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Microbial degradation of methanesulphonic acid (Nature 1991)
LETTERS TO NATURE
Microbial degradation of
a missing link in the
biogeochemical sulphur cycle
Simon C. Baker*, Don P. Kelly?& J. Colin Murrell*
* Department of Biological Sciences,University of Warwick.
Coventry CV4 7AL, UK
I' Natural Environment Research Council, Polaris House,
Swindon SN21EU. UK
ATMOSPHERIC dimethyl sulphide, arising from marine algae,
cyanobacteria and salt marsh plants such as Spartina, is the
principal sulphur compound entering the atmosphere from terres-
trial and aquatic environments'". Methanesulphonic acid
(CH,SO,H; MSA) has been identified as a major product of the
photochemical oxidation in the atmosphere of dimethyl sul-
phide'-3,5,"9 .Dimethyl sulphide and MSA are thus predominantly,
if not exclusively, biogenic in origin, and are the main gaseous
links in the biogeochemical sulphur cycle. MSA is a stable com-
pound, not undergoing photochemical decompositionq so its
removal from the atmosphere is by wet and dry deposition. MSA
partitions into the aerosol phase, as well as nucleating droplet
formation, and is deposited in rain and snow. Analysis of Antarctic
ice cores1' gives evidence of its global deposition over many
thousands of years. The subsequent fate of MSA deposited on
land was unknown. Here we describe terrestrial bacteria that grow
on MSA. Their activities in the natural environment would result
in the mineralization of MSA to carbon dioxide and sulphate,
thus completing our understanding of this part of the sulphur cycle.
Inoculating soil and water samples into media containing
MSA as the sole carbon source did not give rise to organisms
able to grow on MSA. Several culture collection strains, includ-
ing Pseudomonas MS, Bacillus PM6, Methylophilus methyl-
otrophus and Thiobacillus versutus, were also tested but were
unable to use MSA as a carbon source. We then decided to use
a chemostat-enrichment procedure with both methylammonium
and MSA. This would enhance recovery of methylotropic bac-
teria that might use MSA as a sole- or cosubstrate, and would
also allow enrichment of specialist MSA-users unable to use
methylammonium, assuming that these could compete success-
fully with other organisms. Such procedures have been success-
ful in isolation and competition experiments with sulphur-using
and are believed to be able to enrich for several
types of bacteria that can degrade MSA. Soil (100 g) from the
University of Wanvick campus was inoculated into a one-litre
fermenter containing minimal medium E" with 10mM MSA
and 20mM methylammonium chloride (MMA). After batch
culture for 2 weeks at 30 "C (stirring, 500 r.p.m.; aeration, 600 ml
air per min), the culture was given an intermittent supply of
new medium (flow for 1 h at a dilution rate of 0.03 h-', 2 h off)
with 7 mM MSA and 3 mM MMA. Continuous flow of medium
containing 15mM MSA alone was then started. After 3 weeks,
samples were streaked onto 1.5% (w/v) agar medium, containing
15mM MSA. Organisms showing seven different colony forms
were subcultured from these plates. Nine cultures were obtained
(Ml-M9), six of which were Gram-negative rods (including a
pink-pigmented facultative methylotroph), and three Gram-
positive strains: a slender rod (M5), a normal rod (M3) and a
A diversity of organisms able to grow using MSA as sole
energy substrate was thus present in a randomly chosen soil
sample to which the only known input of MSA or other one-
carbon compounds was from natural sources.
One Gram-negative strain (M2) was studied further. It grew
aerobically in batch culture on MSA (10-20 mM) with a doub-
NATURE . VOL 350 . 18 APRIL 1991
ling time of 7-8 h. Formate (-1 mM) was detected during
growth, and there was a decrease in pH, consistent with the
formation of sulphuric acid. Strain M2 showed similar growth
rates on MSA and mono-, di-, and trimethylamines (MMA,
DMA and TMA), and grew rapidly on methanol. In MSA-
limited chemostat culture, the maximum specific growth rate of
M2 was 0.091 h-', and the biomass concentration was doubled
by doubling the input concentration of MSA, proving depen-
dence on MSA. Growth on MSA produced about l o g dry
biomass per g mol. Yields on MSA and MMA were similar, and
on DMA and TMA were roughly two and three times greater,
respectively. It also grew aerobically on formaldehyde, formate,
methyl nitrite, acetate, propionate, pyruvate, lactate, succinate,
glutamate, serine and glucose, but not on methane, thiosul-
phate, methane phosphonate, dimethyl sulphide or dimethyl
disulphide. Strain M2 is thus a facultatively heterotrophic
We tested the ability of MSA-grown organisms to oxidize
MSA in the oxygen electrode cell by centrifuging and resuspen-
ded them in substrate-free medium. Rapid oxidation occurred
until 1.5 mol oxygen were taken up per mol MSA; the rate then
declined to about 7% of that seen initially. Total oxygen con-
sumption did not reach the 2: 1 stoichiometry required for
complete oxidation of MSA to carbon dioxide and sulphate,
but indicated that formatewas accumulated under the conditions
of the experiments. The oxygen :substrate molar stoichiometries
for methanol, formaldehydeand formatein the oxygen electrode
were 1.0, 0.5 and 0, respectively, consistent with oxidation of
the former compounds only to formate. The Michaelis constant
(K,) values (substrate concentration for half-maximal oxidation
rates) for MSA and formaldehyde was estimated as 0.02 and
0.08 mM, respectively. Cyanide inhibited oxidation of MSA
(50% by 0.5 mM KCN) and of formaldehyde (90% by 1mM).
MSA-grown bacteria showed poor affinity for formate, con-
sistent with formate accumulation during growth on MSA, oxi-
dizing it only at concentrations above 16mM.
Growth on MSA was not chemolithotrophic or autotrophic.
Strain M2 could not grow on thiosulphate, so is unlikely to
obtain energy from sulphonate oxidation. During growth on
MSA or MMA it fixed only about 15% of its cellular carbon
from [14C] carbon dioxide. The autotrophic enzyme, ribulose
bisphosphate carboxylase, was not detected. M2 is thus unlike
T. versutus and T. thioparus, which use inorganic sulphur com-
pounds for energy and grow autotrophically using compounds
like MMA or methylated sulphides as energy source^^.'^.
Cell-free extracts contained no hexulose phosphate syn-
thase14,15,which was easily detected in Methylococcus capsulatus,
assayed as a control. Strain M2 cannot, therefore, use the
ribulose monophosphate cycle for carbon assimilation16.
Hydroxypyruvate reductase14 was detected: extracts showed
hydroxypyruvate-dependent NADPH oxidation activities of
589 nmol NADPH per min per mg protein, but no activity with
NADH. We conclude that carbon assimilation by strain M2 is
by the serine pathway for assimilation of formaldehyde and
MSA degradation by strain M2 does not involve hydrolysis
to produce methane, and is thus not analogous to the C-P
cleavage of methane phosphonate seen in P. testosteroni17. Initial
cleavage of MSA may be by: (1) formation of methyl-tetrahy-
drofolate and sulphite, analogous to Pseudomonas MS growing
on trimethylsulphonium (2) attack by an MSA-
specific mono-oxygenase to produce formaldehydeand sulphite,
as for the NADH- and oxygen-dependent cleavage of C4-C12
n-alkane-1-sulphonates by P s e u d ~ r n o n a s ~ ~ ~ ~ ' ;or (3) hydrolysis
to yield methanol and sulphite.
Clearly many bacterial types can degrade MSA, probably
through diverse routes and at different rates. Such bacteria help
to complete the earth-atmosphere-earth cycle of DMS+ MSA+
C 0 2 +sulphate+ DMS, and will doubtless be shown to be
ubiquitous in the natural environment. 0
LETTERS TO NATURE
Recelved 7November 1990:accepted 22 February 1991.
I . Watts. S. F..Brimblecombe,P. & Watson. A. J. Afmos Envm 24 A. 353-359 (1990).
2. Hataeyama.S., muds. M & Akimoto.H. Geophys Res Lett.9. 583-586 (1982).
3. Andreae. M. 0. In The Role of AirSea Exchange in Geochemical Cycling (ed.Buat-Menard.P.)
5-25.331-362 (Reidel.New Yo*. 1986).
4. Oacey,J. W. H.. King.G. M. & Wakeham. S. G. Nature330.643-645 (1987).
5. Kelly.0. P. & Smith.N. A. Adv microb Ecol ll.345-385 (1990).
6. Ferfk,R. J.. Chatfield.R. B. & Andreae, M. 0. Nature320, 514-516 (1986).
7. Berreshe~m.H. J. I geophys Res 92. 245-262 (1987).
8. Berresheim.H. et a/.J. atmos Chem 10, 341-370 (1990).
9. Grosjean. 0. Envir Sci Techno118.460-468 (1984).
10. Saigne.C. & Legrand.M. Nature330, 240-242 (1987).
11. Smith. A. L. & Kelly. D. P. I gen. Microb~ol.US. 377-384 (1979).
12. Kelly, D. P. & Kuenen. J. G. in Aspects of M~crobialMetabolism and Ecology (ed.Codd.G. A,)
211-240 (Academic. London.1984).
13. Owens,J. 0. & Keddie.R.M. I appl Bact 32,338-347 (1969).
14. Kelly, 0. P & Wood. A. P. in Microbial Growth on C-1 Compounds. Fourth Symposium (eds
Crawford,R. L. & Hanson.R. S.) 324-329 (AmericanSociety for Microbiology. Wash~ngtonDC.
15. Oahl, J. S., Mehta.R J. & Hoare,0. S I Bact. 109.916-921(1972).
16. Colby, J.. Dalton.H.& Whtttenbury,R. A. Rev.M~crobiol33, 481-517 (1979).
17. Oaughton,C. G.,C d , A. M. & Alexander. M. EMS microb Lett 5, 91-93 (1979).
18. Kung.H. F. & Wagner.C. Biochem J ll6,257-265 (1970)
19. Wagner.C..Lusty,S. M.. Kung, F. & Rogers.N. L. I b~olChem 242. 1287-1293 (1967).
20. Thysse.G. J. E.& Wanders.T. H. Antonie vanLeeuwenhdI microb Serol 38, 56-63 (1972).
21. Thysse.G. J. E.& Wanders.T. H. Antonie van LeeuwenMJ microb. Serol. 40.25-37 (1974).
synthesis and eliminate the possibility of cleavage by ribozyme-
catalysed hydrolysis during binding assays.
Chimaeric product oligonucleotides were synthesized con-
taining single deoxyribose residues at successive positions from
the 3' end o f the chain a n d were tested for binding affinity to
the L-21 ScaI ribozyme. The data fitted the theoretical curve
for binding of a single oligonucleotide to a single molecule of
ribozyme (Fig. 1). Deoxyribose substitution at position 3
(GGCCCd(U)CU), three nucleotides from the ribozyme
cleavage site, had the most deleterious effect on binding, fol-
lowed by position 2. Substitution at position 1, 4 or 5 had no
observable effect on binding. The all-RNA product bound to
the ribozyme with 4.1 kcal mol-' extra binding energy (beyond
that calculated for simple RNA.RNA base-pairing; see legend
to Table 1). Removal o f the 2'-OH at position 3 eliminated
1.6 kcal molp' (39%) of this extra bindingenergy (Table 1).The
effects of the single deoxyribose substitutions add up to 56% of
the 4.1 kcal mol-'. The differential effect of individual 2'-OH
groups i s not unique to the lOmM M ~ ~ +binding condition.
ACKNOWLEDGEMENTS.We thaw the Science and EngineeringResearchCouncil (SEWfor support
Ribozyme recognition of RNA by
tertiary interactions with specific
ribose 2'-OH groups
Anna Marie Pyle & Thomas R. Cech*
Howard Hughes Medical Institute and Department of Chemistry and
Biochemistry.University of Colorado. Boulder. Colorado 80309, USA
SHORTENEDforms of the group I intron from Tetrahymena- -
catalyse sequence-specific cleavage of exogenous oligonucleotide
~ubstrates"~.The association between RNA enzyme (ribozyme)
and substrate is mediated by pairing between an internal guide
sequence on the ribozyme and complementary sequence on the
s~bstrate'.~".RNA substrates andcleavage productsassociatewith
a binding energy greater than that of baie-pairing by -4 kcal-
mol-' (at 42OC), whereas DNA associates with an energy around
that expected for b a ~ e - ~ a i r i n ~ ~ ~ .It has been proposed that the
difference in binding affinity is due to specific 2'-OH groups on
an RNA substrate forming stabilizing tertiary interactions with
the core of the ribozyme, or that the RNA-RNA helix formed
upon association of an RNA substrate and the ribozyme might be
more stable than an RNA-DNA helix of the same sequence6.To
differentiate between these two models, chimaericoligonucleotides
containing deoxynucleotide residues at successive positions along
the chainwere synthesized, and their equilibrium binding constants
for association with the ribozymewere measured directly by a new
gel electrophoresis technique5. We report here that most of the
extra binding energy can be accounted for by discrete RNA-
ribozymeint&actio&, the 2'-OH group on the sugar residuethree
nucleotides from the cleavage sitecontributingthe most interaction- -energy. Thus, in addition to the well documented binding of RNA
to RNA by ba~e-~airin~'"'~,2'-OH groups within a duplex can
also mediate association between RNA molecules.
The L-21 Sea1 form of the Tetrahymena ribozyme (E)
catalyses the endonucleolytic cleavage of the oligon;cleotide
substrate pGGCCCUCUA, by guanosine, giving products
pGGCCCUCU (matched product, or MP) and GA5 (ref. 2).
The product pGGCCCUCU binds to the ribozyme with an
equilibriumdissociation constant (K, = [E][MP]/[EMP]) very
similar to that of the substrate5". The product rather than the
substrate oligonucleotide was used in this study to facilitate
*TO whom correspondence should be addressed
8 1 2 16 0
FIG. 1Equilibrium binding curves representing association of chimaeric
oligonucleotideswith the Tetrahymena ribozyme at 42 "C and 5 mM ~ g ~ + .
Binding was measured using the gel-mobility shift method described pre-
viously5. Note that the bindingcurve for GGCCCd(U)CUhas a different x-axis
scale than the others. The solid line represents the fit to a theoretical
binding curve. All curves were fitted to 100% final binding. The K, for each
oligonucleotidewas obtained by solving a nonlinear least-squares equation
describing the best fit of the data to the theoretical lines shown.
METHODS.GGCCCUCUandL-21Scal ribozymewere preparedby transcription
using T7 RNA polymerase2.Chimaeric oligonucleotides were synthesized
on an Applied Biosystems 3808 DNA synthesizer as described in the
~ i t e r a t u r e ~ ~ ~ ~ ~using phosphoramidites(Milligen Biosearch and ABN). Oligo-
nucleotideswere labelled at their 5' end with 3 2 ~and purified by PAGE. The
synthetic oligonucleotidesran as single bands on a gel, with electrophoretic
mobilities comparable with that of transcribed RNA of the same composition.
The presence of 2'-5' linkages and residual protecting groups on the bases
or 2'-OH groups was ruled out by the completeness of digestion with
ribonucleases T2 and A. Synthetically prepared oligonucleotides, like those
prepared by transcription, bound to >90% in all cases and gave good fit
to the theoretical binding curve (S, <7.5%. S,, standard error of estimate
as described previously5).The electrophoresis buffer used for the binding
experiments was 34.5 mM Tris. 65.5mM HEPES, 0.1mM disodium-EDTA
and 5 mM MgCI,, a solution which is pH 7.5 without any adjustment. This
was previously termed TH,,E&Mg,pH 7.5.
NATURE . VOL 350 . 18 APRIL 1991