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Previous Article | Next Article 
Applied and Environmental Microbiology, October 1998, p. 4098-4102, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Loss of Ammonia Monooxygenase Activity in Nitrosomonas
europaea upon Exposure to Nitrite
Lisa Y.
Stein and
Daniel J.
Arp*
Department of Botany and Plant Pathology,
Oregon State University, Corvallis, Oregon 97331
Received 16 April 1998/Accepted 28 July 1998
 |
ABSTRACT |
Nitrosomonas europaea, an obligate ammonia-oxidizing
bacterium, lost an increasing amount of ammonia oxidation activity upon exposure to increasing concentrations of nitrite, the primary product
of ammonia-oxidizing metabolism. The loss of activity was specific to
the ammonia monooxygenase (AMO) enzyme, as confirmed by a decreased
rate of NH4+-dependent O2
consumption, some loss of active AMO molecules observed by polypeptide
labeling with 14C2H2, the
protection of activity by substrates of AMO, and the requirement for
copper. The loss of AMO activity via nitrite occurred under both
aerobic and anaerobic conditions, and more activity was lost under
alkaline than under acidic conditions except in the presence of large
concentrations (20 mM) of nitrite. These results indicate that nitrite
toxicity in N. europaea is mediated by a unique
mechanism that is specific for AMO.
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TEXT |
Ammonia oxidation by
Nitrosomonas europaea is mediated by two enzymes, ammonia
monooxygenase (AMO), which catalyzes the oxidation of NH3
to hydroxylamine (NH2OH), and hydroxylamine oxidoreductase (HAO), which catalyzes the oxidation of NH2OH to
NO2
(26). For every molecule of
NH3 oxidized, one molecule of NO2
is produced. Additionally, ammonia oxidation is an acidogenic reaction.
As the pH of the incubation medium decreases during ammonia oxidation,
the NH3-NH4+ equilibrium is shifted
away from NH3, the true substrate of AMO (23).
Ammonia oxidation ceases at about pH 6, partially because of the
reduced concentration of NH3. At the point of maximal cell growth in phosphate-buffered medium with an initial ammonium
concentration of 50 mM, the concentration of nitrite is typically
around 20 to 25 mM, and the rest of the ammonium remains unoxidized. In a previous study, we showed that this remaining
NH3-NH4+ pool, or other substrates
of AMO, had a specific protective effect on the AMO activity of
N. europaea cells (22). In incubations where
the ammonium was completely consumed, the cells lost up to 80% of
their ammonia-oxidizing activity over a 24-h period (22). In
the present paper, we show that this specific loss of AMO activity in
cells of N. europaea is due to the toxicity of
accumulated nitrite in the incubation medium.
Surprisingly, only a few reports describing the toxicity of
nitrite in ammonia-oxidizing bacteria have been published, and the
mechanism of toxicity remains unclear. In one study, nitrite toxicity
in Nitrosomonas sp. occurred only at very high
concentrations (greater than 30 mM) of nitrite, and this effect, as
measured by the ability of the cells to consume O2, was
greater during the lag phase than during the log phase of growth at
several different pH values tested (16). Nitrite was also
toxic for cells in the log phase of growth, but only at acidic pH
values, and the loss of activity was reversible upon washing of the
cells (16). Other studies have shown that ammonia oxidizers
are sensitive to nitrite accumulation in sewage treatment plants, but
the mechanism of this sensitivity was not examined (2).
Because batch cultures of N. europaea accumulate large
concentrations of nitrite, greater than 20 mM, and reach a pH of about 6, it is surprising that the cells are not more susceptible to nitrite
toxicity. In cultures of Clostridium sporogenes, a food spoilage bacterium, 10 mM nitrite has profound bacteriocidal effects, especially at pH values below 7 (6). Many species of
bacteria are susceptible to nitrite toxicity because of the formation
of metal-nitrosyl complexes that occurs when NO+ or NO
radicals interact with bacterial enzymes (28). The radicals, NO+ and NO, form spontaneously from nitrite most favorably
in acidic media.
The present study shows that nitrite can cause a specific loss of
ammonia-oxidizing activity in N. europaea cells at
lower concentrations than those previously considered (5 to 20 mM). The
loss of activity is specific to AMO and occurs under both acidic and
alkaline conditions. In the presence of substrates of AMO, the loss of
activity was not observed. Furthermore, the loss of activity was not
reversible by washing the cells. Lastly, nitrite appears to
specifically target the AMO enzyme in a manner different from that of
characterized inactivators of AMO.
Analysis of batch incubations of N. europaea
containing ammonium or nitrite.
Cells of N. europaea (ATCC 19178) were grown to late log phase, harvested, and
washed as described previously (22). Cells (ca.
109 cells ml1) were incubated in medium (25 ml) initially containing between 0 and 50 mM ammonium for 24 h.
The changes in the ammonia and hydroxylamine oxidation activities, as
measured by NH4+- and
NH2OH-dependent O2 uptake rates, respectively
(12), and the nitrite concentrations in the incubation
medium (9) were monitored. A decrease in the ammonia
oxidation activity was observed over 24 h at intermediate ammonium
concentrations, between 0 and 50 mM, with the greatest loss occurring
at 15 to 25 mM (Fig. 1A). For example, in
the incubation that contained no ammonium, the ammonia oxidation
activity decreased to about 81% of the original level after 24 h.
With 15 mM ammonium, the point of maximal activity loss, the ammonia
oxidation activity decreased to approximately 18% of the initial
level. In the incubation containing 50 mM ammonium, the ammonia
oxidation activity decreased to only about 63% of the initial level.
In all of the incubations, there was little change in the hydroxylamine
oxidation activity after 24 h, confirming the previous observation
that incubations containing different concentrations of ammonium
specifically affect the ammonia oxidation activity of the cells (Fig.
1A) (22).
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FIG. 1.
Ammonia and hydroxylamine oxidation activities and
concentrations of nitrite and ammonium after a 24-h incubation of
N. europaea cells in ammonium-containing media. Cells
were incubated in growth medium containing from 0 to 50 mM ammonium.
Washed cells were sampled for O2 consumption rates, and the
supernatant was assayed for pH and nitrite accumulation. Error bars
represent the standard deviations from the averages of six replicate
experiments. (A) Remaining NH4+-dependent ( )
and NH2OH-dependent ( ) O2 uptake rates for
cells incubated in media containing from 0 to 50 mM
NH4+ for 24 h relative to the amount of
initial activity. The rates of NH4+- and
NH2OH-dependent O2 uptake at 100% activity
were approximately 120 and 35 nmol of O2 consumed
min1 ml of cells1, respectively. (B)
Measured concentration of nitrite ( ) and calculated concentration of
ammonium ( ) (assuming minimal incorporation into cellular biomass)
in the medium from the incubations in panel A after 24 h.
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The trend of ammonia oxidation activity loss was correlated with the
relative proportions of nitrite produced and ammonium remaining in the
incubation medium after 24 h (Fig. 1B). The accumulation of
nitrite in the medium was proportional to the initial concentration of
ammonium up to 20 mM. In incubations containing 25 to 50 mM ammonium,
nitrite accumulation ceased after reaching about 21 mM. At this point,
the limiting pH for ammonia oxidation, 5.7 to 6.0, was reached. The
greatest losses of ammonia oxidation activity were observed with the
largest concentrations of nitrite, from 15 to 21 mM, and the smallest
concentrations of ammonium, from 0 to 5 mM, remaining in the medium
after the 24-h incubation.
Incubations were also conducted in the absence of ammonium and in the
presence of increasing concentrations of nitrite, from 0 to 20 mM, in
medium at a constant pH of 8. Increased loss of ammonia oxidation
activity with increased nitrite concentration was observed over the
24-h incubation, although several hours were required for substantial
losses of activity to occur (data not shown). The greatest loss of
ammonia oxidation activity, approximately 62%, occurred with 20 mM
nitrite in the incubation. Again, the hydroxylamine oxidation activity
was not strongly affected by nitrite in any of the incubations. After
the incubations with nitrite, cells were sedimented, washed, and
resuspended in sodium phosphate buffer for 48 h to determine if
the effect of nitrite was reversible. No activity was recovered during
these incubations, suggesting an irreversible inactivation of the
ammonia oxidation activity by nitrite (data not shown).
The effect of O2 and CH4 on the loss of
ammonia oxidation activity.
To further characterize the mechanism
of nitrite toxicity for ammonia oxidation activity, we incubated cells
in concentrations of nitrite ranging from 0 to 20 mM both aerobically
and anaerobically and with or without CH4, an alternative
substrate for AMO (Fig. 2). The
incubations without O2 were conducted in glass serum vials (160 ml) sealed with butyl rubber stoppers and aluminum crimp seals.
The vials were evacuated and reequilibrated five times with
O2-free N2. Complete anaerobiosis was monitored
by gas chromatography (thermal conductivity detector) and the lack of
nitrite production in the vials upon addition of ammonium. The amount
of ammonia oxidation activity remaining after 24 h was measured in
each treatment. The cells retained about 10% more activity on average
in the absence than in the presence of O2 in the
incubations without CH4; however, the differences were not
substantial (Fig. 2A). Increasing amounts of nitrite resulted in
increased losses of ammonia oxidation activity in both the aerobic and
the anaerobic incubations. Thus, it did not appear that O2
was required for the disabling effect of nitrite on ammonia oxidation
activity.
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FIG. 2.
Response of the ammonia oxidation activity in cells
exposed to nitrite under aerobic or anaerobic conditions with or
without CH4. N. europaea cells were
incubated in media containing the indicated concentrations of nitrite.
(A) Incubations were conducted both aerobically (solid bars) and
anaerobically (shaded bars). (B) Incubations were conducted aerobically
(open bars) and anaerobically (hatched bars) in the presence of 25%
(vol/vol of the gas headspace) CH4. The ammonia oxidation
activity after a 24-h incubation relative to the initial activity is
shown. Error bars represent the standard deviations from the averages
of three replicate experiments.
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Incubations with and without 10 mM nitrite were conducted aerobically
and anaerobically in the presence of 25% (vol/vol of the gas
headspace) CH4 to verify the protective effect of
alternative substrates on ammonia oxidation activity as shown in a
previous study (22). In all of the incubations, an average
of 78% of the ammonia oxidation activity remained, indicating that
CH4 protected the activity both aerobically and
anaerobically and in the presence of nitrite (Fig. 2B).
Effect of pH on nitrite-mediated loss of ammonia oxidation
activity.
The loss of ammonia oxidation activity was 20% greater
in incubations initially containing 15 mM ammonium than in incubations containing 20 mM nitrite alone. Because of this discrepancy and the
dynamic changes in pH that occur in incubations with ammonium, the
possibility of pH being a mediator of activity loss was investigated. Incubations containing 0, 5, 10, or 20 mM nitrite were conducted at a
range of fixed pH values from 5.5 to 8. Without nitrite, the ammonia
oxidation activity remaining after 24 h was the same regardless of
the initial pH (Fig. 3). However, when
nitrite was included in the incubations, the ammonia oxidation activity
decreased more at alkaline (pH 7 to 8) than at acidic (pH 5.5 to 6.5)
pH values, with up to 10 mM nitrite. Activity loss also occurred with a
high concentration of nitrite, 20 mM, at an acidic pH (5.5 to 6). Only
a slight loss of activity was observed with 10 mM nitrite at pH 5.5, indicating that, although nitrite toxicity does occur at both alkaline
and acidic pH values, it is dependent upon the concentration of nitrite
in the incubation medium.
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FIG. 3.
Effect of nitrite on ammonia oxidation activity at
different pH values. N. europaea cells were incubated
in media with 0 (), 5 ( ), 10 (), or 20 () mM nitrite at a
range of pH values from 5.5 to 8.0. The ammonia oxidation activity
remaining after a 24-h incubation was determined relative to the
initial activity. Error bars represent the standard deviations from
four replicate experiments.
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The extent of activity loss at alkaline pH was unexpected because most
of the chemistry that transforms nitrite into active compounds, e.g.,
those that form metal-nitrosyl complexes, occurs at an acidic pH
(28). Thus, traditional nitrite chemistry can explain the
concentration-dependent loss of activity at pH 5.5, but another
mechanism must also exist for the activity loss observed at pH 8. Furthermore, in incubations containing 15 mM ammonium (Fig. 1), the pH
declined to about 6.6 and 80% of the ammonia oxidation activity was
lost. In contrast, with 20 mM nitrite at the same pH, only about 40%
of the activity was lost (Fig. 3). These results suggest that the
interaction between pH and nitrite must be more complex during the
dynamic process of ammonia oxidation.
Quantification of the active AMO polypeptide pool by
14C2H2.
The pool of active AMO
enzyme molecules was examined by radiolabeling with
14C2H2 to determine whether the
loss in ammonia oxidation activity, characterized in the preceding
experiments, was due to the inactivation of the AMO enzyme. Exposure of
N. europaea cells to
14C2H2 results in the specific
incorporation of radiolabel into the 27-kDa, active-site containing
subunit of AMO (12). The amount of radiolabel incorporated
is proportional to the amount of active AMO enzyme at the time of
exposure to 14C2H2. Cells of
N. europaea were incubated with 0, 15, or 50 mM ammonium; 10 mM nitrite; or 10 mM nitrite without O2. Cells
(1 ml, sedimented) were taken from each of these treatments at an initial time point and after 24 h and were incubated for 2 h
in sodium phosphate buffer (900 µl),
14C2H2 (~300 µCi), and either
hydrazine (2 mM), tetramethylhydroquinone (TMHQ; 0.5 mM), or endogenous
reductant. Incubations containing 10 mM NO2
and 15 mM ammonium were also analyzed after incubation with
14C2H2 for 1 and 3 h to verify
that the labeling reactions had reached completion after a 2-h
incubation.
Hydroxylamine provides reductant to AMO via HAO (26), TMHQ
likely provides reductant through a ubiquinone pathway (20), and endogenous reductant is likely mediated through NADH
(25). Because each of the reductant sources may involve
different electron carriers at some point in the electron transport
pathway, a difference in label incorporation would suggest that an
electron transport molecule between AMO and HAO, such as cytochrome
c554 or a quinone, may be damaged by nitrite
rather than by AMO itself. After exposure to
14C2H2, the cells were sedimented,
resuspended in sodium dodecyl sulfate-polyacrylamide gel
electrophoresis loading buffer (100 µl), and frozen at 
80°C. Cell
extracts were thawed and vortexed (1 min), and the polypeptides were
separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(12% polyacrylamide). Incorporation of 14C into
polypeptides was analyzed on a PhosphorImager (Molecular Dynamics,
Sunnyvale, Calif.). The apparent masses of labeled polypeptides were
determined by comparison with Rf values for
molecular mass markers as described previously (12).
Densitometric values were determined with ImageQuant software.
The amount of radiolabel incorporation into polypeptides after 24 h relative to the amount at the initial time point was determined (Fig.
4). Treatments containing 15 mM ammonium
or 10 mM nitrite with or without O2 (treatments 2, 4, and
5, respectively) resulted in about a 15 to 40% decrease in the amount
of label incorporation into the 27-kDa polypeptide after 24 h.
However, these same treatments exhibited a 50 to 80% decrease in
ammonia oxidation activity as measured by the O2 electrode.
These results indicate that nitrite does not initially cause a complete
inactivation of AMO molecules but rather that nitrite lowers the rate
of AMO activity. However, some molecules of AMO are completely
inactivated, as indicated by the loss of 15 to 40% of radiolabeled
polypeptide in these same treatments.
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FIG. 4.
14C2H2 labeling of
cells exposed to ammonia or nitrite for 24 h. Cells of
N. europaea were incubated in media with the following
treatments: 1, no additions; 2, 15 mM ammonium; 3, 50 mM ammonium; 4, 10 mM nitrite; and 5, 10 mM nitrite and without O2.
Aliquots of cells were exposed to
14C2H2 at the beginning of the
treatments and after 24 h, as described in the text, to radiolabel
the active AMO population. The difference in radiolabel accumulation
between the initial and 24-h time points is shown for each treatment as
an average of two replicate experiments. Labeling reaction mixtures
contained 2 mM hydrazine (solid bars), 0.5 mM TMHQ (shaded bars), or
endogenous reductant (open bars).
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There were no major differences in label incorporation with the
different reductant sources for each treatment. The same amount of
label was incorporated with endogenous reductant as with added hydrazine or TMHQ, indicating that very little reductant was necessary for the complete labeling of the AMO polypeptide population by 14C2H2. Therefore, even if an
electron carrier was debilitated by nitrite, the low level of
endogenous reductant flow would circumvent the crippled pathway. This
result alone was unable to verify whether AMO or an accessory molecule
was the target for nitrite. As a better test for the involvement of
AMO, cells were incubated in the presence of the copper chelator
thiourea.
The role of copper in mediating the toxicity of nitrite.
Based
on several lines of evidence, AMO is proposed to contain copper
(7, 19, 27). Thiourea chelates cuprous copper, which leads
to the inactivation of copper-containing enzymes, including AMO
(3, 10). Cells were incubated with 0, 10, or 20 mM nitrite
either in the presence or in the absence of thiourea (100 µg
ml1). The loss of AMO activity by thiourea is partially
reversible by stringent washing and addition of copper. Once the
thiourea-treated cells were washed and copper was reintroduced, about
50 to 60% of the ammonia oxidation activity was recovered (Fig.
5). The initial activity of the cells
which were not exposed to nitrite or thiourea was designated as 100%,
and the activity remaining after 24 h in all of the incubations
was relative to this 100% activity level. Cells exposed to thiourea
maintained approximately the same amount of activity after 24 h
with or without nitrite in the incubations, indicating that copper had
to be present for nitrite to inflict damage on the rate of
O2 consumption by the cells. Because copper plays a major
role in maintaining AMO activity, it is likely that the removal of
copper from AMO resulted in the inability of nitrite to inactivate the
ammonia-oxidizing activity of the cell.
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FIG. 5.
Effect of thiourea on the loss of ammonia oxidation
activity in cells exposed to nitrite. N. europaea cells
were incubated in media containing 0, 10, or 20 mM nitrite either with
or without the copper chelator thiourea (100 µg ml1).
The initial activity of cells incubated without thiourea (solid bars)
and with thiourea (open bars) and the amount of activity remaining
after 24 h without thiourea (shaded bars) and with thiourea
(hatched bars) are shown. Error bars represent the standard deviations
from the averages of three replicate experiments.
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Specificity of nitrite in mediating the loss of ammonia-oxidizing
activity.
We attempted to determine whether the loss of ammonia
oxidation activity was specific to nitrite or if other chemicals
related to nitrite chemistry could also induce activity loss. Nitric
oxide (NO) and nitrous oxide (N2O) are produced by
N. europaea from either the reduction of nitrite or the
oxidation of hydroxylamine during ammonia metabolism (1, 11,
24). In many systems, NO is known to be toxic, especially through
the formation of metal-nitrosyl complexes when NO interacts with
metal-containing enzymes (5, 28). Furthermore, it has been
reported recently that a putative iron moiety within the AMO enzyme can
interact with NO in cell extracts (27). There is no evidence
that N2O is toxic to N. europaea, but
because it is a product of nitrite reduction, we tested whether it
could mediate activity loss. Addition of NO or N2O to
incubations had little effect on the ammonia oxidation activity of
N. europaea, and the observed effects were not specific for AMO (data not shown). Similarly, no loss of activity was observed in the presence of NO3, a compound which is
similar to nitrite in size, composition, and charge. Therefore,
these results suggest that nitrite itself, rather than a known
product of nitrite chemistry, causes the activity loss.
Taking all of the results together, we suggest that nitrite
lowers the ammonia oxidation activity of N. europaea by specifically debilitating AMO. Mechanism-based
inactivators of AMO require the presence of O2, because the
product of oxidation, rather than the substrate itself, is the
toxic agent (21). Therefore, nitrite cannot be considered a
mechanism-based inactivator of AMO, and the low rate of inactivation
suggests that nitrite also does not belong to two other known classes
of AMO inactivators, copper chelators (e.g., thiourea) or metabolic
inactivators (e.g., nitrapyrin) (3, 17). Furthermore,
nitrite does not cause a complete loss of activity as do many of these
other inactivators. However, inactivation of AMO by CS2
does not require O2 and potentially involves a reversible interaction with a nucleophilic amino acid that is in close proximity to the putative copper molecule in the AMO active site (13). Although nitrite might target AMO in a manner similar to that of
CS2, the loss of activity mediated by nitrite is not
reversible and occurs much slower than inactivation by CS2,
suggesting again that nitrite is a unique inactivator of AMO.
Although we cannot discount the possibility that nitrite may interact
with AMO as NO2, it seems more logical that
nitrite would be converted to a reactive species before interacting
with the enzyme, especially if the target is a metal-containing moiety
(4, 28). One possible mechanism is that nitrite could be
reduced by AMO to a reactive species (e.g., NO or a nitrosyl radical)
which targets a copper or iron molecule in close proximity to the
enzyme active site, forming a covalently bound metal-nitrosyl complex.
Once the complex is formed, the catalytic rate of the enzyme may be
lowered initially by a debilitation of O2 binding and
activation, substrate binding and oxidation, or reduction of the active
site. Furthermore, the debilitation of AMO by nitrite may render the
enzyme more susceptible to complete inactivation under the
appropriate conditions (Fig. 4). Nitrite most likely interacts
with the reduced form of AMO because, under anaerobic conditions,
most of the enzyme will be in a reduced state and nitrite is
still toxic in this situation (Fig. 2). Although there is only indirect
evidence for the involvement of AMO as the target of nitrite, the
ability of substrates to protect the activity and the role of copper in
mediating toxicity imply that AMO, and not an accessory enzyme
(e.g., an electron carrier), is the target.
The loss of ammonia-oxidizing activity experienced by N. europaea upon exposure to nitrite may influence the ecology of
ammonia oxidizers. For example, although the interactions between
ammonia- and nitrite-oxidizing bacteria are dependent upon several
factors including substrate availability, O2, and pH
(14), the toxic effect of nitrite on ammonia oxidizers could
be ameliorated by the close physical association between the two
bacterial groups as observed in soils and bioreactors (15,
18). The close physical positioning of ammonia and nitrite
oxidizers in the natural environment is useful for both energetic
reasons and the prevention of substrate accumulation that could be
toxic or could lead to the formation of toxic by-products, especially
in environments where nitrite cannot simply diffuse away
(8). Therefore, this study suggests that the challenge for
ammonia-oxidizing bacteria in natural systems is not only survival with
an inconstant energy source but also avoidance of toxic product
formation once the ammonia has been converted to nitrite.
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ACKNOWLEDGMENTS |
This work was supported by EPA grant R821405 to D. J. Arp and
P. J. Bottomley.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Botany and Plant Pathology, 2082 Cordley, Oregon State University,
Corvallis, OR 97331. Phone: (541) 737-1294. Fax: (541) 737-3573. E-mail: arpd{at}bcc.orst.edu.
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Applied and Environmental Microbiology, October 1998, p. 4098-4102, Vol. 64, No. 10
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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