Previous Article | Next Article 
Appl Environ Microbiol, May 1998, p. 1933-1936, Vol. 64, No. 5
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Chloromethane Metabolism by
Methylobacterium sp. Strain CM4
Todd
Vannelli,
Alex
Studer,
Michael
Kertesz, and
Thomas
Leisinger*
Mikrobiologisches Institut, ETH Zürich,
CH-8092 Zürich, Switzerland
Received 8 December 1997/Accepted 16 February 1998
 |
ABSTRACT |
Methylobacterium sp. strain CM4 metabolized
chloromethane quantitatively with a molar yield of 2.8 g of
whole-cell protein/mol of C. This value was similar to that observed
after growth with methanol (2.9 g of protein/mol of C) and about three
times larger than the yield with formate (0.94 g of protein/mol of C).
Chloromethane dehalogenation activity was inducible.
MiniTn5 transposon insertion mutants with altered growth
characteristics with chloromethane and other C1 compounds
were isolated and characterized. Nine of these were unable to grow with
chloromethane but were able to grow with methanol, methylamine, or
formate. Seventy-three transposon mutants that were defective in the
utilization of either methanol, methylamine, methanol plus methylamine,
or formate could still grow with chloromethane. Based on the protein
yield data and the properties of the transposon mutants, we propose a
pathway for chloromethane metabolism that depends on methyltransferase
and dehydrogenase activities.
| |
TEXT |
Chloromethane is of environmental
concern since it is believed to be responsible for about 15% of the
destruction of the stratospheric ozone layer (14). The
primary sources of the compound are from biological and nonbiological
processes that occur in nature (5). Chloromethane can be
cometabolized both oxidatively (15, 16) and hydrolytically
(7) by bacteria. In addition, several methylotrophic bacteria have been characterized which are able to utilize
chloromethane as a growth substrate. These include the strictly
anaerobic homoacetogenic bacterium strain MC (12) and
several aerobic methylotrophs of the genera Hyphomicrobium
and Methylobacterium (3, 6). Anoxic dehalogenation of chloromethane by strain MC has been shown to be
catalyzed by an enzyme that transfers the methyl group of chloromethane onto tetrahydrofolate and thereby releases inorganic chloride and
methyl tetrahydrofolate (13). It is not known how the
aerobic methylotrophs metabolize chloromethane, although closely
related species are known to dehalogenate dichloromethane by a
glutathione-S-transferase-dependent mechanism
(9). Therefore, we have initiated physiological and genetic
studies to explore the mechanism of chloromethane metabolism in
Methylobacterium sp. strain CM4, a recently isolated
chloromethane-utilizing methylotrophic bacterium (3). This
organism was chosen since in preliminary experiments it has proven
accessible to transposon insertion mutagenesis (17).
Growth and cell yields with C1 substrates.
Methylobacterium sp. strain CM4 (3) was kindly
provided by Y. A. Trotsenko (Institute of Biochemistry and
Physiology of Microorganisms, Russian Academy of Sciences, Pushchino,
Russia). It was grown in minimal mineral medium which contained (per
liter of distilled water) KH2PO4 (1.9 g),
Na2HPO4 (5.1 g),
(NH4)2SO4 (2.0 g), and
MgSO4 · 7H2O (0.1 g) at a pH of 7.2. After sterilization, 1 ml of a trace element solution containing (in
grams/liter) FeSO4 · 7H2O (1.0),
MnSO4 · H2O (1.0),
Na2MoO4 · H2O (0.25),
H3BO3 (0.10), CuCl2 · H2O (0.25), ZnCl (0.25), NH4VO3
(0.10), Co(NO3)2 · 6H2O (0.25), NiSO4 · 6H2O (0.10),
H2SO4 (9.2), and 1 ml of a 25-g/liter solution
of Ca(NO3)2 was added to 1 liter of medium.
Cultures (1 liter) were grown at 30°C on a rotary shaker (180 rpm) in 5-liter Erlenmeyer flasks sealed with rubber stoppers. For
growth with chloromethane, the chloromethane gas was added directly to
the headspace through the rubber stopper with a syringe to a final concentration of 2% (vol/vol) and was further added during growth as
needed. Sterile NaOH (5 M) was also added periodically to maintain the
pH at 7.2. For growth on other carbon sources, methanol, methylamine, or formate was added to a final concentration of 40 mM from sterile filtered solutions. The cells were harvested (10,000 × g, 15 min) in the late-exponential-growth phase, washed with
minimal medium twice, and either used immediately or frozen at 
20°C
as a cell pellet. These cells were then used to measure O2
uptake activities (10), chloride liberation (1),
and whole-cell protein as described previously (11).
During growth, Methylobacterium sp. strain CM4 metabolized
chloromethane with the concurrent production of chloride and protein. The production of bacterial protein precedes chloromethane utilization and chloride formation, and this lack of proportionality may be due to
initial growth at the expense of endogenous reserve material (Fig.
1). When 2% (vol/vol) chloromethane was
supplied in the headspace, the specific growth rate (µ) was 0.12 h
1. At higher concentrations of chloromethane the growth
rate decreased. Nitromethane, dichloromethane, trichloromethane, and
n-haloalkanes were not growth substrates, and no chloride or
nitrite was released from these compounds by the cells. At a
concentration of 2% (vol/vol), bromomethane was rapidly dehalogenated
by resting cells grown with chloromethane. Attempts to use
dibromomethane (2% [vol/vol]) as the sole substrate failed, probably
due to toxicity. Iodomethane was also dehalogenated by these cells, but
it was not tested as a growth substrate since it was readily hydrolyzed
to methanol in the medium used. For resting cells of
Methylobacterium sp. strain CM4, the molar ratio of chloride
produced to chloromethane utilized was 1.03 ± 0.08, whereas
1.52 ± 0.06 mol of oxygen was consumed per mol of chloromethane.
These values are in agreement with 1 mol of chloromethane being
metabolized to produce 1 mol of CO2 and 1 mol of
hydrochloric acid while consuming 1.5 mol of O2.
View larger version (19K):
[in this window]
[in a new window]
|
FIG. 1.
Metabolism of chloromethane by
Methylobacterium sp. strain CM4. Chloromethane disappearance
in the headspace was analyzed by gas chromatography. Chloride and
protein appearance in the solution were measured colorimetrically.
Symbols:  , chloromethane (mM);  , chloride (mM);  , protein
(µg/ml).
|
|
With a methylotrophic bacterium such as
Methylobacterium sp.
strain CM4, the observed reaction stoichiometry is consistent
with
three possible mechanisms that involve either a hydrolase,
a
monooxygenase, or a methyltransferase-dehydrogenase in the
chloromethane
metabolic pathway (Fig.
2).
In the first step of a putative hydrolytic
pathway, methanol and
chloride are produced from chloromethane
in an oxygen-independent
manner. Methanol is then oxidized to
formaldehyde and further to
formate and to carbon dioxide by methanol,
formaldehyde, and formate
dehydrogenases, respectively, yielding
a total of six reducing
equivalents. For this pathway, the growth
yield with chloromethane
would therefore be the same as that for
growth with methanol. As is
evident from Fig.
2, the methyltransferase-dehydrogenase
pathway would
also yield a total of six reducing equivalents per
chloromethane
metabolized and have a growth yield with chloromethane
that would be
the same as that for growth with methanol. We observed
growth yields of
2.8 ± 0.1 g of protein/mol of C with chloromethane
and
2.9 ± 0.1 g of protein/mol of C with methanol and thus
cannot
discriminate between the hydrolase and the
methyltransferase-dehydrogenase
pathways.
View larger version (17K):
[in this window]
[in a new window]
|
FIG. 2.
Possible mechanisms for the metabolism of chloromethane
by methylotrophs. Each of the possible pathways, i.e., hydrolytic,
monooxygenase dependent, and methyltransferase-dehydrogenase dependent,
follows a stoichiometry of CH3Cl + 3/2 O2
 CO2 + H2O + HCl.
|
|
In contrast, from growth yield data for
Methylobacterium sp.
strain CM4, it is unlikely that chloromethane is metabolized
via the
putative monooxygenase pathway (Fig.
2). In the first
step of this
pathway, chloromethane is oxidized to chloromethanol,
which then breaks
down abiotically to form formaldehyde and chloride
in an
oxygen-dependent manner. The formaldehyde is then further
oxidized to
formate and to carbon dioxide, yielding a total of
four reducing
equivalents. Because the initial monooxygenase-catalyzed
step consumes
two of these four reducing equivalents, a net yield
of only two
reducing equivalents is expected, and the growth yield
with
chloromethane should be the same as that for growth with
formate. With
formate, however, we have observed a growth yield
of 0.94 ± 0.02 g of protein/mol of C, a value amounting to one-third
of that
obtained with chloromethane. A degradative pathway including
a
monooxygenase reaction hence appears to be highly unlikely.
The growth yield values measured for
Methylobacterium sp.
strain CM4 are comparable to the values reported for different bacteria
which assimilate C
1 compounds via the serine pathway. Yield
constants
(given as grams of cell dry weight/mol of C) range from 9.8 to
13.1 on methanol, 7.2 to 9.6 on formaldehyde, and 3.35 to 6.95
on
formate (
4).
Inducibility of chloromethane utilization.
Resting cells of
Methylobacterium sp. strain CM4 grown with chloromethane
oxidized methanol, formaldehyde, and formate at rates comparable to
those of cells grown with methanol (data not shown). However, cells
grown with methanol were unable to metabolize chloromethane, indicating
that chloromethane metabolism was inducible. Since all attempts to
demonstrate chloromethane dehalogenase activity with extracts of
chloromethane-grown cells failed, we examined the protein pattern in
crude extracts by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE), looking for proteins that were specifically
synthesized during growth with chloromethane. Cells grown with
methanol, chloromethane, or a mixture of these two carbon sources were
suspended (20% wet weight/volume of phosphate buffer [50 mM, pH
7.2]) and disrupted by three passages through a French pressure cell
(55 mPa). After centrifugation to remove cell debris (10,000 × g, 15 min) the cell extracts were subjected to SDS-PAGE
according to the method of Laemmli (8) on 12% gels, and the
proteins were visualized by staining with Coomassie blue R-250. As
shown in Fig. 3, extracts of cells grown
with chloromethane and extracts of chloromethane-methanol-grown cells
contained a 65-kDa protein and a 35-kDa protein, indicating that growth
with chloromethane involves specific induction of at least two
proteins.
View larger version (101K):
[in this window]
[in a new window]
|
FIG. 3.
SDS-PAGE (12%) of chloromethane-induced proteins from
Methylobacterium sp. strain CM4. Lanes: WT, 10 µg of
protein from extracts of wild-type; MA, cells grown on methanol; CM,
cells grown on chloromethane; MA/CM, cells grown on methanol and
chloromethane; 38A10 and 38G12, 10 µg of protein from extracts of
Cmu mutant cells, i.e., 38A10 (a group 1 mutant) and
38G12 (a group 2 mutant), grown with methanol in the presence of
chloromethane. The arrows indicate the 65- and 35-kDa
chloromethane-induced proteins.
|
|
Phenotypic characterization of chloromethane utilization-negative
mutants.
The data described so far show that chloromethane
metabolism proceeds by an inducible pathway based on either a
hydrolytic or a methyltransferase-dehydrogenase dehalogenation
mechanism. To discriminate between these two mechanisms, chloromethane
utilization-negative (Cmu) transposon insertion mutants
were isolated and phenotypically characterized. Transposon mutagenesis
of Methylobacterium sp. strain CM4 was carried out by using
the miniTn5 system (2). The transposon was
introduced into the methylotroph, with a transposition frequency of
107, by plate conjugation on Luria-Bertani medium at
30°C for 24 h with a donor/recipient ratio of 1:4.
Escherichia coli S17-1 
pir (pUT-miniTn5Km) was
used as the donor strain. Exconjugants were selected at 30°C on
minimal agar plates containing 10 mM methylamine and 10 mM formate plus
25 µg of kanamycin per ml and were incubated in a closed desiccator
containing chloromethane and methanol in the headspace.
Kanamycin-resistant colonies of Methylobacterium sp. strain
CM4 appeared after 7 days and were inoculated into microtiter plate
wells containing 100 µl of minimal medium with methanol, methylamine,
and formate (10 mM each) and 25 µg of kanamycin per ml. After
overnight growth at 30°C, a multipin replicator was used to transfer
each mutant onto four separate agar plates, each containing kanamycin
and only one of the four growth substrates (in the plate or in the
headspace). Mutants unable to grow with one or more of the
C1 compounds were repurified on plates and tested further
in liquid media.
From a total of 4,032 kanamycin-resistant
Methylobacterium
exconjugants tested, 53 were unable to grow with methanol, 7 did
not
utilize methylamine, 11 were defective in the utilization
of both
methanol and methylamine, 9 were Cmu
, and 2 could not
grow with formate and accumulated formate when
grown with
chloromethane. The Cmu
mutants were all able to grow with
the other C
1 substrates tested
and all the mutants unable
to grow with the other C
1 substrates
were Cmu
+.
This shows that methanol, the product of a putative hydrolytic
dehalogenation mechanism, is not an intermediate of chloromethane
metabolism (Fig.
2). Therefore, the most likely route for chloromethane
metabolism is a pathway based on a methyltransferase-dehydrogenase
dehalogenation mechanism. Although the first step of a putative
methyltransferase-dehydrogenase pathway is oxygen independent,
the
second step is dependent on oxygen as a terminal electron
acceptor for
the reducing equivalents produced by the dehydrogenase.
This may
explain why resting cells grown with chloromethane did
not liberate
chloride from chloromethane in the absence of oxygen
but instead
immediately upon the addition of air to the incubation
mixture began to
release chloride at a constant rate (data not
shown). Under anoxic
conditions, the methylated intermediate produced
in the first step of
the methyltransferase-dehydrogenase pathway
may accumulate, preventing
further dehalogenation and limiting
the amount of chloride produced to
levels below the detection
limit of the chloride assay.
The nine Cmu
mutants could be divided phenotypically into
two groups. Group 1 mutants (Table
1)
could not grow with or dehalogenate
chloromethane. Group 2 mutants, by
contrast, released chloride
from chloromethane but were also unable to
use it as a growth
substrate. All group 1 mutants produced the 35-kDa
chloromethane-induced
protein, whereas three mutants of this class were
defective in
the formation of the 65-kDa protein (Fig.
3). Southern
blot analysis
(
15) of genomic DNA isolated from the mutants
indicated that
the transposon insertions of two group 1 mutants, 22B3
and 38A10,
were probably in the same DNA region (Table
1). The five
group
2 Cmu
mutants produced the 65-kDa protein but two
of them, 30F5 and
38G12, did not form the 35-kDa chloromethane-induced
protein (Fig.
3). From Southern blot analysis, these two mutants
probably had
transposon insertions in the same DNA region. Growth with
chloromethane
and the ability to dehalogenate this compound therefore
seem to
be encoded at several different loci. Thus, the two
chloromethane-induced
proteins observed represent only part of the
chloromethane utilization
system that is present in this bacterium.
In summary, the growth experiments and the mutant characterization
reported here suggest that
Methylobacterium sp. strain
CM4
metabolizes chloromethane by initial dehalogenation via a
methyl
transfer reaction, followed by a series of dehydrogenase-based
steps
which are different from those involved in the downstream
steps of
methanol or methylamine metabolism in the same organism.
Our studies
thus provide a testable hypothesis for the as-yet-unknown
dehalogenation reaction involved in aerobic chloromethane metabolism.
So far the nature of the dehalogenating enzyme which is thought
to
transfer the methyl group of chloromethane onto an acceptor
has
remained unknown. Addition of glutathione, which is involved
in the
dehalogenation of dichloromethane by
Methylobacterium sp.
strain DM4 (
9), did not lead to stable chloromethane
dehalogenase
activity in cell extracts of
Methylobacterium
sp. strain CM4,
so another nucleophilic cofactor may be necessary for
the dehalogenation
of chloromethane. Analysis of the genes inactivated
by transposon
insertion in the Cmu
mutants is in progress
and may shed light on the biochemistry
of the chloromethane degradative
pathway.
| |
ACKNOWLEDGMENTS |
This work was supported by a grant from the Swiss Federal Institute
of Technology, Zürich, Switzerland.
We thank Yuri A. Trotsenko who, in the course of INTAS project 94-3122, made Methylobacterium sp. strain CM4 available to us.
| |
FOOTNOTES |
*
Corresponding author. Mailing address:
Mikrobiologisches Institut, ETH-Zentrum, LFV, CH-8092 Zürich,
Switzerland. Phone: (41) 1 632 33 24. Fax: (41) 1 632 13 85. E-mail:
leisinger{at}micro.biol.ethz.ch.
| |
REFERENCES |
| 1.
|
Bergmann, J. G., and J. Sanik.
1957.
Determination of trace amounts of chlorine in naphtha.
Anal. Chem.
29:241-243.
|
| 2.
|
De Lorenzo, V., and K. N. Timmis.
1994.
Analysis and construction of stable phenotypes in gram-negative bacteria with Tn5- and Tn10-derived minitransposons.
Methods Enzymol.
235:386-405[Medline].
|
| 3.
|
Doronina, N. V.,
A. P. Sokolov, and Y. A. Trotsenko.
1996.
Isolation and initial characterization of aerobic chloromethane-utilizing bacteria.
FEMS Microbiol. Lett.
142:179-183.
|
| 4.
|
Goldberg, I.,
J. S. Rock,
A. Ben-Bassat, and R. I. Mateles.
1976.
Bacterial yields on methanol, methylamine, formaldehyde, and formate.
Biotechnol. Bioeng.
18:1657-1668[Medline].
|
| 5.
|
Harper, D. B.
1994.
Biosynthesis of halogenated methanes.
Biochem. Soc. Trans.
22:1007-1011[Medline].
|
| 6.
|
Hartmans, S.,
A. Schmuckle,
A. M. Cook, and T. Leisinger.
1986.
Methyl chloride: naturally occurring toxicant and C-1 growth substrate.
J. Gen. Microbiol.
132:1139-1142.
|
| 7.
|
Keuning, S.,
D. B. Janssen, and B. Witholt.
1985.
Purification and characterization of hydrolytic haloalkane dehalogenase from Xanthobacter autotrophicus GJ10.
J. Bacteriol.
163:635-639[Abstract/Free Full Text].
|
| 8.
|
Laemmli, U. K.
1970.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:680-685[Medline].
|
| 9.
|
Leisinger, T.,
S. La Roche,
R. Bader,
M. Schmid-Appert,
S. Braus-Stromeyer, and A. M. Cook.
1993.
Chlorinated methanes as carbon sources for aerobic and anaerobic bacteria, p. 351-363.
In
J. C. Murrell, and D. P. Kelly (ed.), Microbial growth on C1 compounds. Intercept Ltd., Andover, United Kingdom.
|
| 10.
|
Locher, H. H.,
T. Leisinger, and A. M. Cook.
1991.
4-Sulphobenzoate 3,4-dioxygenase. Purification and properties of a desulphonative two-component enzyme system from Comamonas testosteroni T-2.
Biochem. J.
274:833-842.
|
| 11.
|
Mägli, A.,
F. A. Rainey, and T. Leisinger.
1995.
Acetogenesis from dichloromethane by a two-component mixed culture comprising a novel bacterium.
Appl. Environ. Microbiol.
61:2943-2949[Abstract].
|
| 12.
|
Messmer, M.,
G. Wohlfarth, and G. Diekert.
1993.
Methyl chloride metabolism of the strictly anaerobic methyl chloride-utilizing homoacetogen strain MC.
Arch. Microbiol.
160:383-387.
|
| 13.
|
Messmer, M.,
S. Reinhardt,
G. Wohlfarth, and G. Diekert.
1996.
Studies on methyl chloride dehalogenase and O-demethylase in cell extracts of the homoacetogen strain MC based on a newly developed coupled enzyme assay.
Arch. Microbiol.
165:12-25.
|
| 14.
|
Montzka, S. A.,
J. H. Butler,
R. C. Myers,
T. M. Thompson,
T. H. Swanson,
A. D. Clarke,
L. T. Lock, and J. W. Elkins.
1996.
Decline in the tropospheric abundance of halogen from halocarbons: implications for stratospheric ozone depletion.
Science
272:1318-1322[Abstract].
|
| 15.
|
Rasche, M. E.,
R. E. Hicks,
M. R. Hyman, and D. J. Arp.
1990.
Oxidation of monohalogenated ethanes and n-chlorinated alkanes by whole cells of Nitrosomonas europaea.
J. Bacteriol.
172:5368-5373[Abstract/Free Full Text].
|
| 16.
|
Stirling, D. I., and H. Dalton.
1979.
The fortuitous oxidation and cometabolism of various carbon compounds by whole-cell suspensions of Methylococcus capsulatus (Bath).
FEMS Microbiol. Lett.
5:315-318.
|
| 17.
| Vannelli, T., and T. Leisinger. Unpublished data.
|
Appl Environ Microbiol, May 1998, p. 1933-1936, Vol. 64, No. 5
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Freedman, D. L., Swamy, M., Bell, N. C., Verce, M. F.
(2004). Biodegradation of Chloromethane by Pseudomonas aeruginosa Strain NB1 under Nitrate-Reducing and Aerobic Conditions. Appl. Environ. Microbiol.
70: 4629-4634
[Abstract]
[Full Text]
-
Marx, C. J., O'Brien, B. N., Breezee, J., Lidstrom, M. E.
(2003). Novel Methylotrophy Genes of Methylobacterium extorquens AM1 Identified by using Transposon Mutagenesis Including a Putative Dihydromethanopterin Reductase. J. Bacteriol.
185: 669-673
[Abstract]
[Full Text]
-
Studer, A., McAnulla, C., Buchele, R., Leisinger, T., Vuilleumier, S.
(2002). Chloromethane-Induced Genes Define a Third C1 Utilization Pathway in Methylobacterium chloromethanicum CM4. J. Bacteriol.
184: 3476-3484
[Abstract]
[Full Text]
-
Goodwin, K. D., Varner, R. K., Crill, P. M., Oremland, R. S.
(2001). Consumption of Tropospheric Levels of Methyl Bromide by C1 Compound-Utilizing Bacteria and Comparison to Saturation Kinetics. Appl. Environ. Microbiol.
67: 5437-5443
[Abstract]
[Full Text]
-
Woodall, C. A., Warner, K. L., Oremland, R. S., Murrell, J. C., McDonald, I. R.
(2001). Identification of Methyl Halide-Utilizing Genes in the Methyl Bromide-Utilizing Bacterial Strain IMB-1 Suggests a High Degree of Conservation of Methyl Halide-Specific Genes in Gram-Negative Bacteria. Appl. Environ. Microbiol.
67: 1959-1963
[Abstract]
[Full Text]
-
McAnulla, C., Woodall, C. A., McDonald, I. R., Studer, A., Vuilleumier, S., Leisinger, T., Murrell, J. C.
(2001). Chloromethane Utilization Gene Cluster from Hyphomicrobium chloromethanicum Strain CM2T and Development of Functional Gene Probes To Detect Halomethane-Degrading Bacteria. Appl. Environ. Microbiol.
67: 307-316
[Abstract]
[Full Text]
-
Schaefer, J. K., Oremland, R. S.
(1999). Oxidation of Methyl Halides by the Facultative Methylotroph Strain IMB-1. Appl. Environ. Microbiol.
65: 5035-5041
[Abstract]
[Full Text]
-
Coulter, C., Hamilton, J. T. G., McRoberts, W. C., Kulakov, L., Larkin, M. J., Harper, D. B.
(1999). Halomethane:Bisulfide/Halide Ion Methyltransferase, an Unusual Corrinoid Enzyme of Environmental Significance Isolated from an Aerobic Methylotroph Using Chloromethane as the Sole Carbon Source. Appl. Environ. Microbiol.
65: 4301-4312
[Abstract]
[Full Text]
-
Vannelli, T., Messmer, M., Studer, A., Vuilleumier, S., Leisinger, T.
(1999). A corrinoid-dependent catabolic pathway for growth of a Methylobacterium strain with chloromethane. Proc. Natl. Acad. Sci. USA
96: 4615-4620
[Abstract]
[Full Text]
-
Connell Hancock, T. L., Costello, A. M., Lidstrom, M. E., Oremland, R. S.
(1998). Strain IMB-1, a Novel Bacterium for the Removal of Methyl Bromide in Fumigated Agricultural Soils. Appl. Environ. Microbiol.
64: 2899-2905
[Abstract]
[Full Text]
Top
Abstract
Text
