onto agar plates containing kanamycin:
incubated over night 37° for determination of number of transformants. An
identical sample was plated on antibiotic-free nutrient agar to determine
total viable cells. Transformation frequency is expressed in terms of the
number of kanamycin resistant bacteria relative to total number of viable
cells.
Expression of Antibiotic Resistance by Competent
Cells
• The kinetics of transformation of drug-sensitive
Escherichia coli to kanamycin resistance by closed R6
DNA is shown in Fig 1. Resistance to Kanamycin was
seen immediately by a small fraction of cells treated
with Calcium chloride after uptake of R-factor DNA.
Effect of DNA Concentration
• Transformation ability of several different
concentrations of closed circular R6 DNA is shown in
fig 2. A linear relationship of transformation frequency
to DNA concentration was seen, meaning higher
concentration of DNA platted equals higher
transformation to resistant cells. About 105 transformed
bacteria were obtained per micro gram of R-factor
DNA.
Requirements for Transformation by R-Factor DNA
• The experiment also showed that closed circular R6
DNA have the best transformation ability. Although
circular open DNA have the ability to transform, the
denaturing abolish the transforming ability of open
circular R-factor DNA.
Expression of Different Drug Resistance Markers in
Transformed Bacteria
• Table 2 shows transformation of Escherichia coli for
various antibiotic resistance markers carried by R6
DNA. Escherichia coli transformed and became
resistant to all antibiotics except Streptomycin. It is
possible that expression of Sn marker in transformed
bacteria may require more incubation in antibiotic free
medium then used in this experiment.
• This experiment shows that purified R-factor DNA can
transform Escherichia coli treated with CaCl2 to
multiple antibiotic resistance.
• Closed and open circular forms of R-factor DNA are
both capable of transforming cells into antibiotic
resistant but open cells may denaturize.
• Escherichia coli cells that have been transformed by
R-factor contain an independently replicating closed
circular DNA species identical to R-factor parent and
can also transfer this by conjugation.
• This shows that DNA from both F-like and I-like R
factor is capable of transforming Escherichia coli into
antibiotic resistant cells.
• The observation that DNA obtained from non-
transmissible plasmid is able to transform Escherichia
coli indicates the important use of this method for future
investigation of non transferable independently
replicating species of plasmid DNA.
• Further investigation and knowledge about the
transformational capabilities of other bacterial
organisms using these methods can possibly help
diminish bacterial resistance of antibiotics.

References
Fig 2. Effect of concentration of R-factor DNA on transformation frequency. Stanley N. Cohen, Annie C.Y Chang,Leslie Hsu, Nonchromosomal Antibiotic
Various concentrations of covalently-closed R6 DNA were assayed for their ability to Resistance in Bacteria: Genetic Transformation of Escherichia coli by R-Factor
transform calcium chloride treated Escherichia coli cells to kanamycin resistance. DNA, Proc.Nat.Acad.Sci.USA Vol.69, No.8,pp.2110-2114, August 1972
Transformation frequency was determined after 120-min incubation in antibiotic-free Barry E. Zimmerman, David J. Zimmerman, The Killer Germs, USA, McGraw-hill
Table 2: After transformation of calcium chloride- treated
medium to allow complete expression of Km resistance. Companies, september 2002.
Escherichia coli by R6, samples of bacterial cultures were
spread onto plates containing the antibiotics indicated.
Separate bacterial clones isolated from each antibiotic
plate were examined for the presence of other antibiotic
resistance determinants by stabbing colony samples onto
appropriate drug-containing plates.