STUDIES OF SULFUR METABOLISM OF BRUCELLA SUIS

L. J. RODE, C. E. LANKFORD, AND V. T. SCHUHARDTThe Brucellosis Research Laboratory of the Clayton Foundation and the Department of

Bacteriology, University of Texas, Austin, Texas

Received for publication June 18, 1951

Those chemically defined media which have been proposed for cultivation ofBrucella vary widely, both qualitatively and quantitatively, with respect totheir content of sulfur compounds (McCullough and Dick, 1943; McCulloughet al., 1947; Gerhardt and Wilson, 1948; Rode, Oglesby, and Schuhardt, 1950).Since the early studies of Zobell and Meyer (1932) there has been no criticalinvestigation of Brucella relative to their capacities for utilizing those sulfurcompounds now known to be involved in the pathways of sulfur metabolism.Experience in this laboratory suggests that more information will be requiredin order to define the nutritional requirements for optimum growth of theseorganisms. This report deals with studies of the sulfur nutrition of the suissection of 40 strains of Brucella under investigation.

METHODS

The 10 cultures studied were typical Brucella suis with respect to morphology,cultural characteristics, bacteriostatic action of dyes (basic fuchsin and thionin),and the production of hydrogen sulfide. They were obtained from the followingsources: 3 strains (1C, 2C, and 10C) from Dr. Grace Beal, the University ofChicago; 3 strains (1W, 4W, and 8W) from Dr. J. B. Wilson, the University ofWisconsin; 2 strains (32P and 36P) from Dr. L. M. Hutchings, Purdue Uni-versity; and 2 strains (492 and 1722) from our stock collection.The basal medium used throughout the investigation (table 1) was essentially

that of McCullough and Dick (1943) modified by omission of available nitrogenand sulfur compounds and by the substitution of chlorides for sulfates. L-Aspara-gine, 500 pug per ml, was employed as the nitrogen source in much of the in-vestigation. For certain studies, however, a more complex nitrogen source wasdesirable. In such studies Difco casamino acids, freed of available sulfur com-pounds by treatment with hydrogen peroxide (Lyman et al., 1946), were foundto be suitable. When added to the basal medium at a concentration of 1,000,ug per ml, H202-treated casamino acids failed to support visible growth ofany of the 40 Brucella cultures within the customary 5 days' incubation. Itshould be noted, however, that "adaptation" to growth in this medium wasobserved after 7 to 30 days' incubation of certain cultures of Brucella suis andBrucella melitensis. Several of these "adapted" cultures were found to haveacquired the capacity to utilize cysteic acid and cysteine sulfinic acid, but notsulfate or sulfite. Toennies and Callan (1939) and others have reported thatH202 oxidizes cysteine to cysteic acid and sulfate, and methionine to its sulfoxide

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or sulfone. The wild type of B. suis utilized neither cysteic acid, methioninesulfone, nor sulfate.

In certain tests glutamic acid was used as the nitrogen source at a concentrationof 500 ,ug per ml. DL-Glutamic acid supported no visible growth of the test culturesin the absence of added sulfur compounds, but several commercial samples ofL-glutamic acid were so contaminated with utilizable sulfur that they supportedactive growth when added to the sulfur-free base. The contaminating sulfurcompound was removed by repeated recrystallization of the L-glutamic acid orwas rendered unavailable for growth by oxidation with H202. In the lattermethod, 50 g of L-glutamic acid were dissolved in 500 ml of water with a mini-mum volume of HCI. After the addition of 1 ml of 30 per cent H202, the solutionwas allowed to stand overnight, then steamed 1 hour. The solution was adjustedto pH 2.5 with HCO and refrigerated overnight. Crystals were collected on a

TABLE 1Composition of the basal medium

MATERAL4L MG PER 3M MATERIAL pG PER ML

"S-free" nitrogen source* 0.5-1.0 Fe+ (as C1-) 1.0Glucose 2.0 Mn+ (as Cl-) 0.1NaCl 7.5 Thiamin 0.2K,HPO, 1.0 Nicotinic acid 0.2MgCl, 0.1 Calcium pantothenate 0.04

Biotin 0.001pH 7.0

* See text.

filter, washed three times with absolute methanol, and dried in vacuo. Othernitrogen sources used which did not require preliminary purification were DL-glutamic acid, DL- and L-asparagine, DL-histidine, and NH4Cl. L-Histidlne andL-glutamine were found to be contaminated with available sulfur.The basal medium containing the chosen nitrogen source was tubed in 5 ml

amounts in 20 by 150 mm pyrex tubes and autoclaved for 15 minutes at 115 C.Inorganic sulfur compounds were of reagent or cp grade. L-Cystine, DL-cystine,

L-cysteine hydrochloride, DL-methionine, L-methionine, D-methionine, cysteicacid, taurine, and glutathione were obtained from recognized commercial sources.L-Cystathionine and DL-methionine sulfoxide were supplied by Dr. Lester J.Reed of the Biochemical Institute, University of Texas. Cysteine sulfinic acidand cystine disulfoxide were supplied by Dr. T. F. Lavine of the LankenauHospital Research Institute, Philadelphia. L-Cysteine-S-sulfonate was preparedby the method of Clarke (1932), djenkolic acid by the method of Armstrongand du Vigneaud (1947), and thiazolidine-4-carboxylic acid after the procedureof Ratner and Clarke (1937). All sulfur compounds were compared on the basisof equivalent sulfur concentration. Stock solutions of the compounds, preparedimmediately before use, contained 1 mg sulfur per ml of solution in distilledwater acidified with HCI. These solutions were sterilized by filtration through

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fritted glass UF filters and were added aseptically to the basal medium in ap-propriate concentrations. This precaution was found to be essential, since theresponse to a given sulfur compound when autoclaved often was directly con-trary to the response obtained with the filtered compound. For example, auto-claved thioglycolate was utilized as a single sulfur source, whereas filtered thio-glycolate was not assimilated even in high concentration. This is also in linewith the observations of Schuhardt et al. (1950) on the effect of heat and filtersterilization upon the utilization of cystine by brucellae.

Seed cultures for the preparation of inocula were grown 48 to 72 hours ontryptose agar slants. The growth was suspended in sterile saline and diluted toa scale reading of 12 on the Klett-Summerson electrophotometer. One-tenth mlof this suspension was inoculated to each tube of medium. Although this com-paratively large inoculum (1 to 2 million cells per ml, final density) was desirablein order to produce prompt growth with a suitable sulfur source, it consistentlyfailed to yield detectable growth in 5 days in the sulfur-free basal media. Alltests were incubated without shaking at 37 C for 3 to 5 days. Turbidities wereestimated visually each day, and terminal measurements were made with theKlett-Summerson electrophotometer with blue filter (400 to 465 mMA). Beforethe turbidity measurements were made, the cultures were placed in flowing steamfor 1 hour.

RESULTS

Utilization of inorganic sulfur compounds. Individual inorganic sulfur com-pounds first were tested in the peroxide-treated casamino acids medium at2.5 Mug of sulfur per ml. The amount of growth (turbidity) after 5 days' incubationserved to indicate the relative capacity of the cultures for utilizing each com-pound as the sole source of sulfur (table 2). All of the 40 Brucella cultures testedwere remarkably uniform in their inability to utilize inorganic sulfur at anoxidation level above hydrosulfite (S207). We are thus unable to confirm thereport of Zobell and Meyer (1932) that Brucella utilize sulfate and sulfite.Hydrosulfite, thiosulfate, and sulfide were utilized by all B. suis cultures; thio-sulfate invariably supported best growth at equivalent sulfur concentrations(table 2 and figure 1). At 2.5 Mug sulfur per ml, hydrosulfite was assimilatedsomewhat less efficiently than sulfide by all cultures, and sulfide was less effectivethan a like concentration of thiosulfate sulfur. Each of these was less activethan cystine in supplying the sulfur requirements for growth (table 2). Thequantitative relationship between growth of culture no. 1C and the sulfurconcentration in the medium further bears out this comparison (figure 1) andis representative of the other cultures tested. Sulfide effected only 50 to 60per cent of the growth response produced by thiosulfate at low concentrations,although with excess sulfide the maximum growth may approach that obtainedwith thiosulfate. It should be noted that the minimum in the sulfide curve whichoccurs between 1 and 2.5 ,ug is indicative of partial inhibition within this range.In the asparagine medium the maximum cell density was found to be only one-third to one-half that in the casamino acids base, but the growth response to

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Na2S204, Na2S203, Na2S, and L-cystine was proportionate to that obtained inthe latter medium.

These observations do not permit any conclusions as to the inorganic sulfurcompound which serves as the immediate precursor of organic sulfur. It hasbeen assumed by Lampen, Roepke, and Jones (1947) that sulfide is condensedwith a C3 compound to form cysteine, and they and others have suggested thatcysteine or homocysteine desulfhydrase may catalyze the reaction of sulfidewith an unsaturated keto or amino acid. Hockenhull (1949), however, presentsevidence from studies of Aspergillus nidulans mutants which utilize thiosulfate

TABLE 2Comparative utilization of inorganic sulfur compounds, L-cystine and DL-methionine as the

sole source of sulfur by 10 strains of Brucella suis

RELATIVE RESPONSE TO DIViDUAL COMPOUNDS AS SOLE SOURCZ OF S

STR2AIN NO. so;-, so;-,STRAINNO.433 5slOi S201S S- L-Cystinet DL-Methionine

1C 0 4 8 7 10-(164) 72C 0 4 7 6 10-(171) 710C 0 4 6 6 10-(164) 71W 0 6 8 7 10-(146) 74W 0 3 7 5 10-(168) 78W 0 4 7 7 10-(167) 732P 0 6 9 8 10-(124) 836P 0 3 8 6 10-(140) 8492 0 5 10 7 10-(140) 81722 0 3 9 7 10-(145) 7

* H202-treated "casamino acids" basal medium with 2.5 pg S per ml, incubated at 37 Cfor 5 days.

t Numbers in parentheses are scale readings on the Klett-Summerson electrophotometerfor L-cystine (which produced maximum turbidity for each strain). Other figures are rela-tive to cystine which is assigned a reference value of 10 in each case.

but not sulfide, that thiosulfate may condense with serine to form cysteine-S-sulfonate, an organic precursor of cystine. The quantitative aspects of inorganicsulfur utilization by B. suis might appear to favor slightly the latter hypothesis.However, none of the 10 strains of B. suis produced more than slight growth inthe presence of 2.5 ug sulfur per ml when cysteine-S-sulfonate was supplied asits sole source. The possibility has been considered that the relatively poorresponse to sulfide might be due to its partial oxidative conversion to unassimi-lable or even toxic products. Indeed, the toxicity of certain oxidation products ofsulfide (elemental sulfur?) may account for the failure of Zobell and Meyer(1932) to obtain growth with a relatively high concentration (200 jag per ml)of sulfide as the sole sulfur source. However, it is equally plausible that apparentutilization of sulfide in our tests may actually reflect utilization of certain ofits oxidation products, e.g., thiosulfate.

Colloidal sulfur has been tested as the sole source of sulfur for 2 strains of

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B. msis. If the colloidal aqueous dispersion is sterilized separately from themedium, it is completely ineffective and is, in fact, highly toxic (Schuhardtet al., 1951). However, when colloidal sulfur is autoclaved in the culture medium,it produces a smooth growth response curve with a maximum around 5 ,&g perml. It is assumed that in the presence of certain constituents of the medium,the colloidal sulfur is converted by heat to an assimilable compound.

The utilization of cystine, cystathionine, and methionine. The ability of B.suis to utilize organic compounds as the sole source of sulfur first was tested inthe casamino acids base medium. All B. suis cultures utilized L-cystine, L-

oz*lo-ol /X fe

X//, / * ~~~~~DL-METHIONINE',IZ/EX * ~~~~~L-CYSTATHIONINE

bIOTHIOSUOAl

0

MICROGRAMS S PER. ML

Figure 1. The growth response of Brucella suis, strain 1C, to inorganic and organic sulfurcompounds in H202-treated casamino acids base. Cultures were incubated 5 days. Tur-bidity was determined with the Klett-Summerson electrophotometer.

cystathionine, and DL-methionine. Culture no. 1C typifies the growth responseto these compounds in the casamino acids mediulm (figure 1). L-Cystine at 1 to1.5 ,ug sulfur per ml supported maximum growth, and from the standpoint ofm Unimlm cell density was consistently more effective than DL-methionine(table 2). The latter promoted growth more efficiently in lower concentrations(half-maximum growth: cystme, 0.5; methionine, 0.2 jig sulfur per ml), but thegrowth curve leveled abruptly below the maximuim with cystine. The responseto L-cystine and L-cysteine was found to be qualitatively very similar, but forall 10 cultures the R--H compound was slightly less effective than the R--SS-R compound. In 1 to 3 day cultures DL-cystine was approximately one-halfas effective as L-cystine, but in older cultures utilization of racemic cystine ap-proached that of the natural isomer, particularly at higher concentrations(figure 2A). The pronounced lag in assimilation of D-cystine indicates either aninefficient mechanism for inversion through the corresponding symmetricalketo or imino acid; or perhaps there is a more circuitous route to the L-isomerthrough desulfuration of D-cystine with subsequent assimilation of the inorganicsulfur. In either case the utilization of D-Cystine for growth, even though delayed,

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appears to be an unusual capacity of, microorganisms, since Rydon (1948) hascited no similar instance in his review.By contrast with cystine, DL- and L-methionine produced very nearly identical

growth response at equivalent concentrations at all stages of culture growth(figure 2B). The equivalence of DL-methionine implies an efficient system inB. suis for converting the D- to the L-isomer, probably by way of the correspondingketo acid. However, cultures 1 to 2 days old failed to assimilate D-methionine atconcentrations below 0.3 to 0.15 pg sulfur per ml, although higher concentrations

v .4 ._ ,._

MICROGRAMS S PER ML MICROGRAMS S PER ML

Figure B. A. (left) Utilization of L- and DL-cystine by Brucella suis, strain 1C.B. (right) Utilization of L-, DL-, and D-methionine by Brucella suis.Base medium: H202-treated casamino acids. One set of cultures was incubated 2 days,

the other set for 4 days. Turbidity (ordinate) was determined with the Klett-Summersonelectrophotometer.

promoted growth approaching that obtained with DL- or L-methionine. The pro-nounced sigmoidal character of the dose response curve to D-methionine becameless apparent in older cultures, until growth finally approximated that of L- orDL-methionine after 4 to 6 days (figure 2B). From these results it was concludedthat L-methionine promotes utilization of its enantiomorph. This was confirmedin subsequent experiments in which the addition of very small quantities (0.01to 0.02 ,ug sulfur per ml) of L-methionine to the D-methioninel produced growthin the latter fully equivalent to that obtained with like concentrations of L-methionine alone. More detailed consideration of this phenomenon will bepresented in a subsequent publication.

1 It is not certain that this sample of D-methionine was free of all traces of the L-isomer.However, identical results have been obtained with a highly purified preparation of D-me-thionine (< 0.1 per cent L-methionine) obtained from Dr. Jesse P. Greenstein of the Nat-ional Cancer Institute.

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Combinations of L-cystine and DL-methionine, such that the total sulfurconcentration remained constant, proved to be more effective than either alone.Growth was initiated more promptly, and maximum cell density was increased.Since this effect is much more pronounced with strains of B. melitensis, it willbe reported in detail in relation to these organisms.The mechanism for utilizing L-cystathionine was found to be less efficient

than that for cystine and methionine. Growth was somewhat delayed with cysta-thionine, the sensitivity of response was less than that to cystine, and the doseresponse curves exhibited a sigmoidal tendency (figure 1). This behavior is notdue to toxicity of such possible cleavage products as homoserine or serine, asobserved by Teas (1950a) with serine-sensitive Bacillus subtilis mutants, sincethese compounds did not inhibit growth of B. suis in the presence of cystine.This atypical response to L-cystathionine might be interpreted as indicative ofa lag in synthesis of one or more additional intermediate, possibly isomeric withor closely related to L-cystathionine. Although L-cystathionine is the only isomeryet to be isolated from living systems (Horowitz, 1947), some evidence may beinterpreted as suggestive that an isomeric cystathionine (L-allo-?) also may beinvolved as an intermediate in the cystine =- methionine transformation (Bink-ley, 1950; Anslow, Simmonds, and du Vigneaud, 1946). In order to explain thebehavior of a certain Neurospora mutant, Teas (1950b) has proposed a "pseudo-cystathionine" containing a symmetrical (keto- or imino-) a-carbon in the C4moiety. At least these results suggest that the position of L-cystathionine as thesole intermediate in the cysteine =, homocysteine transformation should receivemore critical investigation.

Utilization of homocystine and homocysteine by B. suis has been studied inconsiderable detail, but the results were of such complexity that it is considereddesirable to treat them in a separate report.

Utilization of methionine and cystathionine in asparagine medium. When aspara-gine, L- or DL-, was substituted for casamino acids as the nitrogen source,methionine or L-cystathionine failed to support appreciable growth within the5 days' incubation period, although cystine was utilized readily. The defectappears not to involve transformation of methionine to L-cystathionine, or thereverse, since mixtures of the two are no more effective than either alone. More-over, tests for the sparing effect of L-cystathionine in the presence of low con-centrations of cystine indicated that the former could be utilized to the extentof satisfying the methionine requirement, even though it did not substitute forcystine (table 3). Since cystine obviously can be transformed to methionine, itappears likely that the cystathionine = cystine transformation is inhibited inthis medium, if the current concept of sulfur transformation is to be accepted(Emerson, 1950; Rachele et al., 1950):

~~~~~~~~P C A

L-CYSTEINE , L-CYSTATHIONINE L-HOMOCYSTEINE L-METHIONINE

CI Cs C,

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It is apparent also that some component of casamino acids relieves this blockedreaction. The maximum effect of casamino acids in promoting utilization ofmethionine or cystathionine was obtained-with 100 to 500 ,ug per ml. In anattempt to identify the responsible component(s), a variety of amino acids andmetabolic intermediates were screened for activity at 100 and 500 ug per ml.Of the amnino acids, only glutamic acid, DL- or -, had appreciable activity, andthis was limited to 5 cultures (table 4). Below 100 ug per ml its activity droppedoff precipitously with some cultures or diminished gradually with others (figure3A). Glutamic acid permitted utilization of methionine or cystathionine by 5of the 6 cultures which utilized it effectively as a source of nitrogen in the presenceof cystine. This behavior points to an intermediate of glutamate oxidation(Gerhardt, Tucker, and Wilson, 1950) as the substance actually responsible

TABLE 3Sparing action of cystathionine on cystine requirement in asparagine base*

ADDITIONAL SULFWU SOURCZCYSTINF5, pG S/1L

0 L-Cystathionine, 0.5 pig S/ml DL-Methionine, 2.5 pg S/ml

0.4 47 79 790.2 25 54 570.1 12 46 620.05 6 40 190 0 3 3

* Cell density expressed in terms of Klett-Summerson scale readings on 4 day culturesof Brucella sui8, strain 492.

for methionine utilization. L-Glutamine appeared to be active also but wasfound to be contaminated with available sulfur. Slight utilization of methionineby 4 cultures occurred in the presence of 500 ,g DL-histidine, but the effect wasminor. Other amino acids, including glycine, serine, and alanine, were withoutappreciable effect, although it is possible that the combination of amino acidsin casamino acids exerts an additive effect.

Little or no consistent effect was elicited with citrate, malate, fumarate,succinate, oxalacetate, DL-a-amino-n-butyrate, 'y-aino-n-butyrate, formate, eth-anol, methanol, glycerol, or adenosine triphosphate. Alpha-ketoglutarate pos-sessed slight activity for 2 cultures. Acetate exhibited definite activity with somecultures but was not effective with others. The effect of acetate was not en-hanced by formate. Pyruvate was consistently more effective than glutamateor acetate, and was active at lower concentration. Of all compounds tested,lactate was most effective in stimulating utilization of methionine and cysta-thionine, and was active in the lowest concentration (figure 3A). As little as10 ,ug lactate per ml affected near-m um utilization of methionine and cysta-thionine in the asparagine base (figure 3B). It is noteworthy also that 10 ,uglactate produced appreciably better growth with cystine as the sulfur source.This concentration (i.e., 10 Mg per ml) of lactate is considerably below that

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usually considered to be required of a compound functioning primarily as anenergy source. For example, in their glycerol asparagine medium, Gerhardt andWilson (1948) found 5,000 ,ug lactate per ml to be the optimum concentrationfor energy requirements of Brucella abortus. A more reasonable supposition inthis instance appears to be that lactate is linked specifically in a reaction ofcystathionine yielding cysteine. From quantitative considerations there seemsalmost a stoichiometric relationship between the quantity of methionine orcystathionine assimilated and the lactate required for its utilization. Although

SUPPLEMENT, JLG PER ML LACTATE, MG PER ML

Figure 8. A. (left) The effect of lactate, pyruvate, glutamate, and acetate upon methio-nine utilization in asparagine base.

B. (right) The effect of lactate upon utilization of cystine, cystathionine, and methio-nine in asparagine base.

Cultures of Brucella SUi8, strain 1C, were incubated 5 days. Turbidity (ordinate) wasdetermined with the Klett-Summerson electrophotometer.

this phase of the investigation has just begun, it might be supposed that therole of lactate is that of a specific hydrogen donor. This would explain its relativeeffectiveness as compared with pyruvate and other possible precursors such asglutamate and acetate. The inability of several other potential hydrogen donors(e.g., methanol, ethanol, formaldehyde, glycerol, succinate, and ascorbic acid)to replace lactate indicates a degree of donor specificity in the reaction. Withone (32P) of three cultures tested, glutathionine and thioglycolate were moder-ately effective in promoting methionine utilization in the asparagine base. Forthe other two cultures, glutathionine and thioglycolate were inactive at test lev-els of 20, 100, and 200 Mg sulfur per ml.

Utilization of other sulfur compounds. The capacity of B. suis to assimilateoxidation products of cysteine and methionine has been investigated in the

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asparagine and the casamino acids mediums. The sulfonic acids, cysteic acid andtaurine, were not utilized in concentrations as high as 10 jug sulfur per ml andwere nontoxic in the presence of low concentrations of cystine. Cysteine sulfinicacid also failed to support growth, whereas cystine disulfoxide was utilized readilyby all cultures. Methionine sulfoxide was utilized quite well in the casaminoacids medium, but like methionine, was not assimilated in the asparagine base inthe absence of lactate. Methionine sulfone was not utilized. These results indicatethat B. suis is capable of reducing sulfoxides (and perhaps cysteine sulfenic acid)to their analogous amino acids, but not sulfones, sulfinic acids, or sulfonic acids.

TABLE 4Utilization of cystine and methionine in L-asparagine (500 ,ug per ml) basal medium

GROWTHE RESPONSE

STRAIN NO. Drmethionine,* 2.5 pg per ml plus:xL-cystine,DmehOl,

2.5 Ag per ml 0 Glutamatell Pyruvatell Acetatell Lactateti

492 + - + + _ +32P + - + + +36P + - + + +10 + - + 41: +1W + - + +10c +t - 4 :4 +20 + - 4-:1 :1: +4W +t -4 -_ +8W +t - -I 4 +1722 +t - +

* L-Cystathionine (0.5 ,ug S per ml), when substituted for DL-methionine, producedgrowth essentially parallel with the latter except for lower cell density.

t Slight growth of these organisms occurred in a glutamic basal medium with L-cystineas the sulfur source.

$ Alpha-ketoglutarate, 500 pg per ml, promoted slight growth with methionine.§ DL-Histidine, 500,ug per ml, promoted slight growth with methionine.11 Tested at levels of 100 and 500 pg per ml.+ = Growth at concentrations tested.

= Growth at 500 but not at 100lg per ml.- = No growth.

When thioglycolate and glutathione were tested as the sole source of sulfurat 1 to 100 ,ug sulfur per ml, they were assimilated by only one (1OC) of the 10cultures. Djenkolic acid was utilized by all but one culture (4W). Thiazolidine-4-carboxylic acid, although utilized by 7 of the 10 cultures at 1 Mg sulfur perml, was partially or completely inhibitory at 10 MAg sulfur per ml. In tryptose,however, the thiazolidine was nontoxic at 200 ,ug per ml.

SUMMARY

The sulfur nutrition of 10 strains of Brucella suis has been investigated in asulfur-free, chemically-defined medium, and in a casamino acids medium renderedfree of available sulfur compounds by treatment with hydrogen peroxide.

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None of the cultures assimilated inorganic sulfur at an oxidation level abovehydrosulfite (S20), and all cultures utilized thiosulfate, sulfide, and hydro-sulfite. Of the inorganic sulfur compounds, thiosulfate was utilized most effi-ciently and hydrosulfite least efficiently. Elemental sulfur was not assimilated.Cysteine-S-sulfonate is not likely to be a precursor of cysteine since it was notutilized by any of the 10 strains.

L-Cystine and L-cysteine were utilized readily in the asparagine medium, aswas thiosulfate. DL-Methionine and L-cystathionine, alone or in combination,were not utilized in the asparagine base. Substitution of peroxide treated cas-amino acids for asparagine as the nitrogen source promoted utilization of methi-onine or cystathionine as the sole sulfur source. Addition of glutamic acid,acetate, or pyruvate to the asparagine base stimulated methionine utilization bysome strains. Lactate, at 10 to 50 ug per ml, permitted efficient utilization ofmethionine and cystathionine by all strains. Many other amino acids and meta-bolic intermediates, including potential hydrogen donors, were ineffective. Therole of lactate in promoting utilization of L-cystathionine appears to be relatedspecifically to a reaction of the latter yielding cysteine.The growth response to L-cystathionine in the casamino acids medium was

atypical, if it is to be considered the only intermediate in the cysteine = homo-cysteine transformation.Both DL- and L-methionine were assimilated with equal efficiency in asparagine-

lactate or casamino acids medium. In the absence of added L-methionine, how-ever, D-methionine was poorly assimilated by young cultures. DL-Cystine,although one-half as active as L-cystine in young cultures, approached thelatter in effectiveness in older cultures.The combination of cystine and methionine induced more prompt growth than

either amino acid alone.B. suis utilized cystine disulfoxide and methionine sulfoxide, the latter only

in the presence of lactate or casamino acids. Methionine sulfone and cysteinesulfinic acid or sulfonic acid were not utilized. Thioglycolate and glutathionewere assimilated by only one culture; djenkolic acid and thiazolidine-4-carboxylicacid were utilized by the majority of strains, although the latter was toxicat 10 Ag sulfur per ml.

REFERENCES

ANSLOW, W. P., JR., SIMMONDS, S., AND DU VIGNEAUD, V. 1946 The synthesis of the iso-mers of cystathionine and a study of their availability in sulfur metabolism. J. Biol.Chem., 166, 35-45.

ARMSTRONG, M. D., AND DU VIGNEAUD, V. 1947 A new synthesis of djenkolic acid. J.Biol. Chem., 168, 373-377.

BINKLEY, F. 1950 Enzymatic cleavage of thioethers. J. Biol. Chem., 186, 287-296.CLARKE, H. T. 1932 The action of sulfite upon cystine. J. Biol. Chem., 97, 235-248.EMERSON, S. 1950 Competitive reactions and antagonisms in the biosynthesis of amino

acids by Neurospora. Cold Spring Harbor Symposia on Quantitative Biology, XIV,40-47.

GERHARDT, P., AND WILSON, J. B. 1948 The nutrition of brucellae: growth in simplechemically defined media. J. Bact., 56, 17-24.

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GERHARDT, P., TUCKER, L. A., AND WILSON, J. B. 1950 The nutrition of brucellae: utili-zation of single amino acids for growth. J. Bact., 59, 777-782.

HOCKENHULL, D. J. D. 1949 The sulfur metabolism of mould fungi: the use of "biochemi-cal mutant" strains of Aspergillus nidulans in elucidating the biosynthesis of cystine.Biophys. et Biochem. Acta, 3, 326-335.

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.

LYMAN, C. M., MOSELEY, O., WOOD, S., AND HALE, F. 1946 Note on the use of hydrogenperoxide-treated peptone in media for the microbiological determination of aminoacids. Arch. Biochem., 10, 427-431.

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