Metabolism of Sulfur Amino Acids by Rumen Microorganisms

JOHN P. ZIKAKIS and R. L. SALSBURY Department of Animal Science and Agricultural Biochemistry

University of Delaware, Newark 197] ]

Abstract

The production of methanethiol and ethanethiol from l~-methionine, s-methyl- L-cysteine, ethionine, and s-ethyl-~.-cysteine during in vitro fermentation by tureen microorganisms was determined by employ- ing gas-liquid chromatography to analyze the head-space gas produced by the fer- mentations. Each substrate with rumen fluid was incubated at 39 C in serum bot- tles equipped to permit syringe sampling of the head-space gas evolved. All sub- strafes were used at a concentration of 1.67 mg/ml rumen fluid. I t was found that methanethiol was formed during the in vitro fermentation of S-methyl-L-cysteine and of methionine and that ethanethiol was pro- duced from s-ethyl-L-eysteine and from ethionine. When dimethyl thetin chloride was used as substrate, dimethyl sulfide was formed, s-ethyl-L-cysteine inhibited pro- duction of methanethiol from s-methyl- L-cysteine in the early stages of fermenta- tion, but inhibition was largely overcome as fermentation proceeded. Some indication of utilization of methanethiol was obtained and appeared to be related to diet. This is in accord with the production of methanethiol by the reaction: methionine

> s-methyl-L-cysteine > methane- thiol. However, it is possible that both methionine and s-methyl-L-cysteine were directly dethiomethylated.

Plants consumed by dairy cows are known to contain various forms of sulfur-containing compounds. F o r instance, legumes contain s-methyl cysteine (SMC) (27), alfalfa contains methionine (7), and grass and corn silage con- tain dimethyl sulfide (19). Upon fermentation of plant material by rumen microorganisms, part of the sulfur-containing compounds (e.g., sulfur amino acids) fulfill some of the require- ments of the microorganisms and eyentually of

Received for publication July 31, 1969. 1 Published with the approval of the Director

of the Delaware Agricultural Experiment Station as Miscellaneous Paper no. 613, Contribution no. 4, of the Department of Animal Science and Agricul- tural Biochemistry, University of Delaware, New- ark.

the host animal, while another portion of these are released as volatile sulfurous rumen gases (e.g., dimethyl sulfide, methanethiol, hydrogen sulfide, etc.).

Methionine is believed to be an essential amino acid which, if not supplied, limits rumi- nant growth (9, 10, 12, 16), and it has been shown recently that an appreciable amount of this amino acid is synthesized in the rumen. Conrad et al. (6) have reported that individual cows receiving alfalfa hay and silage synthe- sized between 31 and 59 mg methionine per kilogram body weight per day. The biosynthe- sis and degradation o£ methionine by various microorganisms have been reported. Schlenk and Tillotson (24) found that yeast synthesized methionine from methanethiol (MESH). Wolff et al. (30) showed that by incubating MeSH plus T,-serine with a par t ia l ly purified enzyme prepared from a yeast extract yielded SMC (the lower homolog of methionine). In view of this finding, Maw (15) suggested that SMC may be a precursor of methionine by a route which excludes cysteine. In addition, Mitsu- hashi (17) has obtained an enzyme from soil bacteria that degrades methionine, with a re- sultant production of MESH, a-amino butyric acid, and NHa. Similar results have been re- ported with enzymes from Escherichia coli (20, 21), Clostridium sporogenes (29), and a Pseu- domonas sp. (18) obtained from soil. Also, it has been shown that the fungi Scopulariopsis brevicaulis (4), Microsporum gypseum (26), and a gram-negative bacterium isolated from soil (17) degrade methionine and SMC into MESH.

Although the synthesis of methionine by rumen microorganisms has been reported (3, 6, 13), little, if any, knowledge is available con- cerning its biosynthetic route in the rumen. The object of this study was to investigate some possible precursors and intermediates as well as some competitive inhibitors, such as s-ethyl cysteine (SEC) and ethionine, of methionine synthesis by tureen microorganisms in an in vitro system. Since this amino acid is neces- sary for ruminant growth, knowledge concern- ing its biosynthetic route in the bovine rumen might permit adjustment of the ration to pro- duce a greater supply of methionine for the animal.

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Experimental Procedure

Inocula for the laboratory fermentation were obtained from four fistulated bovine females maintained in the University of Delaware herd: two ovariectomized nonlactating animals (Guernsey 111 and Holstein 154), and two intact milking cows (191 and 222, both Hol- steins). Unless otherwise indicated, Cows 111 and 154 received a ration of hay (brome grass alfalfa) , corn silage, and pasture, while Cows 191 and 222 received hay, corn silage, pasture, and a commercial dairy concentrate.

Samples of the fluid portion of rumen ingesta were obtained using the procedure of Salsbury et al. (22) at 8 to 9 A~ on the day of experi- ment. No attempt was made to restrict the feeding habits of the animals to obtain a more uniform inoculum.

Gas-liquid chromatography (GLC) was used to determine the amount of methane, MESH, ethanethiol (E tSH) , and dimethyl sulfide in the head-space gas evolved during fermentation of the various substrates by rumen fluid. The analysis of organic compounds in the head- space gas by GLC has been studied by several investigators (1, 2, 14, 28). This method was employed recently by Field and Gilbert (8) to measure the amount of MeSH in urine. Their method was modified to adapt it to the condi- tions of our experiments.

The method used in this study was as fol- lows : 15 ml of tureen fluid plus known amounts of various substrates were placed in 60-ml serum bottles and sealed with channel rubber stoppers to permit syringe sampling of the head-space gas. One bottle without substrate served as a control, s-methyl cysteine, L-methionine, SEC, DL-ethionine, and dimethyl thetin chloride were used as substrates 2 at a concentration of 1.67 mg/ml rumen fluid. The serum bottles were incubated at 39 C in a water bath. At intervals, a sample of the head-space gas was obtained from each serum bottle by using a gas-tight Hamilton syringe, and injected directly into the chromatograph. Gas injections ranged from 50 to 1,000 ~liters, but the most satisfactory volume was found to be 250 ~literS. The gas- chromatographic response was measured as integrator units times attenuation factor.

A dual hydrogen flame gas-liquid chromato- graph ( F & M Scientific, Model 810) equipped with a 6.4-ram by 3.7-m stainless steel column with 10% silicone oil DC-200 on 60 to 80 mesh Diatoport S was used for analysis of the head-

2 Reagents used were obtained from Nutritional Bioehemieals Corp., 21010 Miles Ave., Cleveland, Ohio.

space gas. The flows of flame gases were: hydrogen at 62.5 ml/min and air at 460 ml / min. The carrier gas was helium and used at a flow rate of 47.2 ml/min. Ranges and attenuations most frequently employed were 10 by 64 to 10 by 4 at a chart speed of 2.54 cm/min. All analyses were performed isothermally with the detector temperature maintained at 300 ± 5 C, the injection por t at 285 ---+ 5 C, and the column at 60 ± 2 C. Results

Head-space gases were tentatively identified by comparing retention times on the gas chro- matographic column with those of known com- pounds (Table 1). Also the chromatographic peaks obtained with head-space gases were aug- mented by adding to the samples with known

TABLE 1. Comparison of retention times of known compounds with those obtained from the in vitro fermentation of rumen inoculum, a

Retention time Peak number Unknown Known Known compounds

(rain) (rain) 1 1.4 1.4 CH4

(methane) 2 3.0 3.0 CH3SH

(methanethiol) 3 4.6 4.6 CH3CH.2-SH

(ethanethioI) 4 5.2 5.2 CH3-S-CHa

(dimethyl sulfide)

a Each value is the average of eight determi- nations.

TABLE 2. Production of methanethiol from s- methyl cysteine and from L-methionine.

Incubation Treatment a time Methanethiol b

Rumen fluid

(Relative (rain) production)

4O 0 454 100

1,430 0

Rumen fluid + 54 320 s-methyl 590 5,800 eysteine ],547 6,400

Rumen fluid + 110 160 L-methionine 598 680

1,540 1,640

a Source of rumen fluid: Cow 154. b Each value is the average of two experi-

ments. JOURlVAL OF DAIRY SCIENCE VOL. 52, NO. 12

2016 Z I K A K I S A N D S A L S B U R Y

compounds. Figure 1 shows a representative chromatogram containing all four compounds. Incubation of lumen fluid with SMC yielded a gaseous product which had the same retention time as that of known MeSH (Table 2). Addi- tion of L-methionine in place of SMC also yielded MESH, but at a lower level than that attained with SMC as the substrate. Thus, it is possible that the closer proximity of SMC to MeSH in the pathway shown in Figure 2 may account for the larger production of MeSH

Methan, Ethonethiol

Methonethiol

l~za. 1. Head-space gas over rumen fluid plus s-methyl-L-cysteine plus s-ethyl-L-cysteine plus dimethyl thetin chloride.

MeSH ser SMC 4c p HETHIONINE~, . • SAM ~ 5"-MTA

~IG. 2. Scheme for the biosynthesis of methionine and related compounds from methane- thiol (15, 24, 30). MESH, methanethiol; SMC, s-methyl-L-cysteine; SAM, s-adenosylmethionine; 5'-MTA, 5'-methylthioadenosine; ser, serine; 4C, a four-carbon compound; ATP, adenosine triphos- phate.

in the presence of SMC. I t was indicated that, under some conditions,

MeSH may be utilized. The experiments shown in Table 3 were performed with rumen fluid obtained from Cow 111. When fresh grass (pasture) was the sole intake of the animal, the rumen fluid was characterized by a green color and a high viscosity. When the animal was maintained on hay and corn silage, its rumen fluid was much lighter in color and less viscous. The data presented in Table 3 show that when pasture only was ingested, there was a greater utilization of MESH, with a maximum relative amount produced at approximately five hours fermentation averaging 4,120 with a range of 1,720 to 6,520. At the end of fermentation (approximately 25 hr) the relative amount of MeSH left in the fermentation vessel averaged 260, with a range of 0 to 520. This is contrasted with the less MeSH utilization when hay and corn silage was the sole diet. At about five hours of fermentation, the rate of MeSH utilization was roughly half (7,680 versus 4,120) that obtained when pasture was the diet. At the end of fermentation (about 27 hr), the amount of MeSH left in the fermentation vessel was more

TABLE 3. Effect of diet on the apparent utilization of methanethiol.

Diet Treatment a Incubation time Methanethiol b

Rumen Pasture fluid

(fresh Rumen fluid +

grass) s-methyl cysteine

(rain) (Relative production) 33 0

308 0 1,511 0

48 600 314 4,120

1,528 260

Rumen Hay + fluid

corn Rumen fluid +

silage s-methyl cysteine

20 0 306 0

1,556 0

39 540 327 7,680

1,612 4,600

a Source of rumen fluid: Cow 111. b Each value obtained in the pasture diet is the average of two experiments.

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than 17 times higher than that remaining when pasture was the sole diet.

1 4

o---o--o MeSH . ~ SMC

12

o " i

o o

10

+.:. p, 8

6

e " 4

~ z .,_1

0 ,

0 1 2 3 4 5 6

F'ERHENTATION TIHE (FIRS,) Fro. 3. Methanethiol (MESH) production from

s-methyl cysteine (SMC) and SMC + s-ethyl cysteine (SEC). Inoeula for the fermentations were obtained from Cow 222 and each value is the average of two experiments.

Incubation of rumen fluid with SEC or DL- ethionine released a gaseous product identified as EtSH. The production of EtSH from SEC was much greater than that attained with DL- e~hionine, a situation analogous to the SMC- methionine pathway in the production of MESH.

Because of the well-known antagonism of ethionine for methionine, it was of great inter- est to ascertain whether SEC had an antago- nistic effect on SMC fermentation. The results shown in Table 4 do indeed indicate such an inhibition of MeSH production in the early stages of the fermentation, but this inhibition appears to be largely overcome as the fermen- tation is allowed to continue. The experiments shown in Figure 3 verify this inhibition during relatively short-term fermentations. On the other hand, it was observed that DL-ethionine inhibited the production of MeSH from SMC in one case (rumen fluid from Cow 191 on a ration of hay, corn silage, concentrate, and some pasture), but not in another (rmnen fluid from Cow 111 on pasture only).

I t was observed that some of the chromato- grams showed a small peak with the same reten- tion time as that for the known dimethyl sulfide. This was usually found with fermentations to which T.-methionine had been added, but also

TABLE 4. Effect of s-ethyl eysteine on methanethiol production from s-methyl cysteine.

Experiment number Treatment Incubation time ~ethanethiol a

( min ) (Relative production) Rumen 27 0 fluid 303 50

1,488 40

1 Rumen fluid A-

Cow 154 SMC

47 694 332 5,800

1,486 2,540

Rumen 93 120 fluid -t- 393 3,160 SMC A- SEC 1,472 5,440

Rumen 25 0 fluid 312 0

1,549 0

2 Rumen fluid A-

Cow 191 SMC

a Each value obtained

48 680 341 4,520

1,570 5,440

Rumen 148 80 fluid -t- 446 3,000 SMC -t- SEC 1,512 5,240

with Cow 154 is the average of two experiments.

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2018 ZIKAKIS AND SALSBUR¥

occasionally occurred with some samples of rumen fluid with no added substrate. The di- methyl sulfide peak was always relatively small and MeSH was the main volatile product from both SMC and L-methionine. Since the thetins are a fairly common plant constituent, dimcthyl thetin chloride was also tried as a likely pre- cursor of dimethyl sulfide. Table 5 confirms this observation that dimethyl sulfide was ob- tained when dimethyl thetin chloride was used as a substrate.

Discussion

In this study, both S-methyl-L-cysteine and L-methionine were shown to be metabolized by rumen microorganisms with resultant release of MESH. This observation indicated that these two amino acids are closely linked metabolically. Further evidence for a close metabolic link was previously reported with in vitro cellulose diges- tion studies (23), and with certain yeast cul- tures (15). In both of these studies it was shown that the action of SMC mimicked that of methionine. The findings of Wolff et al. (30) demonstrated that SMC was synthesized by yeast from MeSH plus L-serine and that this reaction was not measurably reversible. The present study, however, indicated that the rumen microorganisms were capable of producing MeSH from SMC and from methionine and also of utilizing MeSH (Table 3 and Experi- ment 1 in Table 4). This finding is in agreement with the results reported by Schlenk and Tillot- son (24), who isolated methylthioadenosine from yeast cells grown in the presence of MESH, which indicated the utilization of MeSH during the formation of methionine and the subse- quent synthesis o£ s-adenosyimethionine and methylthioadenosine.

TABLE 5. Production of dimethyl sulfide from dimethyl thetin chloride.

Incubation Treatment a time Dimethyl sulfide b

(Relative (rain) production)

20 360 Rumen 290 100 fluid 1,755 40

Rumen fluid + 20 360 dimethyl thetin 435 2,540 chloride 1,810 1,160

a Source of tureen fluid: Cow 154. b Each value is the average of two experi-

ments.

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Two possible explanations to account for MeSH liberation from SMC and from methio- nine by rumen microorganisms have been con- sidered, a) Both methionine and SMC may be enzymatically dethiomethylated in one step with SMC yielding MeSH plus a three-carbon com- pound, while methionine yields MeSH plus a four carbon compound, b) Methionine is bro- ken down enzymatically in two steps, with SMC being the intermediate, as shown in Figure 2.

Support for the dethiomethylation of methio- nine and SMC comes from the works of Miwa- tani et ah (18) and Kallio and Larson (11), who have shown that enzymes of Pseudomonas sp. dethiomethylated methionine anaerobically. That methionine is dethiomethylated directly by rumen microorganisms is possible, but the probability that this reaction is reversible seems less likely (i.e., MeSH and a four-carbon com- pound forming methionine in one step). From energetic considerations it would appear more reasonable that formation of methionine from MeSH would pass through one or more inter- mediate compounds, such as SMC.

Support for the stepwise breakdown of methionine comes primarily from work done with yeast cultures (30, 24), and our study. Since under some conditions (Table 3 and Ex- periment 1 in Table 4) MeSH not only appears to be produced but also utilized by rumen mi- croorganisms, the possibility that the pathway shown in Figure 2 may be functional in the rumen cannot be excluded. I t should be empha- sized that as many as nine species of yeasts belonging to three different genera have been isolated from the bovine rumen (5) and this would support such reasoning. Also, the pos- sibility that SMC may be an intermediate in methionine synthesis is suggested, because more MeSH is produced when SMC is the substrate than with L-methionine. Since SMC is a step closer to MESH, and also because the equilib- rium may favor the conversion of methionine to S A ~ (activated form of methionine), lends additional support for this hypothesis.

By analogy, the two explanations given for the production of MeSH from SMC and from methionine could also apply for the production of EtSH from SEC and from ethionine. I n fact, the analogous pathway EtSH > SEC

> ethionine------> SAE > 5'-ETA has been demonstrated in yeast (25).

Acknowledgment

The authors are indebted to Dr. Eric Kissmeyer- Nielsen and Professor Clinton W. Woodmansee for their valuable assistance with the gas chromatograph.

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