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LETTERS TO NATURE
Microbial degradation of
methanesulphonic acid:
a missing link in the
biogeochemical sulphur cycle
Simon...
LETTERS TO NATURE
Recelved 7November 1990:accepted 22 February 1991.
I . Watts. S. F..Brimblecombe,P. & Watson. A. J. Afmo...
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Microbial degradation of methanesulphonic acid (Nature 1991)

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Microbial degradation of methanesulphonic acid (Nature 1991)

  1. 1. LETTERS TO NATURE Microbial degradation of methanesulphonic acid: 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 coccus (M8). 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 methylotroph. 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 carbon dioxide16. 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. 0 627
  2. 2. 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. 1984). 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 628 GGCCCUCU 8 1 2 16 0 [El (nM) 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

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