JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1977, p. 187-194Copyright C 1977 American Society for Microbiology

Vol. 6, No. 3Printed in U.S.A.

Methyl Mercaptan and Dimethyl Disulfide Production fromMethionine by Proteus Species Detected by Head-Space Gas-

Liquid ChromatographyN. J. HAYWARD,'* T. H. JEAVONS,1 A. J. C. NICHOLSON,2 AND A. G. THORNTON2t

Microbiology Department, Monash University Medical School, Prahran, 3181,1 and Division of ChemicalPhysics, Commonwealth Scientific and Industrial Research Organization, Clayton, 3168,2 Victoria,

Australia

Received for publication 4 August 1976

Head-space gas-liquid chromatography and mass spectrometry were used todetect and identify products formed by Proteus vulgaris, P. mirabilis, P. mor-

ganii, and P. rettgeri from a defined medium supplemented with either phenyl-alanine, methionine, valine, leucine, histidine, lysine, ornithine, threonine,asparagine, aspartic acid, or tryptophan. In a detailed study of the productsformed by 68 strains ofProteus spp. from L-methionine, the production of largeamounts of both dimethyl disulfide and methyl mercaptan was found to be acharacteristic of the genus. Both sulfur products appeared within a few hours ofinoculation. Dimethyl disulfide was a more sensitive indicator of growth thanthe spectrometric determination of optical density. This suggests that it could beuseful for the rapid, automated detection of any species of Proteus.

In recent years there has been increasinginterest in rapid methods for detecting microor-ganisms, especially if they can be automated.Gas-liquid chromatography (GLC) is a sensi-tive, quantitative method ofseparating and dis-tinguishing the components ofmixtures of vola-tile substances. So much of microbiology de-pends on detecting chemicals, either from theorganisms themselves or their metabolic prod-ucts, that GLC would seem likely to be applica-ble for rapid techniques.A variety of rapid, discriminatory microbio-

logical tests using GLC have been described (3-5, 11, 15-18, 21, 24, 29, 31), but head-spacesampling seems to have been overlooked andother methods of sample preparation preferred.However, head-space GLC appears to be partic-ularly worthy of investigation as the basis ofrapid techniques. Sample preparation is sim-ple, and volatile compounds are fast to elutefrom the gas chromatograph. In addition, head-space GLC can be automated. In the presentinvestigation the technique has been used tostudy metabolic products whose source hasbeen indicated by using defined growth media.

MATERIALS AND METHODSProteus cultures. The 68 strains used in the in-

vestigation included 18 Proteus vulgaris, 15 P. mi-

t Permanent address: Dairy Research Laboratory, Divi-sion ofFood Research, Commonwealth Scientific and Indus-trial Research Organization, Highett, 3190, Victoria, Aus-tralia.

rabilis, 14 P. morganii, 11 P. rettgeri, 6 P. incon-stans subgroup A, and 4 P. inconstans subgroup B.Culture collections provided 32 strains, and 36 werefreshly isolated. The identities of all strains wereconfirmed (9).

Culture media. The basal defined medium (LAS)was prepared from four solutions which were storedseparately and mixed when required: 1 liter of LAScontained (i) 100 ml of a 2% (wt/vol) potassiumlactate solution sterilized at 110°C for 25 min; (ii) acomplex amino acid mixture (23) dissolved in 100 mlof water and sterilized by filtration through a Gal-lenkamp Sinta glass filter of porosity 5; (iii) a saltmixture (23) dissolved in 780 ml, adjusted to pH 7.4,and sterilized at 121°C for 15 min; and (iv) 20 ml of afilter-sterilized solution of nicotinic acid (0.5 mg)and calcium pantothenate (0.5 mg).

Nutrient broth (Oxoid) and MacConkey agar (Ox-oid) were prepared as directed by the manufac-turers.LAS medium or nutrient broth was supplemented

with amino acids by dissolving them in the basalmedium and sterilizing by filtration.

Incubation conditions. Samples (20 ml) of theliquid media were dispensed aseptically into sterile125-ml Erlenmeyer flasks stoppered with cotton-wool plugs. All liquid cultures were incubated at37°C either still or shaking on a rotary shaker at 160rpm.Measurement of bacterial growth. The optical

densities (ODs) of bacterial suspensions were deter-mined on a Bausch & Lomb Spectronic 20, at 500 nmfor LAS cultures and at 600 nm for broth cultures, ina cell of 1-cm lightpath. A graph to convert OD todry weight of cells was constructed from determina-tions on duplicate samples from six serial twofolddilutions each of eight Proteus cultures, two P. vul-

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188 HAYWARD ET AL.

garis, two P. morganii, and one each ofP. mirabilis,P. rettgeri, P. inconstans A, and P. inconstans B.The results from all eight cultures lay on the same

curve, which was linear for dry weights up to about0.25 mg/ml.

Viable counts (14) were the mean from 9 drops(between 45 and 450 colonies), 3 on each of threewell-dried MacConkey plates, incubated overnightat 30°C to restrict colony size and increase the num-ber of well-isolated colonies for accurate viablecounts.

Preparation of head-space samples for GLC. Apublished technique (1) was modified. A 4.8-g por-tion of anhydrous potassium carbonate was trans-ferred to a "10-ml" glass serum vial (capacity, 14ml); 8 ml of standard solution or culture was added;and the rubber cap was fitted immediately. The vialwas shaken mechanically for 5 min at 370C to mixthe salt in the liquid, but care was taken not to soilthe cap with liquid. The vial was then incubated ina 60°C water bath for 3 min. While the vial remainedimmersed in the water, a 0.5-ml gas syringe fittedwith a Teflon gas-sealing gland (Scientific GlassEngineering, no. 500A-RN-GSG) was rinsed by fill-ing and emptying five times with the gas above thesurface. The syringe was filled the sixth time with=0.6 ml of head-space gas and withdrawn from thevial. The needle was wiped with a tissue, and thesample was adjusted to exactly 0.5 ml and immedi-ately injected into the gas chromatograph.The reproducibility of results of head-space analy-

sis is dependent on the care with which samples are

prepared. In this investigation special precautionswere taken to prevent contamination ofthe gas sam-

ple by splashing from the underlying liquid. Loss ofgas either from around the large-bore needle of thegas syringe when it pierced the rubber cap of thevial or the rubber septum of the gas chromatographor from around the sealing gland of the gas syringeitself was prevented by replacing caps, septa, andsealing glands frequently and regularly.GLC. The instrument used was a Perkin-Elmer

model 881 fitted with a flame ionization detector anda stainless-steel column (366 cm by 3.2 mm), packedwith 5% Carbowax 20 M on Chromosorb G, acidwashed, dimethyldichlorosilane treated, and of60/80-mesh size. The instrument was operated at100°C with a nitrogen carrier gas flow of 22 ml/minand hydrogen and air pressure of 114 kPa (16.5 lb/in2) and 255 kPa (37 lb/in2), respectively. The in-jector temperature was 150°C, and the detectortemperature was 120°C.

Identification of products. Methyl mercaptan, di-methyl disulfide, diethyl disulfide, isoamyl alcohol,isovaleraldehyde, isobutanol, isobutyraldehyde, 2-phenylethanol, phenylacetaldehyde, and benzalde-hyde were identified by using a GLC-mass spectrome-try combination (27). Separated components were

identified by comparing their mass spectra with a

reference library file (8). This identification was

confirmed by comparison of the GLC retention timeof the unknown with that of an analytical-gradesample of the pure compound for all compoundsexcept methyl mercaptan and isobutyraldehyde. Inthe case of methyl ethyl disulfide, neither pure sam-

ple nor library spectrum was available, but an al-

most certain identification was made from the mass

spectrum alone. The molecule had molecular weight108, and isotope ratios showed that it contained twosulfur atoms: the major fragments had molecularweights of 61 and 47, respectively, and each con-

tained one sulfur atom. Ethyl mercaptan was identi-fied by GLC retention time data alone.The amounts of compounds are expressed as peak

heights or areas and were used only to make com-

parisons.Standard. An aqueous solution of dimethyl disul-

fide at a concentration of 0.01% (vol/vol) was ana-

lyzed with each day's samples to ensure that chro-matographic conditions and the sensitivity of thedetector remained constant. The limit of detectionof dimethyl disulfide was about 1.06 ug in 8 ml ofsolution (0.13 jig/ml).

RESULTS

Selection of salt and comparison of head-space sampling with direct injection. Sodiumsulfate has been used to increase the concentra-tion of bacterial products in head-space gassamples (1) and potassium carbonate has beenused for samples of blood gases (10). The effectsof 4.8 g of K2CO3, anhydrous MgSO4, or Na2SO4,more than enough to saturate 8-ml solutions,on some of the volatile compounds encounteredin this study were compared (Table 1), andK2CO3 was chosen. A comparison was made be-tween direct injections of 1.0 ,ul of 0.01% (vol/vol) solutions of various classes of compounds(s)with boiling points of less than 2000C and in-jections of 0.5 ml of K2CO3 head-space gasesfrom the same solutions. A 1.0-,ul amount ofliquid approaches the practical limit of samplesize, beyond which overloading occurs in a

packed column of 3.2-mm diameter. Table 2shows that the GLC response of the 0.5-mlhead-space sample is greater than that of thel.0-,ul liquid sample.Accuracy and reproducibility of head-space

analyses. When 0.5-ml samples of head-spacegases were injected from 10 separate prepara-

TABLE 1. Effectiveness of salts for increasing theconcentrations of compounds in head-space gas

samples

Peak ht x attenuation factor

Compound (cm)aK2CO3 MgSO4 Na2SO4

Ethanol 60 20 18Acetone 409 107 57Ethyl methyl ketone 88 27 9Dimethyl disulfide 15 3 3Ethyl acetate 90 123Acetaldehyde 42 42Diethylamine 700 0

a Average of duplicate determinations.

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METHIONINE CATABOLISM BY PROTEUS 189

TABLE 2. Peak areas of responses to 1.0-pI liquidsamples-and 0.5-ml head-space gas samples

Peak area (cm2)

Compound Liquid Head-space Relativesample gas sample response

(A) (B) (B/A)

Ethanol 18 82 4.6Isoamyl alcohol 24 542 22.6Isovaleraldehyde 28 3,342 119.4Ethyl methyl ketone 19 1,062 55.9Ethyl acetate 15 497 33.2Dimethyl sulfide 26 2,880 110.0Dimethyl disulfide 28 957 34.2Diethylamine 22 600 27.3

tions of a standard solution of dimethyl disul-fide, the standard error of the mean of the peakareas was 5.4%. When 10 replicate cultures ofP. morganii in LAS medium supplementedwith 0.1 M L-methionine were analyzed byhead-space, the mean peak areas and theirstandard errors were 593 20 (3.4%) cm2 formethyl mercaptan and 472 27 (5.7%) cm2 fordimethyl disulfide, which is the same order ofreproducibility as for the head-space techniqueitself.Head-space products formed by Proteus

spp. from 11 selected amino acids. The aminoacids, 0.1 M L-methionine, L-leucine, L-valine,L-phenylalanine, L-histidine hydrochloride, L-lysine, L-ornithine hydrochloride, L-threonine,and L-asparagine, and 0.05 M L-aspartic acidand L-tryptophan, were tested as individualsupplements in LAS medium with one straineach of P. vulgaris, P. mirabilis, P. morganii,and P. rettgeri. Inocula were grown in LAS me-

dium and added to give approximately 105 orga-nisms per ml both in still cultures incubatedfor 24 h and in shaken cultures incubated for18 h. All species grew well, with P. mirabiliscultures reaching the highest OD.In general, all four species yielded the same

major head-space products (Fig. 1): from phen-ylalanine, benzaldehyde; from methionine,methyl mercaptan and dimethyl disulfide; fromvaline, isobutyraldehyde and isobutanol; andfrom leucine, isovaleraldehyde and isoamyl al-cohol. Some of the products were detected insmaller amounts from control cultures in un-

supplemented LAS medium since it containssome leucine, valine, and methionine, but theincrease in products on adding any of thesethree amino acids was clear-cut. No additionalhead-space products were detected in culturesgrown on the other amino acids, and trypto-phan suppressed the products normally pro-duced in LAS medium. However, some cultureshad distinctive odors that suggested there were

other volatile products not detected by thishead-space technique; when culture filtratesfrom phenylalanine medium were examined bydirect injection, phenylacetaldehyde and 2-phenylethanol were detected. In general, thestill cultures formed lower yields of the sameproducts as the shaken cultures.Head-space products formed by several

strains of all species of Proteus from methio-nine. Sixty-seven of the 68 strains of Proteusyielded similar products from shaken culturesin LAS medium supplemented with 0.1 M L-methionine after incubation for 18 h. The twochromatograms shown in Fig. 1 for P. mirabilisand P. morganii are typical. The remainingstrain, P. morganii, grew very poorly andyielded only traces of a few products. Methylmercaptan was detected only when cultureswere grown in LAS medium supplemented withmethionine, whereas small amounts of di-methyl disulfide were produced by cultures inunsupplemented LAS medium. Various otherproducts, including isobutyraldehyde, isovaler-aldehyde, and isoamyl alcohol, were producedin similar amounts from both methionine-sup-plemented and unsupplemented LAS medium.It was concluded that methyl mercaptan anddimethyl disulfide arose with methionine. Thiswas confirmed when shaken cultures of onestrain each of P. vulgaris, P. mirabilis, and P.morganii failed to form either methyl mercap-tan or dimethyl disulfide in 15 h in a simplifiedLAS medium consisting of lactate, salts, vita-mins, and only glutamic acid and cystine. Theaddition of 0.1 M L-methionine to this mediumresulted in the production of high yields of bothmethyl mercaptan and dimethyl disulfide.The yields ofmethyl mercaptan and dimethyl

disulfide from LAS medium supplemented with0.1 M L-methionine expressed both as GLC peakareas per dry weight per milliliter of cultureand as GLC peak areas are shown in Table 3.When yields are correlated with degree ofgrowth (dry weight per milliliter), small spe-cies differences are apparent. P. mirabilis, P.rettgeri, and all but two strains of P. morganiireached high OD values and gave lower yieldsin relation to dry weight per milliliter than P.vulgaris, P. inconstans A, and P. inconstans B,which reached only moderate OD values. Twostrains ofP. morganii grew poorly so that theiryields related to dry weight per milliliter wereexceptionally high, although in terms of GLCpeak areas the yields were similar to those ofother Proteus spp. These apparent species dif-ferences, related to degree of growth, do notimply any inherent difference in the organisms'ability to decompose methionine. GLC peakareas of sulfur products formed by all 67 strains

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190 HAYWARD ET AL.

a

I*

J. CLIN. MICROBIOL.

Methionine LASP morganii

b 1100

UL\jLAS

;wor P mirabilis

FIG. 1. Chromatograms of head-space products from shaken cultures ofP. mirabilis and P. morganii inLAS medium alone or supplemented with 0.1 M amino acid incubated for 18 h at 3700. Stationary phase,Carbowax 20 M; column temperature, 100°C; attenuation setting, x5 unless otherwise marked, x50 being 10times less sensitive than x5. Peak a, Benzaldehyde; b, methyl mercaptan; c, dimethyl disulfide; d, isobutyral-dehyde; e, isobutanol; f, isovaleraldehyde; g, isoamyl alcohol.

from methionine do not show any apparent spe-cies differences and do not vary widely, most ofthose for mercaptan falling between 200 and600 cm2 and, for disulfide, between 300 and 900cm2 (Fig. 2).Product yields from methionine in relation

to incubation time. Analyses at 2-h intervalsfor products from shaken cultures ofP. mirabi-lis in nutrient broth supplemented with 0.1 ML-methionine with initial inocula of 3 x 102 and3 x 105 organisms per ml are shown in Fig. 3. Amoderate amount of dimethyl disulfide was de-tectable at 2 h from an inoculum of 3 x 105organisms per ml and increased to an apparentlimiting value at 6 h, when growth had reachedthe early stationary phase (3 x 109 organismsper ml). Methyl mercaptan was first detectableat 4 h and quickly increased in yield. A large

amount of dimethyl disulfide was detectableafter 6 h from an inoculum of 3 x 102 organismsper ml, although there was no apparent changein OD at this time. When LAS medium re-placed nutrient broth the lag phase-was longer,and the growth rate was slower. Dimethyl di-sulfide again appeared first and methyl mer-captan later. The detection of dimethyl disul-fide from either medium was found to be a moresensitive indicator of growth than the spectro-metric determination of OD. The fact that theformation of sulfur products from methioninestarts in the earliest stages of growth and iscomplete by the time the early stationary phaseis reached explains why yields of sulfur prod-ucts are unrelated to degree of growth afterincubation for 18 h (Table 3). It appears that allProteus spp. are equally efficient in decompos-

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METHIONINE CATABOLISM BY PROTEUS 191

TABLE 3. Yields ofsulfur products from LAS medium supplemented with 0.1 M methionine by all species ofProteus

Peak area/dry wt per ml (cm2/mg per Peak area (cm2)No. of ml)

Organismstrains Methyl Dimethyl Methyl Dimethylmercaptan disulfide mercaptan disulfide

P. vulgaris 18 511 ± 21 640 + 58 450 ± 30 599 + 59P. mirabilis 15 354 ± 18 427 ± 39 428 ± 22 526 ± 62P. morganii 11 234 ± 27 356 ± 37 323 ± 31 488 ± 34

1 2,400 7,474 228 7101 550 1,316 105 250

P. rettgeri 11 467 ± 24 498 ± 39 477 ± 26 512 ± 45P. inconstans A 6 715 ± 54 566 + 106 479 ± 36 380 ± 69P. inconstans B 4 478 46 684 52 515 36 739 36

0

*0 *

O 00

O * O00

£0 0 * *-

0 0£A

00 00 0

CP *

£0 A

asA .

*

0

. Pvulgariso Pmirabilis£ Pmorganii& Prettgerio Pinconstans A. Pinconstans B

200 400 800 8D0

D6METHYL DISULPHIDE Peok orea (cm)1000 1200

FIG. 2. GLC peak areas of sulfur products formed by strains ofProteus from shaken cultures in 0.1 M L-methionine-supplemented LAS medium incubated for 18 h at 37°C.

ing methionine and, since most of the growthoccurs after methionine decomposition hasceased, yields of sulfur products are unrelatedto it.Head-space products from ethionine. The

synthetic amino acid ethionine was investi-gated to enlarge the results from methionine.

Concentrations of DL-ethionine in LAS me-

dium greater than 0.01 M were shown to inhibitthe growth ofP. mirabilis, although some cellsremained viable. Tests for head-space productsfrom 18-h shaken cultures of eight strains ofProteus in LAS medium supplemented with0.01 M DL-ethionine yielded ethyl mercaptan,diethyl disulfide, and another compound, prob-ably methyl ethyl disulfide, which were not

formed from similar cultures in LAS mediumalone (Table 4).Analyses at various times up to 24 h of dupli-

cate shaken cultures of P. mirabilis in LASmedium supplemented with 0.01 M DL-ethio-nine are shown in Fig. 4. Diethyl disulfide wasdetected after incubation for 4 h when the cul-ture was in the early exponential phase ofgrowth, 2 h before the first detection of ethylmercaptan. The detection of mercaptan coin-cided with a leveling off in the production ofdisulfide, and thereafter the yield of ethyl mer-

captan increased rapidly. The results from me-

thionine are supported because analogous prod-ucts are formed in the same order and at thesame stage of growth from ethionine.

FB00

OE1600

Iz4

4400

z

2001-

0

0

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192 HAYWARD ET AL.

DISCUSSIONAll previous studies of production of sulfur

compounds from methionine by microorga-

nisms or their enzymes have been undertakenwith biochemical objectives so that singlestrains, often incompletely identified, have suf-ficed (2, 13, 19, 20, 22, 25, 26, 30). The onlyinformation about any Proteus species is thatthree strains of P. vulgaris produced methylmercaptan from methionine (19, 26). The pres-

ent investigation has been concerned with themicrobiological significance of methioninebreakdown and has shown that 67 strains ofthe

200 -

0

100-/

0 2 4 6Time (h)

FIG. 3. Time course of formation of sulfur prod-ucts from 0.1 M L-methionine-supplemented nutri-ent broth by shaken cultures of P. mirabilis withan initial inoculum of 3 x 102 organisms per ml(small) or 3 x 105 organisms per ml (large). Sym-bols: * *, dimethyl disulfide, large inoculum;O-O0, methyl mercaptan, large inoculum, *----O0,dimethyl disulfide, small inoculum.

J. CLIN. MICROBIOL.

six species P. vulgaris, P. mirabilis, P. mor-

ganii, P. rettgeri, P. inconstans A, and P. in-constans B were alike in possessing powerfulenzymes for the production of methyl mercap-tan and dimethyl disulfide from methionine.Both sulfur products were formed early in theexponential phase of growth and thereforecould be ideal markers for the rapid detection ofProteus spp. by head-space GLC.

It is most probable that Proteus spp. pro-

240

no ~~~~~~~~~~~~~~~~10

160 E

C7

01

120

oL

0~~~~~~~~~~~~~~~~~~~4

FIG 4. 0iecusfgothadfraino

mented~~~~LALeimb.hkncutrso.mrb

~~~~~~E

2 4 6 a 10

Time (h)

FIG. 4. Time course of growth and formation of

sulfur products from 0.01 M DL-ethionine-supple-mented LAS medium by shaken cultures ofP. mirab-

ilis: mean values of duplicate cultures. Symbols: 0,

viable count; El, diethyl disulfide; 0, ethyl mercap-

tan; A, methyl ethyl disulfide.

TABLE 4. Growth and product formation by Proteus spp. in LAS medium supplemented with0.01 M DL-ethionine

Peak area (cm2) of products

Species; GrowthSpecle(mg,dry wt/ml) Ethyl mercaptan Diethyl disulfide disulfidea

P. vulgaris 0.005 0 6 00.066 110 55 3

P. mirabilis 1.135 223 209 5P. morganii 0.013 0 11 1

0.045 16 75 6P. rettgeri 0.895 156 162 5P. inconstans A 0.485 344 144 5P. inconstans B 1.135 182 388 8a Mass spectrometric identification only (see text).

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METHIONINE CATABOLISM BY PROTEUS 193

duce mercaptans from methionine and ethio-nine by fission of the bond between the alkylsulfide group and its adjoining carbon atom andthat the mercaptans are then oxidized by dis-solved oxygen to dialkyl disulfides (6), a reac-tion shown to be very fast (12). Disulfides werealways detected before mercaptans, their sup-posed precursors. It was possible that failure ofearly detection of methyl mercaptan was dueeither to its low boiling point or to insensitivityof the flame ionization detector to one-carboncompounds, but the identical results with ethylmercaptan make this unlikely. However, thedetection of a product before its precursor al-ways occurs in a reaction mechanism in whichthe rate of formation of the successor is verymuch faster than the rate of formation of itspredecessor; for example, in any chain reactionthe free radical intermediates are normally un-detectable. Whether either one or both mercap-tan and disulfide are found in a culture is deter-mined by the time ofsampling relative to deme-thiolase activity and to access of oxygen to themedium; disulfide alone is found when diffusionof oxygen is sufficient to oxidize mercaptan asfast as it is formed, mercaptan alone underanaerobic conditions, and both sulfur productsunder aerobic conditions when demethiolase ac-tivity yields more mercaptan than can be oxi-dized immediately.Dimethyl sulfide has been reported as a mi-

crobial metabolic product (7, 28). However, itwas not detected by us as a breakdown productfrom methionine. The GLC-mass spectrometrytechnique would have detected it and distin-guished it from methyl mercaptan and di-methyl disulfide if it had been present inamounts comparable to them.Apart from benzaldehyde, the aldehydes and

alcohols from phenylalanine, valine, and leucinecan be postulated to have arisen after an initialstep of decarboxylation and oxidative deamina-tion of their amino acid groups. The aldehydeand alcohol that would have arisen from a simi-lar breakdown of the amino acid group of me-thionine and ethionine, propionaldehyde andpropanol, were not detected. Their boilingpoints, 97 and 490C, respectively, are not lowenough for them to have been lost during incu-bation, particularly from still cultures. It mustbe concluded that Proteus spp. do not decom-pose the amino acid groups of methionine andethionine in the same way as those of the otherthree amino acids. It is probable that 2-ketobu-tyric acid was formed as described by Segal andStarkey (26).

ACKNOWLEDGMENTSWe thank the curators ofthe National Collection of Type

Cultures, London, the Queensland University Culture col-

lection, the Institute of Medical and Veterinary Science,Adelaide, and the International Shigella Centre, CentralPublic Health Laboratory, London, for cultures from theircollections and Jennifer Spragg for her capable technicalassistance.

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19. Mitsuhashi, S. 1949. Decomposition of thioether deriva-tives by bacteria. I. Methylmercaptan formation andthe properties of the responsible enzyme. Jpn. J. Exp.Med. 20:211-222.

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