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Applied and Environmental Microbiology, September 2000, p. 4136-4138, Vol. 66, No. 9
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Starvation Alters the Apparent Half-Saturation
Constant for Methane in the Type II Methanotroph
Methylocystis Strain LR1
Peter F.
Dunfield* and
Ralf
Conrad
Max-Planck-Institut für terrestrische
Mikrobiologie, Karl-von-Frisch Strasse, 35043 Marburg, Germany
Received 5 May 2000/Accepted 30 June 2000
 |
ABSTRACT |
When cells of a type II methanotrophic bacterium
(Methylocystis strain LR1) were starved of methane, both
the Km(app) and the
Vmax(app) for methane decreased. The specific
affinity (aos) remained nearly
constant. Therefore, the decreased Km(app) in
starved cells was probably not an adjustment to better utilize low-methane concentrations.
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TEXT |
Microbial oxidation of atmospheric
methane (CH4) takes place in most aerobic upland soils
(5, 10). Because these soils exhibit a lower half-saturation
constant [Km(app)] for CH4 than do
pure cultures of methanotrophic bacteria, it has been postulated that
the active bacteria are unknown species. These have been popularly
dubbed "high-affinity" methane oxidizers (5, 10).
Recently, a novel group of pmoA-like sequences was detected in several soils which oxidize atmospheric CH4 (11,
12), and incubation of soils under 14CH4
resulted in the labeling of signature phospholipid fatty acids which
differed from those of known type II methanotrophs (18). It
is therefore likely that as-yet-uncultured species are involved in
atmospheric CH4 uptake. However, it remains unknown whether atmospheric CH4 oxidation is limited to particular species
and whether these possess a specialized high-affinity CH4
oxidation enzyme.
We previously demonstrated that high-affinity CH4 oxidation
is probably not limited to uncultured methanotrophic groups. We enriched a methane-oxidizing bacterium (strain LR1) from an organic soil and identified it based on 16S rDNA, pmoA, and
mxaF gene sequences as a type II methanotrophic species of
the Methylosinus/Methylocystis cluster (8). Mixed
cultures containing strain LR1, when grown under <275 ppm volume
CH4, had a low Km(app) for
CH4 (56 to 188 nM) similar to the value measured in soil.
This increased to >1 µM when cells were grown under >1%
CH4. In the present study, we investigated the kinetics of
the isolated bacterium (culture is available upon request). Instead of
the time-consuming process of growing the organism under low
CH4 mixing ratios, we tested the effect of starving cells
of CH4.
Kinetics of strain LR1.
Culture was grown in liquid
nitrate-mineral salts (NMS) medium (8) under 10%
CH4. Purity was controlled microscopically and by plating
onto NMS agar, R2A agar, 10% strength Nutrient Agar, and 10% strength
AC Broth (Difco). After 1 to 2 months, the culture was diluted to about
109 cells ml
1 with 0.5 mM phosphate buffer
(pH 6.0), and 7.5-ml amounts were added to 13-ml serum vials. The vials
were capped with sterile butyl rubber stoppers and incubated with
gentle shaking (6 rpm) at 25°C without added CH4. After 1 to 2 weeks, some vials were injected with CH4 to a final
mixing ratio of 1% and incubated for a further 24 h
("unstarved"). Others remained without CH4 ("starved"). Cell counts were made using a Neubauer chamber and showed that no population growth occurred during the 24-h incubation with 1% CH4 (data not shown).
For determination of kinetic properties, CH4 was injected
into these vials to final mixing ratios ranging from 5 to 1,500 ppm
volume. The unstarved vials still contained >0.5% CH4
after 24 h and were first flushed well with air. Vials were shaken
at 280 rpm. Starting 5 min after CH4 addition and at 30-min
intervals thereafter, CH4 was measured by gas
chromatography-flame ionization detection (8). Methane
oxidation rates and kinetic parameters were estimated as previously
described (8).
Unstarved cells had both a higher
Km(app) and a
higher
Vmax(app) for CH
4 than did
starved cells (Table
1). Addition
of KCl
may also have decreased the
Km(app). The
increase
of both
Km(app) and
Vmax(app) in unstarved culture was
about 1 order
of magnitude, and the specific affinity
(
aos)
(
Vmax(app)/
Km(app))
therefore remained nearly constant.
The specific affinity is the
initial slope of the hyperbolic curve,
or the pseudo first-order rate
constant, and directly indicates
how rapidly the culture metabolized
limiting substrate (
4).
At low CH
4
concentrations, the rate of CH
4 uptake was therefore
similar in starved and unstarved culture.
These results extend our previous observations on strain LR1 by
demonstrating that (i) the
Km(app) can vary in
pure
culture, (ii) starvation of CH
4 decreases the
Km(app),
(iii) the specific affinity
(
aos) changes little with
Km(app), and (iv)
Km(app) varies in rapid (<2 h) tests without added chloramphenicol (previous
tests were run over several days and chloramphenicol was necessary
to
prevent enzyme production). As previously discussed (
8),
the
variable
Km(app) could have resulted because
type
II methanotrophs possesses different forms of methane
monooxygenase
(MMO): a particulate (pMMO) and a soluble (sMMO) form.
Multiple
catalytic forms of pMMO also exist, depending on Cu
availability
(
15,
17). However, while the
Km(app) values of these
MMOs are different
(
10,
15,
21), all measured values in
pure culture are above
0.8 µM. The values measured in strain LR1
are as low as 56 to 336 nM
(present results and reference
8).
Multiple enzymes may be responsible for the variable
Km(app) in LR1, but because the
aos per cell remained constant, it
cannot be concluded that a high-affinity
enzyme was induced to better
utilize limiting CH
4. This possibility
is consistent with
the data only if reactivation of inactive cells
and expression of a
lower-affinity enzyme compensated for each
other in the unstarved
culture and caused the
aos to remain
constant. However, if this were so, a biphasic kinetic
curve should
have been evident, and this was never observed. A
better explanation
for our results is that measured
Km(app) values
do not always represent true, constant enzyme properties.
Because of
diffusion limitation in experimental systems, many
reported
Km(app) values are gross overestimates
(
14).
In order to control for this in our experiments, the
CH
4 oxidation
rate constants in the linear portion of the
hyperbolic curve were
also often measured with culture diluted 50%.
These rate constants
were close to 50% of the rate constants in
undiluted culture (in
six cases, rate constants were 42, 54, 61, 73, 74, and 81%), indicating
that there was only a slight limitation in
CH
4 movement from the
gas phase to the liquid phase. We
also used the minimum incubation
times which yielded reproducible data
(<2 h) to estimate CH
4 oxidation
rates. To illustrate the
effect of longer incubation times, we
measured an initial 0 to 2 h rate
at five CH
4 concentrations in
a 10-day-starved culture, and
then after 4 h (during which the
CH
4 declined <40%)
reinjected enough CH
4 to bring each vial back
to its
initial CH
4 concentration and measured a 4 to 6 h rate
(Fig.
1). The CH
4 oxidation
rate increased with time, most strikingly
at the higher CH
4
concentrations. This trend leads to an overestimation
of the
Km(app) with increasing incubation times.
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FIG. 1.
CH4 oxidation rates at five CH4
concentrations in a 10-day-starved culture of Methylocystis
strain LR1, measured 0 to 2 h ( ) and 4 to 6 h ( ) after
adding CH4.
|
|
Although we minimized these methodological problems, there are further
ways in which an apparent
Km in a complex system
can
vary. The
Km(app) of MMO for CH
4
is affected by the CH
4 association and dissociation
constants, the rate of CH
4 diffusion
across the cell
envelope, and the concentrations of cosubstrates
(O
2 and
reductant). One explanation for the lower
Km(app) in starved cells is low reductant
availability. The mechanism
of pMMO is not well elucidated, but sMMO
follows a catalytic sequence
in which O
2, CH
4,
and NADH sequentially bind (
9,
20). If
in starved cells
CH
4 binds normally but the overall catalytic
cycle is
slowed by NADH limitation, the rate-limiting step may
cease to be the
association and dissociation of CH
4 to the enzyme
(i.e.,
the affinity constant) and instead become the reaction
rate (i.e., the
kinetic constant). In such a case, both the
Vmax(app) and the
Km(app)
for CH
4 decrease (
19). To illustrate
this, the
following reaction diagram has been simplified to consider
only
CH
4 as a reactant. The binding of O
2 and NADH
and the formation
of methanol are considered by the rate constant
kp:
Here,
Km(app) = [
k(s) +
kp]/[
ks +
k(p)].
Limitation of reductant will decrease the reaction rate constant
kp and in turn decrease
Km(app) (i.e., cause a higher
apparent
affinity). The above model is a very simple explanation
of the observed
variability. The truth may of course be more
complex.
Bender and Conrad (
2) observed that incubation of various
soils under 20% CH
4 increased methanotrophic
Km(app) and
Vmax(app)
values by 1 to 3 orders of magnitude, but increased
methanotrophic cell
counts only 3- to 10-fold. When specific affinities
were calculated
(Table
2), two of these soils (a meadow
cambisol
and a cultivated cambisol) had a similar pattern as LR1
that
aos varied little despite large
changes (30- to 100-fold) of
Km(app).
A third
soil (forest luvisol) had a much lower
aos after enrichment than before,
suggesting that a different population
had become active in
CH
4-enriched soil. Comparisons must be cautiously
made, but
these data suggest that the pattern noted in LR1 is
applicable to some,
but not all, soils.
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TABLE 2.
Michaelis-Menten apparent half-saturation constants for
CH4 oxidation in three soils, and calculated specific
affinities from data given in
reference 2a
|
|
It is clear from this and other work (
3,
8) that the
Km(app) for CH
4 changes with culture
conditions. Nevertheless,
our lowest measured
Km(app) is still higher than the
lowest measured
values in soil of about 10 nM (
8), so we hesitate
to
conclude that no true high-affinity MMO exists. Calculations
based on
maintenance energy requirements suggest that methanotrophic
bacteria
cannot survive on atmospheric CH
4 without a more efficient
CH
4-oxidizing system (
6). However, soil
methanotrophs may not
consume only atmospheric methane but also
alternate substrates,
such as methanol (
3,
13), or
CH
4 produced in anaerobic soil
microsites (
1,
7,
16,
22). Our present results show
that the observed high-affinity
activity in soil cannot in itself
be taken as proof that the
responsible bacteria are novel oligotrophic
species with a specialized
form of
MMO.
| |
ACKNOWLEDGMENTS |
P.F.D. was supported by a stipendium from the Max Planck Society
and a grant from the EC RTD Programme Biotechnology.
| |
FOOTNOTES |
*
Corresponding author. Mailing address:
Max-Planck-Institut für terrestrische Mikrobiologie,
Karl-von-Frisch Strasse, 35043 Marburg, Germany. Phone: (49)
6421-178-733. Fax: (49) 6421-178-809. E-mail:
dunfield{at}mailer.uni-marburg.de.
| |
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Applied and Environmental Microbiology, September 2000, p. 4136-4138, Vol. 66, No. 9
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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