BIOSYNTHESIS OF CHLORAMPHENICOL

IV. INCORPORATION OF CARBON14-LABELED PRECURSORS

DAVID GOTTLIEB, H. E. CARTER, P. W. ROBBINS,' AND R. W. BURG2

Department of Plant Pathology, University of Illinois, Urbana, Illinois

Received for publication May 26, 1962

ABSTRACT

GOTrLIEB, DAVID (University of Illinois,Urbana), H. E. CARTER, P. W. ROBBINS, AND

R. W. BURG. Biosynthesis of chloramphenicol.IV. Incorporation of carbonl4-labeled precursors.J. Bacteriol. 84:888-895. 1962.-Metabolism ofDL-phenylalanine stimulated antibiotic synthesisby Streptomyces venezuelae, and resulted in thefixation of carbons 1 and 2 into the carbonyl groupof chloramphenicol. It probably occurs by theoxidation of these carbons to carbon dioxide,followed by incorporation. Carbon 3 and theadjacent ring carbon were found in the dichloro-methyl and the carbonyl carbons, respectively,of the dichloroacetyl portion of the molecule. Thephenyl group of the amino acid is not transferredto the ring in chloramphenicol. Another stimu-latory amino acid, DL-norleucine, contributedcarbon 2 only as the carbonyl carbon. DL-Leucineis metabolized so that two adjacent carbonsappear as the carbons in the dichloroacetyl moietyof the antibiotic. From acetic acid, carbon 1 isfound only in the carbonyl group of chloram-phenicol; carbon 2 of the acid is more generallydistributed among the ring and side chain of thep-nitrophenylserinol l)art of the antibiotic, butis in greatest concentration in the dichloroacetylfraction. Formic acid and carbon dioxide alsoare transformed only to the carbonyl group.Glycerol, the main source of metabolized carbonin the medium, has a general role and contributesto all parts of the molecule. In addition, it has aspecific role in supplying an intact three-carbonfragment which enters into the molecule ofchloramphenicol as a unit.

Previous studies on the biosynthesis of chlor-amphenicol by,r Streptomyces venezuelae in chemi-

1 Present address: Massachusetts Institute ofTechnology, Cambridge.

2 Present address: Merck Sharp & Dohme Re-search Laboratories, Rahway, N.J.

cally defined media have shown that certainconstituents might be specifically involved in thesynthesis. Though glucose was a good carbonsource for the growth of the microbe and allowedthe formation of chloramphenicol, glycerol wasoften a better carbon source for the formation ofantibiotic in some media. Lactic acid also wasneeded for this same purpose (Gottlieb andDiamond, 1951).

Nitrate ion served for both growth and anti-biotic synthesis, whereas ammonium ion allowedgrowth but no synthesis of antibiotic. Further-more, certain amino acids stimulated the forma-tion of chloramphenicol (Gottlieb et al., 1954).The structure of some of them, such as phenyl-alanine, could be related to that of the antibioticitself and point to its direct precursor role,whereas others, such as leucine, were not easilyrationalized into such a role. Attributing directprecursor activity to a compound on the basis ofability to stimulate a synthesis is not alwaysreliable. In the case of the intense stimulatoryaction of p-nitrophenylserinol, which is a majorl)ortion of the chloramphenicol molecule, therewas no incorporation into chloramphenicol andthe observed increase in antibiotic activity inthe medium was due to an acetylation of theprecursor to form another biologically activecompound (Gottlieb, Robbins, and Carter, 1956).The structure of chloramphenicol (Fig. 1) is

D - threo-p-nitrophenyl - 2 - dichloroacetamido-1, 3-p)ropanediol (Rebstock et al., 1949). In the pres-ent study of the role of precursor, the incorpora-tion of the following C'4--labeled compounds thathad been shown to stimulate the synthesis ofantibiotic were used: phenylalanine, norleucine,leucine, glycerol, and lactic acid. Incorporationof acetate and carbon dioxide were investigatedbecause these materials are metabolic productsof S. venezuelae that might also be utilized in thesynthesis into component carbons of the anti-biotic.

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h-CH-CH-CH20H CH-CH-CH20H02N 6HNH /\,) OH NH2

1O 02N

CHCl2 I

02N/ -CHO + HCOOH + CH20methone

CO2 methylene-bis-methone

02N -COOH

02N/ + C02

FIG. 1. Scheme for degradation of chloramphenicol.

MATERIALS AND METHODS

S. venezuelae isolates R-1 and 3022A were usedin these studies; they were stored in soil for long-time storage and were transferred, as needed, toslants of either Emerson's (Emerson et al., 1946)or glycerol-Tryptone medium (Oyaas, Ehrlich,and Smith, 1950) to which 2% agar was added.The slant cultures were incubated 3 to 7 days,and about 4 ml of distilled water containing0.01 % Vatsol (American Cyanamid Co., NewYork, N.Y.) were then added. The spores were

gently scraped from the culture surface to forma spore inoculum. Preparation of mycelial inocu-lum, conditions for the fermentation, and assaywere done in a manner previously described(Gottlieb et al., 1954).For most studies, an open-train type of fer-

mentation system was used. Its essential ele-ments, in order, were: a sodium hydroxide flakeabsorption chamber for the removal of atmos-pheric carbon dioxide, a wash bottle containingbarium chloride, and a cotton filter to preventcontamination of the medium. The filter was

connected to a 2-liter Erlenmeyer flask on a

reciprocal shaker containing 300 ml of glycerollactate medium (Gottlieb et al., 1954). Labeledprecursors were added to the medium either as

solids prior to sterilization or as aqueous solutionswhich had been passed through an ultrafinebacteriological filter and added after sterilization.The medium was inoculated with a 3% mycelialsuspension. An outlet from the Erlenmeyer flaskin turn was connected to a series of three gas-

+ C12HC-COOH

11C-

HCOOH + CO2

CO2

washing bottles containing 5 N sodium hydroxide,to remove radioactive carbon dioxide, and finallyto a vacuum pump. The fermentation was carriedout at 26 C for 72 hr. The mycelium was thenseparated from the culture fluid and the anti-biotic was extracted from the liquid (Gottliebet al., 1956).

Resting-cell experiments on the incorporationof methyl-labeled acetate were made using 40-hrshake cultures grown in the glycerol-tryptonemedium. The mycelium was separated by cen-trifugation and then washed twice with distilledwater. The cells were incubated at 26 C for 8 hron a rotary shaker in 300 ml of 0.01 M phosphatebuffer (pH 7.4) containing the labeled precursor.A closed fermentation system was used for

labeled bicarbonate and leucine. The fermenta-tion was carried out on the reciprocal shaker ina 2-liter Erlenmeyer flask. On the top was fitteda gas bulb and stopcocks to allow the storageand removal of gases. After the sterile mediumwas inoculated, the pressure in the system wasreduced to 35 cm, and a commercial tank mixtureof 35% oxygen and 65% nitrogen was allowedto flow into the fermentation vessel until thepressure reached 1 atm. Whenever the pressuredropped more than 3 cm, the gas in the systemwas replaced with 35% oxygen. Samples wereintermittently withdrawn from the gas bulb intopreviously evacuated 100-ml glass bulbs. Finally,the reaction chamber and collection vessel wereconnected to a series of gas-washing bottles con-taining 5 N sodium hydroxide to remove anyresidual labeled gas.

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The medium was assayed for antibiotic at theend of the fermentation period. Crystallinechloramphenicol was then added to the mediumto dilute the radioactive antibiotic. The totalchloramphenicol was extracted and purifiedaccording to method B of Bartz (1948). Thtchloramphenicol obtained from all tracer experi-ments conformed to that of authentic samples inmelting point and RF. The carbon labeling wasdetermined by degrading the antibiotic as out-lined in Fig. 1. In general, the degradation fol-lowed the procedure of Rebstock et al. (1949).All radioactivity determinations were made inan ionization chamber on a carbon dioxide sam-ple from the degradation of chloramphenicol.Procedures for a typical degradation of chlor-amphenicol are given below.

Hydrolysis to p-nitrophenylserinol and dichloro-acetic acid. Chloramphenicol (276.3 mg) washeated under reflux with 7 ml of 1.5 N hydro-chloric acid for 4 hr. The solution was cooledand extracted five times with 7-ml portions ofether. The ether extract was dried over magne-sium sulfate and evaporated to an oily residuewhich was then dissolved in 1.5 ml of benzene. Tothis solution were added 160 mg of p-toluidinedissolved in 2 ml of benzene. The p-toluidine saltof dichloroacetic acid crystallized almost im-mediately; it was filtered and washed severaltimes with cold benzene. The dried productweighed 160.5 mg (79% yield) and had a meltingpoint of 134.5 to 138 C.The aqueous layer, remaining after ether ex-

traction, was lyophilized to a solid, dissolved in afinal volume of 4.5 ml of water, and filtered. Thesolution was cooled in an ice bath, and 1 N sodiumhydroxide was added until the pH was about 11.The p-nitrophenylserinol, which crystallized fromthe alkaline solution, was filtered and washedthree times with distilled water. The yield ofdried product was 158.4 mg (87% yield) and hada melting point of 162 to 163.5 C.

Degradation of the dichloroacetic acid. Thep-toluidine salt of dichloroacetic acid (145.7 mg)was dissolved in 5 ml of 1.5 N hydrochloric acidand extracted six times with equal volumes ofether. The ether was evaporated and the residuedissolved in 1 ml of water. The solution wastitrated with 5.9 ml of 0.0926 N sodium hydroxideto a methyl-red end point which corresponded toan 89% conversion of the salt to the free acid.An extra 0.20 ml of the alkali was added and the

solution was autoclaved at 165 C (80 psi) for4.5 hr. After standing overnight, the solution wastitrated again with 0.0926 N sodium hydroxide;the volume of alkali required to raise the pH to4.5 corresponded to a 98% conversion of di-chloroacetic acid to glyoxylic acid.The glyoxylic acid solution was filtered into

the reaction vessel of the gas train and sweptwith helium for 45 min. Sodium metaperiodate(250 mg) was added and, after it had dissolved,the solution was swept for 40 min. The yield ofcarbon dioxide was 102%, based on the secondtitration.Barium hydroxide was added to the solution

to precipitate the iodate and periodate. Themixture was filtered and the filtrate waslyophilized. The residue was dissolved in 10 mlof 8% mercuric chloride in acetate buffer, andthe solution was filtered into the reaction vesselof the gas train. Another 20 ml of mercuric chlo-ride solution were added to the reaction vessel,and the solution was swept for 20 min to removeresidual carbon dioxide. The reaction vessel washeated in a boiling-water bath for 20 min. Thehelium sweep was continued during this periodand for 40 min after the heating. The yield ofcarbon dioxide was 45% based on the weight ofdichloroacetic acid salt.

Degradation of p-nitrophenylserinol. p-Nitro-phenylserinol (141.8 mg) was suspended in 4 mlof water. To this was added sodium m-periodate(350 mg in 4 ml of water), and the mixture wasallowed to stand at room temperature for 2 hr.The acidity of the solution was raised to pH 1with 10% phosphoric acid, and the solution wasextracted five times with equal volumes of ether.The ether solution was titrated to a phenol-

phthalein end point with 0.0988 N sodium hydrox-ide. The yield of formic acid based on this titrationwas 39%. An extra 0.5 ml of the alkali was addedand the aqueous layer lyophilized. Carbon di-oxide was generated from the formate by theprocedure already described. The yield of carbondioxide from the formic acid was 96%.The ether solution, which remained after

removal of the formic acid, was washed oncewith a small volume of 0.01 N sulfuric acid andthree times with small volumes of water. Theether was dried over magnesium sulfate andevaporated to dryness. The yield of crystallinep-nitrobenzaldehyde (melting point, 105 to 107 C)was 57.6 mg (57% yield).

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The pH of the aqueous solution remainingafter the periodate oxidation and ether extractionwas adjusted to about 8.5 with 0.1 N sodiumhydroxide. Enough 0.1 N sodium arsenite was

then added to destroy the excess periodate, andthe solution was allowed to stand at room tem-perature for 2 hr. The pH was adjusted to about4.5 with 10% phosphoric acid, and 300 mg ofmethone reagent, dissolved in about 5 ml ofethanol, were added. After standing at 3 C for24 hr, the methylene-bis-methone was filteredand washed with water. It was recrystallized from7 ml of 70% (v/v) ethanol. The final productwas washed twice with 50% (v/v) ethanol andonce with absolute ethanol. The product weighed59.2 mg (30% yield) and melted at 192 to 193 C.

Degradation of p-nitrobenzaldehyde. p-Nitro-benzaldehyde (175 mg) was dissolved in 10 mlof 88% formic acid. It was placed in a cold room,

and 1.5 ml of 30% hydrogen peroxide were added.The mixture was kept in the cold room for 12 hrand then lyophilized.

Copper chromite catalyst (250 mg) and 15 mlof dry redistilled quinoline were placed in thereaction vessel of the gas train and swept withhelium for 30 min. The crystalline p-nitrobenzoicacid was added and the solution swept for anotherfew minutes. The quinoline was refluxed andswept with helium for 30 min, after which thehelium flow was continued for 15 min. The yieldof carbon dioxide was 85% based on p-nitro-benzaldehyde.

RESULTS

Typical data on the distribution of C'4 inchloramphenicol when separate experiments were

made with DL-phenylalanine labeled individuallyas 1-C"4, 2-C'4, 3-C14, and U-C'4 are given in Table1. Radioactivity from phenylalanine-1- or -2-C14was contained only in the carbonyl carbon of thedichloroacetyl portion of the molecule. Fromphenylalanine-3-C'4, on the other hand, thehighest specific activity was in the dichloromethylcarbon and only 9.8% in the carbonyl carbon.All the carbons of the p-nitrophenylserinol por-tion together contained only 15.3% of the activ-ity. Uniformly labeled phenylalanine contributedalmost equally to both carbons of the dichloro-acetyl, with slightly higher values in the carbonylcarbon. The remainder of the carbons in themolecule contained only 2% of the total specificactivity.

TABLE 1. Incorporation by Streptomyces venezuelae*of C14 into chloramphenicol from variously labeled

DL-phenylalanine

Specific activity (pc/mmole)from phenylalanine labeled as

Product

-l-Cl4t -2-C414 -3-CI14 _U_C14t

Chloramphenicol ..... 2.78 - - 21.60p-Nitrophenylserinol.. 0.03 0.03 0.58 0.56Dichloroacetic acid.. 2.74 0.37 3.22 21.00Carbonyl carbon... 2.57 0.34 0.37 10.40Dichloromethylcarbon .. . 0.03 0.01 2.85 9.90

*S. venezuelae strain R-1 was used and 0.450mmole of phenylalanine was added to 300 ml ofmedia.

t Specific activity, 422 jc per mmole.t Specific activity, 222 sc per mmole.

Table 2 contains the data from the DL-norleu-cine-2-C14 and DL-leucine-U-C'4. With norleucine,only the carbonyl carbon of the antibiotic wasradioactive. The pattern of labeling from leucinewas almost identical with that from uniformlylabeled phenylalanine. The dichloroacetyl carbonswere equally labeled with some slight labeling inthe p-nitrophenylserinol.Though exogenous acetate does not stimulate

the biosynthesis of chloramphenicol, acetateitself is a very common biosynthetic intermediate.The distribution of its carbons in the antibioticmolecule might give information to aid in under-standing the previous labeling data, or on thesynthesis of chloramphenicol without exogenousprecursors. Data in Table 3 indicate that almostall labeling from acetate-i-C'4 was transformedinto that of the carbonyl carbon of the antibiotic;there was only 2% in the rest of the molecule. Inexperiments with resting cells, acetate-2-C'4produced a more general distribution of C14.The greatest specific activity was found in thedichloroacetyl moiety. In this case, more labelingoccurred in the dichloromethyl than in the car-bonyl carbon. The labeling of the individual car-bons of the side chain in the p-nitrophenylserinolportion was small but significant, and, if thespecific activity in the phenyl group were equallydistributed, these carbons would be as active asthose of the side chain of p-nitrophenylserinol.Some of the labeling data could be better

understood if one knew whether or not the C14carbons were first oxidized to carbon dioxide and

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TABLE 2. Incorporation by Streptomyces venezuelaeof C14 into choloramphenicol from DL-nor-

leucine-2-CI4 and DL-leucine-U-CI4

Specific activity(juc/mmoles)

ProductNorleucine- Leucine-

2_C14* U_C14t

Chloramphenicol..... 1.09 2.70p-Nitrophenylserinol..... 0.00 0.56Dichloroacetic acid.1.13 2.11

Carbonyl carbon... 1.00 0.94

Dichloromethvl carbon.... 0.00 0.99

* Specific activity, 487 ,uc/mmole; 445 mmolesadded per 300 ml of media.

t Specific activity, 443 uc/mmole; 445 mmolesadded per 300 ml of media.

TABLE 3. Incorporation by Streptomyces venezuelaeof C14 into chloramphenicol from acetate-I-C 4

and acetate-2-C14

Specific activity(juc/mmole)

ProductAcetate- Acetate-I_C14* 2-Cl4t

Chloramphenicol... 1.86 37p-Nitrophenylserinol..... 0.04 23Nitrophenyl 15-CH20H.- 2-CHNH 2 3-CHOH. 3

Dichloroacetic acid. 1.78 14

Carbonyl carbon. 1.61 5

Dichloromethylene carbon. 0.02 8

* Specific activity, 71.3 Mc/mmole; 1.86 mmolesadded to 300 ml of media.

t Specific activity, 3,510,uc/mmole; 0.057 mmoleadded to 300 ml of phosphate buffer in a resting-cell experiment.

this compound then fixed into chloramphenicol.Metabolic carbon dioxide from the precursors

was recovered in the sodium hydroxide gas ab-sorption towers, isolated as barium carbonate,and the radioactivity measured. In each case,

metabolic carbon dioxide was formed from thelabeled carbons. The following are examples ofpercentages of total activity recovered in thecarbon dioxide: acetate-i-C'4, 66; acetate-2-C14,44; DL-phenylalanine-1-C14, 46; DL-phenylalanine-U-C'4, 68; L-leucine, 27; DL-norleucine-2-CI4, 36;sodium formate-C14, 92.Sodium bicarbonate-C'4 was used to determine

the capability of fixing this carbon dioxide di-

TABLE 4. Incorporation by Streptomyces venezuelaeof C14 from NaHCO3-C'4 and formate-C'4 into

chloramphenicol

Specific activity(uc/mmole)

ProductNaHCO3- Na formate-

C14* C14t

Chloramphenicol...... 22.20 4.17p-Nitrophenylserinol..... 0.01 0Dichloroacetic acid . 22.06 4.06Carbonyl carbon... . 21.09 4.04Dichloromethyl carbon...... 0.07 0

* Total of 500 ,uc was added to 300 ml of media.t Specific activity, 1,530 Ac/mmole; 0.131 mmole

was added to 300 ml of media.

rectly. It was added to the closed fermentationsystem to allow maximal time of contact withthe metabolizing organism. Data in Table 4 showthat carbon dioxide was readily fixed, but onlyas the carbonyl group of chloramphenicol.The specific activity of the atmospheric carbon

dioxide in the gas bulb decreased logarithmicallywith time until 72 hr, when the experiment wasterminated. At that time, the specific activityof the carbon dioxide in the gas bulb was equalto that of the carbonyl carbon of chloramphenicol(22 Auc/mmole).Formate-C14 incorporation was studied in an

open system to ascertain whether a one-carboncompound which was readily converted to carbondioxide could also be incorporated. Again, thelabeled carbon was fixed only in the carbonylof the antibiotic (Table 4).The addition of glycerol-1,3-C'4, the major

metabolized carbon source in the medium, re-sulted in an interesting pattern of labeling.Unlabeled glycerol was contained in the mediumfrom the start of the fermentation, but the labeledcompound was added after S. venezuelae hadgrown for 18 hr, so as to minimize the utilizationof labeled glycerol for general metabolic functionsthat occur before the chloramphenicol is syn-thesized. The results of two experiments areshown in Table 5. Both carbons of the dichloro-acetyl are labeled, but greater activity wascontained in the dichloromethyl carbon. Thelabeling pattern in the p-nitrophenylserinol sidechain resembled that in the glycerol which wasadded: there was equal specific activity in thetwo end carbons and only very slight labeling inthe middle carbon. Glycerol, in addition, con-

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TABLE 5. Incorporation by Streptomyces venezuelaeof glycerol-i,3-C'4 and lactate-U-C'4 into

chloramphenicol

Specific activity(uc/mmole)

ProductGlycerol- Lactate-1, 3-C14* U_C14t

Chloramphenicol. - 153p-Nitrobenzoic acid.65 - 0.21p-Nitrophenylserinol.. - 118C... 12 16.0 0.04C2. 0.6 0.9 0.04C3. 12 16.8

Dichloroacetic acid. - 33.6 0.23Carbonyl carbon.......... 10 13.7 0.10Dichloromethyl carbon. 13 18.4 0.13

* Radioactive glycerol (1.0 mmole; specificactivity, 57.8 ,c/mmole) was added 18 hr afterthe start of the fermentation.

tRadioactive lactate (0.2 mmole; specific ac-tivity, 6.76 ,uc/mmole) was added 24 hr after thestart of fermentation.

tributed carbons to the phenyl group even in thepresence of lactic acid, the other carbon source.

Lactic acid-U-C'4 also distributed its carbonwidely: heaviest labeling occurred in the dichloro-acetyl fraction of the molecule, and relativelyslight labeling in the p-nitrophenylserinol sidechain compared with that contributed by glycerol(Table 5). The specific activities of the phenylcarbons were generally in the same order as thosein the side chain, if one assumes an equal dis-tribution of labeling among the carbons in thering.Attempts to determine whether the phenyl

group of chloramphenicol arises via shikimic acidwere unsuccessful. When shikimic acid was addedto the culture medium, only small amounts dis-appeared and there was no increase in synthesisof the antibiotic. The lowest pH to which themedium could be adjusted without depressinggrowth of the streptomycete (pH 6) did notgreatly facilitate its absorption. Use of the methylester of shikimic acid also failed to aid in removalfrom the medium. The addition of radioactiveshikimic acid to the medium did not effect thebiosynthesis, for no radioactivity was presentin the antibiotic.

Despite many investigations on the mechanismof chlorination and repeated attempts to isolatesome organic halogen that might be an intermedi-

ate in this process, no important progress wasmade. The use of radioactive chlorine as sodiumchloride in the medium resulted in the productionof trace amounts of organic halogen, as shown byradiographs of paper chromatograms from ex-tracts of the culture fluid. Attempts to producelarger quantities for characterization were notsuccessful.The origin of the nitro group was studied by

seeking to isolate organic nitro compounds otherthan chloramphenicol and by ascertainingwhether arylamines were oxidized to arylnitrocompounds. No organic nitro compounds otherthan chloramphenicol were found in normalcultures. X-ray or ultraviolet mutants that pro-duced no antibiotic also did not accumulate nitrocompounds in the medium. At least three arylam-ines were found in small amounts; one of thesehad been identified by paper chromatography asp-aminobenzoic acid.

DISCUSSION

Despite the general similarity in the structureof phenylalanine and the p-nitrophenylserinolportion of chloramphenicol, the stimulatoryeffect of L-phenylalanine on the biosynthesis ofthe antibiotic is not due to the incorporation ofphenylalanine skeleton into the p-nitrophenylseri-nol fraction of the antibiotic. Even with DL-phenylalanine-U-C'4, no labeling was contained inthis portion of the chloramphenicol molecule.Data from phenylalanine labeled as -1-C14, -2-C14,and -3-C'4 indicate that the major contributionof the amino acid is to the dichloroacetyl portionof the antibiotic. The first two carbons of thephenylalanine side chain were transformed onlyinto the carbonyl carbon of chloramphenicol. Itis reasonable to postulate that both the carbonsare oxidized to carbon dioxide and that thiscompound in turn is incorporated during theformation of the dichloroacetyl moiety. Datafrom incorporation of labeled carbon dioxideshow that this compound is very readily fixedand appears only in the carbonyl carbon. Atthe end of 62 hr, the specific activity of the labeledcarbon of chloramphenicol is in equilibrium withthat of the carbon dioxide in the culture flask.The data from the formate experiment alsosupport the concept of fixation of carbon dioxide.Carbon 3 of phenylalanine contributes pri-

marily to the dichloromethyl carbon of the di-chloroacetyl portion of chloramphenicol. Its

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labeling is eight times greater than the carbonylcarbon. Thus, carbon 3 is probably incorporatedtogether with its adjacent carbon from the ringwhich forms the carbonyl carbon. That someoxidation of carbon 3 to carbon dioxide andfixation could occur is indicated by the minorlabeling in the carbonyl carbon. Further evidencethat the acetyl carbons can be derived as a unitis the fact that the carbons of the dichloroacetylderived from phenylalanine-U-C14 have aboutthe same specific activity. From the specificaetivities of the phenylalanine-3-C'4 and -U-C14,one would expect the specific activity in thedichloromethyl carbon to be two times as greatwhen derived from the uniformly labeled aminoacid as from the singly labeled one. It was threetimes as great; but since the experiments werenot carried out at the same time, the results arenot strictly comparable.

Another stimulatory compound, leucine, alsocontributes carbons to the dichloroacetyl moiety.Since both these carbons when derived fromDL-leucine-U-C'4 have equal specific activities,they probably are derived as a unit. Some of itscarbons also are found in the p-nitrophenylserinolportion of chloramphenicol.The general metabolite, acetate, contributed its

methyl carbon throughout the molecule, withgreatest activity in the dichloromethylene carbon.Its carboxyl carbon was found only in the car-bonyl carbon of chloramphenicol. The contribu-tion of those amino acids which stimulate thebiosynthesis of the antibiotic is to the dichloro-acetyl portion of chloramphenicol. Acetic acid,which does not stimulate the synthesis of chlor-amphenicol, also contributes its carbons in asimilar manner. Thus, the mechanism of stimula-tion by the amino acid cannot be attributed totheir effect on the formation of the carbon skele-ton of the dichloroacetyl portion of the antibiotic.The role of glycerol in stimulating the syn-

thesis of chloramphenicol is more apparent. Itis a precursor for the serinol portion of chloram-phenicol. The labeling pattern in the serinolcarbons was the same as that in the glycerolwhich was added to the medium. Exogenousglycerol was labeled only in the 1 and 3 positions.The serinol portion of the antibiotic also hadcarbons 1 and 3 labeled with a high and equalspecific activity, whereas carbon 2 had only rela-tively slight labeling. The slight labeling of carbon

2 is probably a reflection of some dissimilation ofglycerol to two-carbon compounds with a smallfraction of the side chain arising from them. Thus,it is probable that the serinol carbons are incor-porated into the antibiotic as a three-carbon unit.At present it is not known whether glycerol isincorporated directly or is first metabolized tosome other three-carbon intermediate. Levinand Sprinson (1962) have shown that the three-carbon compound, phosphoenolpyruvate, can bea direct precursor of the side chain of phenylala-nine. Thus, a model exists for the attachment ofa three-carbon chain to a phenyl group. Glycerolis the major carbon source in the medium andis rapidly metabolized, while only very smallamounts of lactic acid are used (Gottlieb andLegator, 1953). It contributes carbons to allportions of the molecule; labeling is found in thenitrophenyl, serinol, and dichloroacetyl parts ofchloramphenicol. The labeling pattern in thedichloroacetyl moiety resembles that frommethyl-labeled acetic acid. From glycerol, S.venezuelae can form acetic acid which has beenidentified as the hydroxamate (unpublished data).Thus, one of the terminal carbons of glycerolwould give rise to the methyl carbon of an acetylgroup and the resultant labeling pattern wouldbe similar to the acetate.

ACKNOWLEDGMENT

This investigation was supported in part byresearch grant E-618 from the National Institutesof Health, U.S. Public Health Service.

LITERATURE CITED

BARTZ, Q. R. 1948. Isolation and characterizationof Chloromycetin. J. Biol. Chem. 172:445-450.

EMERSON, R. L., A. J. WHIFFEN, N. BOHONAS,AND C. DEBOER. 1946. Studies on the produc-tion of antibiotics by actinomycetes andmolds. J. Bacteriol. 52:357-360.

GOTTLIEB, D., H. E. CARTER, M. LEGATOR, ANDV. GALLICCHIO. 1954. The biosynthesis ofchloramphenicol. I. Precursors stimulatingthe synthesis. J. Bacteriol. 68:243-251.

GOTTLIEB, D., AND L. DIAMOND. 1951. A syntheticmedium for Chloromycetin. Bull. TorreyBotan. Club 78:56-60.

GOTTLIEB, D., AND M. LEGATOR. 1953. The growthand metabolic behavior of Streptomycesvenezuelae in liquid culture. Mycologia 45:507-515.

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GOTTLIEB, D., P. W. ROBBINS, AND H. E. CARTER.1956. The biosynthesis of chloramphenicol.II. Acetylation of p-nitrophenylserinol. J.Bacteriol. 68:153-156.

LEVIN, J. G., AND D. B. SPRINSON. 1962. Theenzymatic formation and isolation of 3-enol-pyruvylshikimate-5-phosphate (ESP). Fed-eration Proc. 21:88.

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OYAAS, J. E., J. EHRLICH, AND R. M. SMITH. 1950.Chemical changes during chloramphenicol(Chloromycetin) fermentation. Ind. Eng.Chem. 42: 1775-1776.

REBSTOCK, M. C., H. M. CROOKS, JR., J. CON-TROULMS, AND Q. R. BARTZ. 1949. Chloram-phenicol (Chloromycetin). IV. Chemicalstudies. J. Am. Chem. Soc. 71:2458-2462.

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