Vol. 53, No. 9APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1987, p. 2111-21180099-2240/87/092111-08$02.00/0Copyright © 1987, American Society for Microbiology

Enzymatic Methylation of Sulfide, Selenide, and Organic Thiols byTetrahymena thermophila

ANNAMARIE DROTAR, LANA R. FALL, ELIZABETH A. MISHALANIE, JENIFER E. TAVERNIER,AND RAY FALL*

Department of Chemistry and Biochemistry and Cooperative Institiute for Research in Environmental Sciences,

University of Colorado, Bouilder, Colorado 80309

Received 24 February 1987/Accepted 1 June 1987

Cell extracts from the ciliate Tetrahymena thermophila catalyzed the S-adenosylmethionine-dependentmethylation of sulfide. The product of the reaction, methanethiol, was detected by a radiometric assay and bya gas-chromatographic assay coupled to a sulfur-selective chemiluminescence detector. Extracts also catalyzedthe methylation of selenide, and the product was shown by gas chromatography-mass spectrometry to bemethaneselenol. The sulfide and selenide methyltransferase activities copurified with the aromatic thiolmethyltransferase previously characterized from this organism (A.-M. Drotar and R. Fall, Pestic. Biochem.Physiol. 25:396-406, 1986), but heat inactivation experiments suggested the involvement of distinct sulfide andselenide methyltransferases. Short-term toxicity tests were carried out for sulfide, selenide, and theirmethylated derivatives; the monomethylated forms were somewhat more toxic than the nonmethylated or

dimethylated compounds. Cell suspensions of T. thermophila exposed to sulfide, methanethiol, or their seleniumanalogs emitted methylated derivatives into the headspace. These results suggest that this freshwater protozoanis capable of the stepwise methylation of sulfide and selenide, leading to the release of volatile methylated sulfuror selenium gases.

Biological systems are known to produce and emit meth-ylated sulfur compounds (1, 2, 5, 14, 26). Two major routesfor the biological formation of these compounds are thedegradation of methionine (14, 15, 19, 21) and, in aquaticenvironments, the cleavage of algal dimethylsulfoniopro-pionate (1, 2, 23). Another possible pathway for the forma-tion of these compounds is through the methylation ofsulfide. Indeed, tissues from mammalian cells (24), as well asa spectrum of bacterial species (7), are capable of thistransformation. Thiol methyltransferase, the enzyme re-sponsible for this reaction, has been purified from rat liver(24). Many organisms, including fungi, bacteria, plants, andmammals, also excrete methylated selenium compoundswhen exposed to excess selenium (3, 6, 22). This transfor-mation has been viewed as a detoxication mechanism inmammals. The metabolism of selenite to dimethyl selenide inrats has been shown to involve reaction with glutathione,reduction to selenide, and, finally, methylation of selenide(11). Therefore, in some cases the formation of bothdimethyl sulfide and dimethyl selenide in the environment ispossibly the result of this direct methylation of sulfide orselenide, respectively.The chemical properties of sulfur and selenium are very

similar, and biochemical systems do not always distinguishbetween the two elements (22). Since a variety of organismsexcrete both methylated sulfur and selenium compounds, wewondered whether the same enzyme could be involved intheir formation. Indeed, methanethiol and methaneselenolcan be formed from methionine and selenomethionine, re-spectively, by the enzyme L-methionine -y-lyase from Pselu-domonas putida (11). Another possible candidate for thisdual role would be the direct methylation of sulfide andselenide by the enzyme thiol methyltransferase.

* Corresponding author.

The ciliate Tetrahvmena thermophila has an aromatic thiolmethyltransferase which methylates a variety of xenobioticthiols (10). The organism also takes up foreign thiols,methylates the sulfhydryl group, and excretes the productinto the medium (8). This sequence often leads to detoxica-tion of the reactive thiol, a role similar to that assumed forthe enzyme in rat tissue (25). In this report the ability of T.thermophila extracts to methylate sulfide and methanethioland their selenium analogs is demonstrated, and preliminaryevidence is presented for the presence of distinct sulfidemethyltransferase and selenide methyltransferase. We alsodescribe the ability of whole cells to release volatile, meth-ylated derivatives after exposure to these compounds.

MATERIALS AND METHODSChemicals. [methyl-3H]S-adenosylmethionine (15 mCi

mmol-V) was obtained from New England Nuclear Corp.,Boston, Mass.). Ammonium sulfide was obtained from Sci-entific Gas Products, Denver, Colo., and dimethyl sulfidewas obtained from Sigma Chemical Co., St. Louis, Mo.Sodium selenide, dimethyl selenide, dimethyl diselenide,sodium tetrahydroborate, and 2-nitrophenyl disulfide wereobtained from Aldrich Chemical Co., Inc., Milwaukee, Wis.

2-Nitrobenzenethiol was prepared from 2-nitrophenyl di-sulfide by reduction with a basic glucose solution. Disulfide(1 g) and glucose (1.35 g) were dissolved in 5 ml of 95%ethanol. The solution was heated to 60°C, and 1.5 ml of 10 Nsodium hydroxide was added. The solution was maintainedat 60°C for 10 min, diluted with 20 ml of water, and thenfiltered into a mixture of 5 ml of concentrated hydrochloricacid and ice. The product was recrystallized from boilingethanol.

Methaneselenol was prepared by reduction of dimethyldiselenide with sodium tetrahydroborate. The neat liquid(0.3 ml) was dissolved in ethanol (1 ml) in a vial. Sodium

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tetrahydroborate (0.18 g) was dissolved in ethanol (1 ml),and the solution was slowly added to the vial containing thediselenide. Nitrogen was bubbled through the solution, andthe effluent was collected in 0.1 M potassium phosphatebuffer (pH 8.0). The concentration of selenol was determinedby the reaction with 5,5'-dithiobis(2-nitrobenzoic acid) (17).Growth of cells. T. thermophila Bll (10) was subcultured

from a bacterium-free stock culture and was grown withoutshaking at 25°C in a medium containing 1.5% (wt/vol) DifcoProteose Peptone (Difco Laboratories, Detroit, Mich.) and30 mg of ferric EDTA liter-' for 48 h to a cell density of 6 x105 to 8 x 105 cells ml-'. The cells were harvested bycentrifugation and washed once in 10 mM Tris hydrochloride(pH 7.4) (buffer T). The cell pellet was suspended in avolume of buffer T equal to the original culture volume. Thecell density was determined by diluting a portion 1:2 with 4%(vol/vol) formaldehyde and counting with a hemacytometer.

Whole-cell incubations. Washed cells were dispensed in5-ml aliquots into 25-ml Erlenmyer flasks. Ammonium sul-fide, sodium selenide, methanethiol, methaneselenol, dimeth-yl disulfide, or dimethyl diselenide was added to concentra-tions from 0.01 to 10 mM. The flasks were tightly cappedwith rubber septum stoppers. For toxicity studies, aliquotsof the cell suspension were removed at various times, dilutedwith an equal volume of4% (vol/vol) formaldehyde, and thencounted as described above. Originally, the number of viablecells was determined by using an ink uptake test (10) asdescribed previously. However, 100% of the cell populationwas found to be viable by this test, even as the total cellcount decreased. Therefore cells which were killed bytoxicant apparently lysed, and the cell number was subse-quently used as the number of surviving viable cells. A plotwas made of the log of the concentration of test compoundversus the percentage of surviving cells. The concentrationof toxicant producing 50% mortality in a 3-h exposure (3-hLC50) was determined by using linear interpolation betweentwo successive points representing >50% and <50% sur-vival (10). For the methylation studies 1 ml of headspace wasremoved at various times with an airtight syringe and ana-lyzed by packed-column gas chromatography with the chem-iluminescence sulfur detector as described previously (7).Control reactions were carried out by using cells withoutadded methyl acceptor or boiled cells plus methyl acceptor.For studies of the product of methylated sulfur com-

pounds by untreated cultures of T. thermophila and by cellsexposed to low levels of sulfide, the flux of sulfur compoundsinto the headspace of the culture was measured. Washedcells were dispensed in 25-ml aliquots into 250-mi polycar-bonate bottles. The bottles were sealed with Teflon filmsecured with a rubber band. The film was punctured to allowinsertion of the carrier gas and Teflon sampling tubing. Forstudies involving the use of low concentrations of sulfide, thewashed-cell suspension was placed inside a closed foamcontainer alongside an open bottle of 0.1 M ammoniumsulfide for 2 h before the bottles containing suspension weresealed with Teflon film. Both samples were analyzed byusing a gas chromatograph coupled to a flame photometricdetector (7).

Preparation and fractionation of cell extracts. We preparedcell extracts from harvested and washed cells by freezing thecell pellet on dry ice and then thawing it. The pellet wassuspended in a volume of cold buffer T equal to that of theoriginal culture. The cells were homogenized by using 10passes in a Teflon-glass homogenizer. The extract wascentrifuged at 12,000 x g for 10 min at 4°C, and thesupernatant was used for enzyme assays.

In some experiments cell extracts were fractionated at 4°Cby gel filtration and ion-exchange chromatography. Detailsare described in the text and in the legend to Fig. 3.Heat inactivation. A preparation of the mixture of methyl-

transferases, obtained by fractionation on Sephacryl S-200as above, was exposed to temperatures ranging from 37 to68°C for various times, and aliquots were removed andcooled to ice temperature. Then all the samples were as-sayed by the standard radiometric assay with sulfide,selenide, or 2-nitrobenzenethiol as the substrate.

Protein assay. Protein was determined by the method ofBradford (4).Enzyme assays. Two procedures were used to determine

enzyme activity. The first measured the transfer of 3H-methyl groups from S-adenosylmethionine to 2-nitroben-zenethiol. The second monitored the production of methyl-ated product by gas chromatography. For the radiometricassay, 0.45-ml portions of crude extract were dispersed into0.5-ml microcentrifuge tubes. Methyl acceptor was added atconcentrations indicated for each experiment. The reactionwas started by the addition of 0.1 mM [methyl-3HJS-adenosylmethionine (5.5 mCi mmol-') and was analyzed asdescribed previously (7). For the gas-chromatographic as-says, 5.0-ml aliquots of the crude extract, 0.1 mM S-ade-nosylmethionine, and methyl acceptor were dispensed into25-ml Erlenmyer flasks, which were sealed with rubber septaand incubated at 25°C. The headspace of each reaction flaskwas analyzed by packed-column gas chromatography withthe chemiluminescence sulfur detector as previously de-scribed (7). When 2-nitrobenzenethiol was used as a sub-strate, 1.0 ml of reaction mixture, made basic with 0.2 ml of2N NaOH, was extracted with 2 ml of hexane (UV grade).The hexane layer was then analyzed by capillary column gaschromatography with a flame ionization detector (9). In bothassays, control reactions were always run. For theradiometric assay, control reaction mixtures lacked eitherthe thiol substrate or enzyme or contained a boiled extract.For the gas chromatography assay, control reactions lackedmethyl acceptor or S-adenosylmethionine or contained aboiled extract.

Identification of products. With sulfide as a substrate, theidentity of the methylated product was shown in two ways.First, a cell extract was assayed by the gas chromatographyprocedure with the chemiluminescence detector. The reten-tion times of the reaction products were compared with theretention times of authentic standards on the same chro-matographic column. Second, a typical radiometric reactionmixture was made basic with NaOH and then extractedtwice with toluene. The organic layers from both the basicand acidic extractions were counted in a scintillationcounter.The identity of the enzymatic methylation product of

selenide was determined by the method of Ganther andKraus for the analysis of volatile selenols (13). A typicalradiolabeled reaction was run in a test tube fitted with astopper and Teflon tubing for inlet and outlet. The outlettubing led to a trap containing 1-fluoro-2,4-dinitrobenzenedissolved in dimethyl formamide and aqueous sodium bicar-bonate solution (13). After a 15-min reaction time wasallowed, the reaction mixture was acidified with HCl and N2was bubbled through the system for 10 min. The contents ofthe trap were extracted with benzene, evaporated to neardryness with a stream of N2 and run on a silica gel thin-layerchromatography plate with benzene-pyridine (80:20, vol/vol)as solvent. A strip on one side of the thin-layer chromatog-raphy plate was divided into 1-cm sections. Each section

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was scraped and eluted with benzene until no more radioac-tivity was detected in the wash. The benzene extract was

evaporated to near dryness and streaked across the origin ofa second plate, which was developed in n-heptane-chloro-form-pyridine (5:5:1, vol/vol/vol). The radioactive area was

determined as above, scraped, and eluted with benzene. Thebenzene was evaporated to near dryness, and the solutionwas analyzed by gas chromatography-mass spectrometry.

The gas chromatograph (model 5290A; Hewlett-PackardCo., Palo Alto, Calif.) had a cross-linked SE54, fused silicacapillary column (length, 25 m; internal diameter, 0.2 mm).A temperature program was run from 230 to 300°C at 4°min-'. The mass spectrometer was a model 7070 EQ-HF(VG Analytical Ltd., Manchester, England) with a 70-eVelectron ionization. Mass-spectral resolution was 1,000, witha mass range of 500 with a 40 cutoff. The spectrum of theradioactive derivative was compared with that of authenticdinitrophenyl methylselenide synthesized by the procedureof Ganther and Kraus (13).Gas chromatography. The 2-nitromethylthiobenzene prod-

uct was detected by using a capillary column (length, 12 m;

internal diameter, 0.2 mm) of methyl silicone in a model5790A Hewlett Packard gas chromatograph equipped with a

flame ionization detector. A splitless injection mode was

used, and a temperature program from 80 to 250°C was run.

The detector temperature was 300°C, and that of the injec-tion port was 250°C. Peak areas were determined using a

Hewlett Packard model 3390 A integrator. A standard curve

was generated with a 2-nitromethylthiobenzene standardprepared by methylating the thiophenol with methyl iodide.

Methanethiol, dimethyl sulfide, and their selenium analogswere detected by using a packed glass column in a model5710A Hewlett-Packard gas chromatograph attached to a

chemiluminescence detector with selectivity for reducedsulfur compounds. The detector and instrumentation havebeen described previously (18) and used for quantitation ofthe biological production of methylated sulfur gases (7).The presence of volatile sulfur gases above unamended

cultures was also confirmed by an analysis with a gas

..~~~~1 5

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ro

am5~~

2 3 4

TIME (h)FIG. 1. Time course of production of methanethiol by crude

extracts of T. thermophila. Crude extract containing 0.75 to 1.5 mgof protein was incubated in the presence or absence of 0.1 mMS-adenosylmethionine and 1 mM ammonium sulfide in a sealedflask. The headspace was sampled at the specific times and analyzedby gas chromatography. Symbols: A, complete reaction with 0.75mg of protein; *, complete reaction with 1.5 mg of protein; 0,

reaction minus ammonium sulfide; O, reaction minus S-adenosylmethionine; A, complete reaction, but with boiled crudeextract.

TABLE 1. Methyltransferase activities in crude extractsof T. thermophila

Methyltransferase

Substrate" activity (10-2nmol min-' mg of

protein -)

Ammonium sulfide ................................. 4.43Sodium selenide ................................... 4.31Methanethiol....................................... 3.74Methaneselenol .................................... 11.5Dimethyl disulfide ........... 1.72Dimethyl disulfide (plus GSH)b ....................3.88Dimethyl diselenide ................................ 2.43Dimethyl diselenide (plus GSH) ........ ........... 6.062-Nitrobenzenethiol ................................ 3.76

a Each substrate was added at 1 mM, except 2-nitrobenzenethiol. whichwas added at 5 x 10-5 M.

I GSH, Reduced glutathione.

chromatograph coupled to flame photometric detection asdescribed previously (7).

RESULTS

Detection of sulfide methyltransferase activity in cell ex-tracts. Cell extracts of T. thermophila were capable ofmethylating sulfide in an S-adenosylmethionine-dependentreaction. Two techniques were used to demonstrate thisreaction. A radiometric assay measured the transfer of a3H-methyl group from [methyl-3H]S-adenosylmethionine tosulfide, following the appearance of toluene-soluble radioac-tivity (i.e., methanethiol plus dimethyl sulfide). The secondtechnique involved the use of gas chromatography with asulfur-selective detector to measure the appearance ofmethanethiol in the headspace of closed reaction vessels;this detector responds to organosulfur compounds but not tohydrogen sulfide (18). Figure 1 shows the results of typicalassays using the latter technique; the production of meth-anethiol was linear for several hours in this case. Thereaction was dependent upon the presence of both themethyl donor, S-adenosylmethionine, and the methyl accep-tor, ammonium sulfide. The amount of product formed wasproportional to both the time elapsed and the amount ofextract present. Similar results were obtained by using theradiometric assay (data not shown).To compare the results of these two methods we used a

partition coefficient of 8.0 (water/air) for methanethiol. (Thedistribution of methanethiol between phases depends uponthe ionic strength and the pH of the liquid phase; partitioncoefficients of 8.0 to 9.9 in favor of the liquid phase havebeen published for water/air and seawater/air [20]). Cor-rected sulfide methyltransferase activities for cell extractswere 4.6 x 10-2 and 5.6 x 10-2 nmol min-' mg of protein-'for the radiometric and gas-chromatographic assays, respec-tively. Thus after 4 h, 0.26% of the added substrate wasconverted to product. The lower value for the radiometricassay suggests that some product loss to the gas phase hadoccurred during the solvent extraction used in the assay.Other methyltransferase substrates. Crude extracts were

also assayed for S-adenosylmethionine-dependent methyla-tion of selenide and other thiols and selenols. The specificmethyltransferase activity toward these substrates is listedin Table 1. Also assayed for comparison was the aromaticthiol 2-nitrobenzenethiol, a substrate for the thiol methyl-transferase of T. thermophila (10). Each of these thiols andselenols was methylated (Table 1). Also methylated were

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151

I1Iit 1 IIIIA l .I ...1....1- .........iL~~~~~~~~~~~~~~~~~~~~~~~~I I I l l l20 40 60 80 100

262

I

180 200 220 240m/e

247

.111,1 .111

120 140 160

260 280 300 320

FIG. 2. Mass spectrum of the dinitrophenyl derivative of the enzymatic product resulting from the incubation of crude extracts of T.thermophila with sodium selenide and S-adenosylmethionine. The parent ion (mle 262) and some selected key fragments are indicated on thefigure.

dimethyl disulfide and dimethyl diselenide when the reduc-ing agent glutathione was present, presumably because theywere first reduced to the thiol or selenol form, respectively,and then methylated.The product of the reaction with sulfide was determined to

be methanethiol on the basis of its acid-base properties andits retention time on a gas chromatography column. Theproduct could not be extracted from a basic solution, butcould be recovered from an acidic solution. Thus it con-tained an acidic group. The product also cochromatographedwith authentic methanethiol during gas chromatography andwas detected by the sulfur-selective chemiluminescencedetector. Since the product was known to be radioactive andits formation depended upon the presence of [methyl-3H]S-adenosylmethionine, it had to contain a methyl group. Thusthe fact that the product contained sulfur, a methyl group,and an ionizable hydrogen atom and coeluted with meth-anethiol strongly supports its identity as methanethiol.The identity of the product of the enzymatic reaction with

selenide was determined by making the 2,4-dinitrophenylderivative, purifying it by thin-layer chromatography, andsubjecting it to analysis by gas chromatography-mass spec-trometry. The mass spectrum of the derivative (Fig. 2) wasidentical to that published for 2,4-dinitrophenyl methylse-lenol, demonstrating that the enzymatically methylatedproduct was methaneselenol.

Partial purification of methyltransferases. We wished todetermine whether thiols and selenols were methylated byone nonspecific methyltransferase or by different specificenzymes. Therefore a crude extract was applied to a Seph-acryl S-200 column, and fractions were assayed for methyl-transferase activity. On this column the activity towardsulfide, selenide, and 2-nitrobenzenethiol were all eluted in a

single peak (Fig. 3). The middle two-thirds of the peak was

pooled, applied to a column of DEAE-Sephacel, and elutedwith a linear salt gradient (0 to 0.5 M NaCl). Again, all threemethyltransferase activities were eluted in a single peak(data not shown). The calculated specific enzyme activitieswith these substrates are presented in Table 2, showing that

the ratios of the specific activities toward sulfide, selenide,methanethiol, and methaneselenol were constant throughthese two purification steps, which produced a 50-fold in-crease in specific activity.Heat inactivation. To determine whether the sulfide and

selenide methyltransferases that copurified were due to asingle enzyme capable of sulfide and selenide methyltrans-ferase activities, a partially purified preparation was sub-

10 25

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elte wit 20m oasu popae05mMET p .)

2-5

50 60 70 80

FRACTION NUMBER

FIG. 3. Gel filtration chromatography profile of methyltrans-ferase activity toward sulfide, selenide, and 2-nitrobenzenethiol. A

crude extract was prepared from a 500-ml culture and applied to a

column of Sephacryl S-200 by 50 cm) at 40C. The column was

eluted with 20 mM potassium phosphate-0.5 mM EDTA (pH 7.4),

and 5-ml fractions were collected. Fractions were assayed formethyltransferase activity as described in Materials and Methods.Symbols: 0, activity with sulfide; *, activity with selenide; A,activity with 2-nitrobenzenethiol.

100[-CH3-Se - NO,

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401

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2114 DROTAR ET AL.

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TABLE 2. Copurification of sulfide, selenide, methanethiol, andmethaneselenol methyltransferase activities of T. thermophila

Sp act (nmol min-' mg of protein-')(purification factor) with:

StepSulfide Selenide Methane- Methane-Sulfide Selenide thiol selenol

Crude extract 0.032 (1) 0.043 (1) 0.029 (1) 0.074 (1)Sephacryl S-200 0.28 (8.8) 0.38 (8.8) 0.26 (9.2) 0.59 (8.0)DEAE-Sephacel 1.57 (49.7) 2.30 (53.3) 1.66 (57.6) 4.27 (58.1)

jected to a series of heat inactivation experiments. Theresults of heating the preparation at 37 and 47°C are shown inFig. 4. At 37°C the sulfide methyltransferase was activated40%, while the selenide methyltransferase was progressivelyinactivated. At 47°C both enzyme activities were inactivatedmore than 50% in 30 min, but with different thermal inacti-vation curves. Assays for aromatic thiol methyltransferasein these preparations revealed a thermal inactivation patterndistinct from either the sulfide methyltransferase or selenidemethyltransferase activities (data not shown).

Determination of apparent kinetic parameters. Using thestandard radiometric assay, apparent Km values for theenzyme-catalyzed methylation of sulfide, methanethiol,selenide, and methaneselenol were determined with thepartially purified enzyme preparation. S-Adenosylmethio-nine was held at a fixed concentration of 0.1 mM while therate of the reaction was determined at various concentra-tions of the methyl acceptor substrates. Apparent Km valuesfor the four substrates were obtained from direct linear plotsof the initial velocity data: 1.4 mM for sulfide, 1.9 mM forselenide, 0.75 mM for methanethiol, and 0.85 mM formethaneselenol.

Toxicity studies. Before determining whether whole cells

370 470

140-

1000

z~~~

w

60

wA

20

I0 10 20 30 0 10 20 30

TIME (min)FIG. 4. Heat activation of T. thermophila sulfide and selenide

methyltransferases. A partially purified enzyme preparation con-

taining sulfide and selenide methyltransferase activities was incu-bated at 37°C (left) or 47°C (right) for the times indicated and thenassayed as described in Materials and Methods. Two experimentsare shown (open and solid symbols); each datum point is the averageof duplicate assays. Symbols: 0, 0, activity with sulfide; A, A,

activity with selenide.

[( N H4)2 SI

O mM

Z \0 2 mMz

W 60

w

0

20

5mM

< i A ~~~10mM1 2 3 4

TIME (h)

FIG. 5. Loss of viability of T. thermophila after short-termexposure to various concentrations of ammonium sulfide. Cells wereharvested, washed, and suspended in 10 mM Tris hydrochloride (pH7.4) and dispensed in 5-ml aliquots into stoppered vials at a celldensity of 105 cells ml-'. Ammonium sulfide was added at theindicated concentrations, the vials were stoppered and incubated at22°C, and the number of viable cells was determined at specifictimes as described in Materials and Methods.

could methylate thiols and selenols, we determined thetoxicity of these substances toward T. thermophila. Typicalsurvival curves (for sulfide in this case) are shown in Fig. 5.Three-hour LC50 values were determined from this type ofdata for several sulfur and selenium analogs (Table 3). Theselenium compounds were slightly more toxic than theanalogous sulfur compounds, and methanethiol and meth-aneselenol were somewhat more toxic than sulfide andselenide, respectively. The dimethylated compounds (i.e.,dimethyl sulfide and dimethylselenide) were the least toxicof those studied.

Whole-cell methylation experiments. To prevent cell deathduring biotransformation experiments, whole cells were

exposed to 1 mM sulfide, 0.2 mM selenide, 0.5 mM meth-anethiol, or 0.5 mM methaneselenol, which are sublethallevels of these toxicants. After exposure of whole cells of T.thermophila, suspended in a simple buffer, to these sulfurand selenium compounds, the methylated products were

TABLE 3. Values of 3-h LC50 for sulfide, selenide, and relatedmethylated derivatives in T. thermophila

Compound 3-h LC50 (mM)a

Ammonium sulfide. 4Methanethiol.1.5Dimethyl sulfide.30Dimethyl disulfide.1.5Sodium selenide.1.5Methaneselenol.0.6Dimethyl selenide.19Dimethyl diselenide. 32-Nitrobenzenethiol.0.06

a LC5, values were determined as described in Materials and Methods. Thevalues shown are the averages of duplicate determinations.

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detected in the headspace above the culture by gas chroma-tography with a chemiluminescence sulfur detector (18).This detector also responded to the selenium analogs; how-ever, the detection limits were higher than those for thesulfur compounds, as discussed by Mishalanie and Birks(16).For cells exposed to ammonium sulfide the major volatile

sulfur-containing product in the headspace was methane-thiol; it accumulated linearily for up to 4 h, and its produc-tion was dependent upon the number of cells present.Dimethyl sulfide, the product of a second methylation step,was not detected. However, if methanethiol was added tothe cell suspension, dimethyl sulfide was detectable in theheadspace. The rates of methylated product accumulationwhen cells were exposed to 1 mM sulfide or 0.5 mMmethanethiol were 56.3 and 42.7 pmol h-1 106 cells-' ml ofheadspace-1, respectively. This represents conversion of0.32 and 0.16% of the test compound to its product eachhour. Also, methaneselenol or dimethyl selenide accumu-lated in the headspace at a rate of 9 and 11 pmol h-1 106cells-' ml of headspace-1 when cells were exposed to 0.2mM sodium selenide or 0.5 mM methaneselenol, respec-tively, which represents conversion of 0.058 and 0.038% ofthe selenides to their methylated products per hour. The rateof accumulation of methaneselenol after exposure of cells toselenide is only an estimate, since reagent-grade methane-selenol (for calibrations) was not available. It was assumedthat the ratio of molar detection limits of methaneselenol tomethanethiol was the same as that of dimethyl sulfide todimethyl selenide. Therefore, intact cells emitted methane-thiol when exposed to sulfide, and emitted dimethyl sulfideat similar rates when exposed to methanethiol. In a similarway, but at lower rates than with sulfur analogs, cellsemitted methaneselenol and dimethyl selenide when ex-

posed to selenide and methaneselenol, respectively.To verify the production of methylated sulfur gases by

unamended cultures of T. thermophila and by culturesexposed to physiological levels of sulfide, we measured theflux of gases from the culture by using a flame photometricdetector and gas chromatographic separation. Hydrogensulfide, methanethiol, dimethyl sulfide, and dimethyl disul-fide were all given off by unamended cell suspensions of T.thermophila. The flux of these gases in pmol (standardliter)-' was: hydrogen sulfide, 15.1 ± 2; methane thiol, 24.3+ 3; dimethyl sulfide, 3.1 + 0.1; and dimethyl disulfide, 4.1

1. When cell suspensions were exposed to exogenoushydrogen sulfide gas before analysis, the flux of all thesesulfur gases increased. Hydrogen sulfide flux increased by afactor of 5.5, methanethiol flux increased by a factor of 2,and dimethyl sulfide flux increased by a factor of 1.3. Byusing a partition coefficient of 10 in favor of the liquid phasefor hydrogen sulfide (25), the concentration of sulfide in theliquid phase in this experiment can be estimated to be in thenanomolar range.

DISCUSSION

Many groups of aerobic microorganisms, including bacte-ria and fungi, have been shown to release volatile, methyl-ated sulfur and selenium gases into the environment (5, 6,14). Such processes in terrestrial and aquatic environmentsare of interest in the global cycling of sulfur and selenium (1,5). In this study we have shown that the aerobic, free-livingciliate T. thermophila also can emit such gases and havedemonstrated that the enzymatic basis for these emissions

involves intracellular S-adenosylmethionine-dependent sul-fide and selenide methyltransferases that catalyze the meth-ylation of sulfide and selenide as well as thiols and selenols.Two techniques were used to show the occurrence of

S-adenosylmethionine-dependent methylation of sulfide bycell extracts of T. thermophila. The first was an establishedradiometric assay (25) that measures the transfer of a triti-ated methyl group from S-adenosylmethionine to sulfide.The second method was an analysis of the gas phase aboveclosed reaction vessels by using a gas chromatograph with asulfur-selective chemiluminescence detector (18). Since hy-drogen sulfide and its methylation products, methanethioland dimethyl sulfide, are gases, losses of products to the gasphase during manipulations of the assay leads to underes-timates when using the radiometric method; the amounts ofthe two methylated products are also not determined in thisassay, since the products are not separated in the solventextraction step. The use of gas-chromatographic analysisovercomes these limitations. Furthermore, the use of thechemiluminescence detector in place of the standard flamephotometric detector for detection of sulfur gases has themajor advantage of responding to the organosulfur productsbut not to hydrogen sulfide (18). Therefore, tailing from theabundant substrate peak (i.e., hydrogen sulfide) does notobscure the relatively smaller product peaks (i.e., meth-anethiol and dimethyl sulfide). The validity of the gas-chromatographic assay was confirmed by showing that therate of methylated product formation, after correction forpartitioning between the liquid and gas phases, was compa-rable to that obtained in the radiometric assay.The ability of cell extracts to methylate selenide was also

demonstrated by the radiometric assay, and the product wasconfirmed to be methaneselenol by gas-chromatogra-phic-mass-spectrometric analysis of the 2,4-dinitrophenylderivative. A headspace analysis assay was also possible,because it was discovered that the chemiluminescence de-tector also responded to selenium compounds. However,because the detection limit with the chemiluminescencedetector was higher for the selenium analogs than for theirsulfur counterparts, this assay was not routinely used forassaying selenide methylation. This problem might be alle-viated in the future by changing the optical filter used in thedetector and optimizing it for selenium detection.

Methyltransferase activity with five methyl acceptors,sulfide, selenide, methanethiol, methaneselenol, and 2-nitro-benzenethiol, was found to copurify during gel filtration andion-exchange chromatography, suggesting that one methyl-transferase with broad substrate specificity was present.However, heat inactivation experiments suggested that atleast two enzymes were responsible: a sulfide methyltrans-ferase and a selenide methyltransferase. The presence of adistinct aryl thiol methyltransferase was also suggested.Clearly, the purification of the enzyme(s) will be required toestablish this point.When sulfide was used as the methyl acceptor substrate in

crude extracts, methanethiol but not dimethyl sulfide wasdetected by gas chromatography. In partially purified en-zyme preparations the apparent Km values for hydrogensulfide and for methanethiol were both near 1 mM. How-ever, the amount of methanethiol formed in reactions inwhich sulfide was the methyl acceptor substrate was maxi-mally 2.8 FiM. Therefore the amount of dimethyl sulfide thatcould be formed in a second methylation step would bebelow the detection limits of our system. The thiol methyl-transferase from mammalian sources can methylate bothsulfide and methanethiol as well as a variety of organic thiols

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TETRAHYMENA METHYLTRANSFERASES 2117

(24, 25). Details of the T. thermophila methyltransferasesubstrate specificity and selectivity for S and Se methylationwill have to await purification and kinetic analysis of theenzyme(s).The physiological role of the sulfide and selenide methyl-

transferases in T. thermophila is not known with certainty.In mammals the conversions of hydrogen sulfide tomethanethiol and dimethyl sulfide (25) and of selenite todimethyl selenide have been viewed as detoxication mecha-nisms (12, 22). The methylation of aromatic thiols by T.therm)ophila also affords a protective effect (10). Toxicitytests with T. thermophila (Table 3) demonstrated thatmethanethiol and methaneselenol were slightly more toxicthan sulfide or selenide, but that dimethyl sulfide anddimethyl selenide were 1 order of magnitude less toxic. Thisis not surprising, since methanethiol and methaneselenolboth maintain a reactive thiol or selenol group that is notpresent in the dimethyl derivatives. Dimethyl disulfide anddimethyl diselenide had toxicities similar to those of theproducts methanethiol and methaneselenol, respectively.This could be explained by cellular reduction of the disulfideor diselenide to the corresponding thiol or selenol; this wasobserved for dimethyl disulfide where exposed cells rapidlyrelease methanethiol. It is possible that the toxicity ofsulfide, selenide, and their methyl derivatives was due inpart to anoxic stress caused by oxygen depletion. This effectwas not systematically investigated.

If the process of methylation of thiols and selenols is tofunction in detoxication, whole cells should carry out thetransformation. Whole cells were therefore exposed to con-centrations of these substrates below LC50 levels that causecell death. Under these conditions and as detected bygas-chromatographic analysis with the chemiluminescencedetector, the cells released the methylated derivatives intothe gas phase. Thus they were able to convert sulfide tomethanethiol and then methanethiol to dimethyl sulfide. Thesame two-step sequence was found with the selenium ana-logs.

Control cell suspensions that had not been exposed tosulfide emitted variable amounts of methanethiol at thedetection limits of the system. To confirm the production ofmethylated sulfur compounds by these control suspensions,the flux of sulfur gases was analyzed by using a gas chro-matographic system with a flame photometric detectorwhich was routinely calibrated and dedicated for the detec-tion of a variety of biogenic sulfur gases. The control cellsindeed produced methanethiol, hydrogen sulfide, dimethyldisulfide, and dimethyl sulfide. When cell suspensions wereexposed to very low levels of hydrogen sulfide via the gasphase, the subsequent flux of methanethiol and dimethylsulfide from the culture was increased by a factor of 1.3 to 2.Thus it seems that cultures of T. tlzerinophila are able toconvert sulfide to methanethiol and dimethyl sulfide whenexposed to physiological levels of exogenous sulfide.The rates of in vivo and in vitro methylation of these sulfur

and selenium compounds are difficult to compare, since thetwo types of experiments were performed by different meth-ods and with different substrate concentrations. The concen-trations used in the cell-free experiments were at toxic levelsfor the cells and so could not be used for the whole-cellexperiments. However, the data for the methylation ofsulfide are comparable. Since 106 cells ml-' yield a proteinconcentration of 3 mg ml-1, the rate of whole-cell methane-thiol production was 4.8 x 10-2 nmol min' mg of protein-'after correction for partitioning of the product betweenphases. This compares reasonably well with the estimate of

5.6 x 10-2 nmol min-' mg of protein-' for the reaction ratein crude extracts.

Further insight into these methylation reactions in vivowill be gained by use of isotopically labeled sulfide orselenide combined with measurement of their uptake andincorporation into cellular pools and eventual release fromcells as methylated products. Measurement of cell poolconcentrations of sulfide or selenide would allow compari-son with K,,, values for these methyltransferase substratesand allow some firmer conclusions to be made about the roleof methylation in vivo as a pathway for detoxication ofreactive thiols and selenols.The major sulfur compounds emitted to the atmosphere

above freshwater ponds, where T. thIermophila is oftenfound, are carbon disulfide, hydrogen sulfide, dimethyl sul-fide, and dimethyl disulfide (2). Our results suggest that T.therrnophila is capable of methylating exogenous sulfide.Thus at least part of the flux of methylated sulfur gases fromthis habitat could be due to the biogenic methylation ofsulfide. The ability to produce methanethiol from sulfide hasalso been demonstrated for numerous aerobic bacteria iso-lated from freshwater sediment (7), and these organismscould also be contributing to the flux of methylated sulfurgases. As in T. thlewrnophzila, this biotransformation appearsto be mediated by a sulfide methyltransferase. Our resultsfurther suggest that these organisms are also able to meth-ylate selenium. Indeed, methylated selenium gases areknown to be evolved from soil and aquatic environmentsamended with various inorganic and organic forms of sele-nium (6). However, the naturally occurring fluxes of thevolatile forms of selenium are unknown.

ACKNOWLEDGMENTS

This investigation was supported by grant ES 02639 from theNational Institute for Environmental Science.We thank John Birks for encouraging us to use his chemilumines-

cence detector, Paul Goldan for helping with gas-chromatographictechniques. and Bob Barkley for obtaining the mass-spectral data.

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