FEMS Microbiology Letters 201 (2001) 151^155
www.fems-microbiology.org
Methyl chloride utilising bacteria are ubiquitous in the
natural environment
Craig McAnulla, Ian R. McDonald *, J. Colin Murrell
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK
Received 9 April 2001; received in revised form 17 April 2001; accepted 18 April 2001
First published online 19 June 2001
Abstract
Enrichment and isolation of methyl chloride utilising bacteria from a variety of pristine terrestrial, freshwater, estuarine and marine
environments resulted in the detection of six new methyl chloride utilising Hyphomicrobium strains, strain CMC related to Aminobacter spp.
and to two previously isolated methyl halide utilising bacteria CC495 and IMB-1, and a Gram-positive isolate SAC-4 phylogenetically
related to Nocardioides spp. All the pristine environments sampled for enrichment resulted in the successful isolation of methyl chloride
utilising organisms. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Methyl chloride ; Aminobacter ; Hyphomicrobium
1. Introduction
Methyl chloride (CH3 Cl) is a volatile organic compound
with an average concentration in the atmosphere of 540
pptv [1]. CH3 Cl is of environmental concern because it
may be responsible for about 13% of the destruction of
the stratospheric ozone layer [2]. Major sources of CH3 Cl
include oceans, biomass burning, wood-rotting fungi, salt
marshes and anthropogenic sources [3]. The main global
sink for CH3 Cl is thought to be the reaction with tropospheric and stratospheric hydroxyl radicals. However, soils
may also be a potentially signi¢cant sink for CH3 Cl [1].
CH3 Cl can be co-metabolised by bacteria, both by oxidation and hydrolysis. In addition, several methylotrophic
bacteria can use CH3 Cl as a growth substrate. These include the strictly anaerobic homoacetogenic bacterium
Acetobacterium dehalogenans [4], several aerobic methylotrophs of the genera Hyphomicrobium and Methylobacterium [5], and the methanotroph Methylomicrobium album
BG8, which can derive some bene¢t from oxidising CH3 Cl
[6] and may therefore contribute to the degradation of
CH3 Cl. Doronina et al. [5] isolated eight strains from solvent contaminated Russian soils. However, 16S rRNA sequencing showed that only two distinct strains, recently
* Corresponding author. Tel. : +44 (24) 765 28362;
Fax: +44 (24) 765 23568; E-mail : [email protected]
designated Hyphomicrobium chloromethanicum CM2T and
Methylobacterium chloromethanicum CM4T , had been isolated [7]. Studies on M. chloromethanicum CM4 have revealed a pathway for CH3 Cl utilisation [8,9]. Two polypeptides of 65 and 35 kDa were induced during growth on
CH3 Cl [8]. CH3 Cl grown cells were also capable of dehalogenating methyl bromide (CH3 Br) and methyl iodide
(CH3 I), but not dichloromethane or higher chloroalkanes.
No growth was observed with CH3 Br, presumably due to
the greater toxicity of this compound. Transposon mutagenesis was used to create mutants unable to grow on
CH3 Cl. Biochemical and molecular analysis revealed a
novel catabolic pathway [9] which involves CmuA, a
65-kDa polypeptide, with a methyltransferase domain
and a corrinoid binding domain. The methyltransferase
domain transfers the methyl group of CH3 Cl to the Co
atom of the enzyme bound corrinoid group. A second
polypeptide, CmuB, then transfers the methyl group
onto tetrahydrofolate, forming methyl tetrahydrofolate.
This folate linked methyl group is then progressively oxidised via formate to CO2 to provide reducing equivalents
for biosynthesis. Carbon assimilation presumably occurs
at the level of methylene tetrahydrofolate. This pathway
may also occur in H. chloromethanicum [10] and Aminobacter strain IMB-1 [11].
The only other reports of aerobic methyl halide degraders include strain IMB-1 isolated from soil fumigated
with CH3 Br [12,13], strain CC495 isolated from woodland
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 2 5 6 - 7
FEMSLE 10010 16-7-01
152
C. McAnulla et al. / FEMS Microbiology Letters 201 (2001) 151^155
soil [14] (these two strains were shown by 16S rRNA
sequence analysis to be of the Aminobacter genus), and
strain MB2 isolated from the marine environment on
CH3 Br [15].
In order to assess the distribution of CH3 Cl degraders
in the environment, we report here on their isolation from
a wide variety of pristine terrestrial, freshwater, estuarine
and marine environments, indicating that the biological
sinks for CH3 Cl are widespread and may therefore have
a signi¢cant role in the mediation of atmospheric CH3 Cl
concentrations.
2. Materials and methods
2.1. Enrichment and isolation of methyl chloride utilisers
Environmental samples were obtained from a variety of
pristine environments (Table 1). For enrichment cultures,
soil samples (1 g) were slurried with 10 ml of ANMS
(ammonium nitrate mineral salts medium) [16], while
aquatic samples were concentrated by centrifugation or
¢ltration through a nitrocellulose ¢lter (0.2 Wm pore
size; Millipore) and resuspended in one hundredth of the
original volume with ANMS. One millilitre of sample was
then added to 50 ml of either ANMS or DM medium [5],
supplemented with vitamin solution [17], in 120-ml serum
bottles. For the initial enrichment at 30³C, either CH3 Cl
(2% v/v), methanol, methylamine or formate (0.1% w/v)
was used as the sole carbon and energy source. Enrichments were then subcultured with 2% (v/v) CH3 Cl as the
sole carbon and energy source. CH3 Cl consumption was
measured by removing 200-Wl samples from the headspace
and analysing CH3 Cl concentration by gas chromatography, using a Porapak Q column at 200³C with nitrogen as
the carrier gas at a £ow rate of 30 ml min31 .
Enrichments showing both growth and consumption of
CH3 Cl after three subcultures were serially diluted and
spread onto plates of the appropriate medium and incubated with 2% (v/v) CH3 Cl for 7 days. Individual colonies
were then streaked onto plates and reincubated. Subsequent colonies were then used to inoculate liquid media.
This process was repeated until pure cultures were obtained. Phenotypic and taxonomic characterisation of all
isolates was carried out using standard methods.
2.2. PCR ampli¢cation and sequencing of 16S rRNA genes
DNA was extracted from isolates using the method of
Marmur [18]. DNA sequencing was performed by cycle
sequencing with the Dye Terminator Kit (PE Applied Biosystems, Warrington, UK) and analysed using a Model
373A automated DNA sequencing system (PE Applied
Biosystems).
16S rRNA sequences were aligned, using the ARB program for sequence alignment (http://www.mikro.biologie.tu-muenchen.de), to representative organisms of the same
and related genera of bacteria, and the phylogenetic position determined using DNADIST, DNAML and DNAPARS programs of the PHYLIP package. Phylogenetic
dendrograms were constructed using the Fitch^Margoliash
method and dendrograms drawn using the TreeView program V1.5. The 16S rRNA sequences of strains studied
have been deposited in GenBank under the accession numbers AF279785^AF279792.
3. Results and discussion
3.1. Enrichment and isolation of methyl chloride utilising
bacteria
Enrichments were set up with samples from ¢ve di¡erent sites: Severn estuary, UK (surface seawater, approximately 1.2% w/v NaCl) ; North Sea (pelagic seawater, 2 m
deep, 3.5% salinity); Tocil Lake, University of Warwick
Table 1
Major characteristics of the isolates
Hyphomicrobium
Enrichment site
Gram stain
Cell morphology
Polar prostheca
Budding at tip of prostheca
Pigmentation
Catalase
Oxidase
Optimum growth temperature (³C)
Growth rate on methyl chloride (h31 )
Growth yield on methyl chloride
(g dry weight mol31 C)
Aminobacter
Nocardioides
S-3
S-4
MAR-1
PMC
SAC-1
SAN-1
CMC
SAC-4
Severn
estuary
3
motile
rods
+
+
3
+
+
30
0.16
10.9
Warwick
soil
3
motile
rods
+
+
3
+
+
30
0.15
10.5
North
Sea
3
motile
rods
+
+
3
+
+
30
0.14
11.1
Tocil
Lake
3
motile
rods
+
+
3
+
+
30
0.17
11.5
Tocil
Wood
3
motile
rods
+
+
3
+
+
30
0.10
8.0
Tocil
Wood
3
motile
rods
+
+
3
+
+
30
0.09
8.5
Contaminant
Tocil Wood
3
motile
rods
3
3
3
+
+
30
0.15
11.9
variable
non-motile elongated
rods
3
3
3
+
+
30
0.08
7.0
FEMSLE 10010 16-7-01
C. McAnulla et al. / FEMS Microbiology Letters 201 (2001) 151^155
153
Fig. 1. Phylogenetic analysis of the Gram-negative (A) and Gram-positive (B) methyl chloride utilising bacterial isolates. The known methyl
chloride utilising bacteria, H. chloromethanicum CM2, M. chloromethanicum CM4, strain IMB-1 and strain CC495, are also included in the phylogenetic analysis. The dendrograms show the results of an analysis in
which DNADIST was used. The bar represents 10% sequence divergence, as determined by measuring the lengths of the horizontal lines
connecting any two species.
6
(freshwater); Tocil Wood, University of Warwick (topsoil); Warwick agricultural soil, UK (topsoil). Thirty-three
enrichments were set up with CH3 Cl, methanol, methylamine or formate as carbon sources. Initially, enrichments
showed a mixture of cell types (Gram-negative, Gram-positive, rods and cocci) independent of the enrichment conditions used. However, after further subculturing on
CH3 Cl a single morphology, Gram-negative rods with a
polar prostheca, was observed, suggesting that Hyphomicrobium was the dominant species in these enrichments.
All enrichments degraded CH3 Cl and had OD540 values
of 0.04^0.1, and each sample site yielded at least one pure
isolate. A further isolate was obtained from a contaminated culture of H. chloromethanicum CM2. In total, eight
isolates were obtained.
3.2. Phylogenetic analysis of isolates
The 16S rRNA gene was PCR-ampli¢ed from each of
the isolates, sequenced (either completely or partially, 900
bp) and analysed phylogenetically. The six Hyphomicrobium-like strains were con¢rmed as members of the
cluster II Hyphomicrobium species (Fig. 1A) [19]. Three
isolates (S-3, S-4 and MAR-1) had identical 16S rRNA
sequences and were most similar to H. chloromethanicum
CM2 (98.7% identical). This is also interesting because
these three isolates come from three geographically isolated environments, the North Sea (MAR-1), Severn estuary (S-3) and Warwick soil (S-4), suggesting that this isolate is widespread in nature. Strains SAC-1 and SAN-1
also had identical 16S rRNA sequences but branched separately from the known Hyphomicrobium species, most
similar to Hyphomicrobium facile subsp. tolerans (99.3%
identical), and may therefore be a novel strain. Strain
PMC, which was the most distant from the known Hyphomicrobium species, is most similar to H. facile subsp. tolerans (98.2% identical), and may also be a novel species of
Hyphomicrobium. Strain CMC was shown to group closely
with strains IMB-1 and CC495 within the Aminobacter
genus (Fig. 1A). Strain SAC-4, the Gram-positive isolate,
grouped within the Nocardioides species, the closest extant
relative being Nocardioides simplex (Fig. 1B).
3.3. Characterisation of isolates
The major characteristics of isolates are summarised in
Table 1. While all isolates were capable of growth on
FEMSLE 10010 16-7-01
154
C. McAnulla et al. / FEMS Microbiology Letters 201 (2001) 151^155
CH3 Cl as the sole carbon and energy source, none were
capable of growth on CH3 Br and CH3 I. This may re£ect
the greater toxicity of either the compounds themselves or
the halide ions produced by degradation of these compounds, or may be due to an inability of these cells to
bind these compounds for e¡ective transformation.
Six strains (S-3, S-4, MAR-1, PMC, SAC-1 and SAN-1)
had an almost identical morphology, being Gram-negative
rods with a polar prostheca from which cell budding occurred. This, plus the ability to grow methylotrophically,
led to their identi¢cation as Hyphomicrobium species. This
identi¢cation was supported by the substrate growth pro¢les of these isolates (Table 2). While growth was observed
on many C1 substrates, and some C2 compounds, no
growth occurred on substrates containing more than three
carbons, a typical feature of Hyphomicrobium species [20].
Colonies formed were small, smooth and faintly beige,
typically appearing after 3^4 days incubation. No signi¢cant cell clumping or rosette formation was observed, suggesting that they were type II Hyphomicrobium strains [19].
Strains S-3 and MAR-1, which were isolated from the
Severn estuary and the North Sea respectively, were tested
for growth at NaCl concentrations ranging from 0 to
3.5%. Surprisingly, the optimum concentration for growth
was found to be 0% in both cases, although both strains
were capable of growth at up to 3.5% NaCl. However,
Hyphomicrobium species are generally very salt-tolerant,
with most strains being capable of growth at up to at least
2.5% NaCl. H. chloromethanicum CM2 was also capable
of growth at 3.5% NaCl (data not shown). It is thus feasible that Hyphomicrobium strains arising perhaps from
run-o¡ of land or freshwater environments can grow in
marine environments, even though the salt concentrations
may not be optimal for growth. The growth yield of these
organisms on CH3 Cl was generally high at 8.0^11.5 g dry
weight mol31 C. These values are similar to those reported
for other chloromethane utilisers [5,8,14].
Two other cells were also isolated with strikingly di¡er-
ent colony morphology from the putative Hyphomicrobium species. Isolate CMC was a Gram-negative, motile rod, which was isolated as a contaminant in a culture
of H. chloromethanicum CM2. It formed discrete, smooth
white colonies (1^2 mm diameter) which appeared after
3^4 days incubation at 30³C. Apart from CH3 Cl, methylamine was the only C1 compound used for growth. Unlike
the Hyphomicrobium strains, CMC was capable of growth
on a variety of multicarbon compounds, including acetate
and glucose. The growth yield on CH3 Cl was 11.9 g dry
weight mol31 C and is comparable with the growth yields
reported for other CH3 Cl utilisers.
Strain SAC-4 was a Gram-variable, non-motile rod isolated from topsoil in Tocil Wood. This organism formed
discrete `fried-egg' type colonies after 3^4 days incubation.
CH3 Cl was the only methylotrophic substrate used,
although it grew on a variety of multicarbon compounds.
Growth yield on CH3 Cl was 7.0 g dry weight mol31 C and
is comparable with growth yields reported for other
CH3 Cl utilisers.
As most of the isolates obtained were Hyphomicrobium
species, Hyphomicrobium type strains were tested for
growth on CH3 Cl to see if this ability was common to
the genus. None of the eight strains tested (H. facile subsp.
facile H-526T (DSM 1565), H. facile subsp. ureaphilum
CO-582T (ATCC 27492), H. facile subsp. tolerans I-551T
(ATCC 27489), H. vulgare MC-750T (ATCC 27500),
H. aestuarii NQ-521T (NCIMB 11052), H. denitri¢cans
HA-905, H. hollandicum KB 677T (ATCC 27498), H. zavarzinii ZV 622T (ATCC 27496)) was capable of growth
on CH3 Cl.
All samples used for enrichment yielded strains capable
of growth on CH3 Cl, consistent with the hypothesis that
CH3 Cl utilising bacteria are widespread in the environment. This is expected since natural processes produce
most CH3 Cl, however it has obvious implications for
the cycling of this compound in the environment in
that CH3 Cl utilisers may be a signi¢cant environmental
Table 2
Substrate growth pro¢les of the isolates
Hyphomicrobium
Chloromethane
Bromomethane
Iodomethane
Methane
Methanol
Methylamine
Methanesulfonic acid
Formate
Acetate
Ethanol
Glycerol
Pyruvate
Succinate
Glucose
Sucrose
Aminobacter
Nocardioides
S-3
S-4
MAR-1
PMC
SAC-1
SAN-1
CMC
SAC-4
+
3
3
3
+
+
3
+
+
3
3
3
3
3
3
+
3
3
3
+
+
3
+
+
3
3
3
3
3
3
+
3
3
3
+
+
3
+
3
3
3
3
3
3
3
+
3
3
3
+
+
3
+
+
3
3
3
3
3
3
+
3
3
3
+
+
3
+
3
3
3
3
3
3
3
+
3
3
3
+
+
3
+
+
+
3
3
3
3
3
+
3
3
3
3
+
3
3
+
+
+
+
+
+
3
+
3
3
3
3
3
3
3
+
3
+
+
+
+
3
FEMSLE 10010 16-7-01
C. McAnulla et al. / FEMS Microbiology Letters 201 (2001) 151^155
sink for this compound. Doronina et al. [5] found that
long-term culturing of H. chloromethanicum CM2 and
M. chloromethanicum CM4 on alternative substrates led
to a loss of the ability to grow on CH3 Cl, so presumably
the ability to grow on this compound is advantageous to
these isolates in their natural habitat [9,14].
Most strains isolated were Hyphomicrobium species, presumably because the enrichment conditions favoured this
genus. It is possible that by varying enrichment conditions,
a greater diversity of isolates could be obtained. However,
even with the biased selection procedure it appears that at
least three di¡erent groups of methyl chloride utilisers exist, Hyphomicrobium species, Aminobacter species and a
Nocardioides species. It should also be noted that in previous work [10] it was shown that the Hyphomicrobium
and Aminobacter species contained the gene cmuA, which
encodes the protein CmuA, which is involved in the degradation of CH3 Cl, indicating that they utilise a similar
system to that which has previously been characterised in
several methyl halide utilising bacteria [9^11] and discussed in Section 1.
To complement the enrichment and isolation work, a
representative sample of extant Hyphomicrobium species
were tested for the ability to utilise CH3 Cl. None of these
strains was capable of growth on CH3 Cl. It is worth noting that Methylobacterium species, such as M. extorquens
AM1, which unlike M. chloromethanicum strain CM2 were
not isolated on CH3 Cl, were also unable to grow on
CH3 Cl (A. Studer and S. Vuilleumier, unpublished data).
There are two explanations for these results. The ¢rst is
that in the natural environment these species are unable to
use CH3 Cl as a growth substrate. The second is that these
species are able to use CH3 Cl as a growth substrate in the
natural environment, but this ability was lost during longterm culturing on alternative C1 substrates. SAC-4 was the
only Gram-positive isolate obtained, and warrants further
study since this is the ¢rst report of a Nocardioides growing on CH3 Cl and it may contain a di¡erent pathway for
the metabolism of CH3 Cl.
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
Acknowledgements
We acknowledge the ¢nancial support provided by the
Natural Environment Research Council (GR9/2192) and
for a studentship for C.M., and INTAS (94-3122).
[16]
[17]
References
[1] Khalil, M.A.K. and Rasmussen, R.A. (1999) Atmospheric methyl
chloride. Atmos. Environ. 33, 1305^1321.
[2] Butler, J.H. (2000) Better budgets for methyl halides? Nature 403,
260^261.
[3] Keene, W.C., Khalil, M.A.K., Erickson, D.J., McCulloch, A., Graedel, T.E., Lobert, J.M., Aucott, M.L., Gong, S.L., Harper, D.B.,
Kleiman, G., Midgley, P.M., Moore, R.M., Seuzaret, C., Sturges,
[18]
[19]
[20]
155
W.T., Benkovitz, C.M., Koropalov, V., Barrie, L.A. and Li, Y.F.
(1999) Composite global emissions of reactive chlorine from anthropogenic and natural sources: reactive chlorine emissions inventory.
J. Geophys. Res. 104, 8429^8440.
Messmer, M., Wohlfarth, G. and Diekert, G. (1993) Methyl chloride
metabolism of the strictly anaerobic methyl chloride-utilizing homoacetogen strain MC. Arch. Microbiol. 160, 383^387.
Doronina, N.V., Sokolov, A.P. and Trotsenko, Y.A. (1996) Isolation
and initial characterization of aerobic chloromethane-utilizing bacteria. FEMS Microbiol. Lett. 142, 179^183.
Han, J.I. and Semrau, J.D. (2000) Chloromethane stimulates growth
of Methylomicrobium album BG8 on methanol. FEMS Microbiol.
Lett. 187, 77^81.
McDonald, I.R., Doronina, N.V., Trotsenko, Y.A., McAnulla, C.
and Murrell, J.C. (2001) Hyphomicrobium chloromethanicum sp.
nov. and Methylobacterium chloromethanicum sp. nov., chloromethane-utilising bacteria isolated from a polluted environment. Int.
J. Syst. Evol. Microbiol. 51, 119^122.
Vannelli, T., Studer, A., Kertesz, M. and Leisinger, T. (1998) Chloromethane metabolism by Methylobacterium sp. strain CM4. Appl. Environ. Microbiol. 64, 1933^1936.
Vannelli, T., Messmer, M., Studer, A., Vuilleumier, S. and Leisinger,
T. (1999) A corrinoid-dependent catabolic pathway for growth of a
Methylobacterium strain with chloromethane. Proc. Natl. Acad. Sci.
USA 96, 4615^4620.
McAnulla, C., Woodall, C.A., McDonald, I.R., Studer, A., Vuilleumier, S., Leisinger, T. and 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.
Woodall, C.A., Warner, K.L., Oremland, R.S., Murrell, J.C. and
McDonald, I.R. (2001) Identi¢cation of methyl halide-utilizing genes
in the methyl bromide-utilizing bacterial strain IMB-1 suggests a high
degree of conservation of methyl halide-speci¢c genes in Gram-negative bacteria. Appl. Environ. Microbiol. 67, 1959^1963.
Miller, L.G., Connell, T.L., Guidetti, J.R. and Oremland, R.S. (1997)
Bacterial oxidation of methyl bromide in fumigated agricultural soils.
Appl. Environ. Microbiol. 63, 4346^4354.
Connell Hancock, T.L., Costello, A.M., Lidstrom, M.E. and 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.
Coulter, C., Hamilton, J.T.G., McRoberts, W.C., Kulakov, L., Larkin, M.J. and Harper, D.B. (1999) Halomethane:bisul¢de/halide ion
methyltransferase, an unusual corrinoid enzyme of environmental
signi¢cance isolated from an aerobic methylotroph using chloromethane as the sole carbon source. Appl. Environ. Microbiol. 65, 4301^
4312.
Goodwin, K.D., Schaefer, J.K. and Oremland, R.S. (1998) Bacterial
oxidation of dibromomethane and methyl bromide in natural waters
and enrichment cultures. Appl. Environ. Microbiol. 64, 4629^4636.
Whittenbury, R., Phillips, K.C. and Wilkinson, J.F. (1970) Enrichment, isolation and some properties of methane-utilizing bacteria.
J. Gen. Microbiol. 61, 205^218.
Kanagawa, T., Dazai, M. and Fukuoka, S. (1982) Degradation of
organo-phosphorus wastes. 2. Degradation of O,O-dimethyl phosphorodithioate by Thiobacillus thioparus TK-1 and Pseudomonas
AK-2. Agric. Biol. Chem. 46, 2571^2578.
Marmur, J. (1961) A procedure for the isolation of deoxyribonucleic
acid from microorganisms. J. Mol. Biol. 3, 208^218.
Rainey, F.A., Ward-Rainey, N., Gliesche, C.G. and Stackebrandt, E.
(1998) Phylogenetic analysis and intrageneric structure of the genus
Hyphomicrobium and the related genus Filomicrobium. Int. J. Syst.
Bacteriol. 48, 635^639.
Harder, W. and Attwood, M.M. (1978) Biology, physiology and biochemistry of Hyphomicrobia. Adv. Microb. Physiol. 17, 303^359.
FEMSLE 10010 16-7-01