Department of Biology, Appalachian State
University, Boone, North Carolina 28608,1 and
Microbiology Unit, Department of Natural Resource Sciences,
McGill University, Macdonald Campus, Ste.-Anne-de-Bellevue, Quebec
H9X 3V9, Canada2
Chloramphenicol, at concentrations greater than 0.1 g/liter (0.3 mM), inhibited the denitrifying enzyme activity (DEA) of slurries of
humisol and sandy loam soils by disrupting the activity of existing
nitrate reductase enzymes. When the concentration of chloramphenicol
was increased from 0.1 to 2.0 g/liter (6.0 mM), the rate of nitrite
production from nitrate decreased by 25 to 46%. The rate of NO
production from nitrate decreased by 20 to 39%, and the rate of
N2O production from nitrate, in the presence of acetylene
(DEA), decreased by 21 to 61%. The predicted values of DEA at 0 g
of chloramphenicol/liter computed from linear regressions of DEA versus
chloramphenicol concentration were 18 to 43% lower than DEA
measurements made in the absence of chloramphenicol and within a few
per cent of DEA rates measured in the presence of 0.1 g of
chloramphenicol/liter. We conclude that DEA assays should be carried
out with a single (0.1-g/liter) chloramphenicol concentration.
Chloramphenicol at concentrations greater than 0.1 g/liter inhibits the
activity of existing denitrifying enzymes and should not be used in DEA assays.
 |
INTRODUCTION |
Measurements of denitrifying enzyme
activity (DEA) were proposed by Smith and Tiedje (19) as a
way of assessing the potential optimum activity of existing
denitrifying enzymes in soil. DEA is determined by measuring the rate
of N2O production, in the presence of acetylene, from soil
samples placed under anaerobic conditions and supplied with excess
carbon source (glucose) and nitrate. The DEA assay has been widely used
(7, 13, 15, 16, 18, 22) and is the recommended method for
measuring potential DEA in soil (21).
Smith and Tiedje (19) suggested that chloramphenicol should
be used in DEA assays to inhibit synthesis of new denitrifying enzymes
while the activity of previously existing denitrifying enzymes was
being measured. Recent work has indicated that the use of
chloramphenicol to prevent the synthesis of new denitrifying enzymes
during the DEA assay may have had previously unrecognized effects on
denitrifying enzymes (3, 5, 17, 23). Several investigators
have suggested that chloramphenicol may disrupt the activity of
previously existing denitrifying enzymes in agricultural soils
(17), aquifer sediments (3), and pure cultures of
denitrifying bacteria (23).
Wu and Knowles (23) noted that chloramphenicol inhibited DEA
at the level of nitrate reduction in pure cultures of both Flexibacter canadensis and "Pseudomonas
denitrificans" and at the level of NO reduction in F. canadensis. We investigated the influence of chloramphenicol on
the activity of the four denitrifying enzymes (24, 25) in
two agricultural soils over a range of chloramphenicol concentrations
commonly used in DEA assays.
| |
MATERIALS AND METHODS |
Soil.
Humisol was collected from the Central Experimental
Farm of Agriculture & Agri-Food Canada, Ottawa, Ontario, Canada, and is highly organic (58 to 62% weight loss on ignition
[6]). St. Bernard sandy loam soil was collected from
the Morgan Arboretum of McGill University, Ste.-Anne-de-Bellevue,
Quebec, Canada. It is a mineral soil with a loss on ignition of 4.5%
(6). Both soils were collected from the surface 10 cm and
stored at 4°C. The soils were sieved through a 1.0-mm-mesh-size sieve
prior to the start of experiments.
Net nitrite production from nitrate and nitrite reduction.
Samples of between 20 (humisol) and 40 (sandy loam) g of soil were
placed in 50 ml of assay solution containing phosphate buffer (50 mM;
pH 7) supplemented with 10 mM nitrate, 10 mM glucose, and
chloramphenicol (Sigma). Chloramphenicol was dissolved in the buffer
solution, and the final concentration was varied from 0.1 to 2.0 g/liter. Incubations were carried out in 150-ml Erlenmeyer flasks
capped with SubaSeals (William Freeman Co., Barnsley, England). Anaerobic conditions were established in the flasks by evacuating and
flushing them three times with N2. The flasks were vented to atmospheric pressure prior to the beginning of experiments and
placed on a rotary shaker at 200 rpm. All treatments were in
triplicate. The flasks containing humisol were sampled each hour for
5 h. The flasks containing sandy loam soil were sampled every
2 h for a total of 10 h. The time course of nitrite
production was linear over these periods.
Nitrite consumption was measured by monitoring the consumption of
nitrite that was produced by reduction of nitrate added in the assay
solution. Nitrite was not added directly to soil slurries because
nitrite added directly to soil can undergo chemical decomposition to NO
(4). Flasks containing soil and assay solution as described
above were evacuated and flushed with N2 and placed on a
rotary shaker overnight. Nitrite concentrations in the humisol and
sandy loam reached a maximum after 28 and 30 h, respectively, at
which time an ethanolic solution of chloramphenicol (between 1 and 2%
[vol/vol], final concentration) was injected through the stoppers
into the flasks. Ethanol at these concentrations did not affect the
rate of nitrite consumption by either soil. Consumption of the
indigenously produced nitrite was measured at 0.5-h intervals, over a
period of 3 h for the humisol and at hourly intervals over a
period of 6 h for the sandy loam. Time courses for nitrite
consumption were linear over the sampling period in both soils.
Net production of NO from nitrate and NO reduction.
NO
production was measured in flasks containing 10 g of soil and 25 ml of assay solution (phosphate buffer, 50 mM; pH 7) supplemented with
10 mM nitrate, 10 mM glucose, and chloramphenicol. The flasks were
evacuated, flushed with N2, vented to atmospheric pressure and shaken at 200 rpm. The headspaces were sampled for NO analysis at
20-min intervals over a period of 100 min, and NO production was linear
over this time interval.
NO consumption was monitored in incubations in which NO produced from
endogenous soil nitrate was depleted. Flasks containing 10 g of
soil and assay solution which was not supplemented with nitrate or
chloramphenicol were evacuated, flushed with N2, and shaken
overnight. The next morning, NO produced from endogenous soil nitrate
had been consumed by microorganisms in the soil, and no NO was present.
NO consumption in the presence of chloramphenicol was then measured by
injecting ethanolic solutions of chloramphenicol (between 1 and 2%
[vol/vol] final concentration) into the flasks and by adjusting the
concentration of NO in the flask headspace to between 3 and 5 parts per
million volume (ppmv). Ethanol at concentrations between 1 and 2% did
not affect the rate of NO consumption by either soil. The NO
concentration was measured at 20-min intervals over a period of 120 to
180 min. NO consumption by the humisol and sandy loam soils was a
first-order reaction. First-order rate constants were calculated from
regression of log NO concentration versus time.
Net production of N2O from nitrate and
N2O reduction.
N2O production was
determined by the DEA assay technique (19). DEA assays were
initiated by placing 10 g (fresh weight) of soil and 25 ml of
phosphate buffer (50 mM; pH 7) containing 10 mM KNO3, 10 mM
glucose, and chloramphenicol into a series of 150-ml Erlenmeyer flasks.
Chloramphenicol concentrations were from 0.1 to 2.0 g/liter. The flasks
were evacuated and flushed with N2 three times and vented
to atmospheric pressure. Ten percent of the gas phase over incubated
soils was replaced with acetylene to give a final partial pressure of
10 kPa. All incubations were shaken at 200 rpm at room temperature. The
time courses of N2O production were linear over a period of
at least 100 min, and the flasks were sampled at 10- to 15-min
intervals between 60 and 100 min after the addition of acetylene. Gas
samples were stored in 2-ml serum vials with crimped seals (Wheaton,
Millville, N.J.).
N2O consumption was monitored in incubations in which
N2O produced from endogenous soil nitrate was depleted.
Flasks containing 10 g of soil and assay solution which was not
supplemented with nitrate, chloramphenicol, or acetylene were
evacuated, flushed with N2, and shaken at 200 rpm
overnight. The next morning, N2O produced from endogenous
soil nitrate had been consumed by microorganisms in the soil and no
N2O was present. N2O consumption in the
presence of chloramphenicol was then measured by adding ethanolic
solutions of chloramphenicol to the flasks and by adjusting the
concentration of N2O in the headspace to 950 ppmv. Ethanol
at final concentrations between 1 and 2% (vol/vol) did not affect
N2O consumption by either soil. The decrease in
N2O concentration was measured at 30-min intervals over a
period of 150 min. N2O consumption by the humisol and sandy
loam soils was linear over the 150-min period.
Analytical methods.
Nitrite was measured by an automated
Griess-Ilosvay method (14). Samples for nitrite analysis
were collected with a syringe, transferred to 1.5-ml Eppendorf tubes,
and centrifuged for 10 min at 15,000 × g, and the
supernatants were frozen until analysis. Samples for nitrite analysis
were diluted 1/10 or 1/50 (vol/vol) before analysis to prevent
interference by humic material extracted from the soil. NO was measured
with a Sievers chemiluminescence analyzer (model 270B NOA) equipped
with an injection port. The N2O concentration was measured
with a Perkin-Elmer gas chromatograph (Porapak Q column; oven
temperature, 80°C; detector temperature, 265°C) equipped with a
63Ni electron capture detector (Valco Instruments Co.
Inc.).
| |
RESULTS AND DISCUSSION |
Net nitrite production from nitrate (nitrate reductase
activity).
There was a significant linear relationship between the
rate of net nitrite production from nitrate and chloramphenicol
concentration in both the humisol and sandy loam soils (Table
1 and Fig.
1). The rates of net nitrite production
measured in the presence of 0.1 g of chloramphenicol/liter were
within ±5% of the values predicted by the regression equations (Table
1). The measured rates of nitrite production in the absence of
chloramphenicol were not accurately predicted by the intercept values
of the regression equations (Table 1). In two of the three cases the
predicted rates were higher than the rates of nitrite production
measured in the absence of chloramphenicol (Table 1). The faster
nitrite accumulation in the presence of chloramphenicol was probably
due to a slowing of nitrite consumption because chloramphenicol
prevented synthesis of nitrite reductase enzyme in the 5- or 10-h
period during which nitrite production was measured (see below).
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TABLE 1.
Enzyme activity in the presence and absence of
chloramphenicol and parameters of linear regression equations of enzyme
activity versus chloramphenicol concentration
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FIG. 1.
Net nitrite production from nitrate (nitrate reductase
activity) at different chloramphenicol concentrations in humisol and
sandy loam soils. The data are the means of triplicate determinations,
and the error bars indicate standard deviations that exceed the
dimensions of the symbols. Circles, nitrite production rates at the
indicated concentrations of chloramphenicol; squares, nitrite
production rate in the absence of chloramphenicol. Linear regression
parameters are presented in Table 1.
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Nitrite reduction (nitrite reductase activity).
The rate of
nitrite consumption was not significantly related to chloramphenicol
concentration (Table 1 and Fig. 2). The measured rates of nitrite consumption in the absence of chloramphenicol were between 1.3 and 8.4 times those measured in the presence of
0.1 g of chloramphenicol/liter (Table 1 and Fig. 2).
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FIG. 2.
Nitrite reduction (nitrite reductase activity) at
different concentrations of chloramphenicol in humisol and sandy loam
soils. The data are the means of triplicate determinations, and the
error bars indicate standard deviations that exceed the dimensions of
the symbols. Circles, nitrite consumption rates at the indicated
concentrations of chloramphenicol; squares, nitrite consumption rate in
the absence of chloramphenicol. Linear regression parameters are
presented in Table 1.
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The higher nitrite consumption in the absence of chloramphenicol
suggests that nitrite reductase was being synthesized, in the absence
of chloramphenicol, during the 3- to 6-h assay period. It is reasonable
to expect that nitrite reductase could be synthesized during the assay
period in the absence of chloramphenicol because nitrite reductase is
known to be constitutively produced under aerobic conditions in six
strains of denitrifying bacteria (8) and nitrite reductase
synthesis in Pseudomonas stutzeri is stimulated by oxygen
depletion (11). Moreover, Baumann et al. (1, 2) have detected synthesis of nitrite reductase mRNA within 1 h of placing a chemostat culture of "Pseudomonas
denitrificans" under anaerobic conditions. The inhibition of
nitrite consumption by chloramphenicol, especially in the sandy loam
(Fig. 2), is consistent with our previous observation of increased
nitrite accumulation from nitrate in the presence of chloramphenicol
(Fig. 1).
Net production of NO from nitrate (nitrate reductase activity plus
nitrite reductase activity).
There was a significant linear
relationship between the rate of NO production from added nitrate and
chloramphenicol concentration (Table 2
and Fig. 3). The measured rates of NO
production in the presence of 0.1 g of chloramphenicol/liter were
within ±3% of the intercept values predicted by the regression
equations (Table 2). The observed decreases in the rates of production of NO from nitrate were strongly related to chloramphenicol
concentration (P values of <0.001 [Table 2]) and are
consistent with our observation of the inhibition of existing nitrate
reductase enzymes by chloramphenicol (Fig. 1). The measured rates of NO
production in the absence of chloramphenicol were between 1.3 and 1.7 times the rates predicted from the intercept values of the regression
equations (Table 2 and Fig. 3).
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TABLE 2.
Enzyme activity in the presence and absence of
chloramphenicol and parameters of linear regression equations of enzyme
activity versus chloramphenicol concentration
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FIG. 3.
Net NO production from nitrate (nitrate reductase plus
nitrite reductase activity) versus chloramphenicol concentration in
humisol and sandy loam soils. The data are the means of triplicate
determinations, and the error bars indicate standard deviations that
exceed the dimensions of the symbols. Circles, NO production rates at
the indicated concentrations of chloramphenicol; squares, NO production
rate in the absence of chloramphenicol. Linear regression parameters
are presented in Table 2.
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NO reduction to N2O (NO reductase activity).
The
first-order rate constants of NO consumption were not significantly
related to chloramphenicol concentration (Table 2 and Fig.
4). The measured rate of NO consumption
in the absence of chloramphenicol was approximately 1.1 to 1.2 times
that predicted by the intercept values of the regression equations
(Fig. 4).
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FIG. 4.
First-order rate constants for NO reduction to nitrous
oxide (NO reductase activity) versus chloramphenicol concentration in
humisol and sandy loam soils. The data are the means of triplicate
determinations, and the error bars indicate standard deviations that
exceed the dimensions of the symbols. Circles, NO consumption rates at
the indicated concentrations of chloramphenicol; squares, NO
consumption rate in the absence of chloramphenicol. Linear regression
parameters are presented in Table 2.
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Net production of N2O from nitrate (nitrate reductase
plus nitrite reductase plus NO reductase activity; DEA).
There was
a significant linear relationship between the rate of N2O
production from nitrate and chloramphenicol concentration (Table
3 and Fig.
5). The inhibition of N2O
production from nitrate by increasing chloramphenicol concentration is
consistent with our observation of the inhibition of existing nitrate
reductase enzymes by chloramphenicol (Fig. 1). The measured
N2O production in the presence of 0.1 g of
chloramphenicol/liter was within ±5% of the intercept values
predicted by the regression equations. The measured N2O
production in the absence of chloramphenicol was 1.2 to 1.8 times that
predicted from the intercept values of the regression equations (Table
3).
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TABLE 3.
Enzyme activity in the presence and absence of
chloramphenicol and parameters of linear regression equations of enzyme
activity versus chloramphenicol concentration
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FIG. 5.
Net N2O production from nitrate (nitrate
reductase plus nitrite reductase plus NO reductase activity) versus
chloramphenicol concentration in humisol and sandy loam soils. The data
are the means of triplicate determinations, and the error bars indicate
standard deviations that exceed the dimensions of the symbols. Circles,
N2O production rates at the indicated concentrations of
chloramphenicol; squares, N2O production rate in the
absence of chloramphenicol. Linear regression parameters are presented
in Table 3.
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Reduction of N2O (N2O reductase
activity).
There was a significant linear relationship between the
N2O consumption rate and the chloramphenicol concentration
in the sandy loam soil but not in the humisol (Table 3 and Fig.
6). The measured rate of N2O
consumption in the presence of 0.1 g of chloramphenicol/liter was
within ±9.0% of the rate predicted by the regression equation. The
measured rates of N2O consumption in the absence of
chloramphenicol were between 1.2 and 1.3 times those predicted from the
intercept values of the regression equations (Table 3 and Fig. 6).
N2O reductase activity is not measured during DEA assays
because N2O consumption is blocked by acetylene (9,
10) during the assay, and N2O accumulation is used as the index of denitrification activity.
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FIG. 6.
N2O reduction (nitrous oxide reductase
activity) versus chloramphenicol concentration in humisol and sandy
loam soils. The data are the means of triplicate determinations, and
the error bars indicate standard deviations that exceed the dimensions
of the symbols. Circles, nitrous oxide consumption rates at the
indicated concentrations of chloramphenicol; squares, nitrous oxide
consumption rate in the absence of chloramphenicol. Linear regression
parameters are presented in Table 3.
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Net production and reduction rates.
It is not possible to
directly compare rates of nitrite, NO, and N2O consumption
between assays because long incubation times (during which bacterial
growth probably occurred) were required to deplete existing pools of
soil nitrate, NO, or N2O before consumption assays could be
initiated. Moreover, rates of N2O production (DEA) cannot
be directly compared to rates of nitrite and NO production because the
rate of N2O production in the presence of acetylene represents the total reduction of nitrate to N2O while the
rates of nitrite and NO production represent the transient accumulation of nitrite and NO in excess of that undergoing reduction to
N2O during the assay period.
Chloramphenicol and the synthesis of DEA enzymes.
The measured
rates of net production and reduction of nitrite, NO, and
N2O in the absence of chloramphenicol were not accurately predicted by the intercept values of regression equations of rate versus chloramphenicol concentration (Tables 1 to 3 and Fig. 1 to 6).
The inability of the regression equations to predict the measured
enzyme activities at 0 g of chloramphenicol/liter could be caused
by the synthesis of new enzymes during the assay period in the absence
of chloramphenicol, or a portion of the total denitrifying population
could be extremely sensitive to low concentrations of chloramphenicol
and exhibit a concentration-independent response at low chloramphenicol concentrations.
While our data cannot rule out the possibility of a
concentration-independent effect at low chloramphenicol concentrations (a possibility relevant to all inhibitor studies with mixed
populations), one would expect that synthesis of denitrifying enzymes
would occur in anaerobic incubations of soil supplemented with glucose and nitrate because synthesis of mRNAs for denitrifying enzymes is
known to occur within 1 h of placing a pure culture of "P. denitrificans" under anaerobic conditions (1) and the
patterns of N2O accumulation from long term soil DEA
assays carried out without chloramphenicol are consistent with
populations undergoing exponential growth (17). We suggest
that 0.1 g of chloramphenicol/liter should be used in DEA assays
to preclude the possibility of denitrifying enzyme synthesis.
Chloramphenicol and the inhibition of existing denitrifying
enzymes.
Chloramphenicol at concentrations greater than 0.1 g/liter reduced the activity of nitrate reductase enzymes in both soils (Table 1 and Fig. 1) and that of N2O reductase enzymes in
the sandy loam soil (Table 3 and Fig. 6). When the concentration of
chloramphenicol was increased from 0.1 to 2.0 g/liter (0.3 to 6.0 mM),
the rate of nitrite production from nitrate decreased by 25 to 46%.
The rate of NO production from nitrate decreased by 20 to 39%, and the
rate of N2O production from nitrate (DEA) decreased by 21 to 61% (Tables 1 to 3 and Fig. 1, 3, and 5). The decrease in enzyme
activity caused by chloramphenicol is most likely the result of the
disruption of previously existing nitrate reductase enzymes, because we
used a chloramphenicol concentration in excess of the 0.3- to 5.0-µM
concentration known to inhibit protein synthesis in cultures of
gram-positive and gram-negative bacteria (12) and
chloramphenicol within the 0.1- to 2.0-g/liter concentration range is
known to disrupt the activity of existing nitrate reductase enzymes in
pure cultures of both F. canadensis and "P.
denitrificans" and in cell extracts of "P.
denitrificans" (23). Moreover, we did not observe a
chloramphenicol concentration effect on the activity of nitrite
reductase or NO reductase enzymes. We suggest that chloramphenicol at
concentrations greater than 0.1 g/liter can disrupt the activity of
previously existing nitrate reductase enzymes in the two soils studied
and should not be used in DEA assays.
Modifications to the DEA assay.
Pell et al. (17)
proposed two possible methods for estimating DEA which would avoid the
problem of chloramphenicol inhibition of existing denitrifying enzymes.
The growth-associated product formation method involves fitting a
series of timed measurements of N2O accumulation in the
absence of chloramphenicol to a product formation equation and solving
the fitted equation for the initial denitrification rate
(20). While the growth-associated product formation approach
is of interest and holds out the possibility of obtaining the in situ
growth rate of denitrifying organisms, the long incubation times
required (between 380 and 600 min) would likely preclude the use of the
technique for large numbers of replicate samples. Moreover, assumptions
underlying the technique, particularly the assumptions that the
denitrifying organisms are undergoing balanced growth and that specific
enzyme activity remains constant during the incubation period
(20), need to be verified for indigenous denitrifying
organisms growing in soil slurries.
The second method proposed by Pell et al. (17) involves
measuring DEA in the presence of a range of chloramphenicol
concentrations and extrapolating the N2O production rate
versus chloramphenicol concentration data to the rate at 0 g of
chloramphenicol/liter. We used this method to investigate the influence
of chloramphenicol on the activity of the enzymes associated with each
step in the denitrification process. Enzyme rate versus chloramphenicol
concentration was measured by using triplicate samples to characterize
the variability of enzyme activity in soil. In all cases where there
was a significant relationship between enzyme activity and
chloramphenicol concentration, the activity at 0 g of
chloramphenicol/liter predicted from regression equations was within a
few percent of the value measured in the presence of 0.1 g of
chloramphenicol/liter (Tables 1 to 3 and Fig. 1, 3, 5, and 6).
A concentration of 0.1 g of chloramphenicol/liter appears to be
high enough to prevent the synthesis of denitrifying enzymes during the
DEA assay period and low enough not to disrupt existing denitrifying
enzymes by more than a few percent. We suggest that DEA assays can be
carried out with a single (0.1-g/liter) chloramphenicol concentration
and that using a series of chloramphenicol concentrations would improve
the estimate only slightly while substantially increasing the time and
effort required to obtain a DEA measurement.
This work was supported by award number 97-35106-4801 from the USDA NRI
Competitive Grants Program to R.E.M. and a grant from the Natural
Sciences and Engineering Research Council of Canada to R.K.
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Abstract
Introduction
