Indian Journal of Experimental Biology
Vol. 38, February 2000, pp. 160-166
Validity of mechanism of gene transfer in the process called
conjugation in bacteria
Malldira Banerjee, ~arna Chakrabarti, D P Acharya, A Roy & A N Chakrabarty
Department of Medical Microbiology and Parasitology, Calcutta University College of Medicine. Calcutta 700020. India
and
Jayashree Bhattacharyya & Sujata G Dastidar
Division of Microbiology, Department of Pharmaceutical Technology. Jadavpur University, Calcutta 700 032, India
Received 10 May 1999; revised 30 August 1999
We have attempted a new evaluation of the process of conjugation in bacteria, because of some hasic dissi mi larit ies observed between this and that of eukaryotes. or plants and animals. Reference donor and recipient strains. widely used to
prove conjugation in bacteria. were chosen; addition of DNase during the conjugation process, led to anllnexpected but
highly reproducible increase in the transconjugant colony counts (TCe; ca. ~ I log), when compared with that of the controls without DNase. Transconjugants were also obtained when the same live donors were substituted with the UV - killed
ones although the TCC was very low initially. Contrarily. donors treated with DNA-intercalating agents. e.g. acridine orange
or ethidium bromide, resulted in a complete failure to produce transconjugants. There was a quantitative relationship between the DNase used on donors and levels of DNA sugars/nucleotides/DNA. which possibly resulted from interaction between the DNase and DNA being present/produced on the donor surface. This may be indicative of what lIlay actually happen in the donor-recipient mixtures in the conjugation test proper, where the recipient DNase may activate a donor DNA
production cycle. The evidences presellted did not suggest that the donor DNA in the conjugation process is actu~llly vestibuled through any intercellular conjugation passages. and is susceptible to the action of DNase or the intercal;ltin1! dyes.
Conjugation in prokaryotes is thought to be a means
of transfer of heredit~ry character(s) by cell-to-cell
contact (e.g. F to F cells) during co-cultivation t. A
pilus or fimbria which ensured a close physical
proximity of these cells, by its retraction, was called a
'sex pilus 2, and the process of gene transfer requiring
this, was termed 'sexual mating' or a conjugation
process , A remarkable feature of conjugation is
believed to be the total insusceptibility of this process
to extracellular DNase', although its corroboration by
an actual use of DNase had been seldom reported 4 .
Such a pilus-mediated process was commonly seen
among members of enterobacteria, and determined by
extrachromosomal or 'conjugative plamids,5; this
system was later extended to characterise the gene
transfer process among many other groups of
bacteria, even though no conjugative plasmid was
actually demonstrable in them 5.
However, a difficulty arose as regards evidences
for transmission of DNA from the donor 'male' to the
recipient 'female' bacterium in the conjugation process; this was initially believed to be through an axial
hole or a canal within the pili 6 , although till today no
evidence could be adduced either for this, or for any
DNA being located within the pi Ius canal :u
An alternate hypothesis was lhat following cell-tocell contact due to the contracting pili or the
clumping agents, intercellular passages between male
and female cells were formed 4.5.x, through which the
donor DNA, unexposed to the environment, could be
vestibuled . This too could never be demonstrated) 7.
Contrarily, other observations raised doubts on an
unequivocal role of fimbriae in facilitating the
..
.
..
conjugatIon
process 910
' . 0 ur 0 bservatton
on III
Slfil
natural transformation I 1.1 2. as well as, that of Cohen (! f
at.", showed that the conjugative and nonconjugative plasmids were equally well transferable
by the transformation process to a wide variety of
Gram-positive and Gram-negative bacteria when their
surface layers were suitably 1ll0dified 5 1~ . These
ob~ervations call for a fresh evaluation of the role of
sex or ot her pI' 1'I as gene trans t'er passages"q . W e
present data on the susceptibility of the conjugation
process to DNase/DNA-binding agents, for an
evaluation of this process in bacteria.
Materials and Methods
The conjugation technique-The donor (5) and
recipient (6) E. coli strains used. are listed in Table I;
161
BANERJEE el al.: VALIDITY OF MECHANISM OF GENE-TRANSFER
these had served as reference strains and had been
widely used in conjugation experiments by earlier
workers. Their minimum inhibitory concentration(s)
l5
(MIC) was determined by agar dilution method
which confirmed those reported by earlier workers
(Table I). The donor and the recipient pairs for
different conjugation experiments were selected on
the basis of their complimentary antibiotic
resistarice(s) and efficiency of conjugational transfer;
these were studied by the 'broth mating' technique l4 :
0.2 ml of donor plus 1.0 ml of recipient culture (18 hr
old) were added to 8 ml of fresh nutrient broth and
incubated for 24 hr at 37°C, when replicate samples
(0.05 ml of each mixture) were plated out using a
calibrated Pasteur pipette-tipped (sterile) automatic
syringe, on selection plates with complimentary
antibiotics, in parallel with unmixed donors or
recipients, as controls l5 . Experiments with killed
l6
donors are described later .
Tests with DNase. RNase. acridine orange etc.Purified sterile DNase (ON - EP, Sigma Laboratories,
USA) at concentrations of 30, 50 or 100 flg/ml with
MgS04 (83 flM/ml, pH 7.2) from a stock solution (1
mg/ml) was added to the donor(s) and recipient(s), as
single, or mixed culture broths, at different time
points, according to the experimental designs (Table
2). DNase activity was determined as described by
l7
Schneider using two additional sources of DNase
subsequently for confirmationl XltJ. In similar tests,
DNase was replaced by heated DNase/acridine
orange/ethidium bromide. In corresponding co ntrol
experiments, these reagents were omitted.
Conjugation experimenrs with UV-irmdiored
donors-Earlier studies 4. 16 showed that the presence
of living donor cells was not essential for transfer of
hereditary characters in the conjugation process, but
donor cells, killed or inactivated by UV -irradiation or
by other means, could also serve. For this purpose, an
18 hr donor culture was subjected to UV -irradiation
(2800 - 2400 A or 280 - 240 mfl) for 15 min, from a
UV source, 10 em away, to give an ' optimal kill' as
determined by previous trial experiments. The effect
of UV -irradiation was studied on the viable counts of:
(I) the donor(s), (2) the transconjugant counts (TCC)
using such donor(s), as well as, (3) on the TCC of
such irradiated donor(s) treated with DNase at
different time points. These 3 types of counts were
obtained by plating on 3 different sectors on the same
selection plates, as well as, on three or more different
plates of same medium to minimise the sampling
errors and variability.
Estimation of DNA in conjugation fluids-Free
DNA was estimated by UV-absorballcel x.l tJ, as \vell
l7
as, by diphenylamine reaction as described .
Estimation of DNA-sugars and Ilucleotides was
carried out according to the method descrihed l7.l'J
Table I-Transconjugants produced with different pairs of donor and recipient E.coli in terms
Donor (phenotype)
Recipient (phenotype)
RP 4 (Ap' Km' Tc')*
K
K
K
K
K
or CFU/Ill I
Antibiotics in media f(.Jr
counter selection
Transconjllgalll (CFUlml)
obtained after 24 hr.
SmTc
SmTc
Tc NI
TcRi
SmTc
I()"H
4.6x IOJ
K 12(FLac'NI')*
K 12MAI40(FLac'Sm' )
Tc NI
SmTc
6.X x 10 '
Inc Fime (AP' Cm' Sm' Km' Tc' SlI' )t
K 12 C600 (F Lac' Ri')
ApRi
1.2 x 10 '
Inc W (Cm' Sm' Km' Su')*'"
K 12 ( F Lac' NI')
Ap NI
0
Com 9 (AP' Cm' Sm' Tc' Su')t
K 12 C600 (FLac Ri')
Tc Ri
3.X x I()3
V517 (Ap' Km' Tc')*
12 MA 140 (F Lac' Sm')*
12AB 1157 (FLac'Sm' )t
12 ( F Lac' NI' )**
12 C600 ( F Lac' Ri')*
12 AB2463 ( F Lac' Sm' )*
IO-l
720
1..1 x 10"
<)40
*Received from S. Palchaudhuri , USA; # from S.K. Mahajan, Bombay; ** from H. Natarajan , Ncw Delhi: :1: rrom r.K. Pillai ,
Delhi
Ap, ampicillin : Cm, chloramphenicol ; Sm, streptomycin; Km, kanamycin; Tc, tetracycline;
Ri, rifampicin; NI , nalidixic acid; Su, sulphanilamide; F, fertility factor; Lac, lactose fermenting gene.
§ CFU counts of donor and recipient with same volume of sample(s).
cw
162
INDIAN J EXP BIOL, FEBRUARY 2000
, Results
The results presented in Table I show that the
different donor or recipient strains used by us, had
been widely used for conjugation experiments by
earlier geneticists, and several cultures were received
from more than one sources . The conjugation
frequency was found to be different with respect to
different donor and recIpIent pairs, despite
experimental conditions remaining the same . (Table
I). The effects of addition of DNase (50 Ilg/ml) to the
donor-recipient conjugating mixtures at different time
points are described in Table 2; this showed that
addition of DNase always resulted in a higher yield of
TCC (CFU/ml), irrespective of when the DNase was
added, either to the donor or the recipient, compared
with the controls where no DNase was added (Figs J
and 2); this hinted that possibly some extracellular
DNase could play/have some inducing o r promoting
role on transconjugant production.
Exposing
recipients to a smaller quantity of DNase
Ilg/ml )
for 30 min (or longer) before co-cultivation with the
donor, we found (in Table 3) with respect to all the
conjugating pairs, that DNase added at given time
points led to an increase in the Tee : ca. I log
compared with when DNase was not used .
Acridine orange/ethidium bromide (50 Ilg/ml) was
added to the donor culture 30 min before the cocultivation step, and the effects are presented: Table
4. The Tee determined 24 hI' later showed a total
absence of these colonies using either acridine orange
or ethidium bromide (Table 4) .
The results of Table 5 show that the experiments in
which the donor cells did not have any UV-radiation,
but only some exposure to DNase. the Tee were
hi'ghest (control counts) ; this was followed by Tee of
thbse donors which were neither exposed to lethal
UV -radiation nor DNase. The Tee with respect to 2
other series. i.e., (I) when donor cells were treated
with UV, but not DNase. and (2) when treated with
both UV and DNase, was extremely low (up to 3 or 4
hours). However. Tee in the last test rose remarkably
by overnight incubation.
nO
Fig, I-Effect of DNase on conjugation ; conjugation experiment
was performed by mating donor Ecoli RP 4 Sm' Tc' and recipient
E.coli K 12 MA 140 Sm' Tc' and plating out on Sm + Tc agar
media, Sector A shows the growth of transconjugant colonies
(CFU) after overnight incubation of the. conjugating mixture at
37°C. Sector B shows increase in the CFU count of transconjugant following DNase (50J.lg/ml) treatment. Sectors C and D
show absence of growth of donor and recipient respectively on the
same plate.
Fig. 2-Mating pair consisted of E.. coli V 5 J 7 (donor) and E.coli
K 12 MA 140 (recipient). Sector A shows colonies of normal
conjugation; Sector B shows increased CFU count of transconjugant after DNase (50 jJg/ml) treatment. No growth of donor (Sector C) and recipient (Sector D) .
Test for presence of DNAIDNA-suguars-DNA
was estimated in different samples of conjugating
mixtures (from the calibration curve drawn with '
different concentrations of calf thymus DNA; Table
6).
Discussion
We have repeated the basic co-cultivation
experiments carried out by other workers. who have
accepted these as 'conjugation ' by concensusl.4 X. 14I X;
thus, bacterial conjugation had been distinguished
BANERJEE et al. : VALIDITY OF MECHANISM OF GENE-TRANSFER
163
Table 2.....,-Effect of addition of DNase (50 l1g/ml) at different time points on conjugation
Conjugating pair (donor x recipient)
Antibiotics in
counter selection media
DNase added to conjugation
mixture at time point
RP 4 x K 12 MA 140
SmTc
o hr
30 min before addition
of recipient
30 min before addition of
donor
V 517 x KI2 MA 140
o hr
SmTc
30 min before addition
of recipient
30 min before addition of
donor
CFU/ml of transl:Onjugants on mcdia with counter selectioll
Without DNase
Wit h DNasc
10 *
104
4
10'
105
104
105
6.8 x 103
6.6 x 10.1
10.)
10.)
6.2 x 10 3
10.)
Sm, streptomycin ; Tc, tetracycline.
*CFU counts being rather high, were rounded off to approximate values.
Table 3-Effect of addition of DNase (30 l1g/ml) to recipient culture 30 min before mixing with donor culturc
for conjugation
CFU/ml of transconjugants on Illedia with counter selectioll
Without DNase With DNasc
Conjugating pair (donor
x recipient) .
Antibiotics in counter
selection media
Time of sampling
(hr)
RP4x KI2 MA 140
SmTc
4
24
420
4
10 *
4
24
560
6.8 x 10.1
V 517 x KI2 MA 140
SmTc
HlJO
10'
~ 10'
10.)
Sm, streptomycin; Tc, tetracycline.
* CFU counts being rather high, were rounded off to approximate values.
Table 4-Effects of 50 l1g/ml of acridine orange (AO) and ethidium bromide (EB) on conjugation
Conjugating pair
(donor + recipient)
Antibiotics in
counter selection
media
RP 4 x K 12 MA 140
V 517 x K 12 MA 140
V517 x KI 2
Com 9 x K 12 C(,{M)
SmTc
SmTc
TcNI
TcRi
CFU/ml of transconjugants on media with counter selection
Without AO or EB After AO treatment After EB treatmcnt
104'
6.4 X 103
920
3.8 x 103
o
o
o
o
o
()
o
o
Sm, streptomycin ; TC,tetracycJine; Ri , rifampicin; NI, nalidixic acid
*Counts rounded off to nearest log integer because of too numerous colonies ; loss in number of coloni es of donors and recipients due to 'curing' was below 2%
from all other means of gene transfer, as it is believed
notionally to be totally insusceptible to extracellular
DNase2o ; this is presumably because t~e donor DNA
is thought to be vestibuled through' some protective
cellular passages, although, for which no concrete
proof, based on the actual use of DNase exists)·5,14,2I,
The essential feature of our observation is that when
the classical conjugation process IS subjected to
extracellular modulating factors like DNase, acridine
orange or ethidium bromide, the TCC could be
significantly affected either way; this suggested that
the donor DNA may not really remain intracellular
throughout the conjugation proce~s, but may indeed
get exposed to, and interact with, the extracellular
INDIAN J EXP BIOL, FEBRUARY 2000
164
Table 5-Effects of DNase on conjugation with UV -irradiated donors
Conjugating pair
(donor x recipient)
RP4xK 12MA 140
Antibiotics in
counter
selection
media
SmTc
Y2
1Y2
3
4
6
24
RP x K 12 AB 2463
SmTc
CFUlml of donor ( 0 ). recipi ent ( R ). transconjugant ( TC' )
On counter selection media
Time of
sampling since
beginning of
contact (hr)
Y2
I
3
4
6
24
Without UV or
DNase
treatment
With UV-irradiated
D. without DNase
treatment
UV -irratliatcu D
treated wit h DN ase
(50 pg/ml ) 30 min
before mi xing D+R
0
R
TC
0
R
TJ
0
0
0
0
0
0
0
0
0
0
0
0
14
206
402
10'
104
10 4
0
0
0
0
0
0
0
0
0
0
0
0
4!l
99
1
10
I O~
()
10~
0
0
0
0
0
0
0
0
0
0
0
0
92
11 0
528
800
10'
104
0
0
0
0
0
0
0
0
0
0
0
0
6l
D
R
0
()
0
0
0
0
0
()
()
()
()
TC'
()
II
34
IO~
I ()'
10'
2
()
()
4
50
.94
129
240
0
()
()
()
0
0
()
()
<)6
0
()
X2()
2
2
15
''J
.'-
Srn, streptomycin; Tc. tetracycline.
Table 6--lncrease in DNA/DNA sugars in conjugation mixture
tluids*
Test condi tion
Colorimetric
reading
Equivalent
DNA/sugars (in flg)
Donor + Recipient
(no DNase added)
0.05
16
Donor + Recipient
+ DNase
0.06
20
Donor + DNa~
(no recipient added)
0.07
27
* Readings obtained from calibration curve prepared on the
basis of data obtained by using 30 flglml of DNase against 10,
20, 40 and 100 flg of calf thymus DNA. giving respective colorimetric readin gs: 0.03; 0.06; 0.08 ; 0.22
modulators at least temporari ly. We thus hypothesise
that a small amount of DNA may be initially present
on the surface of potential donor cells, which signals
a DNase production cycle in the recipient cells,
leading in turn, to degradation of such DNA, thereby
switching on a stepped up DNA production cycle in
the donor, according to laws of enzyme-substrate
kinetics 4 .2J. The subsequent steps cou ld be postulated
to be: a donor pilus anchoring on its recipient
counterpart, juxtaposition of the two by the retracting
2o 2J
pilus, and finally their mutual impaction . .
The DNA now becomes firm ly sandwiched
between the 2 cells, being sea led off from further
DNase action and can enter the rec ipient ce ll in a
transformation - like process. The process is targetted
to achieve transfer of DNA between this ·pair ot' ce ll s
only, without the same donor repeating the transfer
process to other recipients 2.1., .
Our observations on the increase in Tee induced
by DNase do not appear to be due to a dQsedependent transformation-like process (i.e .. number
of transformants varying with the amount of DNA),
but is probably due to additiona l donor cells being
recruited and energised by extraneOllS DNase.
A persistent rise in the Tee in all experiments
invol vi ng DNase, needs to be stringently evaluated. It
has been observed that each operationa l cycle of
DNA versus DNase in the co nju gation process
confines itself to single donor-recipient pairs 24J-+; this
appears to be the limiting factor of this system, even
if induced externally by addition of DNase. Despite
this, the increase in Tee ca .~ 10~; . 1 in aJi of the
numerous DNase-based experiments. confers on it
high frudiciallimits statisticall y, when compared with
all the controls without DNase. This is based on the
BANERJEE et at.: VALIDITY OF MECHANISM OF GENE-TRANSFER
principle of repetition of small (ca,l log) changes in a
highly significantly large number of instances, The
results of Table 6 further corroborate this and we find
that the reaction fluids contained substantial amounts
of the DNA sugars, nucleotides and DNA, whose
presence cannot be explained unless extnlcelIular
DNase and DNA had reacted to produce these end
products,
The UV -irradiated and killed donor cells provide
interesting clues in this regard : conjugation is not
known to be effected by dead donor cells in any other
biological system, Therefore, the transfer of hereditary characters4.'4.'6 that occurs from a dead_ bacterium to a live one in mixed cultivation, raises
doubts about the process being truly conjugational.
However, it is plausible to assume that the hereditary
characters that are transferred from dead donor celIs,
are transmitted by a smaII quantity of preformed
donor DNA, as a dead donor is incapable of carrying
out further DNA metabolism and synthesis, Our data
(Table 5) show that initially only a few
transconjugants are produced, which, in their turn, act
as 'normal' donor cells, and transfer DNA to other
recipient cells till the counts rise considerably by 24
hr. This is elaborated further subsequently,
The finding that acridine orange and ethidium
bromide significantly prevented transconjugant
fonnation needs to be examined in the light of the
above observations, Both these agents are able to
eliminate plasmids (F/R) by intercalating with their
DNA intracellularly and preventing their replication 4,
which usually takes 24 to 48 hr and occurs at
frequencies varying ca, 1-5% , The total inhibition of
transconjugant colonies, however, may be explained
in tenns of easy binding of these dyes with the
prefonned DNA molecules 22,23 present on the surface
of donor cells which makes it unsuitable both for the
conjugation or transformation processes for gene
transfer, According to our earlier20 studies which
showed the 'phage mediated conjugation' also to be
susceptible (at least partialIy) to the action of DNase s,
Thus, the various conjugation processes described
in bacteria involve cell-to-celI contact, mediated
through sex pili, pheromones, impacted donorrecipients within membrane filter pores l4 ,16 or as in
the phage-mediated conjugationS process, appear to
facilitate a transfer of DNA across the recipient ceII
wall, mimicking a transformation process, In nature,
many
groups
of
bacteria
release
DNA
spontaneously5.'9,24.27 on their surface whose
165
significance remains unexplained, while others
excrete DNase 27 , It is plausible to think that these act
as the donors and recipients respectively, for gene
transfer in nature, Griffith's original experiment on
' testt'fy th'IS 4 "S'-ryo.
trans f onnatton
The possibility of a phage involvement accounting
for the process of gene transfer from the donor
(lysogenic) to the recipients needs to be examined,
Our donor and recipient strains have been widely
studied by others, as well as, by us, and no phages,
inducible/non-inducible have been detected, as
plaques, in mixed cultivation or when plated out on
solid media, Moreover, the phages, if any, would not
be susceptible to the action of DNase as seen in our
experiments,
Despite earlier
restricted
applicability
of
transformation 2,3,s,'4 as a gene transfer process,
compared with the conjugation process, subsequent
molecular biological technologies modifying bacterial
permeability had made genetic transformation as one
o f WI'd est app I'Ica b'l'
I Ity 11·14. -?X·10
. .
In one recently reported·1() rare instance where
DNase had been actually used to identify the gene
transfer process occurring between 2 strains of
Helicobacter pylori, it was observed that a fully
DNase-sensitive process conforming to transformation occurred, side by side, with a partially DNase
sensitive one which could not be unequivocally
characterised either as transformation or conjugation.
We have reported that variable responses to DNase
may occur in the so-called "conjugation" process,
which may be a special form of transformation only ;
this may be due to a partial exposure of the donor
DNA (to the endogenous/extraneous DNase)
becoming sandwiched between the donor and
recipient celIs, later in the process, becoming resistant
to DNase. These observations elegantly corroborate
'
10 ,
' d'mgs an d coneIuSlons
our f m
Our results appear to indicate that the mechanisms
of gene transfer postulated here are only preliminary .
This may point out to the relevance of this work for
future understanding of the genetic processes th<!t are
possible as a result of cell-to-cell contact, generally
believed to be a 'conjugation' process , which however lacks essential evidences ,
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INDIAN J EXP BIOL, FEBRUARY 2000
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