Molecular Membrane Biology

ISSN: 0968-7688 (Print) 1464-5203 (Online) Journal homepage: www.tandfonline.com/journals/imbc20

How does plasmid DNA penetrate cell membranes

in artificial transformation process of Escherichia
coli?

Subrata Panja, Pulakesh Aich, Bimal Jana & Dr Tarakdas Basu

To cite this article: Subrata Panja, Pulakesh Aich, Bimal Jana & Dr Tarakdas Basu (2008) How
does plasmid DNA penetrate cell membranes in artificial transformation process of Escherichia
coli?, Molecular Membrane Biology, 25:5, 411-422, DOI: 10.1080/09687680802187765

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Molecular Membrane Biology, August 2008; 25(5): 411
422

How does plasmid DNA penetrate cell membranes in artificial

transformation process of Escherichia coli?

SUBRATA PANJA, PULAKESH AICH, BIMAL JANA, & TARAKDAS BASU

Department of Biochemistry and Biophysics, University of Kalyani, West Bengal, India

(Received 24 December 2007; and in revised form 26 April 2008)

Abstract
Artificial transformation of Escherichia coli with plasmid DNA in presence of CaCl2 is a widely used technique in
recombinant DNA technology. However, exact mechanism of DNA transfer across cell membranes is largely obscure. In
this study, measurements of both steady state and time-resolved anisotropies of fluorescent dye trimethyl ammonium
diphenyl hexatriene (TMA-DPH), bound to cellular outer membrane, indicated heat-pulse (08C0428C) step of the
standard transformation procedure had lowered considerably outer membrane fluidity of cells. The decrease in fluidity was
caused by release of lipids from cell surface to extra-cellular medium. A subsequent cold-shock (428C008C) to the cells
raised the fluidity further to its original value and this was caused by release of membrane proteins to extra-cellular medium.
When the cycle of heat-pulse and cold-shock steps was repeated, more release of lipids and proteins respectively had taken
place, which ultimately enhanced transformation efficiency gradually up to third cycle. Study of competent cell surface by
atomic force microscope showed release of lipids had formed pores on cell surface. Moreover, the heat-pulse step almost
depolarized cellular inner membrane. In this communication, we propose heat-pulse step had two important roles on DNA
entry: (a) Release of lipids and consequent formation of pores on cell surface, which helped DNA to cross outer membrane
barrier, and (b) lowering of membrane potential, which facilitated DNA to cross inner membrane of E. coli.

Keywords: Escherichia coli, plasmid DNA, artificial transformation, membrane fluidity, membrane potential

Introduction method has been improved with time to different

In gene technology, transformation is known as a
basic technique. The process involves binding of
extra-cellular DNA to cell surface followed by
uptake across wall-membrane complex into cell
cytoplasm. Transformation occurs naturally in bac-
terial genus such as Micrococcus, Haemophilus and
Bacillus [1,2]; all these organisms have proteins on
their exterior surface whose function is to bind DNA
in their environment and transport it into the cell.
However, it is still a rare event for most bacteria to
naturally uptake DNA from environment.
The discoveries of transferring phage [3] and
plasmid [4] DNAs into Escherichia coli cells set the
stage for molecular cloning of recombinant DNAs.
According to this transfer process, E. coli cells are
made competent for DNA uptake by suspending in
ice-cold CaCl2 (50
100 mM) and applying a brief
heat-shock at 428C. Even after generation of com-
petence, efficiency of transformation is quite low;
majority of DNA cannot enter any cell and majority
of cells receive no DNA. Therefore, above original
high-transformation-efficiency protocols, employing
magnesium ions, manganese ions, dimethyl sulfox-
ide etc. together with the calcium ions [5,6].
Besides chemical methods, physical treatment like
brief electroshock of high voltage is applied for
DNA uptake in E. coli and other bacteria [7].
Even after all such developments, exact mechan-
ism of CaCl2-mediated artificial transformation
process is still largely obscure. It is only believed
that CaCl2 helps DNA adsorption to cell surface
and the heat-shock step facilitates penetration of
adsorbed DNA into cell cytosol. Our previous
findings [8,9] show in artificial transformation of
E. coli, DNA is bound to lipopolysaccharide (LPS)
receptor molecules on cell surface; divalent cation
Ca2 is suggested to form coordination complex
with the two negatively charged macromolecules

DNA and LPS. However, it is still unknown how
a macromolecule like DNA enters cell cytosol,
when molecular weight exclusion limit of outer
membrane porins to hydrophilic molecules is ap-
proximately 500 daltons [10]. In this context our

Correspondence: Dr Tarakdas Basu, Reader in Biophysics, Department of Biochemistry and Biophysics, University of Kalyani, Kalyani

741 235, West Bengal, India. Tel: 91 (033) 25828750. Fax: 91 (033) 25828282. E-mail: tarakdb@yahoo.com

ISSN 0968-7688 print/ISSN 1464-5203 online # 2008 Informa UK Ltd

DOI: 10.1080/09687680802187765
412 S. Panja et al.
earlier communication [11] reports the heat-pulse
step depolarizes membrane of CaCl2-treated com-
petent cells, which consequently lowers the poten-
tial barrier for movement of negatively charged
DNA molecules into cell interior. As, membrane
potential exists across inner cytoplasmic membrane,
question still remains how DNA penetrates across
outer membrane of E. coli. The present study
suggests the release of lipids from E. coli cell surface
by heat-pulse step creates pores, through which the
adsorbed plasmid DNA crosses outer membrane
barrier.
Of other gram negative bacteria, only Salmonella
typhimurium has been characterized with regard to
different factors that can influence transformation
[12]. Other bacteria are quite unexplored; moreover,
there are many other organisms which are very
difficult to transform. Therefore, as importance of
the present study it can be emphasized that once the
hidden mechanism of DNA transfer is understood in
E. coli system, the concept can be utilized to explore
transformation in other organisms, which are still
difficult to transform.
Materials and methods
A genetically engineered highly transformable strain
E. coli XL1-Blue [13] and a cloning vector plasmid
pUC19 [14] were used in this study. The fluores-
cent dyes trimethyl-ammonium-diphenyl-hexatriene
(TMA-DPH), diphenyl-hexatriene (DPH) and 3,3?-
diphenylthiocarbocyanine iodide were purchased
from Sigma-Aldrich, USA.
Preparation of competent cells and their transformation
with plasmid DNA
Cells were grown to log phase [up to (OD)600nm
0.10 i.e.,
5 107 cells/ml] in LB-medium,
washed with and ultimately concentrated 25 times
in ice-cold 100 mM CaCl2 and kept at 08C for 30
min to make the cells competent for DNA upake
[9,15]. For transformation, pUC19 DNA (no more
than 50 ng in a volume of 10 ml) was added to 200 ml
competent cell suspension and incubated at 08C for
30 min. DNA-adsorbed cell suspension was trans-
ferred to 428C for 90 s and was rapidly chilled in ice
for 5 min. A volume of 800 ml LB was added to the
cells and subsequent incubation at 378C was allowed
for 45 min. Cells were then serially diluted in chilled
tryptone broth [TB] [9]. 100 ml cells from properly
diluted samples were spread on agar-LB medium
with ampicillin (50 mg/ml), to obtain colonies of the
transformed cells. Transformation efficiency (TR)E
was calculated as the number of transformants per
mg of added DNA
Measurement of steady state anisotropy of TMA-DPH
bound to outer membrane of competent cells
Steady state anisotropy of fluorescent dye TMA-
DPH, bound to outer membrane of intact compe-
tent cells, was measured as described in [16].
TMA-DPH (stock solution prepared in DMF)
was added to 200 ml competent cells at final
concentration of 1.0 106 M. The cell suspension
was finally diluted 15 times by adding 2.8 ml of
100 mM ice-cold CaCl2 and was incubated in dark
at 08C for 45 min to allow steady incorporation of
dye. Fluorescence intensity and anisotropy were
measured in a spectrofluoropolarimeter (LS 50B,
Perkin Elmer) and during measurement, tempera-
ture was controlled by attaching a circulating water-
bath to the instrument. The sample was illuminated
with vertically polarized light (34095 nm) and the
steady state anisotropy rs (III GI )/(III2GI  )
and total intensity I III2I were simultaneously
measured at an emission wavelength of 42595 nm;
G is sensitivity factor of the instrument. Corrections
for scatted light for both intensity and anisotropy
were done according to:
Imeasured  Ifluo Iscatter and rs:measured
 f :rs fluo (1f ) rs: scatter
where f (a balanced fluorescence intensity factor) 
Ifluo/(IfluoIscatter). Scattered light intensity (Iscatter)
and anisotropy (rscatter) were determined by mea-
suring an unlabelled control under the same con-
ditions as that of sample.
Measurement of fluorescence life-time and time-resolved
anisotropy of TMA-DPH bound to outer membrane of
competent cells
For measurement of fluorescence life-time and
time-resolved fluorescence anisotropy, TMA-DPH-
labeled cells were excited at 375 nm using a
picosecond diode laser (IBH nanoleds) in an IBH
Fluorocube apparatus [17,18]. The emission was
collected at a magic angle polarization using a
Hamamatsu MCP photomultiplier (5000U-09).
Time-correlated single photon counting (TCSPC)
setup consisted of an Ortec 9327 CFD and a
Tennelec TC 863 TAC. The data was collected
with a PCA 3 card (Oxford) as a multi-channel
analyzer. The typical full width at half-maxima of
the system response using a liquid scatterer was
about 90 ps. The fluorescence decays were decon-
voluted using IBH DAS6 software. The experiments
were done at 08C and 428C. To study fluorescence
anisotropy decay, analyzer was rotated at regular
intervals to get perpendicular (I ) and parallel
(I) components and anisotropy function r(t) was

calculated using the formula r(t) I(t) GI  (t)
/I(t)2GI  (t); the G value of the picosecond set
up was determined to be 1.5, using a probe of very
fast rotational relaxation (e.g., coumarin 480 in
methanol).
Assay of lipid by measuring fluorescence of embedded
DPH
Lipid was assayed by measuring fluorescence of
embedded DPH as described in [19]. According to
this method, lipid content was directly proportional
to intensity of DPH fluorescence. Competent cells
were centrifuged to collect the supernatant. In
200 ml of cell supernatant, 2 ml of DPH (from a
stock of 0.5 mM in DMF) was added. The mixture
was then diluted 15 times by adding 2.8 ml of
100 mM CaCl2 and was incubated in dark for 40
min to allow proper binding of DPH with phospho-
lipid molecules, if any, in the cell supernatant. The
intensity of fluorescence of bound DPH was mea-
sured in Perkin Elmer LS50B spectrofluorimeter
with excitation and emission wavelengths at 350 nm
and 440 nm, respectively, keeping both slits at
10 nm.
Assay of protein by Bradford method
Protein was assayed according to Bradford method,
described in [20]. Competent cells were centrifuged
to collect the supernatant. In 200 ml cell super-
natant, 800 ?ml Bradford reagent was added, mixed
thoroughly and incubated for 5 min. The absor-
bance of the mixed solution was measured at
595 nm in Shimadzu UV-1601 spectrophotometer.
The protein content in cell supernatant, if any, was
proportional to the value of (absorbance)595nm.
Induction and assay of E. coli periplasmic protein
Alkaline Phosphatase (AP)
For induction of AP, cells of E. coli XL1Blue were
initially grown at 378C to log phase [up to
(OD)600nm 0.2, i.e.,
108 cells/ml] in tris-glucose
medium [21]. The grown cells were washed with
and finally suspended in phosphate-free tris-glucose
medium to grow further at 378C for 90 min, when
high pool of AP had been induced and stored at the
periplasmic space of cell membrane. The induced
culture was then washed with and finally re-sus-
pended in 1/25th volume of 100 mM ice-cold CaCl2
to make them competent. Such competent cells were
used to study the release of AP at different steps of
transformation. AP was assayed according to
method of Schlesinger and Levinthal [22]. Compe-
tent cells were centrifuged to collect the supernatant.
In 1.0 ml of cell supernatant, 2.0 ml of substrate
How does plasmid DNA enter into E. coli cell? 413
solution (0.2% paranitrophenyl phosphate in 1 M
tris-HCl, pH 8.2) was added and subsequent
incubation at 378C was allowed for 15 min to
develop yellow colour of paranitrophenol. The en-
zyme-substrate reaction was terminated by adding
0.5 ml of 13% w/v K2HPO4 and the absorbance was
measured at 420 nm. One unit of enzyme was
defined as that amount, which led to a change of
absorbance of 0.1 per 6.0 min [21].
2D gel electrophoresis of proteins
Competent cells, after subjecting the cold shock step
(428C 008C), were centrifuged to collect the super-
natant. The supernatant were precipitated with four
volumes of cold acetone, washed two times with
80% cold acetone and finally suspended in sample
buffer [23]. The IPG strips (pH 4.0
7.0) were re-
hydrated in a mixture of 200 ml re-swelling solution
[23] and 50 ml protein sample. After 12 h in-gel re-
hydration, iso-electric focusing was performed with
Ettan IPGphor 3 unit (GE Healthcare) employing
the voltage profile as described in [23]. The IPG
strips were then equilibrated in two steps, each for
15 min, with 10 ml SDS equilibration buffer solu-
tion [6 M urea, 75 mM tris-HCl (pH 8.8), 29.3%
glycerol, 2% SDS and 0.002% bromophenol blue];
the buffer additionally contained 100 mg DTT in
the first step and 250 mg iodoacetamide in the
second step. Electrophoresis in second dimension
was carried out on 12% SDS-polyacrylamide gels
[24] in Hoefer SE 600 Ruby (GE Healthcare), first
1 h in 25 mA and second 4 h in 50 mA. The gel was
stained with silver nitrate, according to the method
described in [24], scanned by Typhoon 9210 (GE
Healthcare) and the image was analyzed by the ‘2-D
Image Master’ software (GE Healthcare). 2D pro-
tein spots were finally matched with E. coli protein
databank.
Study of competent cell surface by Atomic Force
Microscope (AFM)
Surface morphology of competent cells was studied,
as described in [25], using a scanning probe micro-
scope (Veeco, AP 0100) equipped with a scanner
having a maximum XY range of 10 mm by 10 mm. At
different steps of transformation, 100 ml of cell
aliquot was taken in microfuge tube and any further
change of surface morphology was quenched by
centrifugation and fixing the cells at 48C with 2.5%
gluteraldehyde for overnight. The fixed cells were
washed twice and finally suspended in water. The
drop and dry method was used to transfer the
bacteria from suspension to a solid support (cover
slip), so that a mono-layer of cells is formed. To
414 S. Panja et al.
enhance adhesion of bacterial cells, the cover slip
was pretreated with Piranha solution (1:3 ratio of
30% H2O2/concentrated H2SO4) and was washed
copiously with de-ionized water before adding the
cells. For each specimen, 20 ml bacterial suspension
(total no. of cells
9 106) was placed on 1 cm2
region of cover slip. After placing the cells, surface
was washed thrice with 40 ml distilled water and
allowed to dry in air. Height and phase data were
captured using tapping mode AFM with standard
Vecco phosphorus doped silicon cantilevers having
resonance frequencies of 245
284 kHz. Root mean
square (RMS) values of roughness were calculated
for plane-fitted, flattened images and were used to
measure the standard deviation of the height of a
given line. All the measurements were done using
Vecco ProScan 1.8 software.
Measurement of membrane potential of intact competent
cell
Membrane potential was assayed spectrofluorime-
trically by measuring fluorescence quenching by
intact competent cells as described in [11,26,27],
using fluorescent dye 3,3?-diphenylthiocarbocyanine
iodide. Competent cells were diluted five times by
ice-cold 100 mM CaCl2 and a 2.0 ml amount of
1.0 mM fluorescent dye (dissolved in methanol) was
added to 3.0 ml of cell suspension in a cuvette with
continuous stirring at 48C. After signal stabilization,
fluorescence was measured in spectrofluoropolari-
meter (Perkin Elmer
LS50B) with excitation and
emission at 556 nm and 575 nm, respectively, keep-
ing both slits at 5.0 nm. The protonophore CCCP
(carbonyl cyanide m-chloro phenylhydrazone) was
then added to the dye-bound cells (in the cuvette) at
a final concentration of 70 mM and after a brief
vortexing, fluorescence was measured again as
above. Relative membrane potential was calculated
as: 100[1-(b-a)/(c-a)], where ‘a’ was the fluorescence
intensity with cells only, ‘b’ was that with (cells
dye) and ‘c’ with (cellsdyeCCCP).
The data points in each figure correspond to the
average of at least five independent experiments.
Results and discussion
The sequence of steps involved in standard trans-
formation procedure was: (a) Generation of compe-
tence for DNA uptake by suspending exponentially
growing cells in 100 mM CaCl2 at 08C for 30 min;
(b) addition of plasmid DNA to competent cells
and allowing adsorption of DNA to cell surface for
30 min; (c) administration of a heat-pulse (08C
0428C) to the DNA-adsorbed cells for 90 s; (d)
bringing back the cells further in ice (equivalent to a
cold-shock) and incubating there for 5 min; (e)
allowing growth of the cells at 378C for 45 min for
phenotypic expression of antibiotic resistant gene(s)
of plasmid; and (f) serial dilution and subsequent
plating under selection pressure of antibiotic(s). Of
the above steps, the first four were believed to be
responsible, through unknown mechanisms, for
adsorption and penetration of plasmid DNA into
the cell. In this study, to understand the mechanism
of DNA penetration through competent cell mem-
branes, experiments were carried out to observe
change in characteristics of cell membranes after
each of the following three steps
competence
generation, heat-pulse and cold shock.
In order to study membrane fluidity/rigidity, ice-
cold competent cells were labeled with fluorophore
TMA-DPH and steady state fluorescence anisotropy
was measured at different steps of transformation.
Figure 1 shows for competent cells initially kept at
08C, the value of steady state anisotropy of TMA-
DPH was 0.239 (90.014). After shifting the cells
from 0
428C and keeping there for 90 s, anisotropy
increased to the value of 0.336 (90.021). When
heat-pulsed cells were further brought back to 08C,
anisotropy had decreased to 0.241 (90.013), i.e.,
almost to its initial value. Steady state anisotropy of a
fluorophore in any fluid environment was known to
be inversely proportional to fluidity of the environ-
ment. Therefore, increase of anisotropy signified
heat-pulse step of transformation process had rigi-
dified outer membrane of competent cells. The
result also shows subsequent cold-shock step flui-
dized membrane again to its initial state. Figure 1
further shows when the cycle of heat-pulse and cold-
shock steps was repeatedly administered on compe-
tent cells, rigidification and fluidization of outer
0.38
0.36
Steady state anisotropy
0.34 42°C 42°C
42°C
0.32
0.30
0.28
0.26
0°C 0°C
0.24 0°C 0°C
0.22
1 2 3
Number of cycles
Figure 1. Steady state anisotropy of TMA-DPH bound to outer
membrane of CaCl2-treated competent cells of E. coli XL1Blue,
when the cells were subjected to repetitive heat-pulse (08C
0428C) and cold-shock (428C008C) steps of the transformation
process.
 How does plasmid DNA enter into E. coli cell? 415

membrane had occurred alternatively, and almost same cells were brought back to 08C and kept there
similar extent of changes in steady state anisotropy for 5 min, the life-time of each component had been
implied heat-pulse and cold shock steps had com- decreased to a value, slightly lower than its initial
plete reversible effect on cellular outer membrane. value. This result implied heat-pulse step of trans-
In order to get more detailed information about formation process had made the microenvironment
microenvironment of outer membrane of competent of the fluorescent probe so rigid that fluorescence
cells, fluorescence life-time and time-resolved fluo- decay rate of the probe had been slowed down. On
rescence anisotropy of TMA-DPH were measured other hand, subsequent cold-shock step reverted
after each of the competence generation, heat-pulse back the microenvironment nearly to its initial state,
and cold-shock steps of transformation procedure. i.e., the cold-shock fluidized cell membrane, which
The fluorescence intensity decay of TMA-DPH consequently made fluorescence decay rate faster.
could be satisfactorily recovered in terms of a double Thus the result of fluorescence life-time measure-
exponential decay model, I(t) A1 exp ( t/t1)A2 ment of TMA-DPH, bound to outer membrane of
exp (t/t2). Figure 2A shows the bi-exponential intact competent E. coli cells, corroborated the result
decays of TMA-DPH fluorescence comprised of two obtained from measurement of steady state aniso-
components
one with life-time of faster decay rate tropy.
t2 500
900 picoseconds (ps) and other with life- Study of time-resolved fluorescence anisotropy
time of slower decay rate t1 3
5 nanoseconds (ns). was carried out in such a way that zero time
Figure 2A signifies when competent cells were anisotropy (r0) had been fixed to the value 0.4 (the
transferred from 0
428C for 90 s, life-times of both fluorescence anisotropy of TMA-DPH in frozen
the components had been increased, and when the medium). Figure 2B shows time-resolved decays of
anisotropy at different steps of transformation. For
A competent cells, initially kept at 08C, r0 value
(τ1) decayed with a time constant of 1.34 1010.
(τ2) When such competent cells were administered heat
4 pulse of 428C for 90 s, anisotropy had decayed down
at a slower rate with time constant of 2.35 109.
Fluorescence life time inns

Shifting of heat-pulsed cells further to 08C made

3 decay of anisotropy again faster, lowering down the
value of time constant to 1.59 10 10. In all three
2
cases, anisotropy did not decay to zero, rather had a
residual value (ra). The value of ra at 428C was
greater than both the values
that at initial 08C
1 before heat-pulse and that at final 08C after heat-
pulse. However, residual anisotropy at final 08C was
greater than that at initial 08C. The existence of
0 limiting anisotropy (ra) could be interpreted as the
0°C 42°C 0°C
hindrance in rotational motion of TMA-DPH in
B membrane microenvironment of competent cells. If
0.4 After competence generation at 0°C the rotation of TMA-DPH molecule in membrane
After 90 sec heat-pulse (0° 42°C)
remained unhindered up to a certain angle (uc), then
After 5 min cold shock (42°C 0°C)
ra, r0 and uc were known to be related as: ra/r0 [(3
Cos2u 1)/2]2. At each of the three steps of trans-
Anisotropy

formation process, value of uc was determined to be

0.3 22.98C (at initial 08C), 21.88C (90 s after heat-
pulse) and 22.18C (5 min after cold-shock). The
values of uc signified rotational motion of fluoro-
phore had been maximally hindered at 428C, or in
other words, heat-pulse step made outer membrane
0.2 of competent cell qualitatively rigid. Moreover,
300 400 500 600 700 800 900
Time in ps values of uc for cells kept at 08C, before and after
heat-pulse step, implied fluidization of cell mem-
Figure 2. Pulse excitation fluorescence of TMA-DPH bound to brane.
outer membrane of E. coli XL1Blue cells after the steps of
competence generation (at 08C), heat-pulse (08C 0428C) and
For intact E. coli cells, membrane fluidity was
cold-shock (428C 008C). (A) Fluorescence life-times of TMA- reported to be directly proportional to the lipid to
DPH; (B) Time-resolved anisotropies of TMA-DPH. protein ratio of membrane [28]; i.e., the more was
416 S. Panja et al.

the value of the ratio, the more was the membrane A

60
fluidity and vice versa. Therefore, in order to find 42°C

DPH fluorescence intensity at 440nm

out the reason behind rigidification and fluidization
50 0°C
of outer membrane of transformation-competent E.
coli cells due to heat-pulse and cold-shock steps 40
42°C
respectively, studies were performed to investigate if
0 °C
any changes in lipid and protein profiles of intact 30 42°C
cell membrane had taken place by the said steps of
0°C
transformation process. Instead of direct measure- 20
ment of lipid and protein contents of membrane,
extra-cellular medium was assayed for presence of 10
lipid and protein molecules, if any, that had been
released from membrane. The lipid was monitored 0
0°C
by measuring DPH fluorescence and the protein
was assayed using Bradford method. Figure 3A and B
3B represent the profiles of lipid and protein release 0.28 0°C
respectively. Both Figures show after competence
generation (i.e., after incubation of cells in 100 mM

CaCl2 at 08C for 30 min), release of both lipids and
proteins took place from competent cells to extra-
cellular medium. When heat-pulse (08C 0428C)
was applied to competent cells for 90 s, further
release of lipids (Figure 3A), but no protein (Figure
3B), had occurred. On other hand, application of
cold-shock (428C 008C) to the cells for 5 min had
released proteins only (Figure 3B), but no lipid
(Figure 3A). These results implied release of lipid
molecules by heat-pulse step had decreased lipid to
protein ratio, ultimately rigidifying cell membrane;
in contrast, release of protein molecules by cold-
shock step had apparently increased lipid to protein
ratio, causing fluidization of cell membrane. When
the cycle of heat- and cold-shocks was repeatedly
applied on competent cells, release of lipid and
protein molecules respectively, had occurred in
every cycle (Figure 3), and cumulative effect of
release had caused alternate rigidification and
fluidization of outer membrane in every cycle, as
was observed from the results of steady state
anisotropy experiment (Figure 1).
In order to check whether released proteins were
membrane bound or cytoplasmic in nature, studies
were concentrated over three proteins of E. coli

inducible periplasmic protein alkaline phosphatase
(AP), inducible cytoplasmic protein D-serine dea-
minase and inner membrane protein F0/F1 ATPase.
Figure 4A shows when previously induced AP-
containing cells were made competent by suspend-
ing them in 100 mM ice-cold CaCl2 for 30 min, AP
had been secreted out to extra-cellular medium.
The application of heat-pulse to competent cells did
not further increase the extent of AP secretion,
whereas subsequent application of cold-shock to the
heat-pulsed cells had increased the level of AP
secretion. When the experiment was carried out on
previously induced D-serine deaminase-containing
Absorbance at 595 nm
0.26
0°C
0.24 42°C
0°C
0.22 42°C
0.20
0°C
0.18 42°C
Figure 3. (A) DPH fluorescence, as a measure of lipid release
from competent cell surface. (B) Result of Bradford assay, as a
measure of protein release from competent cell surface, when
cells were subjected to repetitive heat-pulse (08C 0428C) and
cold-shock (428C 008C) steps.
cells (prepared as described in Sarkar et al. [8]),
neither of competence generation, heat-pulse nor
cold-shock steps had released any D-serine deami-
nase (result not shown). Similarly, when cell super-
natant was assayed for the presence of F0/F1
ATPase, as described in [29], no release of the
enzyme had occurred in any of the three steps of
transformation process (data not shown). The
above results implied artificial transformation pro-
cess had disintegrated outer membrane of E. coli
cells, for which release of periplasmic AP took
place; however, inner cytoplasmic membrane had
remained intact, for which no release of either
the cytoplasmic protein D-serine deaminase or
the inner membrane protein F0/F1 ATPase took
place. Moreover, disintegration of outer membrane
caused release of many other periplasmic/outer
membrane proteins, like that of AP. This was
observed when cell supernatants, collected at
different steps of transformation, were electrophor-
esed in SDS-polyacrylamide gel [24]. Multiple
protein bands in lane A of Figure 4B signified
release of proteins had occurred at the initial steps

Figure 4. (A) Release of AP from the surface of E. coli XL1Blue
cells after the steps of competence generation (at 08C), heat-pulse
(08C 0428C) and cold-shock (428C008C). (B) SDS-Polyacryla-
mide gel (12%) electrophoretic pattern of cell supernatants. Lane
a: supernatant of cells suspended in 100 mM CaCl2 and kept at
08C for 30 min; lane b: supernatant of CaCl2-treated competent
cells after heat-pulse step and lane c: supernatant of competent
cells after cold-shock step. (C) 2D gel electrophoresis pattern of
the supernatant of competent cells after the cold-shock step.
of competence generation. Protein bands in lane B
were equally intense as those in lane A, which
indicated no additional release of proteins had
taken place after heat pulse step on competent
cells. On other hand, enhanced intensity of bands in
lane C implied further release of proteins after the
cold shock step.
Furthermore, the 2D gel electrophoresis pattern
of the supernatant of the competent cells, after the
cold shock step, showed the presence of the outer
membrane proteins like Ag43 (1), OmpA (2, 3),
OmpC (4), OmpF (5), NmpC (6), Tsx (7), TeaF
(8), OmpW (9) and the periplasmic proteins like
How does plasmid DNA enter into E. coli cell? 417
malE (10), rbsB (11), dsbA (12), OsmY (13), yjbP
(14).
The release of lipid and protein molecules was
expected to produce pores on competent cell sur-
face. The surface morphology of E. coli XL1 Blue
cell was, therefore, studied using AFM at different
steps of artificial transformation. To observe the
effect of CaCl2-mediated competence generation on
cell morphology, control experiment was done with
cells, suspended in ice-cold phosphate buffer. In
order to select desired cells for higher resolution
imaging, a large scan area (2 2 mm2) was first done.
For cells suspended in ice-cold phosphate buffer,
individual bacterium, represented by image A of
Figure 5, contained its rod-shaped character with a
typical length of 2
3 mm; the corresponding width
and height were 1 and 0.8 mm respectively. The two-
dimensional image (Figure 5B) of Figure 5A, at
nanometer resolution (0.35 0.35 mm2), exhibited
bumps with lateral dimension of 40
80 nm and
average surface roughness of 5.890.14 nm on outer
membrane of cells. The bumps were reported to be
due to LPS assembly in outer membrane and
difference in heights of the bumps was suggested to
be due to different O-antigen units at the termini of
LPS molecules [25]. In comparison to control cells
in phosphate buffer (Figure 5A), the shape and size
of competent cells in 100 mM CaCl2 remained
unchanged (Figure 5C); however, packing of LPS of
competent cells changed considerably (Figure 5D).
Image D shows after generation of competence,
average surface roughness increased to 7.990.19
nm. The heat-pulse step made cell surface more
rough (8.590.43) with appearance of pores (Figure
5E); average width and depth of pores were 70 nm
and 7 nm, respectively. When the heat-pulsed cells
were subsequently subjected to cold-shock step,
average roughness of cell surface further increased
to 11.490.62, but pores disappeared (Figure 5F); a
large area of outer membrane had decreased in
height. Fludization of outer membrane (due to
release of proteins) by cold-shock step was perhaps
responsible for disappearance of the pores. The
result of this study indicated that in the artificial
transformation process of E. coli, plasmid DNA
crossed outer membrane barrier possibly through
the pores formed by heat-pulse step.
When the cycle of heat- and cold-shocks was
repeated on competent cells, release of lipids and
proteins had taken place in each cycle (Figure 3).
Therefore, with gradual increase of the number of
cycles, the pore formation on outer membrane had
also increased. In the process of transformation, if
these pores truly made the passage for DNA
penetration across outer membrane, transformation
efficiency (TR)E of cells with plasmid DNA should
418 S. Panja et al.

Figure 5. AFM images of the native E. coli XL1 Blue cell in phosphate buffer (images A & B), after generation of competence in 100 M
CaCl2 at 08C (images C & D), after heat pulse at 428C for 90 s (image E) and after cold shock at 08C for 5 min (image F). The scanning
area of the images A and C of entire bacteria were 22 mm2. Two dimensional images B, D, E and F were acquired by zooming into the
boxed area (0.70.7 mm2) of the images A and C. In every case 15 individual cells were studied. This Figure is reproduced in colour in
Molecular Membrane Biology online.

have been increased with repetition of alternate
heat-pulse and cold-shock steps. The experimental
results, shown in Figure 6, corroborated this propo-
sition, i.e., with repetition of cycles, (TR)E increased
at least up to third cycle, above which saturation in
(TR)E appeared. Figure 6 shows when DNA-ad-
sorbed competent cells at 08C, without applying
heat- and cold-shock steps, was directly brought to
the next step of transformation, i.e., shifted to LB for
allowing growth at 378C, the cells had shown certain
value of (TR)E. This result apparently implied heat-
pulse step had no exclusive role on DNA entry
rather, pores on competent cell surface were suffi-
cient to uptake DNA. However, even after the
omission of heat-pulse (08C 0428C) step, cells
experienced a considerable heat-shock when shifted
from 0
378C (to allow growth for phenotypic
expression of antibiotic resistance marker on plas-
mid) and this step had perhaps facilitated transfer of
adsorbed DNA into cell cytosol. In support of this
view, it would be justified to refer our earlier
communication [11], where we reported membrane
depolarization by the heat-pulse step had facilitated
DNA entry into the cell and the depolarization had
occurred nearly to the same extent by shifting
competent cells from 0
428C or 378C. Figure 6
also shows the value of (TR)E after heat-pulse step
was nearly twice of that under no heat-pulse condi-
tion and the cold-shock step, by which the pores
were found to disappear (Figure 5F), had no
additional effect on (TR)E. Moreover, when the
cycle of heat-pulse and cold-shock was repeated
on DNA-adsorbed competent cells, (TR)E at second
cycle was almost double of that at first cycle and
Heat pulse (0°to 42°C)
Cold shock (42° to 0°C)
14
12
[TR]E X 10-5 / µg of DNA
10
8 %(1mM CaCl2)
6 0.30
4
2 42°C
0 0.22
After
Cycle 1 Cycle 2
competence
generation
Figure 6. (TR)E of the CaCl2-treated competent cells of E. coli
XL1Blue, when the cells were subjected to repetitive heat-pulse
(08C 0428C) and cold-shock (428C008C) steps.
How does plasmid DNA enter into E. coli cell? 419
(TR)E at third cycle was slightly greater than that
at second cycle, indicating a saturation effect.
Excessive pore formation after third cycle perhaps
made the (TR)E to attain its maximum saturation
value.
The original method of competence induction by
CaCl2 treatment was a low transformation-effi-
ciency-protocol, where (TR)E was found to be

106 transformants per mg of DNA. In order to
improve the order of efficiency, different chemical
methods had been developed later on. One of the
high transformation efficiency chemical methods
was developed by Inoue et al. [30], where (TR)E
was
108 transformants per mg of DNA. The
standard transformation buffer of 100 mM CaCl2
was replaced, in the Inoue method, by a buffer
containing 15 mM CaCl2, 55 mM MnCl2, 250 mM
KCl and 10 mM PIPES (pH 7.6). Here also,
rigidification of outer membrane of competent cells
due to exclusive release of lipids by heat-pulse step
and fluidization of the membrane due to exclusive
release of proteins by cold-shock step had taken
place (data not shown).
There is a report [5] showing (TR)E to be
maximum when cells were made competent with
CaCl2 of strength 40
100 mM; with lowering of the
concentration of CaCl2 below 40 mM, gradual
decrease of (TR)E took place. In order to investigate
the reason, the steady state anisotropy study was
carried out at each step of transformation process by
treating cells with CaCl2 of different molarities.
Figure 7 shows that in between 40 and 100 mM
CaCl2, the extent of changes of steady state aniso-
tropy by heat-pulse and cold-shock steps were nearly
the same at every molarity of CaCl2. The measure of
release of lipids and proteins (data not shown) also
corroborated this result. However, at 10 mM CaCl2,
anisotropy increased by heat-pulse step; but the
extent of increase was much less compared to that
%(100mM CaCl2)
%(40mM CaCl2)
0.36
%(10mM CaCl2)
Steady state anisotropy
0.34
0.32 % (Phosphate buffer)
0.28
0.26
0.24
0°C 0°C
Cycle 3
0.20
Figure 7. Steady state anisotropy of TMA-DPH bound to outer
membrane of E. coli XL1Blue cells (suspended in CaCl2 of
different molarities) after the steps of competence generation (at
08C), heat-pulse (08C0428C) and cold-shock (428C008C).
420 S. Panja et al.

in case of 40
100 M CaCl2 (Figure 7). This was due A

0.38 Control
to proportional fall of lipid release at 10 mM CaCl2 Benzyl alcohol
(data not shown) and thus less rigidification of outer 0.36 Ethyl alcohol
membrane caused less (TR)E. Figure 7 also shows at

Steady state anisotropy

0.34
1 mM CaCl2, a reversal of change of anisotropy had
0.32
taken place, i.e., the membrane had been fluidized
by heat-shock step and rigidified by cold-shock step. 0.30

Similar changes of anisotropy was also observed 0.28 42°C

when, instead of 1 mM CaCl2, cells had been 0.26
suspended in ordinary phosphate buffer. Moreover,
0.24
in case of 1 mM CaCl2 or phosphate buffer, even
after change of anisotropy, no release of lipids or 0.22
0°C 0°C
proteins were found to occur (data not shown), 0.20
which ultimately made the (TR)E to be nil in either
of the cases. In these cases, transfer of cells from 08C Control cell
B BA pretreated cell
to a temperature 428C, higher than the lipid transi- 16
15 Ethanol pretreated cell
tion temperature, perhaps made the lipids more
14
fluid, which had caused outer membrane fluidization

[TR]E X 10-5 / µg of DNA

13
by the heat-pulse step, even after no release of lipid 12
11
or protein from cell surface.
10
Our results indicated the more was the extent of 9
rigidification of outer membrane of E. coli cell, the 8
7
more was its (TR)E. For more evidential support of 6
this phenomenon, experiments like measurement of 5
steady state anisotropy and (TR)E were carried out 4
3
on cells previously treated separately with two 2
chemicals such as: (i) ethanol, an agent known to 1
rigidify [28], and (ii) benzyl alcohol, an agent known 0

to fluidize [31] E. coli outer membrane. For the
studies, CaCl2-treated competent cells were sepa-
rately incubated with 5% v/v ethanol and 30 mM
benzyl alcohol for 30 min at 08C. Cells were then
centrifuged down and were re-suspended in fresh
100 mM CaCl2. A part of each cell suspension was
subjected to heat-pulse (08C 0428C) and cold-
shock (428C 008C) steps and steady state aniso-
tropy of TMA-DPH was measured at each of the
steps; whereas residual parts of the cells were used to
measure (TR)E. Figure 8A shows at each different
temperature (08C 0428C 008C), the value of steady
state anisotropy in case of ethanol-pretreated cells
was greater than that in case of untreated control
cells; on other hand, the values in case of benzyl
alcohol-pretreated cells were less than those in case
of untreated control cells. This result indicated
ethanol-pretreated cells were more rigid, where as
benzyl alcohol-pretreated cells were more fluid than
control cells at every temperature between 08C and
428C. Figure 8B shows (TR)E of control cells was
lower than that of ethanol-pretreated cells, but
higher than that of benzyl alcohol-pretreated cells.
The result of this study clearly implied that in case of
E. coli cells, transformation efficiency increased with
rigidification of outer membrane.
In an earlier communication [11] we reported
that by heat-pulse step (08C 0428C for 90 s) of
Figure 8. (A) Steady state anisotropy of TMA-DPH bound to
outer membrane of E. coli XL1Blue cells (previously treated with
5% (v/v) ethanol and 30 mM benzyl alcohol separately) after the
steps of competence generation (at 08C), heat-pulse (08C 0428C)
and cold-shock (428C008C). (B) (TR)E of E. coli XL1Blue cells
previously treated with 5% (v/v) ethanol and 30 mM benzyl
alcohol separately.
transformation process, membrane potential of in-
tact competent cells suddenly dropped down from
(-) 120 mV to (-) 10 mV; and by subsequent cold-
shock step (428C 008C for 5 min), potential further
increased to its original value. Here, we report that
when CaCl2-treated competent cells were subjected
to repeated cycles of heat-pulse and cold-shock
steps, alternate depolarization (decrease of poten-
tial) and polarization (increase of potential) of
plasma membrane of intact competent cells had
taken place (Figure 9). Therefore, a close look on
the results of the studies on steady state anisotropy,
membrane potential and (TR)E indicated heat-pulse
step of standard transformation procedure had
simultaneously caused: (a) rigidification of outer
membrane (Figure 1) by releasing lipids from cell
surface (Figure 3A), (b) depolarization of inner
membrane (Figure 9), and (c) enhancement of
(TR)E (Figure 6) or in other words facilitation of
DNA entry in intact competent E. coli cells. The

-120
0°C
0°C
Membrane potential in mV
-100
-80
-60
-40
-20
42°C 42°C
0 [5] Hanahan D. 1983. Studies on transformation of E. coli with
1 2
Number of Cycles
Figure 9. Membrane potential of competent cells of E. coli
XL1Blue, when the cells were subjected to repetitive heat-pulse
(08C 0428C) and cold-shock (428C008C) steps.
subsequent cold-shock step although fluidized outer
membrane and polarized inner membrane, however,
this step had no significant role on change of (TR)E,
i.e., on DNA entry.
The results of this study led us to propose the
following model to explain the obscure mechanism
of DNA entry during artificial transformation of E.
coli with plasmid DNA. According to our proposi-
tion
the heat-pulse step of standard CaCl2-
mediated transformation process rigidified outer
membrane by releasing lipids and thus produced
pores on cell surface through which previously
adsorbed plasmid DNA molecules crossed outer
membrane barrier; moreover, the heat-pulse-
mediated depolarization of inner plasma membrane
eventually lowering the negativity of cell inside
potential, decreased the potential barrier for move-
ment of negatively charged DNA molecules into cell
interior.
Acknowledgements
For financial assistance, we are indebted to the
Department of Biotechnology, (project no. BT/
PR3418/BRB/10/287/2002) and the Department of
Science and Technology (FIST programme, 2001

2011), Govt. of India. We also acknowledge the
Indian Association for the Cultivation of Sciences
(IACS), Kolkata, for providing us the pico-second
laser facility procured from the grant no. IR/I-1/CF-
01/2002 of the Department of Science and Technol-
ogy, Govt. of India.
Declaration of interest: The authors report no
conflicts of interest. The authors alone are respon-
sible for the content and writing of the paper.
How does plasmid DNA enter into E. coli cell? 421
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