CHLORAMPHENICOLMODEOFACTION OF CHLORAMPHENICOL reactions of this general type, such as those catalyzed by the enzymes xanthine oxidase, amino acid oxidase, and cytochrome oxidase,

MODE OF ACTION OF CHLORAMPHENICOL

IIL ACTON OF CHLORAMPHENICOL ON BACTERIAL ENERGY METABOLISM

F. E. HAHN, C. L. WISSEMAN, JR., AND H. E. HOPPSDepartment of Virw and Rickett8ial Diseases, Army Medical Service Graduate School, Washington, D. C.

Received for publication August 9, 1954

The inhibition of protein synthesis in sensitivemicroorganisms (Wisseman et al., 1953, 1954;Gale and Folkes, 1953b) stands out as oneof the most striking effects of chloramphenicolon bacterial metabolism. It occurs at minimalgrowth inhibitory concentrations of the antibioticand constitutes an action of the drug on ametabolic process of sufficient importance tocellular economy to place it in the foregroundas a most likely explanation for the principalmechanism of antibiotic action of chlorampheni-col. Indeed, the action on protein synthesisappeas highly specific since no other majorsynthetic process tested seems to be affectedto the same degree. Those already examinedinclude nucleic acid synthesis (Wiseman et al.,1953, 1954; Gale and Folkes, 1953b), poly-saccharide synthesis (Hopps et al., 1954), andD(-) glutamyl polypeptide synthesis (Hahnet al., 1954), all processe tested in this laboratoryin accordance with the systematic metabolicgroup analysis outlined in a previous publication(Wisseman et al., 1954).While the phenomenon of an action on protein

synthesis is clearly established, the exact mech-anism by which chloramphenicol exerts thiseffect has not yet been explained. There isindirect evidence that it may be through aspecific interference with some phases of the finalassemblage of building blocks into the finishedproteins (Wiseman et al., 1954; Hahn et al.,1954). However, the possibility still remains,as has been discussed previously (Wissemanet al., 1954), that an action on an essentialcontributory process might lead to inhibition ofprotein synthesis as a secondary phenomenon.Since protein synthesis is an endergonic reaction,interference by the antibiotic with "energymetabolism" could result in inhibition of proteinsynthesis.

Despite the growing body of evidence that aphosphate cycle involving adenosine triphosphate

(ATP) is in some way asociated with thesynthesis of proteins (Chantrenne, 1953; Galeand Folkes, 1953a), the exact manner by which itparticipates in the asembly of amino acids intoproteins is unknown and, hence, this particularsegment of the pathway of energy flow in proteinsynthesis is not currently amenable to directexamination for chloramphenicol action. How-ever, some segments of the energy flow pathwayantecedent to this, namely, those which con-tribute to and culminate in the formation ofATP, can be tested for possible action of theantibiotic. Furthermore, widely divergent proc-esse which depend upon utilization of metabolicenergy can be examined for possible interferenceby the drug. This last named indirect approach,though not neessarily precisely representativeof the advanced stages of protein synthesis,might uncover a general interference by chlor-amphenicol with utilization of metabolic energy.In this project, the following three differentexamples of bacterial energy metabolism wereslected for systematic study with chloram-phenicol to supplement the existing information(Smith, 1953) in this important category of cellfunction: (1) the luminescence of Achromobacterfiwcheri which presumably depends on a system ofelectron transport; (2) the adenylic acid-phos-phate cycle in the glycolytic system of Escherichiacoli, strain B, which appears to be a representativeexample of the transformation of energy throughthe synthesis of energy-rich phosphate bonds;and (3) the motility of E. coli which presumablyreflects the conversion of metabolic energy tomechanical energy.The current studies, of which represetative

examples are included in this report, indicatethat none of these processes is influenced signifi-cantly by chloramphenicol even in concentrationswhich are 50 to 100 times higher than theminimal bacteriostatic levels for the testorganisms.

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F. E. HAHN, C. L. WISSEMAN, JR., AND H. E. HOPPS

I. Action of Chloramphenicol on the Luminescenof Achromobacter fischeri

Bacterial luminescence, long known to bedependent upon atmospheric oxygen, hasrecently been demonstrated in a cell-free systemfrom A. fi&cheri to require coenzyme I andflavin mononucleotide for optimal light produc-tion (Strehler, 1953; Strehler and Cormier,1953). Although the details are still unknown,it is likely that the light producing reaction isassociated with the transfer of electrons fromthese cofactors to oxygen, probably through aspecial alternate carrier system (McElroy andKipnis, 1947) which differs from the cytochromesystem of the main respiratory pathway by itsinsensitivity to cyanide (Harvey, 1952).

MATERIALS AND METHODS

A. fischeri, strain 7744, obtained from theAmerican Type Culture Collection, was main-tained at 23 C in the basal medium of Farghaly(1950) fortified with 0.2 per cent peptone. Forpreparation of working suspensions, nutrientagar plates (Difco) containing 0.3 per centglycerol and 3.0 per cent NaCl were inoculatedwith 0.5 ml of an 18 hour liquid culture andincubated overnight at 23 C. The surface growthfrom each plate was suspended in 10 ml of 3 percent NaCl, washed, and resuspended in 7.5 ml ofthe sodium chloride solution. After aeration for2 hours at room temperature these stock suspen-sions were used without further delay for theexperiments on bioluminescence.

Luminescence was measured quantitatively in

1.6

1.4

3usz

1.2

U

.oI0 0

2<0.It-'I .80 NO CHLORAMPHENICOL

CHLORAPHENICOL (1OO,>g./mL)

20 40 so soTIME IN MINUTES

Figure 1. Failure of chloramphe:bacterial luminescence.

arbitrary units with a special photometer whichemployed a photomultiplier tube arrangementresembling that described by McElroy andKipnis (1947) and which accommodated roundcuvettes 19 mm in diameter.The action of chloramphenicol on bacterial

luminescence was studied in quintuplieatesamples of 5 ml volume containing 100 pg perml of the antibiotic, 3.0 per cent NaCl, m/75phosphate at pH 7.4, m/200 glucose, 0.2 percent peptone, and usually 2 ml of a 1:500dilution of the luminous bacterial stock suspen-sion. A control experiment was carried outsimultaneously without added chloramphenicol.The working concentration of bacteria wasadjusted in each individual experiment to alevel which emitted light of an intensity con-venient for measurement in the photometer.The experimental tubes were aerated by continu-ous mechanical agitation which was interruptedonly for the periodic measurement of lumi-nescence at the indicated intervals.The sensitivity of A. fiwheri towards chlor-

amphenicol was determined by inoculating thebasal medium of Farghaly (1950) containingserial dilutions of the drug with the organismand examining bacterial growth visually after24 hours' incubation at 23 C. The minimalbacteriostatic level of chloramphenicol waM1.5jug pe il.

RESULTS

Figure 1 shows the results of a typical experi-ment; in all instances bacterial luminescenceremained practically constant for the first 45 to60 minutes of the experiments and then beganto decline in an exponential manner. One hundredjug per ml of chloramphenicol did not influencesignificantly either the initial intensity ofbacterial luminescence or the rate of its decay.

DISCUSSION

Since chloramphenicol is without influence onN the eion of light by suspensions of A. fischeri

which presumably depends on the transfer ofelectrons from flavin mononucleotide to oxygenthrough a special carrier, it may be inferred that

160 the antibiotic does not interfere with this electron0 60 ISO transport mechanism or with the series of

reactions by which the flavin mononucleotide isnicol to inhibit reduced. The present findings corroborate and

extend the observations reported by others that

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reactions of this general type, such as thosecatalyzed by the enzymes xanthine oxidase,amino acid oxidase, and cytochrome oxidase,are unaffected by chloramphenicol (Smith, 1953).Finally, many bacterial oxidations which requirethe participation of complex sequences of redoxreactions are not influenced by the antibiotic;hence, in all probability, this general categoryof reactions may be dismised as the site of thegrowth inhibiting action of chloramphenicol.

II. Action of Chloramphenicol on Phosphorylationin E. coli

The molecule of chloramphenicol contains a

p-nitrophenyl group, and the action of the drugon certain cellular processes involving proteinformation shows a striking similarity to that ofanother aromatic nitro compound, viz., 2,4-dinitrophenol (DNP). Thus, incorporation ofindividual amino acids into the proteins ofE. coli (Wisseman et al., 1954; Melchior et al.,

1951) and of complex mixtures of amino acidsinto the proteins of staphylococci (Gale andFolkes, 1953b; Hotchkiss, 1947), formation ofadaptive enzymes (Hahn and Wisseman, 1951;Monod, 1944), and multiplication of bacterio-phages (Bozeman et al., 1954; Spizizen, 1943)are inhibited by both substances alike.The remarkable parallelism in the biologica

action of DNP and chloramphenicol suggestedthat chloramphenicol, like DNP (Hotchkis,1946; Loomis and Lipmann, 1948), might act on

phosphorylation. Energy-rich phosphate bondspresumably provide the energy to "drive"protein synthesis (Lipn, 1941; Borsook,1950; Street, 1950; Chantrenne, 1953). Since thenegative finding of Loomis (1950) dealt with thefailure of chloramphenicol to act on a phos-phorylating system in mitochondria, which were

derived from animal cells not generally regardedas susceptible to the action of the antibiotic(L6pine et al., 1950; LePage, 1953), it wasconsidered esential to reinvestigate the action ofchloramphenicol on a phosphorylating systemfrom a microorganism whose growth is stronglyinhibited by the antibiotic.A glucose metabolizing stem from E. coli,

strain B, was chosen for this study since there isevidence that E. coli may metabolize glucoseat least in part according to the Embden-Meyerhof scheme (Elsden, 1952). It was foundthat neither the turnover of radioactive phos-

phate by respiring whole celLs of E. coli nor theesterification of inorganic phosphate by glycolyticenzymes in cell-free bacterial extracts of the sameorganism was influenced significantly bychlofamphenicol.

RIAIE3EL AN METHODS

Organism. E. coli, strain B, grown withconstant shaking in brain-heart infusion broth(Difoo) overnight at room temperature, washarvested and washed with distilled water. Foruse as whole cells in the phosphorus P"-turnoverexperiments, the orgAnis were resuspended in0.85 per cent NaCl solution and aerated for2 hours at room temperature.

Preparation of bactl 8onic extracts. Cell-freeenzyme extracts were prepared by suspendingthe bacteria in 0.1 M potassium bicarbonate toform a thick paste, treating the suspensions for45 minutes in a magnetostriction oscillator(Raytheon, Model S-102A), and centrifugingthe sonic treated supensions at 14,000 X Gfor 30 minutes. The clear yellowish and viscoussupernatants were used for the experiments onesterification of inorganic phosphate and re-mained active for several months if stored at-20 C. Extracts containig 7 mg of totalnitrogen per ml exhibited a high degree ofphosphorylating activity.

Total nitrogen was determined with a modifica-tion of Johnson's procedure (1941) whichemploys sulfuric acid-hydrogen peroxide digestionfollowed by neslerization.

Determination of P2 turnover. The turnover ofinorganic phosphate by respiring whole cells ofE. coli was studied with the aid of exogenousinorganic phosphate labeled with p32. Theexperiments were carried out in series of con-ventional Warburg vessls, each containing in3 ml of bacterial suspensions diluted 1:2, M/900glucose and M/140 phosphate at pH 7.4, labeledwith 7.5 ,uc of PF. Two series of experimentswere carried out simultaneously: (1) a controlseries without added chloramphenicol and (2) aseries containing 100 jAg of chloramphenicolper ml of experimental mixture. Each experimentextended over a total period of 1 hour duringwhich time one flask from each series wasremoved every fifteen minutes for analysis.

Glucose was determined in 0.5 ml aliquotsof the flask contents by the method of Folinand Malmros (1929). To the remni ing 2.5

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ml portions of experimental mixture was addedenough strong formalin to make them 3 percent with respect to formaldehyde. The bacteriawere separated and washed twice by centri-fugation at 10,000 X G for 30 minutes andfinally suspended in 1 ml of water. Labaw et al.(1950) have shown that samples thus treatedare remarkably free of adsorbed radioactivity.Aliquots of 0.1 ml of the suspensions werespread evenly to dry on copper planchets andthen counted in a mica end-window Geigercounter (Tracerlab) with a window thicknesscorresponding to a weight of 3 to 4 mg per cm2.

Determination of phosphate esterification bycell-free extracts. Phosphorylation by sonicextracts from E. coli was studied in conventionalWarburg vessels, each containing 3 ml of 1:2diluted bacterial extract with the followingchemical additions: 25 $M of potassium ortho-phosphate, 50 juM of glucose, 50 jkm of potassiumfluoride, 1.5 mg of coenzyme I, and 1.2 mg ofATP. The pH was adjusted to 7.4 with potassiumhydroxide.The exclusive use of potassium salts was

suggested by Utter's findings (1950) of aninhibition of glycolysis by sodium ions. Two seriesof experiments were carried out: (1) a controlwithout added chloramphenicol and (2) experi-ments with 100 Mg per ml of chloramphenicoladded.Experiments were run for 1 hour at 34 C with

air as the gaseous phase. The contents of eachvessel were then poured into 2 ml of ice cold 17.5per cent trichloracetic acid (TCA); the mixtureswere chilled, centrifuged, and free inorganicphosphate was determined in 0.25 ml aliquots ofthe supernatants by the method of Fiske andSubbarow (1925). The difference between thecontent of free phosphate in zero time controlsand in the experimental mixtures was consideredas phosphate esterified during the experiments.Paper chromatography of phosphate esters.

Samples for the paper chromatography ofphosphorylated compounds were prepared in thefollowing manner: Finely powdered bariumhydroxide was added to the TCA supernatantsfrom the Warburg experiments to bring the pH to8.2 (distinctly pink with phenolphthalein); afteraddition of 25 ml of absolute alcohol to each 5 mlsample, the mixtures were refrigerated at -20 Cfor several hours. The precipitates were collectedby centrifugation and dissolved in 2 ml of 0.1 N

HCI; 0.55 ml of 2 N H2S04 was then added toprecipitate the barium ions. After centrifugationthe supernatants were saved, the barium sulfatesediments were washed with 2 ml of water, andsupematants and washings were combined andmade up to 5 ml.

Aliquots of 0.6 ml of these samples werechromatographed on Whatman no. 1 filter paperby the method of Bandurski and Axelrod (1951)using a methanol-formic acid mixture as themobile phase. The developed and dried chromato-grams were cut into serial consecutive 1 cm widestrips which then were eluted with 3 ml of4 N H2S04. Free inorganic and total phosphoruswere determined in 1 ml aliquots of each of theeluates by the procedure of Fiske and Subbarow(1925) which for total phosphorus determinationwas preceded by evaporating the water from thesamples and heating the acidic residues withhydrogen peroxide in order to digest organicmatter. Esterified phosphorus present in theeluates was calculated from the differencebetween total and free phosphorus.

Detemination of phosphoglyceic acids. Samplesfor the determination of phosphoglyceric acids bythe polarimetric method of Meyerhof and Schulz(1938) were prepared in the same manner as forpaper chromatography, and the specific rotationdata of -650° to -670° for the equilibriummixture of 3-phosphoglyceric and 2-phospho-glyceric acids were used since Utter and Werkman(1942) have shown that cell-free extracts from E.coli establish the same equilibrium between thetwo isomers as muscle or yeast.

Determination of individual glycolytic enzyme8 insonic extracts from E. coli. For the followingstudies the sonic extracts from E. coli weredialyzed exhaustively against distilled water.

Hexokinase was demonstrated spectrophoto-metrically by an adaptation of Kornberg'smethod (1950) for the determination of ATP; thebacterial extract was substituted for the hexoki-nase fraction, and an excess of ATP was supplied,thereby making the bacterial hexokinase the ratelimiting factor. The method of Somers and Cosby(1945) was used to show the presence of phospho-hexose isomerase in the enzyme extracts; that ofDounce and Beyer (1948) for aldolase; and amodification of the same procedure for phospho-fructokinase in which glucose-6-phosphate andATP were substituted for fructose-1,6-diphos-phate as substrate. With phosphohexose isomerase

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and aldolase determined previously andindividually the kinase became the limitingfactor in the formation of phosphotriose.A spectrophotometric application of the

Thunberg technique (Tam and Wilson, 1941) wasused for the determination of phosphotriosedehydrogenase (Still, 1940) and glycerophosphatedehydrogenase. The sodium glycerophosphate(Fisher Scientific Company, no. 644) contained52 per cent a-glycerophosphate.

- T WITHOUT CHLORAMPHENICOL AND WCOSE------- WITHOUT CHLORAWHNICOL, WITH LUCOK

WITH AND GLUCOE

3IL

5 OXYGEN CONSUMPTIO

0f ..o/

fI- I I

RESULTS

Phosphate turnover during aerobic dissimilaionof glucose by E. coli. Glucose dissimilation bynonproliferating suspensions of E. coli proceedsin the presence of low orthophosphate con-centrations without a detectable change in theconcentration of free phosphate in the medium(Aubel and Szulmajster, 1950). However, P39from labeled orthophosphate in the medium wastaken up rapidly by cells of E. coli duringglucose dissimilation in current experiments,indicating an exchange of intracellular phosphatewith that of the medium. This ceased whenall the glucose had disappeared from the experi-mental mixtures although oxygen consumptioncontinued at a low rate. In control experimentswithout added glucose, oxygen consumptionwas small, and the uptake of P'2 by the bacteriarose only to a level of about 30 per cent ofthat attained by cells which actively metabolizedglucose. The presence of 100 jg per ml of chlor-amphenicol in the experimental mixtures hadno significant influence on the consumptionof oxygen, the disappearance of glucose, oron the uptake of phosphorus P'2 by the bacteriaas illustrated by the typical data presentedin figure 2.

Esterification of phosphate and formationof phosphoglyceric acid during glucose dissimilationby ceU-free extracts from E. coli, strain B. Whenglucose was disimilated aerobically by sonicextracts from E. coli, strain B, in the presenceof orthophosphate and fluoride ions, free phos-phate disappeared and equivalent amounts ofphosphoglyceric acid accumulated. No netphosphorylation could be demonstrated underanaerobic conditions.

Analyses for free phosphate and total phos-phorus in eluates from series of consecutive stripsfrom the chromatograms revealed the presenceof a substance containing bound phosphorus

S

I

hi

I,

4

aKw

GLUCOSE DUWSAPPAWANC

WIUTES

Figure B. Failure of chloramphenicol to influenceP32 uptake in Escherichia coli.

which was not detectable by the acid molybdatespray reagent of Bandurski and Axelrod (1951).The resistance to acid hydrolysis, the chromato-graphic behavior, and the characteristic andmarked change in optical rotation upon additionof ammonium molybdate (Meyerhof and Schulz,1938) served to identify the compound asphosphoglyceric acid. No significant quantities ofphosphorylated compounds other than phospho-glyceric acid were detected by the chromato-graphic methods employed.

Chloramphenicol, 100 ,ug per ml, failed toinfluence significantly the esterification ofinorganic phosphate or the consumption ofoxygen. Typical analytical data are presentedin table 1.

Demonstration of individual glycolytic enzymesin cell-free bacterial extracts. The followingenzymes were demonstrated to be present in thedialyzed sonic extracts from E. coli employed inthese studies: hexokinase, phosphohexose iso-merase, phosphofructokinase, aldolase, andphosphotriose dehydrogenase. A glycerophos-

30-

Pn UPTAKE15-

............0.............

I a I I

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TABLE 1

Failure of chloramphenicol to influencephosphorylation by cell-free extracts of

Escherichia coli, strain B

PsM Ortho- O

Experimental Mixtures opted OxygenEstenified Consumedafter 1 hr after 1 hr

Without glucose and chloram-phenicol.5.3 16.5

With glucose and without chlor-amphenicol.22.9 27.9

With glucose and with chlor-amphenicol.23.9 25.1

phate dehydrogenase was also detected in theenzyme extracts by the Thunberg technique;additional coenzyme I did not increase itsactivity.These enzyme studies were carried out in

order to establish the presence of enzymes of theglycolytic system in the experimental mixture.

DISCUSSION

Chloramphenicol does not influence signifi-cantly the turnover of phosphate in whole cellsof E. coli, strain B, and it does not inhibit theesterification of phosphate due to the operationof a part of the glycolytic system in cell-free sonicextracts from the same organism.

Juni et al. (1948) have shown that the turnoverof phosphate in nonproliferating yeast cells,which disimilate glucose in a nitrogen freemedium, can be studied by determining theincorporation of P'2-labeled exogenous phos-phate into the organis. The same methodappears to be applicable to the study of phosphateturnover in cells of E. coli, strain B. The incor-poration of phosphorus P'2 into the bacteriawas not influenced by 100 ,ug per ml of chlor-amphenicol.On the basis of the individual enzymes that

have been demonstrated to be present, phos-phorylation by cell-free extracts from E. coliin the present experiments appears to proceedthrough a part of the Embden-Meyerhof sequenceof reactions; a metabolic block introduced bythe inhibition of enolase by fluoride causes an

accumulation of phosphoglyceric acid equivalentto the amounts of orthophosphate which dis-appear from the medium.

III. Adion of Chloramphenicol on the Motility ofE. coli

The motility of bacteria presumably providesan instance in which the energy derived frommetabolic procs is ultimately converted intomechanical energy. The effect of chloramphenicolon the motility of a sensitive bacterium wasinvestigated to supplement the other studies onbacterial energy metabolism.

MAIS AND METHODS

An actively motile culture of Echerichia coli,strain 67 L-15, obtained from the BacteriologyDepartment of the University of Maryland wasgrown in Difoo brain-heart infusion broth for4.5 hours at 35 C; the culture at this time wasextremely motile and growing rapidly. Nine mlaliquots of the bacterial s on were addedto sterile cuvettes containing 1.0 ml of chlor-amphenicol and 2,4-dinitrophenol solution,respectively, 10 x the final desired concentration.Viable counts were made by the conventionalplate count technique immediately after mixingand at the end of each experiment. Motility wasobserved by the haging drop method andrecorded at 30 minute intervals.

TABLE 2Effect of chloramphenicol and dinitrophenol

on motility and viable cell count ofEscherichia coli, strain 67 L-15

Compounds AddedTime after Addition

of Compounds None Chloram Dinitro-(control) phenicol phenol(100pg/ml) (1 X 10 x)

0 Viable cell 3.0 X 108 3.6 X 10' 1.8 X 108count

Motility* 4+ 4+ 4+

30 Motility* 4+ 4+ 4+

60 Motility' 4+ 4+ 2+

90 Motility* 4+ 4+ 2+

120 Motility' 4+ 4+ 1+Viable cell 1.1 X 10' 2.2 X 10' 2.8 X 108count

* Motility was recorded in units of 0 to 4+ bycomparison with the control which was arbitrarilyassigned the value of 4+.

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RBSULTS

Table 2 presents the observations made in a

typical experiment. Here, tubes containingadded chloramphenicol or 2,4-dinitrophenol are

compared with a control. The control cultureremained actively motile and grew over the twohour observation period. Chloramphenicol,however, at a concentration 50 X the minimalgrowth inhibitory level had no detectableeffect on motility even though growth was

completely inhibited. In contrast, dinitrophenolproduced a progressive decrease in motilitywhich was noticeable after about 30 minutes.This occurred without a decrease in the numberof viable cells.

DISCUSSION

Chloramphenicol does not interfere withbacterial motility at concentrations whichexert a strong bacteriostatic effect. In contrast,2,4-dinitrophenol causes a progressive reductionin motility even though the organisms remainviable. The metabolic req ents for motilityin bacteria are not wel known. However, on thebasis of theoretical calculations, Morowitz (1954)suggested that the energy required for motilitywas of an order of magnitude which could besupplied by reasonable quantities of ATP. Theaction of 2 ,4-dinitrophenol on bacterial motilityas well as ciliary action in animal cells (Seamanand Houlihan, 1951; Werstedt, 1944) is com-patible with such an hypothesis even though itdoes not necessarily constitute proof for theparticipation of a phosphate cycle.The current experiments were restricted to

short term observations in order to avoid as

much as posible the operation of the unknownfactors which influence motility, even in theordinary growth cycle, and to avoid error

introduced by any lethal actionofthecomponents.

GENERAL DISCSSION

None of the three examples of bacterialenergy metabolism investigated in the presentstudies is influenced significantly by concentra-tions of chloramphenicol up to 100 times higherthan the minimal bacteriostatic levels for thetest organisms.While the exact mechanism of energy supply

for protein biosynthesis remains unknown, some

model reactions involving amide bond synthesis,e.g., formation of acetylsulfonamide, hippuric

acid, glutamine, and glutathione, require ATPas a source of energy (Chantrenne, 1953).Furthermore, net protein synthesis in a cell-freesystem of bacterial origin (Gale and Folkes,1953a) utilized ATP and hexose diphosphateas energy source.The idea is widely accepted that protein

synthesis utilizes phosphate energy which inturn is generated by electron transport involvedin oxidations and stored by phosphorylation.Examples of these forms of energy metabolismand of the utilization of metabolic energy havebeen investigated in the present study. It is theopinion of the authors that failure of chlor-amphenicol to influence energy metabolism in thepresent examples strongly suggests that the drugdoes not inhibit protein biosynthesis by inter-fering with the generation of the necesarysupply of metabolic energy and probably doesnot do so by a general inhibitory action on theutilization of such energy.

Three different forms of bacterial energymetabolism have been studied for possibleinterference by chloramphenicol. These included(1) the bioluminesence of Achromobacter fiwcherias an example of electron transport, (2) phos-phorylation involved in the diimilation ofglucose in Escherichia coli, strain B, as a keyprocess of synthesis of high-energy phosphatebonds and their utilization in glucose metabolism,and (3) the motility of E. coli as a special exampleof the utilization of metabolic energy.None of these examples of energy metabolism

is influenced significantly by chloramphenicol.The implications of these findings as regards themode of action of chloramphenicol have beendiscussed.

REFERENCESAuBUL, E., AND SZULMAJ1STER, J. 1950 Contri-

bution a l'6tude de la fermentation et de larespiration de Escherichia coli. III. R6le duphosphore dans le m6tabolisme de E. coli.Action de quelques inhibiteurs sur la phos-phorylation et la dEphosphorylation desoses. Biochim. et Biophys. Acta, 5, 499-514.

BANDURSKI, R. S., AND AXELROD, B. 1951 Thechromatographic identification of some bio-logically important phosphate esters. J.Biol. Chem., 198, 405-410.

BoRsoox, H. 1950 Protein turnover and in-corporation of labeled amino acids into tissue

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proteins in vivo and in vitro. Physiol. Revs.,30, 206-219.

BOZEMAN, F. M., WISSEMAN, C. L., JR., Hopps,H. E., AND DANAUSKAS, J. X. 1954 Actionof chloramphenicol on T-1 bacteriophage.I. Inhibition of intracellular multiplication.J. Bacteriol., 67, 530-536.

CHANTRENNE, H. 1953 Problems of proteinsynthesis. Second Symposium of the Societyfor General Microbiology, Cambridge Uni-versity Press.

DOUNCE, A. L., AND BEYER, G. T. 1948 Anew method for the determination of theenzyme aldolase. J. Biol. Chem., 173,159-173.

ELSDEN, S. R. 1952 Bacterial fermentation.In The enzymes. Vol. II, part 2, pp. 791-843.Edited by Sumner, J. B., and Myrback, K.Academic Press Inc., New York, N. Y.

FARGHALY, A.-H. 1950 Factors influencing thegrowth and light production of luminousbacteria. J. Cellular Comp. Physiol., 36,165-184.

FISKE, C. H., AND SUBBAROW, Y. 1925 Thecolorimetric determination of phosphorus.J. Biol. Chem., 66,375-400.

FOLIN, O., AND MALMROS, H. 1929 An improvedform of Folin's micro-chemical method forblood sugar determinations. J. Biol. Chem.,83, 115-120.

GALE, E. F., AND FOLKES, J. P. 1953a Aminoacid incorporation by fragmented staphy-lococcal cells. Biochem. J. (London), 55, XI.

GALE, E. F., AND FOLKES, J. P. 1953b Theassimilation of amino-acids by bacteria. 15.Actions of antibiotics on nucleic acid andprotein synthesis in Staphylococcus aureus.Biochem. J. (London), 53, 493-498.

HAHN, F. E., AND WISSEMAN, C. L., JR. 1951Inhibition of adaptive enzyme formation byantimicrobial agents. Proc. Soc. Exptl.Biol. Med., 76, 533-535.

HAHN, F. E., WISSEMAN, C. L., JR., AND Hopps,H. E. 1954 Mode of action of chlorampheni-col. II. Inhibition of bacterial D-polypeptideformation by an L-stereoisomer of chloram-phenicol. J. Bacteriol., 67, 674-679.

HARVEY, E. N. 1952 Bioluminescence. Aca-demic Press Inc., New York, N. Y.

Hopps, H. E., WISSEMAN, C. L., JR., AND HAHN,F. E. 1954 Mode of action of chlorampheni-col. V. Effect of chloramphenicol on poly-saccharide synthesis by Neisseria perfiava.Antibiotics & Chemotherapy, 4, 857-58.

HOTCHKISS, R. D. 1946 Gramicidin, tyrocidine,and tyrothricin. Advances in Enzymol.,4, 153-199.

HOTCHKISS, R. D. 1947 The assimilation of

amino acids by respiring washed staphylo-cocci. Federation Proc., 6, 263.

JOHNSON, M. J. 1941 Isolation and propertiesof a pure yeast polypeptidase. J. Biol.Chem., 137, 575-586.

JUNI, E., KAMEN, M. D., REINER, J. M., ANDSPIEGELMAN, S. 1948 Turnover and distri-bution of phosphate compounds in yeastmetabolism. Arch. Biochem., 18, 387-408.

KORNBERG, A. 1950 Reversible enzymatic syn-thesis of diphosphopyridine nucleotide andinorganic pyrophosphate. J. Biol. Chem.,182, 779-793.

LABAW, L. W., MOSLEY, V. M., AND WYCKOFF,R. W. G. 1950 Radioactive studies of thephosphorus metabolism of Escherichia coli.J. Bacteriol., 59, 251-262.

LEPAGE, G. A. 1953 Effects of chloramphenicolon incorporation of glycine-2-C14 into mam-malian tumor cell proteins and purines.Proc. Soc. Exptl. Biol. Med., 83, 724-726.

LP.PINE, P., BARSKI, G., AND MAURIN, J. 1950Action of chloromycetin and of aureomycinon normal tissue cultures. Proc. Soc. Exptl.Biol. Med., 73, 252-255.

LIPMANN, F. 1941 Metabolic generation andutilization of phosphate bond energy. Ad-vances in Enzymol., 1, 99-162.

LooMIs, W. F. 1950 On the mechanism ofaction of aureomycin. Science, 111, 474.

LooMIs, W. F., AND LIPMANN, F. 1948 Re-versible inhibition of the coupling betweenphosphorylation and oxidation. J. Biol.Chem., 173, 807-808.

McELROY, W. D., AND KIPNIS, D. M. 1947The mechanism of inhibition of biolumi-nescence by naphthoquinones. J. CellularComp. Physiol., 30, 359-380.

MELCHIOR, J. B., KEozE, O., AND KLOTZ, I. M.1951 Further studies on the synthesis ofprotein by Escherichia coli. J. Biol. Chem.,189, 411-420.

MEYERHOF, O., AND SCHULZ, W. 1938 Eine neueBestimmungsmethode der Phosphoglyzerin-s&ure. Biochem. Z., 297, 60-65.

MONOD, J. 1944 Inhibition de L'adaptionenzymatique chez B. coli en en pr6sence de2-4 dinitroph6nol. Ann. inst. Pasteur, 70,381-384.

MOROWITZ, H. J. 1954 The energy requirementsfor bacterial motility. Science, 119, 286.

SEAMAN, G. R., AND HOULIHAN, R. K. 1951Enzyme systems in Tetrahymena geleii S. II.Acetylcholinesterase activity. Its relation tomotility of the organism and to coordinatedciliary action in general. J. Cellular Comp.Physiol., 37, 309-321.

SMITH, G. N. 1953 The possible modes of

222 [VOL. 69


.org/D

ownloaded from

http://jb.asm.org/


action of chloromycetin. Bacteriol. Revs.,17, 19-29.

SOMERS, G. F., AND CoBBY, E. L. 1945 Theconversion of fructose-6-phosphate intoglucose-6-phosphate in plant extracts. Arch.Biochem., 6, 295-32.

SPIZIZEN, J. 1943 Biochemical studies on thephenomenon of virus reproduction. II.Studies on the influence of compounds ofmetabolic significance on the multiplicationof bacteriophage. J. Infectious Diseases,73, 212-228.

STLL, J. L. 1940 Triosephosphate dehydro-genase of Bacterium coli. Biochem. J.(London), 34, 1374-1382.

STREET, H. E. 1950 The role of high-energyphosphate bonds in biosynthesis. ScienceProgr., 38, 43-6.

STREHLER, B. L. 1953 Luminescence in cell-freeextracts of luminous bacteria and its activa-tion by DPN. J. Am. Chem. Soc., 75, 1264.

STREHLER, B. L., AND CORMIR, M. J. 1953Factors affecting the luminescence of cell-freeextracts of the luminous bacterium Achromo-bacterficheri. Arch. Biochem. and Biophys.,47, 16-33.

TAM, R. K., AND WILSON, P. W. 1941 Respira-tory enzyme systems in symbiotic nitrogenfixation. III. The dehydrogenase systems ofRhizobium trifolii and Rhizobium legumino-sarum. J. Bacteriol., 41, 529-546.

UTTER, M. F. 1950 Mechanism of inhibition ofanaerobic glycolysis of brain by sodiumions. J. Biol. Chem., 185, 499-517.

UTTER, M. F., AND WERKMAN, C. H. 1942 Dis-similation of phosphoglyceric acid by Es-cherichia coli. Biochem. J. (London), 36,485-493.

WERNSTEDT, C. 1944 Metabolism of gill epi-thelium of a freshwater mussel. Nature,164, 463.

WISSEMAN, C. L., JR., HAHN, F. E., Hopps, H. E.,AND SMADEL, J. E. 1953 Chloramphenicolinhibition of protein synthesis. FederationProc., 12, 466.

WIS8EMAN, C. L., JR., SMADEL, J. E., HAHN, F. E.,AND Hopps, H. E. 1954 Mode of action ofchloramphenicol. I. Action of chloram-phenicol on assimilation of ammonia and onsynthesis of proteins and nucleic acids inEscherichia coli. J. Bacteriol., 67, 662-673.

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CHLORAMPHENICOLMODEOFACTION OF CHLORAMPHENICOL reactions of this general type, such as those catalyzed by the enzymes xanthine oxidase, amino acid oxidase, and cytochrome oxidase,