Antonie van Leeuwenhoek 83: 99–106, 2003.
2003 Kluwer Academic Publishers. Printed in the Netherlands.
Copper resistance in Desulfovibrio strain R2
Olia V. Karnachuk
Denis A. Ivasenko , Elena A. Phyllipenko and Olli H. Tuovinen
1Department of Agriculture and Environmental Science, Tomsk State University, Prospect Lenina 36, Tomsk
Department of Microbiology, The Ohio State University, 484 West 12th Avenue,
Columbus, OH 43210-1292, USA;
Institute of Cytology and Genetics, Siberian Branch of the Russian
Academy of Science, Prospect Academika Lavrent’eva 10, Novosibirsk 630090, Russia;
correspondence (e-mail: email@example.com)
, Svetlana Y. Kurochkina , Duongruitai Nicomrat , Yulia A. Frank ,
Received 27 November 2001; accepted in revised form 5 February 2002
Key words: 16S rDNA gene analysis, Copper resistance, Desulfovibrio spp., Pco genes, Sulfate reduction
A sulfate-reducing bacterium, designated as strain R2, was isolated from wastewater of a ball-bearing manufactur-
ing facility in Tomsk, Western Siberia. This isolate was resistant up to 800 mg Cu/l in the growth medium. By
comparison, Cu-resistance of reference cultures of sulfate-reducing bacteria ranged from 50 to 75 mg Cu/l.
Growth experiments with strain R2 showed that Cu was an essential trace element and, on one hand, enhanced
growth at concentrations up to 10 mg/l but, on the other hand, the growth rate decreased and lag-period extended
at copper concentrations of .50 mg/l. Phenotypic characteristics and a 1078 bp nucleotide sequence of the 16S
rDNA placed strain R2 within the genus Desulfovibrio. Desulfovibrio R2 carried at least one plasmid of
approximately of 23.1 kbp. A 636 bp fragment ot the pcoR gene of the pco operon that encodes Cu resistance was
amplified by PCR from plasmid DNA of strain R2. The pco genes are involved in Cu-resistance in some enteric
and aerobic soil bacteria. Desulfovibrio R2 is a prospective strain for bioremediation purposes and for developing
a homologous system for transformation of Cu-resistance in sulfate-reducing bacteria.
many environmental sources for various applications
(Hao 2000), but the resistance of pure cultures of
sulfate-reducing bacteria to heavy metals and the
underlying biochemical and genetic principles are
poorly understood. Sulfate-reducing bacteria have
been isolated from mine tailings containing elevated
concentrations of Cu and other metals (Wielinga et al.
1999; Fortin et al. 2000). Isolates from uranium acid
mine drainage, capable of growing with methanol,
were reported to tolerate 10 mM CuSO (Hard et al.
1997), but further characterization of the isolates was
not carried out. Other environmental sources of en-
richment and isolation of sulfate-reducing bacteria
include wetland sediments treating mine drainage
from copper and iron mines as well as active and
abandoned coal mines.
Two different genetic systems of Cu-resistance,
cop and pco, have been described for bacteria. The
Potential uses of sulfate-reducing bacteria in the
treatment of metal contaminated environments have
received increased attention over the years. Sulfate-
reducing bacteria have been applied for metal removal
from various industrial wastewaters (Cowling et al.
1992; Dvorak et al. 1992; Barnes et al. 1994; Ham-
mack et al. 1994; Christensen et al. 1996; Chang et al.
2000; Hao 2000; Glombitza 2001). Metal precipi-
tation under sulfate reducing conditions occurs in
anaerobic zones in natural and constructed wetlands
receiving acid mine drainage (Hedin and Nairn 1993;
Webb et al. 1998; Johnson 2000), and may thus have
application in wetland mitigation.
Bacterial resistance to metals may be a major
limitation in these environmental applications. Metal-
tolerant enrichment cultures have been derived from
cop system is based on uptake and efflux of Cu, but
periplasmic binding may also be involved depending
on the organism (Silver and Phung 1996). The cop
genes have been characterized in Pseudomonas syr-
ingae (Bender and Cooksey 1987; Mellano and Cook-
sey 1988a, 1988b; Cha and Cooksey 1991) and other
bacterial plant pathogens isolated from areas where
Cu-salts have been applied for plant pest control
(Cooksey et al. 1990;Voloudakis et al. 1993). The pco
system for Cu-resistance, also involving Cu binding
and efflux, has been characterized in Escherichia coli
isolated from pigs that were fed Cu-supplemented
growth stimulants (Tetaz and Luke 1983) and in other
enteric bacteria (Williams et al. 1993). The present
work describes a Cu-resistant sulfate-reducing bac-
terium R2. A PCR product using pcoR-primers was
amplified from the genomic and plasmid DNA of this
bacterium. The phylogenetic position of the isolate
was also determined. To our knowledge, this is the
first time that genetic determinants were explored in
sulfate-reducing bacteria to clarify the mechanism of
solution to a final concentration of 200, 400, and 500
mg Cu/l. Iron paper clips were placed in culture tubes
as a source of Fe and H for bacteria. FeS precipi-
tation nucleating on paper clips signaled sulfate re-
duction. The sealed culture tubes were headspace free
and incubated under static conditions in the dark at 28
Pure cultures were isolated from a dilution series on
the lactate-sulfate media that were solidified with
1.5% agar. Single colonies were inoculated into liquid
media that contained 50, 100, 200, 300, 375, and 400
mg Cu/l. In addition to microscopic examination, the
culture purity was checked by the lack of growth on
Plate Count Agar (aerobic incubation) and Anaerobic
Agar (anaerobic incubation), both from Difco (De-
Electron donors and acceptors were prepared at final
concentrations of 20 mM thiosulfate, 15 mM ethanol;
7.5 mM each of acetate, formate, succinate, fumarate,
malate, citrate, 3.75 mM benzoate and nicotinate; 1
mM each of palmitate and sulfite. The electron accep-
tors were tested in sulfate-free media with lactate as
the electron donor. Bacteria were subcultured at least
five times to confirm that they utilized the test com-
pounds. The concentration of NaCl required to sup-
port growth in liquid media was also tested. Growth
was monitoring by measurement of protein concen-
tration and H S formation.
Materials and methods
Enrichment and isolation
The source of isolate R2 was a sample of wastewater
from a ball-bearing manufacturing facility in Tomsk,
Siberia. The wastewater was alkaline, pH 9.2, and
contained sulfate (14 mg SO
(0.23 mg S/l). At the time of sampling, the waste-
water contained no detectable dissolved O , the redox
potential (SCE) was 37 mV, and the water sample
contained 2.3 mg PO/l, 8.0 mg NH /l, 0.6 mg
NO /l, and 35 mg NO /l. The high nitrite con-
centration was the result of a chemical reducing agent
used in the process water.
Enrichment cultures were prepared from the waste-
water in a lactate-sulfate medium (Widdel and Bak
1992). The medium was designed for freshwater
sulfate-reducers and contained (per liter) 1.6 ml of
40% solution of lactic acid, 1 g NaCl, 0.4 g MgCl ?
6H O, 0.15 g CaCl ?2H O, 4.0 g Na SO , 0.25 g
NH Cl, 0.2 g KH PO , 0.5 g KCl, 1 ml trace element
solution, 1 ml vitamin solution, and 1 ml selenite-
tungstate solution. The pH was adjusted to 7.2–7.4
with NaHCO solution. Sulfide, added as Na S?9H O
stock solution, was used as a reducing agent. Before
inoculation, Cu was added as CuSO ?7H O stock
/l) and traces of H S
Reference cultures and determination of minimum
inhibitory concentration of Cu
Desulfovibrio desulfuricans ATCC 7757, Desulfobac-
terium macestii B-1958 (Gogotova and Vainstein
1989), and Desulfobacterium strain 63 (Karnachuk
1995) were used as reference strains. The bacteria
were grown with lactate in liquid media in sealed,
headspace-free test tubes. Cu was added to final
concentrations of 10, 20, 50, 75, and 100 mg/l.
Cultures received Na-thioglycollate (0.1 g/l) as a
reducing agent to alleviate initial precipitation of Cu.
Growth of the reference cultures was monitored by
optical density at 600 nm and H S formation. For
strain R2, growth was monitored by measurements of
protein and H S. Before the experiments, all cultures
were adapted to the target Cu concentrations through
multiple, successive subcultures. The cultures were
sampled sacrificially and three replicate cultures were
analyzed at each time point. The minimum inhibitory
concentration of Cu was defined as the lowest test
concentration of Cu in which cells did not grow.
D. desulfuricans ATCC 7757 and Desulfobacterium
strain 63. The primers for pcoR gene were 59-
GACATATC-39 (pcoR2 downstream primer), as re-
ported by Trajanovska et al. (1997). The primers were
acquired from the Institute of Cytology and Genetics,
Novosibirsk, Russia. Amplifications were run for 35
cycles in an automated thermocycler (Bio-Rad, Her-
cules, CA). After the initial denaturation at 95 8C for 3
min, each cycle was run at 95 8C for 0.7 min, 58 8C
for 0.3 min, and 72 8C for 1.5 min. PCR products were
separated by gel electrophoresis using 1% agarose.
Protein samples were digested in 0.5 N NaOH at 100
8C for 30 min before measurement by the Lowry
method (Lowry et al. 1951). Bovine serum albumin
was used as the protein standard.
colorimetrically with N, N-dimethyl-p-phenylen-
diamine (dihydrochloride salt) as the chromophore
(Cline 1969). X-ray diffraction analysis (CuKa radia-
tion) was carried out by Dr. Oleg Kukharenko, Tomsk
Center of the Siberian Branch, Russian Academy of
16S rDNA gene amplification and sequencing
The template DNA was purified by agarose gel elec-
trophoresis in 0.6% low melting agarose gel and
extracted from the gel using a Gel extraction kit
(GIBCO BRL, Rockville, MD) according to the
manufacturer’s recommendations. The PCR amplifi-
cation reaction (total volume of 50 ml) contained 10
to 50 ng of the purified target DNA, 5 ml of l0X
reaction buffer (GIBCO BRL), 2.5 mM MgCl , 200
mM of dNTP mixture, 250 nM each primer, and 2.5 U
of Taq DNA polymerase (GIBCO BRL). The follow-
ing thermocycling program was used for the specific
primers: 94 8C for 45 s, 50 8C for 30 s, and 72 8C for
1.5 min (30 cycles), and 72 8C for 10 min (1 cycle).
The primers for amplifying the nearly full length of
16S rRNA gene were the bacteria universal primers 8f
(Amann and Stahl 1992) and U1492r (Lane 1991).
The 8f primer has the base pair composition of 59-
AGAGTTTGATCCTGGCTCAG-39, E. coli
rRNA positions 8 to 27, and the base composition for
GACTT-39 (E. coli 16S rRNA position 1492 to 1468).
The PCR products were cloned into pCR2.1 vector
using the TA Cloning System (Invitrogen, Carlsbad,
CA) as recommended by the manufacturer. After
overnight incubation at 37 8C, plasmid DNA from
transformants was extracted using a plasmid extrac-
tion kit (GIBCO BRL). Nearly full length 16S rDNA
fragments were submitted to bi-directional sequenc-
ing with an ABI Prism model 377 sequencer (Perkin-
Elmer Applied Biosystems, Foster City, CA). The
primers used for sequencing were M13 universal
forward and reverse primers, which can bind on both
sides of the pCR2.1 vector (Invitrogen).
A comparison of the sequences for similarity with
the database was conducted using (i) nucleotide se-
Preparation of genomic and plasmid DNA
Bacteria in the late exponential phase were harvested
by centrifligation at 5,000 3 g for 15 min and washed
with mineral salts solution. The cells were lysed with
alkaline solution of SDS, and genomic DNA was
isolated as described by Sambrook and Russell
(2001). The lysate was extracted two to three times
with Tris-equilibrated (pH 8) phenol-chloroform-iso-
amyl alcohol (25:24:1), followed by extraction with
chloroform-isoamyl alcohol (24:1). The DNA was
precipitated at -20 8C by the addition of 1/10 volume
of 3 M sodium acetate and 2.5 volumes of ethanol.
The pellet was rinsed with 70% ethanol, dried at 22 8C
and resuspended in TE (10 mM Tris?Cl, 1 mM
EDTA) buffer, pH 8.0. Plasmid DNA was isolated by
modified alkaline lysis method of Birnboim & Doly as
described by Hardy (1987).
DNA base composition
Denaturation temperatures were determined with a
spectrophotometer in 0.1 3 SSC (standard sodium
citrate: 150 mM NaCl, 15 mM Na-citrate) buffer with
E. coli K12 genomic DNA as a standard. The mol-%
G1C was estimated by the equation of Marmur and
PCR amplification of pcoR
Templates for PCR amplification included plasmid
pPA87 (positive reference), plasmid DNA and gen-
omic DNA from strain R2, and genomic DNA from
quences obtained from GenBank/EMBL (Madden et
al. 1996) with BLAST and (ii) nucleotide sequences
obtained from the Ribosomal Database Project (RDP)
with SIMILARITY-RANK tool (Maidak et al. 2001).
The approximately 1,100-bp partial sequence gener-
ated by the cloning technique was aligned with known
species sequences using CLUSTAL W (Thompson et
al. 1994). The nucleotide sequence of the cloned 16S
rDNA reported in this article has been deposited in the
NCBI nucleotide sequence database under accession
lactate-sulfate medium although the optimum NaCl
concentration was 5 g/l. The highest NaCl concen-
tration in the medium still allowing growth was 20 g
NaCl/l. Initial experiments included a vitamin solu-
tion (per recipe of Widdel and Bak (1992)) in the
medium but it was not found to be necessary for
growth of this bacterium. Strain R2 used lactate,
fumarate, succinate, malate, ethanol, alanine, cys-
teine, formate, palmitate, and fructose as electron
donors for sulfate reduction. The fastest growth was
observed with ethanol, lactate, succinate, malate,
fumarate, and formate. The isolate did not grow with
acetate, nicotinate, benzoate, or glucose. In addition to
sulfate, growing cultures could use sulfite, thiosulfate,
elemental sultur, and nitrate as electron acceptors.
The G1C content of the genomic DNA of the
isolate was 58 mol-%. A partial 16S rDNA sequence
was amplified from the genomic DNA of strain R2,
followed by cloning and sequencing of the 1078 bp
PCR product. Phylogenetic analysis of the 1078 bp
sequence placed strain R2 in the genus Desulfovibrio
of the delta-subclass of Proteobacteria. Strain R2 was
phylogenetically related with a 98% sequence identity
to strain KH2 of an unnamed Desulfovibrio sp. (NCBI
X93147) and to three unnamed Desulfovibrio strains
isolated from uranium mining waste piles; JG5 (NCBI
AJ295679), JG1 (NCBI M295678), and IrT-JG1-58
(NCBI A1295670). The next closest identity was
Desulfovibrio termitidis (NCBI X87409) with a
matching identity of 96%. Desulfovibrio sp. KH2 and
D. termitidis were isolated from the digestive tract of
termites (Trinkerl et al. 1990), and Desulfovibrio
strains JG5, JGI, and IrT-JG-58 as well as the present
isolate R2 originated from metal-contaminated en-
Results and discussion
Minimum inhibitory Cu concentration for strain R2
and reference cultures
Cu-resistance of the sulfate-reducing bacteria was
tested by growth in Cu-containing medium. Desul-
fovibrio Desulfuricans ATCC 7757 was the most
sensitive and did not grow at concentrations exceed-
ing 50 mg Cu/l. The highest concentration tolerated
by Desulfobacterium strain 63 and Desulfobacterium
macestii was 75 mg Cu/l. Strain 63 is also par-
ticularly resistant to chromate (Karnachuk 1995).
Growth and H S formation by all cultures were
affected at concentrations as low as 25 mg Cu/l (data
The enrichments for the sulfate-reducing bacteria
were initially grown in the presence of 150 and 200
mg Cu/l. A vibrio-shaped isolate, isolated from the
enrichment containing 200 mg Cu/l and designated as
R2, was chosen for further study. This isolate grew at
Cu concentrations of up to 800 mg/l, which was 10-
to 16-fold higher when compared to the other test
cultures. The minimum inhibitory Cu concentration
for strain R2 was in the same range as that determined
for aerobic growth of E. coli (Williams et al. 1993). It
is noteworthy that Cu co-precipitates with iron as a
mixed sulfide during growth of the bacteria in the
lactate-sulfate medium and therefore the concentra-
tion and bioavailability of Cu in solution change with
Growth kinetics with Cu
Although strain R2 was resistant to 800 mg Cu/l,
growth decreased at Cu concentrations higher than 20
mg/l (Figure 1). The addition of 20 mg Cu/l to the
growth medium had little influence on the growth rate
(m5 0.106 h ), but it extended the lag period for
up to 50 h. The m
was 0.14 h
4.9 h) in the presence of 10 mg Cu/l, and with Cu
only included in trace element solution (0.76 mg Cu/
l), the m
was 0.102 h
6.8 h. For completely Cu-free media, the trace metal
stock solution (Widdel and Bak 1992) was modified
by excluding CuCl ?2H O. The highest growth rate,
0.303 h and generation time of 2.3 h, was
(generation time 5
Characteristics of the Cu-resistant isolate R2
with a generation time of
The R2 culture comprised vibrio-shaped cells measur-
ing 1.6–2.2 mm long and 0.6–1.0 mm in diameter.
The bacteria were motile by means of a single polar
flagellum. The culture grew well in the freshwater
Figure 1. Growth of Desulfovibrio sp. R2 at different Cu con-
centrations in the medium. Data are expressed as the means of three
replicates, with the vertical bars indicating standard deviations.
Before the experiments the culture was adapted to each Cu con-
centration by multiple (at least five times) successive subcultures.
Figure 2. Changes in sulfide concentration in the medium during
growth of Desulfovibrio sp. R2 at different Cu concentrations. Data
are expressed as the means of three replicates, with the vertical bars
indicating standard deviations.
extent of Cu-resistance in subsequent cultures, sug-
gesting that Cu-resistance may be an inducible trait in
observed when the culture medium was completely
devoid of Cu.
H S formed by sulfate-reducing bacteria is reactive
and precipitates Cu in the medium. Thus the precipi-
tation reduces the biologically available concentration
of Cu in the culture solution. The time course of the
formation of dissolved H S by R2 is shown on Figure
2. For strain R2 growing at 20 mg Cu/l, there was
little initial growth and H S was not detected during
the first 50 h. Cu-sulfide precipitation was associated
with H S formation, and the exponential phase of
growth commenced when the Cu concentration in the
medium decreased as the result of Cu-precipitation.
This was evident from the change in the protein/H S
ratio during growth (Figure 3). In the presence of no
or a low concentration of Cu, the protein/H S ratio
was about 4.5 during growth of R2. At Cu con-
centrations of .20 mg/l, the early logarithmic phase
was characterized by high protein content and low
H S concentration. With time, the protein H S ratio
decreased to 4.5 as usual for normal growth con-
X-ray diffraction analysis revealed no crystalline
phases in solids retrieved from spent cultures grown
with Cu. The precipitate was of dark brown color and
was concluded to comprise anhedral Fe- and Cu-
The length of the lag period preceding the growth
was proportional to the concentration of Cu in the
medium (Figure 4). Growth of strain R2 at sub-inhib-
itory Cu concentrations (10 mg Cu/l) increased the
Genetic determinants of Cu-resistance in strain R2
Genetic determinants for Cu-resistance can be associ-
ated with plasmids in aerobic bacteria (Cooksey 1987;
Williams et al. 1993; Trajanovska et al. 1997). For
anaerobes, no previous information is available on the
genetic basis of Cu-resistance. Strain R2 carried at
least one plasmid of approx. 23.1 kb. Plasmid DNA
was not found in Desulfobacterium macestii, Desul-
Figure 3. Changes in the protein:H S ratio during growth of
Desulfovibrio sp. R2 at different Cu concentrations in the medium.
Data are expressed as the means of three replications with the
vertical bars indicating standard deviations.
Strain R2 grew faster in the presence of 100 mg
Cu/l (m5 0.089 h , generation time 8.2 h) as
compared to media containing 50 mg Cu/l (m
0.049 h , generation time 14.1 h). It is possible that
the Cu-resistance mechanism coded by pco-deter-
minants is activated at .100 mg Cu/l concentrations.
At lower concentrations, cells can tolerate Cu because
of its precipitation with H S.
The amplification of a 636 bp sequence from strain
R2 with a pcoR-specific primer warrants further work
for sequencing and genetic analysis of the pco-homo-
logues. The pco operon was originally described for
enteric bacteria and has been subsequently found in
soil bacteria such as Arthrobacter, Corynebacterium,
and Alcaligenes spp. (Trajanovska et al. 1997), and
now in the obligately anaerobic sulfate-reducer, De-
sulfovibrio R2. The pco determinants in soil bacteria
may be the result of horizontal gene transfer, or they
may have originated from conservative sequences that
evolved before the divergence of these groups. Sul-
fate-reducers are ubiquitous in the environment and
are found in the digestive tract in mammals (Morvan
et al. 1996), and thus they are likely to be involved in
horizontal transfer of genes in these and other an-
aerobic open and confined habitats.
Sani et al. (2001) reported that another sulfate-
reducer, Desulfovibrio desulfuricans G20, was sensi-
tive to Cu, with growth being inhibited at ,1 mg
Cu/l, but the underlying genetic or biochemical basis
is not known. This sensitivity is contrasted by the
resistance to Cu in Desulfovibrio R2 in the present
work. Differences in inhibitory concentrations of Cu
between the two strains, G20 and R2, could be due to
genetic determinants such as the pco-homologous
PCR product found in plasmid DNA of Desulfovibrio
R2. The lack of pco homologs was apparent in
genomic DNA of the Desulfovibrio and Desulfobac-
terium reference cultures and, by comparison to R2,
they were more sensitive to Cu. In general, most
plasmids isolated from sulfate-reducing bacteria are
reportedly cryptic. The genetic system of strain R2
may be a useful beginning phase for creating a
transformation system for transfer of Cu and other
resistance traits to sulfate-reducing bacteria that can
be exploited in bioremediation of toxic effluents and
Figure 4. Lag periods of Desulfovibrio R2 before and after adapta-
tion to Cu in growth medium.
fobacterium strain 63, and Desulfovibrio desul-
Using the primers pcoRl and pcoR2, a PCR product
of approx. 636 bp was amplified from both plasmid
and genomic DNA of R2 (Figure 5). The fragment
size was identical to the positive control pPA87 that
corresponds to the pcoR gene of the pco operon
(Trajanovska et al. 1997). No PCR products were
obtained with these primers from genomic DNA of
the reference strains. Plasmid DNA could not be
retrieved from Desulfovibrio R2 growing at Cu con-
centrations lower than 100 mg/l even after first
transfer from Cu-containing media. This observation
indicated that the plasmid may be lost in the absence
of Cu-based selective conditions.
Figure 5. Agarose gel electrophoresis of PCR products of genomic
and plasmid DNA with primer pair pcoRl-pcoR2. Lanes: 1, DNA
size marker (pBlue Skript SK digested with BspRl); 2 positive
control, plasmid DNA pPA87; 3, plasmid DNA of Desulfovibrio
R2; 4, genomic DNA of Desulfovibrio desulfuricans; 5, genomic
DNA of Desulfobacterium 63; 6 and 7, genomic DNA of De-
sulfovibrio R2; 8, negative control.
We thank Jill Williams for the generous gift of
Gogotova G.I. and Vainstein M.B. 1989. Description of sulfate
reducing bacterium Desulfobacterium macestii sp. nov. capable
of autotrophic growth. Mikrobiologiya 58: 76–80.
Hammack R.W., Edenborn H.M. and Dvorak D.H. 1994. Treatment
of water from an open-pit copper mine using biogenic sulfide and
limestone: a feasibility study. Wat. Res. 28: 2321–2329.
Hao O.J. 2000. Metal effects on sulfur cycle bacteria and metal
removal by sulfate reducing bacteria. In: Lens P.N.L. and Hul-
shoff Pol L. (eds), Environmental technologies to treat sulfur
pollution: principles and engineering. IWA Publishing, London,
UK, pp. 393–414.
Hard B.C., Friedrich S. and Babel W. 1997. Bioremediation of acid
mine water using facultatively methylotrophic metal-tolerant
sulfate-reducing bacteria. Microbiol. Res. 152: 65–73.
Hardy K.G. 1987. Purification of bacterial plasmids. In: Hardy K.G.
(ed.), Plasmids: a practical approach. IRL Press, Oxford, UK, pp.
Hedin R.S. and Nairn R.W. 1993. Contaminant removal capabilities
of wetlands constructed to treat coal mine drainage. In: Moshiri
G.A. (ed.), Constructed wetlands for water quality improvement.
Lewis Publishers, Chelsea, MI, pp. 187–195.
Johnson B. 2000. Biological removal of sulfurous compounds from
inorganic wastewaters. In: Lens P.N.L. and Hulshoff Pol L. (eds),
Environmental technologies to treat sulfur pollution: principles
and engineering. IWA Publishing, London, UK, pp. 175–205.
Karnachuk O.V. 1995. Influence of hexavalent chromium on hydro-
gen sulfide formation by sulfate-reducing bacteria. Mik-
robiologiya 64: 262–266.
Lane D.J. 1991. 16S/23S rRNA sequencing. In: Stackebrandt E.
and Goodfellow M. (eds), Nucleic acid techniques in bacterial
systematics. John Wiley and Sons, New York, pp. 115–175.
Lowry O.H., Rosebrough N.J., Farr A.L. and Randall R.J. 1951.
Protein measurement with the Folin phenol reagent. J. Biol.
Chem. 193: 265–275.
Madden T.L., Tatusov R.L. and Zhang J. 1996. Application of
network BLAST sever. Meth. Enzymol. 266: 131–141.
Maidak B.L., Cole J.R., Liburn T.O., Parker C.T. Jr, Saxman P.R.,
Farris R.J. et al. 2001. The RDP-II (Ribosomal Database Pro-
ject). Nucleic Acids Res. 29: 173–174.
Marmur J. and Doty P. 1962. Determination of the base com-
position of deoxyribonucleic acid from its thermal denaturation
temperature. J. Mol. Biol. 5: 109–118.
Mellano M.A. and Cooksey D.A. 1988a. Nucleotide sequence and
organization of copper resistance genes fromPseudomonas syr-
ingae pv. tomato. J. Bacteriol. 170: 2879–2883.
Mellano M.A. and Cooksey D.A. 1988b. Induction of the copper
resistance operon from Pseudomonas syringae. J. Bacteriol. 170:
Morvan B., Bonnemoy F., Fonty G. and Gouet P. 1996. Quantita-
tive determination of H -utilizing acetogenic and sulfate-reduc-
ing bacteria and methanogenic archaea from digestive tract of
different mammals. Curr. Microbiol. 32: 129–133.
Sambrook J. and Russell D.W. 2001. Molecular cloning: a labora-
tory manual. Cold Spring Harbor Laboratory Press, Cold Spring
Sani R.K., Peyton B.M. and Brown L.T. 2001. Copper-induced
inhibition of growth of Desulfovibrio desulfuricans G20: assess-
ment of its toxicity and correlation with those of zinc and lead.
Appl. Environ. Microbiol. 67: 4765–4772.
Silver S. and Phung L.T. 1996. Bacterial heavy metal resistance:
new surprises. Annu. Rev. Microbiol. 50: 753–789.
plasmid pPA87 and E. coli ED8739, Elena V.
Deineko, Maxim L. Pillipenko and Liudmila Yu.
Popova for valuable advice in molecular techniques,
and Natalia M. Maximova for providing data on
chemical characteristics of the industrial wastewater.
This work was partially supported by the Tomsk
Regional Committee of Ecology and a Title VI grant
from the U.S. Department of Education obtained
through the Center of Slavic and East European
Studies, The Ohio State University.
Amann R.I. and Stahl D.A. 1992. Dual staining of natural bac-
terioplankton with 49,6-diamino-2-phenylindole and fluorescent
oligonucleotide probes targeting kingdom-level 16S rRNA se-
quences. Appl. Environ. Microbiol. 58: 2158–2163.
Barnes L.J., Scheeren P.J.M. and Buisman C.J.N. 1994. Microbial
removal of heavy metals and sulphate from contaminated
groundwaters. In: Means J.L. and Hinchee R.E. (eds), Emerging
technology for bioremediation of metals. Lewis Publishers, Boca
Raton, FL, pp. 38–49.
Bender C.L. and Cooksey D.A. 1987. Molecular cloning of copper
resistance genes from Pseudomonas syringae pv. tomato. J.
Bacteriol. 165: 470–474.
Cha J.-S. and Cooksey D.A. 1991. Copper resistance in Pseudo-
monas syringae mediated by periplasmic and membrane pro-
teins. Proc. Natl. Acad. Sci. USA 88: 8915–8919.
Chang I.S., Shin P.K. and Kim B.H. 2000. Biological treatment of
acid mine drainage under sulphate-reducing conditions with solid
waste material as substrate. Wat. Res. 34: 1269–1277.
Christensen B., Laake M. and Lien T. 1996. Treatment of acid mine
water by sulfate-reducing bacteria: results from a bench scale
experiment. Wat. Res. 30: 1617–1624.
Cline J.D. 1969. Spectrophotometric determination of hydrogen
sulfide in natural waters. Limnol. Oceanogr. 14: 454–458.
Cooksey D.A. 1987. Characterization of a copper resistance plas-
mid conserved in copper-resistant strains of Pseudomonas syr-
ingae pv. tomato. Appl. Environ. Microbiol. 53: 454–456.
Cooksey D.A., Azad H.R., Cha J.-S. and Lim C.-K. 1990. Copper
resistance gene homologs in pathogenic and saprophytic bacteri-
al species from tomato. Appl. Environ. Microbiol. 56: 431–435.
Cowling S.J., Gardner M.J. and Hunt D.T.E. 1992. Removal of
heavy metals from sewage by sulphide precipitation: thermo-
dynamic calculations and tests on a pilot-scale anaerobic reactor.
Environ. Sci. Technol. 13: 281–291.
Dvorak D.H., Hedin R.S., Edenborn H.M. and McIntyre P.E. 1992.
Treatment of metal-contaminated water using bacterial sulfate
reduction: results from pilot-scale reactors. Biotechnol. Bioeng.
Fortin D., Roy M., Rioux J. and Thibault P. 2000. Occurrence of
sulfate-reducing bacteria under a wide range of physico-chemical
conditions in Au and Cu-Zn mine tailings. FEMS Microbiol.
Ecol. 33: 197–208.
Glombitza F. 2001. Treatment of acid lignite mine flooding water
by means of microbial sulfate reduction. Waste Managem. 21:
106 Download full-text
Tetaz T.J. and Luke R.K.J. 1983. Plasmid-controlled resistance to
copper in Escherichia coli. J. Bacteriol. 154: 1263–1268.
Thompson J.D., Higgins D.G. and Gibson T.J. 1994. CLUSTALW:
improving the sensitivity of progressive multiple sequence align-
ment through sequence weighting, positions-specific gap penal-
ties and weight matrix choice. Nucleic Acids Res. 22: 4673–
Trajanovska S., Britz M.L. and Bhave M. 1997. Detection of heavy
metal ion resistance genes in Gram-positive and Gram-negative
bacteria isolated from a lead-contaminated site. Biodegradation
Trinkerl M., Breunig A., Schauder R. and Konig H. 1990. De-
sulfovibrio termitidis sp. nov., a carbohydrate-degrading sulfate-
reducing bacterium from the hindgut of the termite Heterotermes
indicola (Wasman). System. Appl. Microbiol. 13: 372–377.
Voloudakis A.E., Bender C.L. and Cooksey D.A. 1993. Similarity
between copper resistance genes from Xanthomonas campestris
and Pseudomonas syringae. Appl. Environ. Microbiol. 59:
Webb J.S., McGinness S. and Lappin-Scott H.M. 1998. Metal
removal by sulphate-reducing bacteria from natural and con-
structed wetlands. J. Appl. Microbiol. 84: 240–248.
Widdel F. and Bak F. 1992. Gram-negative mesophilic sulfate-
reducing bacteria. In: Balows A., Truper H.G., Dworkin M.,
Harder W. and Schleifer K.-H. (eds), The prokaryotes: a hand-
book on the biology of bacteria: ecophysiology, isolation, identi-
fication, applicationsVol. 4. 2nd edn. Springer-Verlag, Berlin, pp.
Wielinga B., Lucy J.K., Moore J.N., Seastone O.F. and Gannon J.E.
1999. Microbiological and geochemical characterization of flu-
vially deposited sulfidic mine tailings. Appl. Environ. Microbiol.
Williams J.R., Morgan A.G., Rouch D.A., Brown N.L. and Lee
B.T.O. 1993. Copper-resistant enteric bacteria from United
Kingdom and Australian piggeries. Appl. Environ. Microbiol.