SULFUR METABOLISM IN ESCHERICHIA COLI

III. THE METABOLIC FATE OF SULFATE SULFUR

ELLIS T. BOLTON, DEAN B. COWIE, AND MARGOT K. SANDS

Department of Terrestrial Magnetism, Carnegie Institution of Washington, Washington, D.C.

Received for publication August 10, 1951

The biochemical transformations of sulfur compounds by microorganisms arecomplex. Nevertheless, the broad patterns of utilization of sulfur have beenknown for some time (Bersin, 1950; Foster, 1949; Porter, 1946) and the detailsof certain biosynthetic pathways worked out (Emerson, 1950; Horowitz, 1947).Recently, it has been found (Cowie, Bolton, and Sands, 1950; Cowie et al.,1951a; Cowie et al., 1951b) that the total sulfur content of Escherichia coli dependsin a large measure upon the sulfur composition of the chemically defined mediumin which the organism is cultured. It was shown that the contribution from anyparticular source to the total sulfur of the cell depends upon the chemical formof the sulfur source itself, the amount of sulfur in the medium, and the type ofcell employed. These studies, demonstrating the effect of the environment uponthe utilization of sulfur by the cell, raised questions concerning the kinds andamounts of sulfur compounds formed by E. coli cultured under various condi-tions.The criteria of growth and sparing action or the sulfur uptake of normal or

mutant cells provides, in general, only indirect evidence as to the nature of bio-logically synthesized sulfur compounds. The older direct chemical analyses of thesulfur content of microorganisms are highly variable (summarized by Porter,1946) and are not interpretable in terms of biosynthetic pathways, other than toindicate the range of products which may be expected to occur. A recent aminoacid analysis of E. coli (Polson, 1948) gives a value for the methionine contentbut does not mention cystine. Therefore, it is desirable to augment these investi-gations by chemical methods which provide direct knowledge of the major sulfurcompounds of the cell and allow correlation of the findings with growth andsulfur uptake data.The present paper reports the results of studies upon the fate of sulfate sulfur

metabolized under a number of experimental conditions. For these studies themethods of paper chromatography, radioactive tracers, growth, and competitionhave been simultaneously employed.

METHODS

The general methods employed for culture and handling of radioactive E. coli,strain B,' have been described previously (Cowie et al., 1950). To determine themetabolic fate of sulfate administered as Na2S3504 the procedures adopted were:

1 Obtained from the Department of Genetics, Carnegie Institution of Washington, ColdSpring Harbor, New York.

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(a) culture of bacteria in appropriate synthetic media, (b) acid hydrolysis of har-vested and washed cells, and (c) chromatographic analysis of the acid hydroly-sates. All cultures were started from light inocula of well-washed cells previouslygrown on agar slants. The M-9 medium of Anderson (1946), in which MgSO4was replaced by MgCl2, was employed as the basal culture medium after enrich-ment with radiosulfate at a level of 0.026 mg sulfur per ml. For some of the ex-periments nonradioactive cystathionine,2 homocystine, or methionine was addedto the modified M-9 medium in place of, or in combination with, radiosulfate.Upon 15 to 18 hours' incubation the cells were harvested and thoroughly washedin saline after sampling to determine radiosulfur uptake and density of growth.

PENCHROMIATOGRAMA

0METrER

rl~~f J

[-I/NTEGRATORTo-9JBC RECORDERl

Figure 1. Scanning apparatus. The radioactive chromatogram is drawn across the slitby means of the recording tape of the meter. The motion of the chromatogram and therecord of radioactivity therefore correspond.

The washed cells were hydrolyzed in 5 volumes of 6 N HCI in sealed glass tubesat 105 to 112 C for 6 hours. The radioactive sulfur compounds of an aliquot ofthe hydrolysate were separated by ascending paper chromatography usingWhatman no. 1 acid washed strips. The solvents employed were: (a) 80 per centethanol; (b) tertiary amyl alcohol-water-formic acid, 6:6:3, v/v; (c) n-butanol-water-acetic acid, 4:5: 1, v/v; and (d) 80 per cent tert-butanol containing 2.0 percent oxalic acid. The latter two solvents were found to be the most generallyuseful.

Instrumentation. Radioactivity along the chromatograms was detected bymeans of the scanning device diagrammed in figure 1. The vertical movement ofthe automatically recorded trace is a logarithmic function of the amount of radio-

2 A generous gift of highly purified cystathionine was provided by Dr. M. D. Armstrong.University of Utah College of Medicine, Halt Lake City, Utah.

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1952] SULFUR METABOLISM IN ESCHERICHIA COLI 311

activity brought in line with the variable slit on the face of the Geiger tube. Thelogarithmic display of information is convenient for the routine analysis of ra-dioactive chromatograms since very large differences among radioactivities arepresented without continual adjustment of the instrument. The chromatogramis moved past the slit, and hence the trace recorded, at a rate of one inch perminute.The chromatographic resolution of the radioactive components of the hydroly-

sates studied is reasonably good, as the dotted line of trace AF of figure 2 shows.

s-_olvwwwent.s: TB = tetbanloli aci; BA =obu w]~tanwol-cti acd AF = ter-amyaloo-fri acd TH80 pe cent ethnlTe arrws inict th sJ0tarting positionsof th choatgapi rus Roa nueas deinte rein hc aveneue

Q t4:r>ce=$=T7-T-i-r-AA

-winTBOthe resut ben shown in figr 3. = J8--

linerpofata raiatvtes afte eluio aneprte contn (the oriae shwdosnt appl to ths cu t'L.,_.,_rve).

e_i~*|. _ = f~s0~ fsS .-

Ai 7Z

^:_,tsz -

ofthecho aorpirunsL Roma nu erl deint rein whc hav bee.eluted.

doe no apply to thi curv,ie).

This dotted line is a linear plot of the actual radioactivity along the paperchromatogram. The radioactivity in this case has been measured with a conven-tional Geiger counter by separately counting 1-cm segments in sequence alongthe strip. Routinely, the shortest movement of the solvent front which will yieldunequivocal separation of the separable radioactive components is employed.

RESULTS

Identification of the radioactive components of hydrolysates. E. coli grown in M-9containing radiosulfate of high specific activity (Cowie et a7., 1951b) as the solesulfur source was hydrolyzed and the radioactive hydrolysates submitted tochromatography in a number of solvents. Typical distributions of radioactivity

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E. T. BOLTON, D. B. COWIE, AND M. K. SANDS

along the chromatograms are shown in figure 2. These chromatograms of radio-active sulfur compounds formed during metabolism or as a result of alterationand degradation during hydrolysis and chromatography are complex. They havebeen resolved into 5 amino acids: methionine, methionine sulfoxide, cyst(e)ine,and cysteic acid.

Comparison of figures 2 and 3 indicates in part the manner in which the radio-active components of figure 2 were identified. The method employed has been toelute and collect individually the compounds separated in the solvents of figure 2and to resubmit the eluted areas to chromatography in other solvents. Figure 3

Figure 3. Radioactive chromatograms of areas I, II, III of the BA run (figure 2) elutedand re-run in TBO. Region BA-I corresponds to TBO-a-b and -c; BA-II to TBO-d; BA-IIIto TBO-e. Region TBO-a is "soluble humin", TBO-b methionine, TBO-c methioninesulfoxide, TBO-d cysteic acid (+ cysteine), TBO-e cyst(e)ine and "insoluble humin".

is an example of the result in tert-butanol-oxalic acid for radioactive areas origi-nally separated in butanol-acetic acid. In this way each radioactive region offigure 2 has been identified in terms of corresponding regions on the remainingchromatograms. Each eluted region was then (a) identified in terms of authenticsamples of sulfur amino acids run in parallel, (b) detected colorimetrically withninhydrin and the platinic iodide reagent (Winegard, Toennies, and Block, 1948),and (c) determined by failure to separate from the appropriate nonlabeled com-pound added as carrier. Radioactive cystine and methionine have also been pre-pared from the hydrolysates. The presence of methionine and cyst(e)ine wasconfirmed by the hydrogen peroxide oxidation method of Dent (1947a).

Hydrolytic destruction of cyst(e)ine. Destruction of cyst (e)ine during acidhydrolysis in the presence of carbohydrates (Block and Bolling, 1945) or pyru-vate (Olcott and Fraenkel-Conrat, 1946) is well known. In the acid hydrolysates

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of the present study hydrolytic destruction of cyst(e)ine is appreciable and isaccompanied by the formation of radioactive "humin" (component 1 of the tert-butanol-oxalic acid runs figures 2 and 3). Since the presence of carbohydratesmust be tolerated in whole E. coli cells, a systematic study was made to determinethe extent of destruction of the radioactive sulfur amino acids during hydrolysis.Radiosulfate-grown cells were sealed in glass test tubes with 6 N HC1 and heatedat 105 to 112 C. Each hour for 8 hours one of the tubes was withdrawn and itscontents chromatographed. Radioactive regions, which corresponded to cyst (e)-

TABLE 1Destruction of cyst(e)ine

PER CENT OF TOTAL RADIOACTIVITY*

Hours of hydrolysis

1 5 8

Cyst(e)ine and insoluble humint(a) 29 27 22(b) 34 34 20

Methionine(a) 53 60 54(b) 56 57 57

Soluble humin(a) 0 13 24(b) 0 9 23

Other (peptide?)(a) 18 0 0(b) 9 0 0

a, b = duplicate experiments.* The measurements were made by directly counting the appropriate radioactive regions

on chromatograms employing a conventional Geiger counter.t Cyst(e)ine and insoluble humin were incompletely separated in these runs and were

counted together.

ine methionine, and soluble humin were measured. The results are summarizedin table 1. Although it is the usual practice to reflux protein in acid for longperiods of time in order to ensure completeness of hydrolysis, these results demon-strate that only a small quantity of radiosulfur is not accounted for in cyst (e)ineand methionine at the end of 1 hour. This radiosulfur may be mainly in peptidesof methionine or cystine. After 5 hours' hydrolysis the cyst(e)ine content beginsrapidly to disappear while the soluble humin portion rapidly increases. At 8 hoursat least one third of the original cyst(e)ine has disappeared while the solublehumin fraction now accounts for one quarter of the total radioactivity. Thedestruction of methionine is slight, although in other experiments losses of 20per cent have been observed.

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E. T. BOLTON) D. B. COWIE, AND M. K. SANDS

The oxidized amino acids, methionine sulfoxide and cysteic acid, are observedin largest quantity whenever a mixture of formic and hydrochloric acids (Blockand Bolling, 1945) is used for hydrolysis, when concentration of the hydrolysateis conducted in an open watch glass on a steam bath, or when the tert-butanol-oxalic acid solvent is used in chromatography.

Estimation of radioactive methionine and cystine content of E. coli. Since the solesulfur source of these cells is radiosulfate of known specific activity, methioninemay be determined by elution of the radioactive methionine from a chromato-gram and counting under standard conditions. A typical estimation showed that84 per cent of the total radioactivity was readily eluted from a strip, 38 per centof the total was found in methionine, the remainder constituting the cyst(e)inegroup or breakdown products. The activity not eluted was found in the insoluble

TABLE 2Growth and sulfur uptake in cystathionine media

NORMAL CELLSt CYSTINE MUTANT§SULFUR SOURCEt

ml gS*/ml mleell/mmgS*/ml cells ml cells!m g*m cellsi mlclslmedium mg~mmedium

S*04-- 3.4 3.6 X 10-3 II <0.03 X 10-3Cystathionine 0.19 0.06S*04-- + cystathionine 3.4 4.7 <0.03Cyst,ine* 3.0 3.9 2.9 2.9Cystine* + cystathionine 3.0 3.5 2.4 3.6Methionine* 1.4 0.51 1 .8 0.6Methionine* + cystathionine 1.2 1.6 1.8 1.6

* The asterisk indicates the S35 labeled component.t Each sulfur source added to M-9 medium at 0.026 mg sulfur per ml.t Strain B.§ Mutant no. 508-462 (Lampen et al., 1947). This mutant was kindly supplied by M. J.

Jones, American Cyanamid Company, Stamford, Conn.11 Sulfur uptake in these tests was negligible. One ml equals 1012 cells.

residue at the start of the chromatogram. Since cyst(e)ine is readily destroyedduring hydrolysis, these acid hydrolysates are unfavorable materials for directisolation and determination of cystine. The cystine content of sulfate-grown cellshas been determined indirectly by the alkaline permanganate procedure ofCallan and Toennies according to Block and Bolling (1945). Thirty-four per centof the total radioactivity was found to be "alkali labile" (Dent, 1947b) whilethe amount of free sulfate in the acid hydrolysates is negligible (about 1 per cent).No other sulfur-containing amino acids than those already noted have been ob-

served in the hydrolysates of E. coli grown on radiosulfate as its sole sulfursource. Those sought have been lanthionine, S-methylcysteine, cystathionine,homocysteine, homocystine, and djenkolic acid. Thiourea and taurine have alsobeen investigated. It is highly improbable that methionine sulfoxide or cysteicacid exists as such in the native proteins of the bacterial cell since they are ob-served to arise as a result of oxidizing conditions during analysis. Cysteic acid will

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neither support growth nor influence the sulfate uptake of strain B of E. coli(Cowie et al., 1951a). It is concluded that the predominant products of themetabolism of sulfate by E. coli are methionine and cyst(e)ine. The small quan-tities of other sulfur compounds probably formed such as thiamin or sulfuricacid esters of carbohydrates would be overlooked by the methods employed.These findings are closely parallel to the results of a similar study on alfalfa leavesby Steward et at., (1951).The chromatograms for sulfate-grown cells shown in figures 2 and 3 serve as

a convenient frame of reference in which the effects of alterations in culture con-ditions upon the distribution of sulfur among metabolic products may be studiedand compared.

Figure 4. Chromatogram of the hydrolysate of Escherichia coli grown on nonradioactivehomocystine and radioactive sulfate. Component "a" is insoluble humin and "b" cystine.The position of methionine is indicated at "c" by the circle at the base line. n-Butanol-acetic acid solvent was employed.

Effect of cystathionine, homocystine, and methionine. The thio-ether cystathio-nine is of special significance in the metabolism of sulfur, for by virtue of itsstructure it may provide a molecular "bridge" in the transport of the sulfur atomfrom a three- to a four-carbon chain during the biosynthesis of methionine(Horowitz, 1947; Emerson, 1950; Lampen, Roepke, and Jones, 1947; Simmonds,1948). Measurements of growth and radiosulfur uptake, shown in table 2 for E.coli grown in cystathionine-containing media, indicate that cystathionine has no

striking effect upon the quantity of cells produced after 15 to 18 hours' growth or

upon the amount of radiosulfur incorporated. These results contrast with those(Cowie et at., 1951a) in which a number of sulfur compounds alter the uptake of a

labeled sulfur source. Chromatograms of hydrolysates of cells grown on radio-sulfate and cystathionine are identical with those shown in figures 2 and 3 wheresulfate was the sole sulfur source.

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The chromatograms of figure 4 are of a hydrolysate of cells grown on M-9 con-taining radiosulfate and nonlabeled homocystine. An identical chromatogramresults for cultures in which methionine is substituted for homocystine. Theradiosulfur content of both groups of cells was about 1.5 mg per ml (1012) cells.The sulfur content for E. coli grown on sulfate as the sole sulfur source is ap-proximately 3.5 mg sulfur per ml cells. Figure 4 demonstrates that the decreasein radiosulfur content of the cells grown on the mixed sulfur substrates is ac-counted for by the absence of biologically synthesized radiomethionine. Theradiosulfur taken up under these conditions appears as nonmethionine sulfur,mainly cystine or its degradation products.

DISCUSSION

It is evident from these and other studies (Cowie et al., 1950, 1951a,b) thatthe results of experiments on growth, sulfur uptake, and the isolation of radio-active sulfur compounds provide independent and complementary data by whichbiosynthetic pathways may be determined in wild type cells.These studies show that for radiosulfate-grown E. coli, strain B, most of the

radiosulfur incorporated is distributed between methionine (38 per cent) andcyst (e)ine (34 per cent). These estimates are minimal since no effort has beenmade to compensate for the observed losses during hydrolysis or chromatography.They may, however, be used as a basis for comparison of the results of experi-ments in which the sulfur sources are varied.

It has been suggested (Cowie et al., 1951a) that cystine is utilized by twopathways in these cells since cystine completely eliminates sulfate uptake, whilemethionine only partially suppresses sulfate or cystine uptake. In one of thesepathways cystine provides the sulfur of methionine and in the other is incor-porated in protein. The result of figure 4 demonstrates the validity of this sug-gestion. The formation of radiomethionine is completely suppressed while theend product of radiosulfate metabolism is radiocyst(e)ine.

Transient metabolic intermediates, such as homocysteine or cystathionine,are not found in the hydrolysates. Nevertheless, their effect upon the incorpora-tion of radiosulfur in metabolic end products may be acutely studied and theirrole in the biosynthesis of sulfur evaluated. Thus, homocystine appears equiva-lent to methionine when examined from the point of view of radiosulfate utiliza-tion. Cystathionine presents a different case.

Cystathionine as the sole sulfur source allows only slightly greater growththan a "sulfur-free" medium (0.4 ,ug sulfur per ml of medium). It has no certaininfluence on the incorporation of radiosulfur when administered simultaneouslywith labeled sulfate, cystine, or methionine. This compound does not alter thepatterns of radioactivity on chromatograms of the hydrolysates of cells grownin a medium containing radiosulfate as the labeled source. It is improbable thatstrain B of E. coli is impermeable to cystathionine since radiocystine readilydiffuses into the water space of these cells while lanthionine and djenkolic acid,which are grossly similar in structure to cystathionine, provide for growth of theorganism or alter the radiosulfur uptake from labeled sources. The data of table

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2 show that cystathionine has little influence on the utilization of radiocystineor radiomethionine by the cystine mutant derived from strain no. 15. Figure 4shows, on the other hand, that homocystine or methionine completely sup-presses the formation of radiomethionine from labeled sulfate. It is, therefore,concluded that cystathionine is not a normal metabolic intermediate in strain Bof E. coli, and thus this organism appears to differ qualitatively from strain K-12of E. coli (Simmonds, 1948), Neurospora (Horowitz, 1947), and from mammals(Binkley, 1944).

SUM-ARY

Radioactive methionine and cyst(e)ine have been separated from acid hy-drolysates of Escherichia coli by paper chromatography. These amino acids ac-count for most of the radioactive sulfur of the cells grown on radiosulfate as thesole sulfur source. Minimal estimates indicate that 38 per cent of the total sulfuris in methionine while 34 per cent is in cyst(e)ine.

Methionine sulfoxide and cysteic acid have been detected in hydrolysatesexposed to mildly oxidizing conditions. Lanthionine, djenkolic acid, homocyst(e)-ine, S-methylcysteine, cystathionine, taurine, and thiourea have not been ob-served in the acid hydrolysates.

Cells grown in radiosulfate and nonradioactive homocystine or methioninecontain no radiomethionine while most of the radiosulfur is in cyst(e)ine. Thisresult confirms an earlier suggestion that cystine takes part in two pathways inthese cells.

Cystathionine appears to be without effect upon growth or sulfur uptake ofE. coli, strain B. It is probably not a normal metabolic intermediate in the bio-synthesis of methionine by this strain.

REFERENCESANDERSON, E. H. 1946 Growth requirements of virus-resistant mutants of E. coli strain

B. Proc. Natl. Acad. Sci. U. S., 32, 120-128.BERSIN, T. 1950 Die Phytochemie des Schwefels. Advances in Enzymol., 10, 223-323.

Interscience Publishers, Inc., New York, N. Y.BINKLEY, F. J. 1944 Cleavage of cystathionine by an enzyme system from rat liver.

J. Biol. Chem., 155, 39-43.BLOCK, R. J., AND BOLLING, D. 1945 Amino Acid Composition of Proteins and Foods,

p. 282. C. C. Thomas, Springfield, Ill.COWIE, D. B., BOLTON, E. T., AND SANDS, M. K. 1950 Sulfur metabolism in Escherichia

coli. I. Sulfate metabolism of normal and mutant cells. J. Bact., 60, 233-248.COWIE, D. B., BOLTON, E. T., AND SANDS, M. K. 1951a Sulfur metabolism in Escherichia

coli. II. Competitive utilization of labeled and nonlabeled sulfur compounds. J.Bact., 62, 63-74.

CowIE, D. B., BOLTON, E. T., AND SANDS, M. K. 1951b The labeling of bacterial cellswith S35 for the production of high specific activity compounds. Arch. Biochem. andBiophys., in press.

DENT, C. E. 1947a The amino-aciduria in Fanconi Syndrome. A study making extensiveuse of techniques based on paper partition chromatography. Biochem. J., 41, 240-253.

DENT, C. E. 1947b The cystine and methionine contents of liver protein in acute hepaticnecrosis. Biochem. J., 41, 314-320.

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EMERSON, S. 1950 Competitive reactions and ant~gonisms in the biosynthesis of aminoacids by Neurospora. Cold Spring Harbor Symnposia Quant. Biol., 14, 40-48.

FOSTER, J. W. 1949 Chemical Activities of Fungi, p. 532. Academic Press, Inc., NewYork, N. Y.

HOROWITZ, N. H. 1947 Methionine synthesis in Neurospora. The isolation of cystathio-nine. J. Biol. Chem., 171, 255-264.

LAMPEN, J. O., ROEPKE, R. R., AND JONES, M. J. 1947 Studies on the sulfur metabolismof Escherichia coli. III. Mutant strains of Escherichia coli unable to utilize sulfate fortheir complete sulfur requirements. Arch. Biochem., 13, 55-66.

OLco-rr, H. S., AND FRAENKEL-CONRAT, H. 1946 Formation and loss of cysteine duringacid hydrolysis of proteins-role of tryptophane. J. Biol. Chem., 171, 583-594.

POLSON, A. 1948 Quantitative partition chromatography and the composition of E. coli.Biochim. et Biophys. Acta, 2, 575-581.

PORTER, J. R. 1946 Bacterial Chemistry and Physiology. John Wiley and Sons, Inc.,New York, N. Y.

SIMMONDS, S. 1948 Utilization of sulfur-containing amino acids by mutant strains ofEscherichia coli. J. Biol. Chem., 174, 717-722.

STEWARD, F. C., THOMPSON, J. F., MILLAR, F. K., THOMAS, M. D., AND HENDRICKS, R. H.1951 The amino acids of alfalfa as revealed by paper chromatography with specialreference to compounds labelled with S35. Plant Physiol., 28, 123-135.

WINEGARD, H. M., TOENNIES, G., AND BLOCK, R. J. 1948 Detection of sulfur-containingamino acids on paper chromatograms. Science, 108, 506-507.

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