ISSN 1022-7954, Russian Journal of Genetics, 2009, Vol. 45, No. 3, pp. 292–297. © Pleiades Publishing, Inc., 2009.
Original Russian Text © T.V. Siunova, A.V. Siunov, V.V. Kochetkov, A.M. Boronin, 2009, published in Genetika, 2009, Vol. 45, No. 3, pp. 336–341.
Nickel-resistant bacteria are widely distributed in
anthropogenically nickel-contaminated and nickel-
enriched serpentine soils. Bacteria resistant to
cobalt/nickel/cadmium or to cobalt/nickel are most fre-
quently encountered among hydrogen-oxidizing bacte-
ria close to the
genus . In
these bacteria, the mechanism providing for cell resis-
tance is related to the efﬂux of toxic cations from the
cell via transmembrane protein complexes RND
(Resistance Nodulation Division), HME (Heavy Metal
Efﬂux), “pumps” . To date, only two operons that
determine resistance to nickel and are referred to the
smallest-number second group of RND HME systems
have been sequenced and studied in detail: the operon
(resistance to Co/Ni) on plasmid
pMOL28 in strain
CH34) , and operon
(resistance to Ni/Co/Cd) on plasmid pTOM9 in strain
31A . Sequences homologous to
operons were detected in bacteria belonging to the
orders Burkholderiales, Pseudomonadales, and Entero-
bacteriales, but further studies of the corresponding
determinants were not conducted .
The aim of this work was to determine the taxo-
nomic afﬁliation of strain BS501 and the type of resis-
tance determinant on plasmid pBS501.
MATERIALS AND METHODS
Bacterial strains and plasmids.
concentrations (MTC) of metals for bacteria were
determined by replica plating to the tris-mineral
medium after a 5-day cultivation.
As a negative control and plasmid recipients, we used
B-1241 (BCM). Plasmids
I fragment of
pSUP202 (4 and 4.5 kb)), pECD110 (Ap
on pT7-5 (10 kb)), pDNA327 (Tc
I fragment of
on pVDZ'2 (5 kb)) provided by
D.H. Nies (Institute of Microbiology, Halle, Germany)
were used to construct probes.
Media and cultivation conditions.
grown in LB medium  and tris-mineral medium
(TMM) . As carbon sources, 0.2% sodium glutamate
was used. Stock solutions of metal salts (100 mM)
O were added to the medium at a final concentra-
tion of 0.01–10 mM.
Induction of resistance.
Bacteria were grown for
12 h in metal-free TMM (control) and with 20, 50, 100,
M cobalt, nickel, zink, and cadmium. The
bacterial culture was diluted with fresh TMM to the
0.05 optical density, next, 2 mM cobalt or nickel and
1.5 mM zink were added, and the mixture was grown
with aeration. Induction was recognized by a decrease
in the lag period during the ﬁrst hours, compared to
control (strain without induction by metals).
Isolation of plasmid DNA and DNA digestion with
restriction enzymes were conducted as in . Transfor-
mation with plasmid DNA was accomplished accord-
ing to . DNA fragments were eluted from an agarose
gel using a QIAEXII Gel Extraction Kit (Qiagen,
United States). Highly-labeled
P-DNA was obtained
using a Decalabel DNA labeling kit (Fermentas,
-Like Operon in Strain
Resistance to Cobalt and Nickel
T. V. Siunova
, A. V. Siunov
, V. V. Kochetkov
, and A. M. Boronin
Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Moscow oblast,
Pushchino, 142290 Russia
Pushchino State University, Moscow oblast, Pushchino 142290;
Received April 02, 2008
—Plasmid pBS501 was detected in the strain
sp. BS501. This plasmid speciﬁes high
level of induced resistance (5 mM) to cobalt/nickel both in the host strain and in related strains
B-1251. Hybridization analysis revealed a homology of pBS501 restriction frag-
ments with the only well-characterized operon
that resides in plasmid pMOL28 from
CH34. Essential differences in the structural organization of the cobalt/nickel resistance
determinant were found between plasmid pBS501 and the
GENETICS OF MICROORGANISMS