FEMS Microbiology Ecology 27 (1998) 319^325
The in£uence of mercury on the antioxidant enzyme activity of
rumen bacteria Streptococcus bovis and Selenomonas ruminantium
Viera Lenaèrtovaè a; *, Katar|èna Holovskaè a , Peter Javorskyè
a
b
Department of Chemistry, Biochemistry and Biophysics, University of Veterinary medicine, 04181 Kosíice, Slovakia
b
Institute of Animal Physiology, Slovak Academy of Science, Kosíice, Slovakia
Received 13 May 1998; revised 14 July 1998 ; accepted 14 July 1998
Abstract
Studies were undertaken to investigate the activity response of the antioxidant enzymes superoxide dismutase (SOD),
glutathione peroxidase (GSHPx), glutathione reductase (GR) and mercury reductase (MR) of rumen bacteria Streptococcus
bovis and Selenomonas ruminantium following exposure to HgCl2 . SOD activity of S. bovis which was considered as Mn-SOD
increased when incubated with 5 Wg Hg2‡ ml31 in anaerobic or aerobic conditions. A significant increase in the aerobic
activities of GSHPx, GR and MR was observed in the presence of Hg2‡ . The anaerobic activities of these enzymes were
unchanged and increased production of thiobarbituric acid reactive substances was observed. S. ruminantium was tolerant to
10 times higher concentrations of Hg2‡ than S. bovis. As a reduction of GSHPx, GR and MR activities after exposure to Hg2‡
was observed, we assume that the production of sulfides prevented the toxic effect of mercury on this bacterium. z 1998
Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Mercury ; Antioxidant enzyme ; Streptococcus bovis ; Selenomonas ruminantium
1. Introduction
The toxicity of mercury and its ability to react
with free sulfhydryl groups is well known [1]. Primary exposure occurs through environmental contamination as the result of mining, smelting and industrial discharge and includes ingestion via
inhalation and the food chain. Ruminant animals
can be exposed to toxic concentrations of mercurials
by the consumption of contaminated feed and water.
* Corresponding author. Tel./Fax: +421 (95) 6334768;
E-mail: [email protected]
Heavy metals can be inhibitory to both the fermentative activity and growth of the organisms present
in the reticulorumen, thereby decreasing the productivity of animals. Alternatively microbes may also
modify the toxicity of the elements to the animal
by decreasing their toxicity, e.g. sul¢de production
resulting in the precipitation of heavy metals [2].
In the environment, mercury can exist in the elemental form, as inorganic monovalent and divalent
salts, and as organomercurials such as methyl mercury. Mercury is a well-known pro-oxidant. Studies
with HgCl2 have demonstrated that it exerts oxidative stress via H2 O2 generation, GSH depletion, and
reactivity with membrane bound protein thiols and
these may lead to lipid peroxidation [3^5].
0168-6496 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 6 4 9 6 ( 9 8 ) 0 0 0 7 7 - 4
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Fig. 1. Levels of antioxidant enzymes and lipid peroxidation products in S. bovis 4/1 under aerobic (A) or anaerobic (B) conditions. The
assays were performed as described in Section 2. The results are expressed in mU mg protein31 (*P 6 0.05; asterisks represent signi¢cant
di¡erences between the control group (0) and bacteria grown in the presence of 5 Wg Hg2‡ ml31 ).
Bacterial resistance to mercury and organomercurials has been reported in some bacteria such as Escherichia coli, Staphylococcus aureus and some Pseudomonas species [6^8]. The e¡ect of heavy metals
including mercury on the fermentation of di¡erent
rumen bacteria was described by Fosberg [2]. Our
study was initiated to determine how the rumen facultatively anaerobic bacterium Streptococcus bovis [9]
and the strictly anaerobic bacterium Selenomonas ruminantium [10] respond to environmental stress
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Fig. 2. Nondenaturing PAGE analyses of SOD activity of S. bovis. After electrophoresis (100 Wg of protein per lane) the gel was
soaked in 10 mM KCN for 30 min, then covered with a solution
containing nitroblue tetrazolium and ribo£avin, and exposed to
light. Cell extracts were prepared from bacteria grown under
aerobic (A) or anaerobic (B) conditions. The samples are: 0, control, and 5 Wg Hg2‡ ml31 .
evoked by the mercury ion. We have studied whether
mercury results in alterations in the activities of antioxidant defence enzymes including superoxide dismutase (SOD), glutathione peroxidase (GSHPx),
and glutathione reductase (GR) and also mercuric
reductase (MR) which can play a critical role in
bacterial resistance to mercuric salts.
2. Materials and methods
2.1. Chemicals
All reagents, of the highest purity, were from Sigma, Merck and Boehringer.
2.2. Bacterial strains and growth conditions
Streptococcus bovis 4/1 [11] and Selenomonas ruminantium E32 [12] used in this study were isolated
from the rumen of sheep and both are maintained
in our microbe collection. S. bovis 4/1 was grown
aerobically overnight at 37³C in Todd-Hewitt broth
(Imuna, Slovakia) containing 0 and 5 Wg Hg2‡ ml31
in the form of mercuric chloride or anaerobically in
321
the same medium prepared with a gas phase of
CO2 containing 0 and 5 Wg Hg2‡ ml31 . S. ruminantium E32 was also grown anaerobically overnight
at 37³C in the selective M medium described by
Tiwari et al. [13] in the presence of 0, 5 or 50 Wg
Hg2‡ ml31 .
The cultures were harvested by centrifugation at
4³C at 10 000Ug for 15 min and washed in potassium phosphate bu¡er containing 0.1 mM EDTA, pH
7.4, pelleted by centrifugation as before, and resuspended in the same bu¡er. The cells were disrupted
by sonication for a 30-s burst for a total of 3 min
with a 1-min cooling period after each burst using a
MSE Soniprep 150 ultrasonic disintegrator at 4³C.
Cellular debris was removed by centrifugation at
12 000Ug for 15 min, the supernatants were dialyzed
against a potassium phosphate bu¡er and used for
the enzyme assays.
2.3. Enzyme assays
Superoxide dismutase activity (SOD, EC 1.15.1.1)
was determined by measuring the inhibition of cytochrome c reduction using xanthine/xanthine oxidase
Oc3
2 generating system at 550 nm [14]. One unit of
SOD activity was de¢ned as that amount of enzyme
causing 50% inhibition of cytochrome c reduction
under the assay conditions.
SOD isoenzymes were separated on 10% nondenaturing polyacrylamide gels [15] and the enzyme activity was visualized as achromatic bands by staining with nitroblue tetrazolium chloride according
to Beauchamp and Fridovich [16]. To identify the
isoenzymes of SOD, gels were treated with 10 mM
KCN or 5 mM H2 O2 in a bu¡er for 30 min to
inactivate Cu/Zn-SOD or Fe-SOD respectively
[17].
Glutathione peroxidase activity (GSHPx, EC
1.11.1.9) was measured by monitoring the oxidation
of NADPH at 340 nm as described by Floheè and
Guënzler [18] in a coupled assay with glutathione reductase. Cumene hydroperoxide (for Se independent
activity) or H2 O2 (for Se dependent activity) were
used as substrates.
Glutathione reductase (GR, EC 1.6.4.2.) was determined by following the decrease in NADPH absorbance at 340 nm due to GSSG reduction [19].
Mercuric reductase (MR, reduced NADP:mercu-
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Fig. 3. Levels of antioxidant enzymes and lipid peroxidation products in S. ruminantium E32. The assays were performed as described in
Section 2. The results are expressed in mU mg protein31 (*P 6 0.05; asterisks represent signi¢cant di¡erences between control group (0)
and bacteria grown in the presence of 5 Wg Hg2‡ and 50 Wg Hg2‡ ml31 ).
ric ion oxidoreductase) was assayed by following
Hg2‡ dependent NADPH oxidation according to
Fox and Walsh [20].
One unit of enzyme activity (MR, GSHPx) was
de¢ned as the amount of enzyme that catalyzes the
formation of 1 Wmol of product per minute under the
assay conditions. Speci¢c activity was de¢ned as the
unit of enzyme activity per mg of protein.
Protein concentration was measured by the method of Bradford [21], using bovine serum albumin as
a standard.
Lipid peroxidation products measured as thiobarbituric acid reactive substances (TBARS) were determined according to Gutteridge [22].
2.4. Statistics
The results are given as means þ S.E.M. of three
independent determinations in three di¡erent
batches. Data were analyzed using Student's t-test
with a signi¢cance level of P 6 0.05.
3. Results
The inhibitory e¡ect of Hg2‡ in the form of HgCl2
on the growth of the facultatively anaerobic bacterium S. bovis was examined in the presence of 5 Wg
of Hg2‡ ml31 when cultivated in aerobic or anaerobic conditions. The obligate anaerobe S. ruminantium grew well in the presence of 5 and 50 Wg
Hg2‡ ml31 .
3.1. Enzyme activities of S. bovis 4-1
SOD activities of S. bovis were similar under anaerobic or aerobic conditions (189.6 þ 1.0 mU mg
protein31 ; 196.8 þ 29.1 mU mg protein31 , respectively). Incubation with Hg2‡ produced a signi¢cant
increase in total SOD activity to 448.4 þ 73.3 mU mg
protein31 in aerobic and 298.8 þ 41.3 mU mg
protein31 in anaerobic conditions (Fig. 1). This activity was not inhibited by 2 mM KCN (data not
shown). When analyzed by native PAGE, a single
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Mn-SOD band, una¡ected by l0 mM cyanide or
5 mM H2 O2 , was seen under aerobic or anaerobic
conditions (Fig. 2). The GSHPx activity determined
was only Se independent and was almost identical
under both aerobic and anaerobic incubation conditions. The aerobic activity of GSHPx was 2.6-fold
higher in the presence of Hg2‡ (Fig. 1). Signi¢cant
increases in both aerobic activity of GR (1.4-fold)
and MR (1.2-fold) in the presence of Hg2‡ were
also observed. The anaerobic activities of the three
antioxidant enzymes examined, GSHPx, GR and
MR, were the same in the presence of Hg2‡ as in
the control group. The changes in the aerobic enzyme activities led us to study whether mercuric
ion evoked oxidative stress in bacteria. We analyzed
malondialdehyde and other lipid peroxidation products by determining the levels of thiobarbituric acid
reactive substances. TBARS contents were unchanged which suggests that higher enzyme activity
was able to prevent the increased production of
TBARS. Anaerobic incubation of S. bovis evoked
an increase in TBARS content in the presence of
Hg2‡ (Fig. 1).
3.2. Enzyme activities of S. ruminantium E32
The obligately anaerobic rumen bacterium S. ruminantium showed no SOD activity. Se independent
GSHPx decreased 3.0-fold and MR 1.56- and 1.76fold in the presence of 5 Wg or 50 Wg Hg2‡ ml31 ,
respectively. GR activity was not in£uenced by
Hg2‡ . TBARS content was signi¢cantly increased
in the presence of 50 Wg Hg2‡ ml31 (Fig. 3).
4. Discussion
In eukaryotes mercury disrupts the structural integrity of the inner mitochondrial membrane, resulting in altered ion permeability and membrane potential, causing leakage of both H2 O2 and Oc3
2 from the
electron transport chain [5]. A variety of bacteria, all
facultative anaerobes, have developed e¤cient enzymatic and nonenzymatic mechanisms to eliminate
the oxygen by-products along with synthesis of
DNA repair enzymes and oxidative defence regulators [23]. The present study was undertaken to investigate how mercury might in£uence the antioxidant
323
defence enzymes SOD, GSHPx and GR of the rumen amylolytic facultatively anaerobic bacterium S.
bovis which represents a part of the rumen epimural
micro£ora [9] and S. ruminantium, an obligate anaerobe that has been reported to represent up to 16%
of the total bacterial £ora in the rumen [10]. We also
followed the activity of MR, an enzyme which reduces mercury salts to volatile, elemental mercury
and which has been identi¢ed as a key component
in mercury detoxi¢cation in many bacteria [20]. The
¢rst line of defence against the generation of toxic
oxygen species is the induction of SOD. In our experiments SOD activity was observed in S. bovis
when incubated under both aerobic and anaerobic
conditions. No SOD activity was found in S. ruminantium. Currently, very little is known about the
oxidative stress response of rumen bacteria, whereas
extensive studies on the regulation and enzymology
of SOD in other microorganisms have been carried
out [24]. Bacterial SODs have been intensively
studied in E. coli. This facultative anaerobe possesses
two cytoplasmic SODs, one of which is cofactored
by iron (Fe-SOD) and the other of which is cofactored by manganese (Mn-SOD), and a periplasmatic
SOD cofactored by copper and zinc (Cu/Zn-SOD)
[25^27]. Mn-SOD is induced in response to a variety
of environmental stress conditions including exposure to oxygen, redox cycling compounds such as
paraquat, iron chelation and oxidants [28]. This enzyme is encoded by the sodA gene, the expression of
which is under rigorous control of a multiregulated
system involving six redox sensitive proteins which
enable the cell to respond quickly and e¤ciently either to activate or to repress Mn-SOD expression
when encountering aerobic or anaerobic environments, respectively [29]. In contrast to E. coli, S.
thermophilus, a Gram-positive facultative anaerobe,
possesses a single manganese-containing SOD. The
speci¢c activity for Mn-SOD was the same under
anaerobic or aerobic conditions and was not induced
by the presence of paraquat under aerobic conditions
[30]. The inhibition study to di¡erentiate SOD isozymes of S. bovis in our experiment indicated that
this activity was a Mn-SOD and was activated by
mercury. To determine whether the Mn-SOD gene
of ruminal strains of S. bovis in our experiment is
also regulated by other environmental signals, further studies are necessary.
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Since mercury is known to deplete free thiols [31],
the levels of the glutathione dependent enzymes
GSHPx and GR were determined. While GSHPx is
widely distributed in animal tissues [5,32], its occurrence in microorganisms is still uncertain. The most
important GSH producing bacteria, e.g. the purple
bacteria and cyanobacteria and E. coli, were found
to lack any signi¢cant GSH peroxidase and transferase activities. In this study both bacteria only exhibited the selenium independent form of GSHPx,
which acts mainly on organic hydroperoxides and
is related to certain GSH S-transferase isoenzymes
[33,34]. We observed increased aerobic and unchanged anaerobic GSHPx activities of S. bovis in
the presence of mercury.
Several studies have con¢rmed that the activity of
GSHPx was suppressed in response to mercury in
vertebrate systems [35,36]. On the other hand, the
induction of GSHPx and GR was demonstrated in
strains of Pseudomonas putida, especially in cells oxidizing trivalent arsenite which is thought to initiate
free radical lipid peroxidation yielding malonic aldehyde [31]. Also in the house£y Musca domestica, an
increase in GSHPx level was observed in response to
HgCl2 [37].
In the rumen bacterium S. bovis a relatively high
NADPH speci¢c GR activity was found. As GR is
thought to play an important role in maintaining
cellular thiol groups in a reduced state, we assume
that the increased level of GR activity in the presence of Hg2‡ was able to reduce GSSG to GSH
necessary for GSHPx activity and prevent the oxidative damage. This scenario could be supported by
unchanged TBARS content whereas in anaerobic
conditions unchanged activities of these enzymes in
the presence of Hg2‡ evoked an increase in TBARS
content. It is also possible that increased aerobic MR
activity participates in mercury detoxi¢cation in S.
bovis. This £avoenzyme is unique in the reduction of
the mercuric ion to elemental mercury which is much
less toxic to the bacterial cell [20,38].
Many studies have shown that anaerobic bacteria
are not uniformly sensitive to oxygen and there is a
broad range of oxygen tolerance [39,40]. The results
presented here show that S. ruminantium possesses
GSHPx, GR and MR activities. It is interesting
that despite the inhibition of GSHPx and MR activities in the presence of Hg2‡ , S. ruminantium was
tolerant to a 10 times higher concentration of Hg2‡
(50 Wg ml31 ) than S. bovis. This low sensitivity to
Hg2‡ is probably a result of sul¢de production,
which was evident from the black precipitates
formed during the growth of this bacterium. Bacteria
responsible for sul¢de production in the rumen produce sul¢de from sulfur containing amino acids or
from sulfate [2,41]. Our results assume that the production of sul¢des prevented the toxic e¡ects of
Hg2‡ to S. ruminantium. To elucidate the role of
antioxidant enzymes in the defence mechanisms of
rumen bacteria against environmental pollutants,
further more complete studies are necessary.
Acknowledgments
This investigation was supported by Grant Agency
VEGA 4003/97.
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