JOURNAL OF BACTERIOLOGY, Aug. 1969, p. 544-551 Vol. 99, No. 2Copyright © 1969 American Society for Microbiology Printed in U.S.A.

Dissimilation of Methionine by Fungi'JOSE RUIZ-HERRERA' AND ROBERT L. STARKEY

Departmenit ofBiochemistry and Microbiology, College ofAgriculture and Enivironmental Science, Rutgers,The State Uniiversity, New Bruniswick, New Jersey 08903

Received for publication 8 May 1969

Soil fungi that attacked methionine required a utilizable source of energy such asglucose for growth. This is an example of co-dissimilation. Experiments with one ofthe fungi, representative of the group, are reported. In the absence of glucose, pre-grown mycelium, even when depleted of energy reserves, oxidatively deaminatedmethionine with accumulation of a-keto-y-methyl mercapto butyric acid and a-hydroxy-,y-methyl mercapto butyric acid. When glucose was provided, all of thesulfur of methionine was released as methanethiol, part of which was oxidized todimethyl disulfide. No sulfate, sulfide, or hydrosulfide products were detected.Evidence was obtained that deaminase and demethiolase were constitutive. De-amination preceded demethiolation and a-keto butyric acid accumulated as a prod-uct of the two reactions. Other carbon residues were a-hydroxy butyric acid anda-amino butyric acid. Inability of the fungus to metabolize a-keto butyrate was re-sponsible for its inability to utilize methionine as a source of carbon and energy.Several other fungi isolated from soil grew on a-amino butyrate but could not growon methionine owing to inability to demethiolate it.

Methionine can be attacked by various micro-organisms such as bacteria, including actinomy-cetes, and filamentous fungi (19, 26, 30), but itserves as a growth substrate for only exceptionalmicroorganisms. Gram-negative, nonsporeform-ing, rod-shaped bacteria are the only cultures forwhich there is definite evidence of growth onmethionine (13, 26). Most studies of methioninebreakdown have been made with nongrowingwashed cells, with cell-free extracts, or withcultures grown in media which contained otherorganic compounds as sources of energy.

Methanethiol is the generally reported sulfurproduct of methionine decomposition but severalothers have been noted. The bacteria of Segal andStarkey (26), which grew on methionine, deami-nated the amino acid and then demethiolated itwith production of methanethiol, part of whichwas oxidized to dimethyl disulfide.

This report is concerned with the dissimilationof methionine by fungi which were unable to usethe amino acid for growth. Factors affecting theirdecomposition of methionine and the products ofdecomposition were determined. The resultsexplain why most of the microorganisms whichdecompose methionine are unable to use it as asubstrate for growth.

I Paper of the Journal Series, New Jersey Agricultural Experi-ment Station, New Brunswick, N.J.

2 Present address: Department of Microbiology, EscuelaNacional de Ciencias Biologicas, Instituto Politecnico Nacional,Mexico 17, D.F., Mexico.

MATERIALS AND METHODS

Cultures and cultural methods. The basal saltsmedium had the following composition: K2HPO4,1 g; KH2PO4, 1.0 g; MgCl2 *6H20, 0.5 g; CaC12 2H20,0.1 g; FeCl3-6H20, 0.02 g; ZnCl,, 0.02 g; distilledwater to make 1,000 ml. The reaction was adjusted topH 6.7. Solid media contained 30 g of agar per liter.The agar medium used to maintain the fungi con-tained the mineral salts plus 0.5%76 methionine and1.0% glucose. Methionine (0.5%) was generallyincluded in the solution media and additions of thefollowing were made as indicated in the protocols ofthe experiments: glucose or other sugars, 1.0%;NH4Cl, 0.5%; K2SO4, 0.05%/7c. The sugars weresterilized separately.The agar media used to recover fungi from methio-

nine-enriched barnyard and forest soils (pH 7.8 and3.5, respectively) contained 30 mg per liter each ofrose bengal and streptomycin and 0.5% methionineor 1.0% glucose or both methionine and glucose.Generally, the fungi were cultivated in 100-ml por-tions of solution medium in 250-ml Erlenmeyer flasksincubated at 28 C on a rotary shaker. The inoculumconsisted of 0.5 ml of a suspension of spores andmycelium removed from an agar slant in 7 ml ofsterile water. For replacement cultures, the funguswas pregrown, in 100-ml portions of the basal mediumcontaining 0.5%lc methionine and 1.0% glucose, for 4days in shaken flasks. Cell material was filtered offaseptically, thoroughly washed with sterile distilledwater, and resuspended in the test medium. Amountof growth is reported as the weight of washed fungusmaterial after drying at 70 C for 24 hr.

Analytical methods. Methionine was determined by544

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two methods. One of these, the Lavine method (16),determines methionine which contains both aminoand thioether groups. Thus, the method would givenegative results when either group is absent. Duringbacterial dissimilation of methionine by a bacterium,deamination preceded demethiolation (26). It is con-

cluded that this occurred with the fungus also because,in the results to be reported, the amount of aminonitrogen lost during dissimilation was nearly the same

as the amount of methionine lost according to theLavine method (Table 1). Furthermore, the amountof demethiolation consistently exceeded the loss ofmethionine according to the Lavine method (Tables 1and 2 and Fig. 1). Therefore, results obtained by thismethod are generally designated deamination ofmethionine. The second method is the followingmodification of the method of McCarthy and Sullivan(12) for determining the thioether group: 1 ml of thetest solution was mixed with 0.2 ml of 3% glycine,0.4 ml of 2% sodium nitroprusside, and 0.2 ml of 1 N

NaOH. The tubes were incubated in a water bath at35 to 40 C for 5 to 10 min and then chilled in an icebath for 2 min. To each tube was added 1 ml of a 1:9mixture of HCI and H3PO4, and the tube was shakenvigorously for I min and cooled in water. A Beckmanspectrophotometer was used to measure the opticaldensity at 530 nm. The results obtained by this methodare referred to as demethiolation of methionine.To determine volatile sulfur products, a stream of

air or oxygen was passed through the culture solution,held at 28 C, into two traps in series held in an icebath. The first was 5% mercuric acetate which recov-ered methanethiol, and the second was 2% HgCl2which trapped dimethyl disulfide. The mercury pre-

cipitates were removed by filtration, washed, driedover CaC12, and used for the following determinations.Methanethiol in both the precipitate and in the solu-tion of mercuric acetate was determined by the micromethod of Sliwinski and Doty (28), modified to use a

final volume of 10 ml. The method was standardizedwith pure (CH3S)2Hg dissolved in 5% mercuricacetate. To determine dimethyl disulfide, weighedamounts of the precipitate formed with HgCl2 wereacidified with 6 N HCI in a closed vessel under a streamof nitrogen gas. When the solution was warmed in awater bath, methanethiol was evolved (5). The gas

was recovered in a solution of mercuric acetate heldin an ice bath. The methanethiol in the clear solutionwas determined as above.

Total amount of keto acid was determined byFriedemann's double-extraction method (9). Standardcurves were prepared from a-keto butyric acid. Ketoacids which accumulated in the culture solution wereidentified as their 2,4-dinitrophenyl hydrazones. Thehydrazones were extracted from the medium withethyl acetate and then reextracted with 10% Na2CO3.The alkaline solutions were acidified with HCI and thehydrazones were reextracted with ethyl acetate. Thesolvent was evaporated and the residue was dissolvedin hexane. This solution was fractionated by adsorp-tion on a silica gel column followed by elution withbenzene and mixtures of benzene and increasing con-centrations of methanol. The fractions were driedand analyzed by paper chromatography using thesolvents indicated in the protocols of the experiments.

Tests for other organic acids were made on theaqueous solution remaining after extraction of theketo acids as the 2,4-dinitrophenyl hydrazones. Thesolution was made alkaline with NaOH, evaporated to5 ml, acidified with H2SO4, adsorbed on a Celitecolumn, and eluted with ether. The ether eluate wasdehydrated with anhydrous Na2SO4 and evaporatedunder vacuum. The dry residue was analyzed by paperchromatography. Sulfur-containing acids were devel-oped with Feigl's solution, and other acids were devel-oped with aniline-xylose (2) or with a mixture of pHindicators and alkaline KMnO4 (23). Ammoniumsalts were developed by spraying with Nessler'sreagent. Keto acids were prepared from their aminoacids according to Meister (17) but crystalline D-amino acid oxidase was used instead of kidney acetonepowder.

Dubin's method (8) was used to determine a-aminonitrogen; the yellow color of the 2,4-dinitrofluoro-benzene derivative of methionine was read at 340 nm.Absorption was maximal at this wavelength. Thevalues were corrected for ammonia, when present,which was determined by Nesslerization. To identifyamino acids, the filtered culture solution was deionizedby the method of Thompson, Morris, and Gering (31)and analyzed by paper chromatography using thefollowing two solvents: (a) water-saturated phenolwhich contained 0.002% 8-hydroxy quinoline; (b)butanol-acetic acid-water, 250:60:250 (2). Sugarswere estimated by the anthrone reaction. Hexosesand disaccharides were determined by the method ofDimler et al. (7) and pentoses by that of Gary andKlausmeier (10).

Total sulfur content of dried cell material and driedculture solution was determined as sulfate after oxida-tion with sodium peroxide in a Parr bomb. The sulfatewas precipitated with BaCl2 and determined by meansof a Klett nephelometer. Sulfate in the culture solu-tion was determined also as the barium salt. Hydro-sulfide was detected with nitroprusside (11).

RESULTS

Isolation of methionine-decomposing fungi. Theabundance of fungi in methionine-enriched forestand barnyard soils was determined by plating ona medium in which methionine was the onlysource of carbon, nitrogen, and sulfur. Othermedia contained glucose (1.0%), NH4C1 (0.5%),and K2S04 (0.05 %) in various combinations withand without methionine. Several thousand fungiper gram of soil were indicated by counts on themedia which contained glucose and in whichmethionine was the only source of sulfur ornitrogen or both. There was meager developmenton media in which methionine was the onlyorganic nutrient. Numerous colonies failed togrow when transferred to slants of basal agarmedium with methionine as the only source ofcarbon, nitrogen, and sulfur, but they developedwell when transferred to slants of a similarmedium containing glucose. Several of the fungiwere used in the following experiments, but the

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results obtained with only one, Aspergillus sp.,RS-la, are reported since these results are repre-sentative of the group.

Decomposition of methionine. When cultivatedin basal salts medium with 0.5%, methionine and1.0% glucose, the fungus grew well, decomposednearly all of the glucose in 3 days, deaminatedabout two-thirds of the methionine, and demethi-olated 41 % of it (Table 1). The value for loss ofamino nitrogen determined by Dubin's methodwas nearly the same as that for loss of methio-nine determined by the Lavine method which wasinterpreted as deamination. Since the loss ofmethionine according to the Sullivan method(demethiolation) was less than that determinedby the Lavine method as well as loss of aminonitrogen, it appears that deamination was morerapid than demethiolation and preceded it. Nosulfate was produced. The increase in acidity isascribed to production of organic acids from glu-cose. Results obtained with several fungi indicatedthat the amount of decomposed methionine wasnot proportional to the amount of growth; theculture which grew least was one of the most ac-tive decomposers of methionine. In anotherexperiment, in which the inoculum consisted of asmall amount of spore-mycelium mixture, therewas increase in growth, loss of amino nitrogen,and loss of methionine with increase in the con-centration of glucose from 0.01 to 1.0%. Inmedium which contained 1.0% glucose, 99% ofthe amino acid was deaminated and 56% wasdemethiolated. Since values for amino nitrogenby Dubin's method and for methionine byLavine's method were nearly the same, only thelatter results are reported in the following experi-ments. These values are designated deaminationof methionine.

Influence of endogenous and exogenous energysources on dissimilation. One portion of pregrownmycelium was suspended in basal medium con-taining methionine but lacking other organiccompounds. An equal amount was suspended indistilled water and shaken 24 hr at 28 C to deplete

TABLE 1. Dissimilation of methionine

Loss ofmethionine

Incu- Dry wt Glucose Loss ofbation pH of my- con- aminoperiod celium sumed N Deamio De-Deati-n methio-nain lation

days mg/JOOml % % % %1 34 12 163 95 44 305 100 48 317 4.9 131 100 67 63 41

the mycelium of stored energy material. The cellmaterial was then removed and resuspended inmethionine medium. Deamination was notaffected by depletion of the endogenous reserves,whereas demethiolation was reduced. Further-more, the amount of deamination greatlyexceeded that of demethiolation; more than90% of the methionine was deaminated in 4 daysby both the depleted and nondepleted mycelium,whereas only 11 and 29% was demethiolated bythe depleted and nondepleted mycelium, respec-tively.Equal portions of similarly pregrown mycelium

depleted of reserves were suspended in solutionmedia containing methionine (0.5%) and methio-nine (0.5%) plus glucose (0.5%). Glucose hadnegligible effect on deamination but increaseddemethiolation (Fig. 1). Additional experimentsindicated that the amount of demethiolation in-creased with glucose concentration up to 3%.The weight of the mycelium decreased duringincubation in media lacking glucose or its equiva-lent and with concentrations of glucose up to0.1%.Pregrown depleted mycelium was homogenized

in a Waring Blendor for 30 sec and 5-ml portionswere added to 20-ml portions of distilled watercontaining 2 mg of methionine and 0, 0.20, or

0N

w

cr0LLCl)zcna:

wzz0IHrLii

0 24 48 72 96

TIME, Hr

FiG. 1. Effect of glucose on deamination anddemethiolation by pregrown fungus mycelium. Deamina-tion in presence (0) and absence (0) of glucose;demethiolation in presence (A) and absence (-) ofglucose.

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METHIONINE DISSIMILATION

1.00 mg of glucose. During incubation, a streamof oxygen was passed through the culture solutioninto mercuric acetate held in an ice bath to trapmethanethiol. Periodic determinations indicatedthat methanethiol was produced only when glu-cose was available (Fig. 2). On adding glucoseafter all had been consumed, more methanethiolwas produced.No methanethiol was produced from methio-

nine when the cell suspension was sparged withnitrogen or carbon dioxide, whereas it was pro-duced when the sparging gas was oxygen or air.Considerably more methanethiol was producedwhen oxygen was used.To determine whether other compounds could

substitute for glucose as energy sources, pre-grown depleted mycelium was suspended inbasaland (shakelium Ilent dthe sicontanine iwhichof deabsen

Co]

>LL

I

0

LUI

I

LUJz

LI

zj_

Fic(A),pregrAt aradded

TABLE 2. Influence ofsome carbohydrates on break -

down ofmethionine by pregrown mycelium

Loss of methionineLoss of Dry wt

Substrate Iusrae of my-celiuma Deamina- Demethio-

tion lation

% mg % %None 83 94 8Glucose 100 255 92 54Fructose 100 291 97 69Galactose 100 298 96 72Arabinose 46 220 97 44Lactose 40 243 90 43Maltose 87 257 92 65

a Initial weight, 112 mg.

salts medium containing 0.2% methionine methionine. Fungus mycelium was grown in).5% of the carbohydrate and incubated as media which contained 1.0% glucose and one of-n cultures for 4 days. The amount of myce- the following: (a) 0.5% methionine, (b) 0.5%which was added to each flask had an equiva- NH4Cl and 0.05% K2S04, (c) 0.5% asparagineIry weight of 112 mg. The fungus grew on all and 0.05% K2S04. The three lots of myceliumugars but lost weight in the medium which were washed and homogenized, and equalined no sugar (Table 2). Most of the methio- amounts were suspended in 25-ml portions ofwas deaminated in all media, even in the one distilled water containing 2.5 mg of glucose andh contained no carbohydrate, but the amount 5 mg of methionine. Determinations for methane-methiolation differed and was small in the thiol were made at short intervals (30 min) toice of an energy source. ascertain whether there was an initial short lagnstitutive nature of capacity to degrade phase of demethiolation. The course of production

of methanethiol was the same irrespective of50 presence of methionine in the growth medium

50 _ _ and it was nearly linear for the 2-hr incubationperiod. Another experiment indicated that bothdeamination and demethiolation by pregrown

40 mycelium were unaffected by lack of methioninein the growth medium.

In various experiments, washed mycelium pre-/0 _ grown in media which contained glucose and

30 - L, methionine failed to grow in methionine mediaV/ which lacked glucose or its equivalent. Actually,

the mycelium lost weight when incubated in these20 replacement media for 1 week.

Sulfur balance and identification of volatileproducts. Pregrown washed cell material wassuspended in 100 ml of basal medium which

10 _ _ contained 0.5% methionine and 2.0% glucose.The solution was incubated as a shaken culturefor 4 days. Sterile air was passed through the

^: ^ medium and into solutions of 2% mercuric ace-

0 60 120 180 240 tate and 2% HgC12 to trap methanethiol anddimethyl disulfide. Methionine content of the

TIME (MIN.) culture solution was determined initially and atthe end of the experiments by the method of

3. 2. Effect of glucose [0.00)4% (0), 0.0008% McCarthy and Sullivan. Nearly half of thenone ([I)] on production of methanethiol by Methi and practall all-own fungus mycelium in 25 ml of culture solution.row, a second increment of I mg of glucose was of the sulfur which was released was recovered asl. the volatile products, methanethiol and dimethyl

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disulfide (Table 3). A small amount of sulfur wascontained in the cell material which was producedduring the experiment. Most of the released sulfurwas recovered as dimethyl disulfide but largeramounts were released as methanethiol in otherexperiments. In two experiments carried out forpurposes other than to determine sulfur balances,100% and49% of the released sulfur was methane-thiol. Means of identification of the productshave been described previously (5, 25). When theprecipitate which had been trapped in the HgCl2was warmed with 5 N NaOH under N2 and thereleased gases were bubbled through 2% HgC92,no precipitate was formed, which indicatedabsence of dimethyl sulfide. Neither sulfide norhydrosulfide compounds were detected as prod-ucts.

Acids produced from methionine. Pregrowndepleted mycelium was suspended in basal saltsmedia which contained the following: (a) 1.0%glucose and 0.5% NH4Cl, (b) 1.0% glucose and0.5% methionine, (c) 0.5% methionine. After thecultures had incubated for 2 days, the 2,4-dinitrophenyl hydrazones of the keto acids in theculture solutions were prepared and analyzed bypaper chromatography using the following sol-vents: (a) butanol-water-ethyl alcohol, 5:4:1;(b) methanol-benzene-butanol-water, 4:2:2:2(18). Hydroxy phenyl pyruvic acid was found inboth media which contained glucose irrespectiveof the presence of methionine, and phenyl pyruvicacid was detected in the glucose medium whichcontained no methionine (Table 4). It is con-cluded from this that they originated from glu-cose. In both culture solutions which containedmethionine, ca-keto-y-methyl mercapto butyricacid (a-keto methionine) was present, and therewas more in the methionine medium which con-tained no glucose. In the culture solution which

TABLE 3. Sulfur balanice of decomposedmethionine

Sulfur content

DeterminationPct

Amt initial S

mg

Initial mediumMethionine.............. 111.6 100

Culture solutionTotal ................... 52.2 47Methionine .............. 50.6Other.. 1.6

Volatile products............ 53.1 48Methanethiol ............ 8.6Dimethyl disulfide ....... 44.5

Mycelium .................. 2.9 3

contained both methionine and glucose, there wasa-keto butyric acid. These results support theconcept that a-keto methionine was the pro-duct of deamination of methionine and that itwas demethiolated to a-keto butyric acid.

After extracting keto acids from the culturesolution which had contained methionine andglucose, determinations were made for other acidsby paper chromatography using the following assolvents: solvent a of the preceding experiment;(c) ethyl alcohol-concentrated NH40H-water,80:5:15; (d) butanol-acetic acid-water, 250:60:250 (2); (e) ethyl alcohol-water-concentratedNH40H, 95:5:1 (14). Both a-hydroxy-'y-methylmercapto butyric acid (a-hydroxy methionine)and a-hydroxy butyric acid were found, but onlya small amount of the latter. Tests for amino acidsindicated the presence of only a-amino butyricacid. There was no homoserine. Pregrown funguscells produced a-hydroxy butyric acid froma-hydroxy methionine.

Influence ofpH on dissimilation. Equal amountsof pregrown washed mycelium were suspended inglucose, methionine, mineral salts media adjustedto reactions from pH 4.5 to 9.0 and incubated for4 days. Over this range of reaction there wereonly slight differences in the amounts of deamina-tion and demethiolation. Minimum and maxi-mum values for deamination were 82 and 100%,respectively, and those for demethiolation were51 and 59%. Deamination decreased graduallywith increase in pH. The pH of media that werealkaline initially decreased.

Carbon residues of methionine dissimilation andtheir effect on growth. Since methionine failed tosupport growth, it seemed likely that its deami-nated and demethiolated products could not bemetabolized. This conclusion was supported bydetection of organic products of dissimilation inculture solutions (Table 4). Furthermore, thefungus was unable to grow on a-amino butyrate,but both it and methionine served as sources ofnitrogen for growth in glucose-containing media.

Additional evidence that a-amino butyric acidwas deaminated and that the resulting keto acidwas not metabolized is shown in Fig. 3. Equalamounts of pregrown washed mycelium weresuspended in 20 ml of 0.05 M phosphate buffer(pH 7.2) containing 4.12 mg of a-amino butyricacid (2 mM) and in two other portions of buffercontaining equivalent amounts of a-keto butyricacid, one adjusted to pH 4.5 and the other to pH7.5. The added keto acid was unaltered by thefungus even at pH 4.5 which would have favoredits penetration into the mycelium. The keto acidproduced by deamination of the amino acidaccumulated. This was identified as a-ketobutyric acid by paper chromatography.

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TABLE 4. Keto acids producedfrom glucose and methionine

RFFraction of

Additions to basal medium keto acid IdentificationDNPHa Solvent Solvent

a b

Glucose + NH4Cl 1 0.73 0.85 Phenyl pyruvic acid2 0.88 0.95 Hydroxy phenyl pyruvic acid

Glucose + methionine 3 0.62 0.82 a-Keto butyric acid4 0.81 0.88 a-Keto methionine5 0.86 0.96 Hydroxy phenyl pyruvic acid

Methionine 6 0.81 0.88 a-Keto methionine

Authentic compounds0.77 0.86 Phenyl pyruvic acid0.90 0.93 Hydroxy phenyl pyruvic acid0.64 0.85 a-Keto butyric acid0.81 0.88 a-Keto methionine

a 2,4-Dinitrophenyl hydrazones.

z0

cr0C)0

0

LLI

-j1

LJLi0

0.8

0.6

0.4 F

0.2

0 1 2 3 4

TIME, HrFIG. 3. Influence of pregrown fungus mycelium on

ca-keto butyric acid at pH 4.5 (0) and pH 7.2 (0)and on a-amino butyric acid with production of am-

monia (A) and a-keto butyric acid (A).

In another experiment, the fungus was inocu-lated into media of pH 4.5 which contained theoiganic compounds (0.05 M) listed in Table 5,NH4Cl, and K2S04. The cultures were incubatedfor 4 days. Growth was limited though significanton pyruvate, propionate, and succinate.

DISCUSSIONWhereas certain bacteria use methionine as a

growth substrate (13, 26), search for filamentous

TABLE 5. Influence of some potential intermediateson growth

Compound Dry wt ofCompound ~~~myceliummg

Glucose.............................. 303Pyruvate.............................. 17Propionate............................. 40Succinate.............................. 26Methyl malonate....................... 0

fungi able to do this failed. Therefore, it is con-cluded that fungi lack the capacity to grow at theexpense of methionine or that this capacity is arare endowment, as yet undetected. Ability todecompose methionine in the presence of growth-supporting organic compounds such as glucose isa property of many fungi recovered from soil aswell as of other fungi (6, 29, 32). This ability wasnoted also by Uchida (33) for Shigelia flexneri.The capability to decompose a compound in thepresence of another compound which supportsgrowth, but not in the absence of the lattercompound, is designated co-dissimilation.When provided glucose, representative fungi

deaminated and demethiolated the amino acid.With small amounts of spores and mycelium asinoculum, both deamination and demethiolationincreased with increase in the glucose concentra-tion, whereas, with replacement cultures usingpregrown mycelium, methionine was attacked inthe absence of glucose or its equivalent. Methio-nine was only deaminated, and the cells lostweight during the incubation. Although noenergy source was required for deamination, asource of energy was needed for demethiolation;in absence of glucose, demethiolation was limited,

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particularly by pregrown mycelium depleted ofendogenous reserves. Oxygen was required fordemethiolation. Likewise, decomposition ofmethionine by a methionine-decomposing bac-terium was promoted by aeration (26). However,others (13, 19-22) reported that certain faculta-tive anaerobic bacteria and their enzymesdemethiolated methionine anaerobically, andevidence was provided (21, 22) that demethiola-tion was more rapid in the absence of oxygen.

Deamination and demethiolation by pregrownmycelium were unaffected by absence of methio-nine in the growth medium, which indicates thatthe amino acid is not required for synthesis ofdeaminase and demethiolase; that is, the enzymesystems are constitutive. To the contrary, abacterial culture which was unable to grow onmethionine initially, was able to do this afterbeing grown in a medium which contained bothmethionine and glucose (26).Methionine was first deaminated and then

demethiolated with release of the sulfur asmethanethiol, part of which was oxidized todimethyl disulfide (Fig. 4). This is the samecourse of events noted by Kallio and Larson (13)and Segal and Starkey (26) for the initial stagesof dissimilation of methionine by bacteria. Noother sulfur products were detected. Othersreported that methanethiol, dimethyl disulfide,and related compounds were products of methio-nine breakdown by several fungi: methanethioland ethyl sulfide by Oidium lactis in presence ofsucrose (1), methanethiol and dimethyl sulfide byScopulariopsis brevicaulis, and methanethiol bySchizophyllum commune on bread media (6), byMicrosporum gypseum, S. brevicaulis, andAspergillus niger with glucose (29), by Penicilliumcaseicolum on skim milk and casein (32), and bydiverse other fungi in a medium which containedglucose and peptone (26).The principal carbon residue of methionine

HOOC*CH(NH2) *CH2*CH2 *S*CH3(methionine)

Oxidativedeamination 4

HOOC*CO*CH2*CH2*S*CH3 + NH3(at-keto methionine)

Demethiolation

HOOC*CO*CH2*CH3 + HS *CH3(a-keto butyric acid) (methanethiol)

"(CH3 S.S.CH3)(dimethyl disulfide)

FiG. 4. Deamination and demethiolation of methio-nine.

dissimilation in the absence of glucose was a-ketomethionine, the product of an amino acid oxidase.This keto acid was identified as the product ofmethionine transformation by animal tissueslices (3, 4) and by an amino acid oxidase ofTrigonopsis variabilis (27). In the presence of bothglucose and methionine, a-keto butyric acidaccumulated. Both keto acids were the anticipatedproducts of oxidative deamination and of de-methiolation with production of methanethiol.In addition, a-hydroxy methionine and a-hydroxybutyric acid were detected. Whether these aredirect products of deamination and demethiola-tion or of reduction of the keto homologues isunknown. Some a-amino butyric acid which wasdetected may have originated by demethiolationof methionine. Others (1, 13, 20, 21, 34) identifieda-keto butyric acid as the product of demethiola-tion of methionine by bacteria and their enzymes.Even though methionine, a-amino butyrate, anda-keto butyrate failed to support growth, thefungus grew on propionic acid and pyruvic acidwhich might be expected to be produced fromthem (15, 24). Therefore, it is unlikely that theseacids were generated from the products ofdemethiolated methionine.Although the fungi which were tested were

unable to oxidize a-keto butyrate, several fungiisolated from soil could use a-amino butyrateas their sole source of carbon and nitrogen.However, they could not use methionine as anenergy source and did not produce volatile sulfurproducts from it. Their inability to develop onmethionine is ascribed to lack of demethiolase.The results lead to the following conclusions.

Various fungi attack methionine, deaminatingand demethiolating it, leaving a-keto butyric acidas the principal carbon skeleton. This cannot bemetabolized. Therefore, other organic compoundsare required as energy sources. Many micro-organisms, including diverse bacteria and actino-mycetes as well as fungi, attack methionine butcannot grow on it. For them, as for the fungistudied, this is probably due to inability tometabolize the deaminated and demethiolatedcarbon residues.

ACKNOWLEDGMENT

This investigation was supported by National Science Founda-tion research grant GI8536.

LITRATURE CITMD

1. Akobe, K. 1936. Darstellung von D- und L-a-oxy-y-methio-buttersaure und damit ausgefuhrte Ermahrungsversuche.Hoppe-Seylers Z. Physiol. Chem. 244:14-18.

2. Block, R. J., E. L. Durrum, and G. Zweig. 1958. A manual ofpaper chromatography and paper electrophoresis, 2nd ed.Academic Press Inc., New York.

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