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 pilus2

, 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 reported4

.

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 them5.

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 pili6

, 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 .. 910 0 b . .. conjugatIon process '. ur 0 servatton 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

h ' 1' t' q W sex or ot er pI I as gene trans er passages" . 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;

BANERJEE el al.: VALIDITY OF MECHANISM OF GENE-TRANSFER 161

these had served as reference strains and had been widely used in conjugation experiments by earlier workers. Their minimum inhibitory concentration(s) (MIC) was determined by agar dilution method l5

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 donors are described later l6.

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 Schneider l7 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 control experiments, these reagents were omitted.

Conjugation experimenrs with UV-irmdiored donors-Earlier studies4

.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 as, by diphenylamine reaction as described

l7.

Estimation of DNA-sugars and Ilucleotides was carried out according to the method descrihed l

7.l'J

Table I-Transconjugants produced with different pairs of donor and recipient E.coli in terms or CFU/Ill I

Donor (phenotype) Recipient (phenotype) Antibiotics in media f(.Jr Transconjllgalll (CFUlml) counter selection obtained after 24 hr.

RP 4 (Ap' Km' Tc')* K 12 MA 140 (F Lac' Sm')* SmTc I()"H

K 12AB 1157 (FLac'Sm' )t SmTc IO-l

K 12 ( F Lac' NI' )** Tc NI 720 K 12 C600 ( F Lac' Ri')* TcRi 1 .. 1 x 10" K 12 AB2463 ( F Lac' Sm' )* SmTc 4.6x IOJ

V517 (Ap' Km' Tc')* K 12(FLac'NI')* Tc NI <)40

K 12MAI40(FLac'Sm' ) 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

*Received from S. Palchaudhuri , USA; # from S.K. Mahajan, Bombay; ** from H. Natarajan , Ncw Delhi: :1: rrom r.K. Pillai , cw 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).

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

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) .

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 or promoting role on transconjugant production. Exposing recipients to a smaller quantity of DNase nO 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.

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

Table 2.....,-Effect of addition of DNase (50 l1g/ml) at different time points on conjugation

Conjugating pair (do­nor x recipient)

Antibiotics in DNase added to conjugation CFU/ml of transl:Onjugants on mc-counter selec- mixture at time point dia with counter selectioll

tion media Without DNase Wit h DNasc

RP 4 x K 12 MA 140 SmTc o hr 104* 10'

30 min before addition 104 105

of recipient 30 min before addition of 104 105

donor

V 517 x KI2 MA 140 SmTc o hr 6.8 x 103 10.)

30 min before addition 6.6 x 10.1 10.)

of recipient 30 min before addition of 6.2 x 103 10.)

donor

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

Conjugating pair (donor Antibiotics in counter Time of sampling CFU/ml of transconjugants on Ille-x recipient) . selection media (hr) dia with counter selectioll

Without DNase With DNasc

RP4x KI2 MA 140 SmTc 4 420 HlJO 24 10

4* 10'

V 517 x KI2 MA 140 SmTc 4 560 ~ 10' 24 6.8 x 10.1 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)

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)

Antibiotics in counter selection

media

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 colonies of do­nors and recipients due to 'curing' was below 2%

163

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

164 INDIAN J EXP BIOL, FEBRUARY 2000

Table 5-Effects of DNase on conjugation with UV -irradiated donors

Conjugating pair Antibiotics in Time of CFUlml of donor ( 0 ). recipient ( R ). transconjugant ( TC' ) (donor x recipient) counter sampling since On counter selection media

selection beginning of media contact (hr) Without UV or With UV-irradiated UV -irratliatcu D

DNase D. without DNase treated wit h DN ase treatment treatment (50 pg/ml ) 30 min

0

RP4xK 12MA 140 SmTc Y2 0 1Y2 0 3 0 4 0 6 0

24 0

RP x K 12 AB 2463 SmTc Y2 0 I 0 3 0 4 0 6 0

24 0

Srn, streptomycin; Tc. tetracycline.

Table 6--lncrease in DNA/DNA sugars in conjugation mixture tluids*

Test condi tion Colorimetric Equivalent reading DNA/sugars (in flg)

Donor + Recipient 0.05 16 (no DNase added)

Donor + Recipient 0.06 20 + DNase

Donor + DNa~ 0.07 27 (no recipient added)

* 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 readings: 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 kinetics4

.2J. The subsequent steps cou ld be postulated

to be: a donor pilus anchoring on its recipient

TJ

before mi xing D+R

R TC 0 R D R TC'

0 14 0 0 6l 0 0 ()

0 206 0 0 4!l () 0 II 0 402 0 0 99 () 0 34 0 10' 0 0 101 () 0 IO~

0 104 0 0 I O~ () 0 I ()' 0 104 0 0 10~ () () 10'

0 92 0 0 2 () () 2 0 11 0 0 0 4 0 () 2 0 528 0 0 50 () 0 15 0 800 0 0 .94 () 0 ''J .'-0 10' 0 0 129 () () <)6

0 104 0 0 240 0 () X2()

counterpart, juxtaposition of the two by the retracting pilus, and finally their mutual impaction 2o

.2J .

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 recipients2

.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 additional donor cells being recruited and energised by extraneOllS DNase.

A persistent rise in the Tee in all experiments invol ving DNase, needs to be stringently evaluated. It has been observed that each operationa l cycle of DNA versus DNase in the conjugation process confines itself to single donor-recipient pairs24J-+; 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 165

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 replication4, 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 molecules22,23 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 DNases,

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

significance remains unexplained, while others excrete DNase27

, It is plausible to think that these act as the donors and recipients respectively, for gene transfer in nature, Griffith's original experiment on

f ' 'f h' 4 S ryo trans onnatton testt y t IS " '- . 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 transformation2,3,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

f 'd I' b'l' 11·14 ?X·10 o WI est app Ica I Ity . - . . 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

f ' d' d I' 10 our m mgs an cone uSlons , 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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166 INDIAN J EXP BIOL, FEBRUARY 2000

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