Previous Article | Next Article 
Applied and Environmental Microbiology, April 2000, p. 1730-1733, Vol. 66, No. 4
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Effect of Copper Speciation on Whole-Cell Soluble
Methane Monooxygenase Activity in Methylosinus
trichosporium OB3b
John D.
Morton,
Kim F.
Hayes, and
Jeremy D.
Semrau*
Environmental and Water Resources
Engineering, Department of Civil and Environmental Engineering, The
University of Michigan, Ann Arbor, Michigan 48109-2125
Received 1 November 1999/Accepted 21 January 2000
 |
ABSTRACT |
Soluble methane monooxygenase (sMMO) activity in Methylosinus
trichosporium OB3b was found to be more strongly affected as copper-to-biomass ratios changed in a newly developed medium, M2M,
which uses pyrophosphate for metal chelation, than in nitrate mineral
salts (NMS), which uses EDTA. When M2M medium was amended with EDTA,
sMMO activity was similar to that in NMS medium, indicating that
EDTA-bound copper had lower bioavailability than pyrophosphate-bound copper. EDTA did not limit the association of copper with the cells; rather, copper was sequestered in a form which did not affect
sMMO activity.
| |
TEXT |
Methanotrophs, a group of
gram-negative bacteria that utilize methane as their sole source of
carbon and energy, can express two forms of methane monooxygenase
(MMO), depending on the amount of copper available. At low
copper-to-biomass ratios, a cytoplasmic or soluble methane
monooxygenase (sMMO) is synthesized by some methanotrophs, while a
membrane-associated or particulate methane monooxygenase (pMMO) is
found at high copper-to-biomass ratios (5). Both forms of
the MMO can degrade priority pollutants, such as trichloroethylene
(TCE), but at much different rates (7, 9). Although the
mechanism(s) of copper uptake by methanotrophs is less well
characterized than the effect of changing copper on methanotrophic
activity, recent studies indicate the existence of specific copper
uptake systems in methanotrophs (1, 2, 4, 12, 14).
It is unknown which forms of copper are bioavailable to methanotrophs,
however, and such information is needed to optimize methanotroph-mediated bioremediation. A system in which metal speciation (i.e., its distribution in chelated, free, and precipitated forms) can be easily controlled must be used to address this issue. Typical microbial media, however, are designed to optimize growth and
often have metal precipitates, poorly understood metal complexation, and ill-defined equilibrium conditions that prevent the assessment of
metal bioavailability. To better understand what forms of copper are
bioavailable to methanotrophs, a new growth medium, M2M medium, was
developed that had few to no copper precipitates and rapid chemical
equilibrium of copper species and that allowed for changes in copper
speciation that did not affect the speciation of other metals,
particularly iron. The bioavailability of different copper species was
assessed using this well-defined growth medium by monitoring whole-cell
sMMO activity in Methylosinus trichosporium OB3b with
different copper concentrations and different chelating agents as well
as through comparisons to whole-cell activity in the commonly used
growth medium nitrate mineral salts (NMS).
Medium preparation and analysis.
NMS medium was prepared as
described previously (13). M2M medium was prepared by
adding, per liter of deionized distilled water, the following: 11 ml of
pyrophosphate stock solution (44.6 g of
Na4P2O7 · 10H2O/liter at pH 6.0), 1.4 ml of 0.5-g/liter
FeSO4 · 7H2O stock adjusted to pH 7.1 to
7.5, 10 ml of a background electrolyte stock (2.46 g of
MgSO4 · 7H2O/liter, 101 g of
KNO3/liter, 1.47 g of CaCl2 · 2H2O/liter), 2 ml of an acidified (pH 3) trace element
stock (0.025 g of CoCl2 · 6H2O/liter,
9.9 × 10
3 g of MnCl2 · 4H2O/liter, 0.2 g of ZnSO4 · 7H2O/liter, 5.1 × 103 g of
NiCl2 · 6H2O/liter, 7.4 × 103 g of H3BO3/liter, 0.25 g
of Na2MoO4 · 2H2O/liter),
2.5 ml of phosphate stock solution (26 g of
KH2PO4 · H2O/liter, 33 g of Na2HPO4 · 7H2O/liter),
and various amounts of copper from an acidified Cu(NO3)2 stock. The pH of the medium was
adjusted to 6.8 using NaOH. To avoid copper contamination, all
glassware was carefully acid washed and medium components of American
Chemical Society grade or better were used. Based on the
manufacturer's specifications, total heavy metal contamination was
less than 5 × 108 and 107 M for M2M
and NMS media, respectively. When prepared without added copper,
however, all media contained no detectable copper (less than 5 × 109 M) as measured by atomic absorption spectrophotometry (AAS).
The growth media were characterized with regard to the time required
for copper speciation to reach an apparent equilibrium as well as the
amounts of precipitated copper and iron. To measure the equilibration
time in each medium, the free copper (Cu2+) concentration
was monitored using a cupric ion-selective electrode in stirred
polycarbonate reactors at 30°C until the change in output voltage was
less than 0.02 mV · min1. Free copper
concentrations at equilibrium are reported as pCu, or
log([Cu2+]). The amounts of copper and iron
precipitated in each medium were determined by centrifuging the media
at 206,000 × g for 40 min at 30°C and measuring the
copper and iron in the supernatant using AAS. The amounts of
precipitated copper and iron were calculated as the differences between
the total and supernatant metal concentrations.
The major differences between the two media were in the buffers and
metal chelating agents used. Buffering and metal chelation
in M2M were
provided by pyrophosphate, while in NMS, phosphate
and EDTA were used
for buffering and metal chelation, respectively.
The substitution of
pyrophosphate for phosphate to provide buffering
capacity in M2M
facilitated medium preparation by preventing phosphate
precipitation.
As a result, the buffer could be autoclaved with
the contents of M2M
medium, and copper speciation immediately
reached equilibrium, as the
free copper concentration did not
change within 25 h after
autoclaving. Free copper concentrations
in NMS medium, however,
decreased by more than 2 orders of magnitude
in the same time period.
The likely cause for this slower equilibration
was the exchange of
copper for iron in EDTA and the subsequent
precipitation of iron. Such
precipitation increased with increasing
copper concentrations in NMS,
as shown in Table
1. Therefore,
it was
not possible to vary copper concentrations over this range
without also
altering the speciation of iron in NMS. In M2M, changing
copper
concentrations changed the amount of free copper, as shown
in Table
1,
but the concentration of soluble iron did not vary
greatly with
changing copper concentrations. These data underscore
the advantage of
using a medium such as M2M, with easily controlled
metal speciation,
when studying the effect of copper on methanotrophs.
sMMO activity assays.
sMMO activity in M. trichosporium OB3b grown in either NMS, M2M, or M2M with EDTA was
assayed using a modified version of the naphthalene assay
(3). The media were inoculated with cells grown on NMS agar
plates with no added copper. Over the tested copper concentrations,
M. trichosporium OB3b had average growth rates of 0.075 h1 in NMS and 0.045 h1 in M2M with and
without EDTA, although the extent of growth was the same in all three
media. The cells were grown to an optical density at 600 nm of 0.2 to
0.5, and triplicate samples of 2 ml each were put in 6-ml serum vials
with naphthalene, which were capped and sealed. The cells were
incubated for 1 h at 270 rpm and 30°C, after which 4 mM
acetylene was added to inhibit sMMO activity. The cell suspension was
then centrifuged for 5 min at 12,000 × g. One hundred
thirty microliters of 4.21 mM tetrazotized o-dianisidine was
placed in a 1.5-ml cuvette with 1.3 ml of the culture supernatant, and
the absorbance at 528 nm was monitored immediately to assay for the
production of naphthol. As the complex that formed between the naphthol
and the tetrazotized o-dianisidine was unstable, the
absorbance increased to a maximum level and then decreased over a
period of time ranging from a few seconds to several minutes, depending
on the sample. By taking the maximum value, a measurement was obtained
that produced consistent standard curves with standard naphthol
solutions ranging from 0 to 7.5 mg/liter. With this procedure, the
triplicate samples had an average standard deviation of ±3.6%. The
amount of cell-associated copper was determined by AAS as the
difference between the total amount of copper in the growth medium and
that remaining in the spent medium.
The trends of sMMO activity measured after growth in NMS and M2M media
are shown in Fig.
1. Generally, maximal
activity decreased
linearly with increasing total copper-to-biomass
ratios in both
media. sMMO activity was apparent at levels of up to
5.64 µmol
of copper · g of protein
1 in NMS that
had EDTA as the chelating agent. In M2M with pyrophosphate
as the
chelating agent, however, no detectable sMMO activity was
observed when
>2.63 µmol of copper · g of protein
1 was
present. Evidently, the amount of bioavailable copper in
NMS medium is
half of that in M2M medium, as it took twice as
much copper per unit of
biomass to reduce sMMO activity to below
measurable levels in NMS as in
M2M.
View larger version (22K):
[in this window]
[in a new window]
|
FIG. 1.
sMMO whole-cell activity in M. trichosporium
OB3b in NMS medium ( ), M2M medium ( ), and M2M with EDTA ( ).
|
|
The changing sMMO activity trends in the two media are likely due to
changes in copper speciation. If free copper were controlling
copper
bioavailability, it would be expected that the media with
smaller
amounts of free copper would have smaller amounts of bioavailable
copper and thus would cause less repression of sMMO activity.
As shown
in Table
2, with the copper
concentrations used to monitor
sMMO activity, M2M had slightly smaller
amounts of free copper
than NMS; therefore, sMMO activity would be
expected to be less
strongly affected by copper in M2M than in NMS.
Whole-cell sMMO
activity, however, was lower in M2M medium than in NMS,
indicating
that the difference in the amounts of free copper in the two
media
cannot account for the observed changes in sMMO activity. The
small difference between the amounts of precipitated copper in
the two
media (2%) cannot account for the differences in the amounts
of
available copper; therefore, it is likely that differences
in amounts
of chelated copper affected sMMO activity. The amounts
of chelated
copper in the two media were similar, suggesting that
the differences
are related to the type of chelating agent.
To test this hypothesis, M2M medium was amended with the same
concentration of EDTA used in NMS. As shown in Fig.
1, the whole-cell
sMMO activity of
M. trichosporium OB3b as a function of
changing
copper-to-biomass ratios in M2M with EDTA was similar to that
in NMS. The copper-to-biomass ratio at which sMMO activity was
no
longer measurable was 5.77 µmol of copper · g of
protein
1 for M2M with EDTA, similar to that for NMS and
twice as great
as that for M2M without EDTA, indicating that EDTA
reduced the
amount of bioavailable
copper.
It is possible that the changes in whole-cell sMMO activity were due in
part to changes in iron speciation. In addition to
the established data
on the effect of copper on expression of
sMMO, some evidence has also
indicated that iron may affect sMMO
activity. For example,
M. trichosporium OB3b grown in NMS with
iron concentrations of <10
µM had low sMMO activity, but sMMO
activity could be increased if the
iron concentration was increased
above 30 µM (
11). Similar
results were found for
M. trichosporium OB3b grown in
Higgins medium with various iron concentrations
(
10). In the
sMMO activity experiments reported here, however,
the soluble iron
concentration was 10-fold higher in NMS medium
than in M2M with EDTA
(Table
2). As the measured whole-cell sMMO
activities were similar in
the two media regardless of the copper-to-biomass
ratio, it appears
that in this study iron bioavailability did
not affect sMMO activity.
Rather, since whole-cell sMMO activities
were markedly different at the
same copper-to-biomass ratios in
M2M with and without EDTA, it appears
that EDTA limited the bioavailability
of copper to
M. trichosporium OB3b compared to pyrophosphate.
Although it is
difficult to quantitatively compare these results
with those of earlier
studies, some interesting trends with relevance
to copper
bioavailability can be discerned. One study found that
adding 84 µM
Na
2EDTA to NMS medium with 1.6 µM copper enhanced
the
rates of measured TCE degradation by a mixed methanotrophic
culture
(
6). As TCE degradation rates are known to be significantly
higher for sMMO than for pMMO, the increased rates may be due
to
decreased copper bioavailability and enhanced expression of
sMMO in the
presence of high EDTA concentrations. The concomitant
expression of
sMMO and pMMO by
Methylococcus capsulatus Bath observed
upon
the addition of 2 µM FeEDTA may also be due to enhanced expression
of
sMMO as a result of lower copper bioavailability (
8).
Copper sorption on inactive cells.
After growth, close to
100% of the copper in all three growth media was cell associated, as
shown in Fig. 2. This is surprising, as
at low total copper-to-biomass ratios, M. trichosporium OB3b had markedly different sMMO activities, depending on the type of
chelating agent present. Apparently, copper in the presence of EDTA was
cell associated but was not in a form that could affect whole-cell sMMO
activity. It is unclear what prevents such cell-associated copper from
being bioavailable. The larger binding constant of CuEDTA (log
k = 18.8) than of copper pyrophosphate (log
k = 10.4) may limit the cells' ability to accumulate
copper in a form necessary to affect sMMO activity.
View larger version (14K):
[in this window]
[in a new window]
|
FIG. 2.
Copper accumulation in M. trichosporium OB3b
in NMS medium (), M2M medium (), and M2M with EDTA (). The
line indicates 100% copper accumulation by the cells.
|
|
To test this hypothesis, the sorption of copper onto
acetylene-inactivated
M. trichosporium OB3b, which was
incapable of actively
sequestering copper, was measured in the presence
and absence
of EDTA.
M. trichosporium OB3b was grown in M2M
with 0.12 µM copper,
inactivated with the addition of acetylene, and
collected by centrifugation.
The cells were then washed with phosphate
buffer (3.1 mM PO
43, 20 mM NaNO
3
[pH 7]) and resuspended in M2M with 0.12 µM Cu and
either 0 or 11 µM EDTA along with acetylene for 24 h at 30°C.
In M2M, nearly
100% of the added copper was cell associated after
24 h. If 11 µM EDTA was added to M2M, however, only 25% of the
total copper was
cell associated, indicating that copper uptake
in the presence of EDTA
requires an energy-dependent extracellular
uptake mechanism, possibly
the recently discovered copper binding
cofactor (CBC) (
4,
12). It is possible that these biogenic
metal ligands bind copper
in CuEDTA complexes but cannot effectively
transport these copper
species into the cell, thus reducing their
bioavailability. It is
interesting that in solutions of EDTA,
copper, and the purified CBC,
68% of the copper was associated
with EDTA. In the presence of
ethylenediamine, a chelating agent
with a copper binding constant
similar to that of pyrophosphate,
only 3.7% of the copper was
associated with ethylenediamine (
12).
These trends, which
are similar to the data presented here, point
to the possibility that
the copper binding sites on the cell surface
have the same affinity for
copper as the CBC and may be cell-bound
CBC.
| |
ACKNOWLEDGMENTS |
Research support from the National Science Foundation (MCB-9708552)
is gratefully acknowledged, as are the assistance of Candice Winful and
helpful discussions with A. A. DiSpirito (Iowa State University).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Civil and Environmental Engineering, The University of Michigan, 1351 Beal Ave., 181 EWRE Building, Ann Arbor, MI 48109-2125. Phone: (734)
764-6487. Fax: (734) 763-2275. E-mail:
jsemrau{at}engin.umich.edu.
| |
REFERENCES |
| 1.
|
Berson, O., and M. E. Lidstrom.
1996.
Study of copper accumulation by the type I methanotroph Methylomicrobium albus BG8.
Environ. Sci. Technol.
30:802-809[CrossRef].
|
| 2.
|
Berson, O., and M. E. Lidstrom.
1997.
Cloning and characterization of corA, a gene encoding a copper-repressible polypeptide in the type I methanotroph Methylomicrobium albus BG8.
FEMS Microbiol. Lett.
148:169-174[CrossRef][Medline].
|
| 3.
|
Brusseau, G. A.,
H. C. Tsien,
R. S. Hanson, and L. P. Wackett.
1990.
Optimization of trichloroethylene oxidation by methanotrophs and the use of a colorimetric assay to detect soluble methane monooxygenase activity.
Biodegradation
1:19-29[CrossRef][Medline].
|
| 4.
|
DiSpirito, A. A.,
J. A. Zahn,
D. W. Graham,
H. J. Kim,
C. K. Larive,
T. S. Derrick,
C. D. Cox, and A. Taylor.
1998.
Copper-binding compounds from Methylosinus trichosporium OB3b.
J. Bacteriol.
180:3606-3613[Abstract/Free Full Text].
|
| 5.
|
Hanson, R. S., and T. E. Hanson.
1996.
Methanotrophic bacteria.
Microbiol. Rev.
60:439-471[Abstract/Free Full Text].
|
| 6.
|
Henry, S., and D. Grbic-Galic.
1990.
Effect of mineral media on trichloroethylene oxidation by aquifer methanotrophs.
Microb. Ecol.
20:151-169.
|
| 7.
|
Lontoh, S., and J. D. Semrau.
1998.
Methane and trichloroethylene degradation by Methylosinus trichosporium OB3b expressing particulate methane monooxygenase.
Appl. Environ. Microbiol.
64:1106-1114[Abstract/Free Full Text].
|
| 8.
|
Nguyen, H. T.,
S. J. Elliott,
J. H. Yi, and S. I. Chan.
1998.
The particulate methane monooxygenase from Methylococcus capsulatus (Bath) is a novel copper-containing three subunit enzyme.
J. Biol. Chem.
273:7957-7966[Abstract/Free Full Text].
|
| 9.
|
Oldenhuis, R.,
J. Y. Oedzes,
J. J. van der Waarde, and D. B. Janssen.
1991.
Kinetics of chlorinated hydrocarbon degradation by Methylosinus trichosporium OB3b and toxicity of trichloroethylene.
Appl. Environ. Microbiol.
57:7-14[Abstract/Free Full Text].
|
| 10.
|
Park, S.,
M. L. Hanna,
R. T. Taylor, and M. W. Droege.
1991.
Batch cultivation of Methylosinus trichosporium OB3b. I. Production of soluble methane monooxygenase.
Biotechnol. Bioeng.
38:423-433[CrossRef].
|
| 11.
|
Sayler, G. S., and J. P. Bowman.
1994.
Optimization and maintenance of soluble methane monooxygenase activity in Methylosinus trichosporium OB3b.
Biodegradation
5:1-11[Medline].
|
| 12.
|
Téllez, C. M.,
K. P. Gaus,
D. W. Graham,
R. G. Arnold, and R. Z. Guzman.
1998.
Isolation of copper biochelates from Methylosinus trichosporium OB3b and soluble methane monooxygenase mutants.
Appl. Environ. Microbiol.
64:1115-1122[Abstract/Free Full Text].
|
| 13.
|
Whittenbury, R.,
K. C. Phillips, and J. F. Wilkinson.
1970.
Enrichment, isolation, and some properties of methane-utilizing bacteria.
J. Gen. Microbiol.
59:2771-2776.
|
| 14.
|
Zahn, J. A., and A. A. DiSpirito.
1996.
Membrane-associated methane monooxygenase from Methylococcus capsulatus (Bath).
J. Bacteriol.
178:1018-1029[Abstract/Free Full Text].
|
Applied and Environmental Microbiology, April 2000, p. 1730-1733, Vol. 66, No. 4
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Yu, S. S.-F., Chen, K. H.-C., Tseng, M. Y.-H., Wang, Y.-S., Tseng, C.-F., Chen, Y.-J., Huang, D.-S., Chan, S. I.
(2003). Production of High-Quality Particulate Methane Monooxygenase in High Yields from Methylococcus capsulatus (Bath) with a Hollow-Fiber Membrane Bioreactor. J. Bacteriol.
185: 5915-5924
[Abstract]
[Full Text]
-
Dunfield, P. F., Khmelenina, V. N., Suzina, N. E., Trotsenko, Y. A., Dedysh, S. N.
(2003). Methylocella silvestris sp. nov., a novel methanotroph isolated from an acidic forest cambisol. Int. J. Syst. Evol. Microbiol.
53: 1231-1239
[Abstract]
[Full Text]
-
Sani, R. K., Peyton, B. M., Brown, L. T.
(2001). Copper-Induced Inhibition of Growth of Desulfovibrio desulfuricans G20: Assessment of Its Toxicity and Correlation with Those of Zinc and Lead. Appl. Environ. Microbiol.
67: 4765-4772
[Abstract]
[Full Text]
Top
Abstract
Text
