1022-7954/05/4105- © 2005 Pleiades Publishing, Inc.
Russian Journal of Genetics, Vol. 41, No. 5, 2005, pp. 479–483. Translated from Genetika, Vol. 41, No. 5, 2005, pp. 601–606.
Original Russian Text Copyright © 2005 by Poluektova, Fedorina, Prozorov.
Exchange of plasmids via conjugation is known for
many species and genera of bacteria and is the most
common mechanism of horizontal gene transfer [1, 2].
There are two variants, transfer of self-transmitting
conjugative plasmids, which are commonly large and
-type replication, and mobilization of small
plasmids, which show
-type replication. Mobilization
always involves large conjugative plasmids; small plas-
mids are incapable of self-transmission.
Conjugation was ﬁrst described in
Conjugative transfer of plasmids in bacilli was reported
far more recently; experiments were performed mostly
(see [3, 4] for review). In
, conjugal transfer
was observed for large plasmid pLS20, which mobi-
lized small plasmid pBC16 . Low-frequency mobili-
zation of small plasmid pUB110 by large plasmid
was studied in our laboratory .
More recently, we found a more convenient conjuga-
tion system based on cryptic plasmid p19 from soil
strain 19. It was observed that p19 mobilizes
small plasmids pUB110 and pBC16 both on a solid
medium and in a liquid medium. The mobilization fre-
quency was relatively high: approximately 3
ient cells received a small plasmid. Several parameters
of mobilization were studied with the use of this system
[7, 8]. However, its considerable drawback is that p19
is a cryptic plasmid; i.e., selective media were suitable
for detecting only mobilization of small plasmids
pUB110 and pBC16, which contained the kanamycin
and tetracycline resistance genes, respectively. To
detect transfer of p19 itself, it was necessary to test
transcipients carrying a small plasmid for the capability
of mobilization or to carry out hybridization with the
cloned p19 replicon, which is a laborious and time-con-
In this work, we used insertional mutagenesis to
label the p19 plasmid with the chloramphenicol acetyl-
) gene, which determines chloram-
phenicol resistance. The resulting plasmid, p19
used to estimate the transfer frequency for the conjuga-
tive plasmid proper, to study the dynamics of plasmid
transfer, and to detect some speciﬁc features of conju-
gation between various
MATERIALS AND METHODS
Bacterial strains and plasmids. Bacillus subtilis
strain 19 (p19 pV) was isolated from forest soil in
Belarus. Chromosomal DNA of this strain transforms
competent cells of
strain 168 . Strain 19
can serve as a recipient in genetic transformation and
contains cryptic plasmids p19 (95 kb) and pV (8.3 kb)
. Its variants containing only one of the two plasmids
or resistant to streptomycin (Str
) are spontaneous
mutants. Plasmids pUB110  and pC194 were intro-
duced in cells of strain 19 by protoplast transformation.
168 strains 39-22
arg(GH)15 leuB8 hsd
purB6 metB5 thr5
were obtained from
a laboratory collection. Str
variants of these strains are
spontaneous mutants. Plasmids p19 and pUB110 were
introduced in strain 39-22 by conjugation with strain 19.
Plasmids pC194 (Cm
, 2910 bp) and pUB110 (Km
4545 bp) are natural plasmids of
[11, 12]; pBD64 (Cm
, 5.0 kb) is a hybrid
plasmid . These plasmids are capable of
cells. In addition, we used
vector pUC19 (Ap
, 2686 bp) .
Plasmid DNA isolation and enzyme treatment.
mid DNA was isolated from
cells by alkaline
lysis . Digestion with restriction enzymes and treat-
ment with T4 DNA ligase were performed as recom-
mended by the manufacturer (Fermentas, Lithuania).
lowed a protocol developed in our laboratory .
strains was carried
out in a liquid medium. Overnight cultures of a donor
and a recipient, which were grown separately in antibi-
Conjugative Transfer of the Large Plasmid p19
E. U. Poluektova, E. A. Fedorina, and A. A. Prozorov
Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, 119991 Russia; e-mail: email@example.com
Received July 13, 2004
—Cryptic conjugative plasmid p19 from the environmental
strain 19 was labeled
gene conferring resistance to chloramphenicol. The resulting plasmid, p19
, was used to estimate
the transfer frequency, to study the dynamics of plasmid transfer, and to detect some speciﬁc features of conju-
gation between various