APPLIED MICROBIOLOGY, Nov., 1966Copyright © 1966 American Society for Microbiology

Action of Chloramphenicol and Its Isomers on

Secondary Biosynthetic Processes of BacillusEUGENE D. WEINBERG AND SUE M. TONNIS

Department of Microbiology, Indiana University, Bloomington, Indiana

Received for publication 23 March 1966

ABSTRACT

WEINBERG, EUGENE D. (Indiana University, Bloomington), AND SUE M. ToNNIS.Action of chloramphenicol and its isomers on secondary biosynthetic processes ofBacillus. Appl. Microbiol. 14:850-856. 1966.-Chloramphenicol or its isomers, ifsupplied within 4 hr after the addition of Mn+2, prevented sporulation of Bacillusmegaterium at concentrations that partially inhibited protein synthesis but whichwere neither bacteriostatic nor bactericidal. Likewise, sub-bactericidal quantities ofthe compounds, if supplied within 2 hr after the addition of Mn 2, suppressed for-mation of bacitracin by B. licheniformis. In contrast to previous reports that chlor-amphenicol is less active than its isomers against sporulation and peptide formation,the results of the present study indicated that the order and extent of these activitiesof the compounds is similar to that of their ability to prevent growth and to suppressprotein synthesis; i.e., D(-)-threo > L(+)-erythro > D(-)-erythro.

Studies of the action of chloramphenicol andits isomers against species of Bacillus have yieldeda collection of rather unexpected and somewhatconflicting observations. For example, althoughthe antibiotic is bacteriostatic for most genera,for the genus Bacillus it is bactericidal (4). TheD(-)-threo isomer, which inhibits protein syn-thesis and has clinical usefulness, predictablysuppresses sporulation at growth inhibitory con-centrations (5, 17); but, surprisingly, a mixture ofthe L(+)- and D(-)-erythro isomers was re-

ported to be more active than the D(-)-threoform in preventing spore formation (3). TheL(+)-erythro isomer, which has only 1 to 2% ofthe bacteriostatic potency of the D( -)-threo com-pound for Escherichia coli (12), suppresses D-

glutamyl peptide formation of B. subtilis at con-

centrations that affect neither growth nor proteinsynthesis, whereas the D( -)-threo form is inactive(7). On the other hand, a mixture of the DL-erythro isomers prevents accumulation of bothD- and L-glutamyl peptide, but only if growth isslowed and the rate of death increased by limitingconcentrations of Mn--2 (9). Formation of baci-tracin, a peptide that contains both D- and L-

amino acids, is suppressed by neither the D(-)-threo nor the DL-erythro isomers (3).At the end of the exponential phase of growth,

a shift to secondary metabolism occurs in culturesof Bacillus if the quantity of available Mn+2 is atleast 1 to 2 log units higher than the concentra-

tion of 5 X 10-8 M required for vegetative growth(18). The onset of secondary metabolism can bedelayed by withholding Mn+2, but, if the periodof withholding is prolonged, death intervenes(10, 18). In the normal sequence of events insecondary metabolism of Bacillus, production ofpeptides that contain both D- and L-amino acidsoccurs first and is followed by sporulation (2, 11);it is not known whether the presence of the pep-tides is a prerequisite for spore formation. Fivepossible functions of the peptides have been -sug-gested, but little evidence is available in supportof any of these (Weinberg, in press).The experiments described in the present study

were performed to answer the following ques-tions: (i) is the ability of the D(-)-threo, L(+)-erythro, and D(-)-erythro isomers of chlor-amphenicol to suppress growth and proteinsynthesis parallel or antipodal to their ability toprevent sporulation; (ii) is the antisporogeniceffect of the compounds simply a consequence oftheir bactericidal activity; and (iii) can any of theisomers influence the quantity of a peptide(bacitracin) that accumulates in the cultures?

MATERIALS AND METHODS

Strains and culture media. B. megaterium strain WS(Indiana University culture collection) was used forthe studies on viability, sporulation, and protein syn-thesis; strain ATCC 10716 of B. licheniformis was usedfor the experiments on production of bacitracin. Cul-tures of B. megaterium were grown and tested in a

850

Vol. 14, No. 6Printed in U.S.A.

on August 17, 2019 by guest

http://aem.asm

.org/D

ownloaded from

VOL. 14, 1966 CHLORAMPHENICOL ACTION ON BIOSYNTHESIS IN BACILLUS

basal medium of the following composition: glucose,1.0 g; KH2PO4, 5.0 g; (NH4)2HP04, 1.0 g; NaCI, 1.0 g;MgSO4-7H20, 0.41 g; FeSO4-7H20, 18 mg; ZnSO47H20, 18 mg; CaCl2-2H20, 6.6 mg; deionized water,1.0 liter. Solutions of each ingredient were sterilizedseparately, and appropriate volumes were combinedfor the complete medium; the latter was then adjustedto pH 7.3 with sterile 1 N NaOH. The composition ofthe medium was similar to that used by Slepecky andFoster (15), except that glucose was substituted forsucrose and MnSO4 was not included. The smallquantity of Mn+1 required for vegetative growth issupplied as a contaminant of glucose and the inor-ganic salts. Cultures of B. licheniformis were grownand tested in nutrient broth which consisted of 0.3%Beef Extract (Difco), 0.5% Polypeptone (BBL), anddeionized water, pH 7.0. This complex medium, pro-vided that deionized water is used, contains a biologi-cally available concentration of Mn-2 of <10-6 M(17; unpublished data); to obtain formation of sporesand detectable quantities of bacitracin, additionalMn+2 must be supplied.

Inocula for the experiments were obtained by sub-culturing three times, in the appropriate test medium,organisms originally obtained from nutrient agarslants. Cultures used in the serial passages, as well as inthe experiments, were grown in 10-ml portions of therespective media contained in 50-ml Erlenmeyer flasksand incubated at 37 C on a reciprocating shaker.Inocula for the actual tests consisted of 0.1-ml quanti-ties of suspensions of the third subcultures obtainedat the end of the period of logarithmic growth.

Assaysfor viable organisms, spores, protein synthesis,and bacitracin. For counts of total viable cells and ofviable spores of either B. megaterium or B. licheni-formis, diluted samples were plated in nutrient agar;for the spore counts, vegetative cells were inactivatedprior to plating by exposure of the cultures to 72 C for30 min. To rule out the possibility that, under someconditions, heat-sensitive spores were being formed,wet mounts of all cultures were examined for sporesunder the phase microscope. In no case were samplesobserved to contain spores that were visible under themicroscope but which could not survive the heatingprocess.

For studies on protein synthesis, 14-hr basaJmedium cultures of B. megaterium were adjusted topH 7.3 and supplemented with 5 X 10-6 M Mn+2. Ap-propriate concentrations of either the D(-)-threo orL(+)-erythro isomer of chloramphenicol were added,and 1-ml samples were then incubated in a water-bathshaker at 37 C for 5 min. A 0.02-ml amount of a solu-tion of uniformly labeled L-leucine-C14 was added toeach tube; the quantity of leucine per sample was0.012 mg, and the activity was 0.1 Mc. A preliminaryexperiment demonstrated that uptake of radioactivityin control cells would be linear with time for at least120 min; in the actual tests, a reaction time of 30 minwas selected. The reaction was terminated by additionof 1 ml of 10% trichloroacetic acid, and the tubeswere steamed for 20 min. The samples were filteredthrough membrane filters (type HA, 25 mm; MilliporeFilter Corp., Bedford, Mass.); the filters were washedwith 3 ml of 5% trichloroacetic acid, glued to plan-

chets, and dried. Radioactivity was counted in aNuclear-Chicago gas-flow counter with thin windowfor at least 1,000 counts.

Bacitracin produced by B. licheniformis was meas-ured by assay with a stock laboratory strain of Staphy-lococcus aureus. Portions of 18-hr nutrient broth culLtures of S. aureus were diluted 1:100 in nutrient agarat 48 C. Portions (3-ml) of the seeded agar were addedto petri dishes containing a base layer of 15 ml ofnutrient agar. To obtain maximal expression of baci-tracin activity, the nutrient agar was enriched with5 X 10-' M Zn-2 (19). The seeded plates were storedat 2 C until needed. Holes were then made in the agarby use of a no. 5 cork borer. Portions (0.1-ml) of solu-tions of authentic bacitracin or of culture supernatantfluids to be assayed were added to the holes; each sam-ple was tested in duplicate. The petri plates were thenincubated at 37 C for 24 hr. Zones of inhibition weremeasured with a Fisher Lilly Antibiotic Zonoe Reader.Under these conditions, a straight-line relationshipwas obtained when zone diameters between 12 and24mm were plotted against the logarithms of doses ofauthentic bacitracin between 10 and 100 ,g (0.54 and5.4 units) per ml. Cultures to which chloramphenicolor its isomers were added were included as controls,to ascertain that these agents would not be present inthe supernatant fluids in sufficient quantity to interferewith the biological assay for bacitracin. Under condi-tions in which bacitracin could not be formed (i.e.,absence of Mnw2 or presence of large amounts ofchloramphenicol or its isomers), no zones of inhibi-tion were obtained.

Test reagents. Various concentrations of MnCl2,4H20 or one of three chloramphenicol isomers, orboth, were added to the cultures of B. megaterium orB. licheniformis at times to be described in Results.

H O

H N-C-CHC12O2 -c§CCH,OH02N C, -CH0

OH H

H H

02N / C-C-CH20HOH N-C-CHC1211

H O

H O11

OH N-C-CHCl2

O N / C-C-CH2OHH H

OH H

02NNoc-CC-CH20HH N-C-CHCI2

H11

H O

D (-)-THREO ISOMER

(CH LORAMP HEN IC OL)

L (+)-ERYTHRO ISOMER

D(-)-ERYTHRO ISOMER

L (+)-THREO ISOMER

FiG. 1. Chloramphenicol and its stereoisomers.

85I

on August 17, 2019 by guest

http://aem.asm

.org/D

ownloaded from

WEINBERG AND TONNIS

The particular manganous salt ("Baker Analyzed"reagent grade, lot 23422; J. T. Baker Chemical Co.,North Phillipsburg, N.J.) was reported by the proc-essor to contain 0.02% zinc and 0.0002% iron, butthese metals were present in both the basal and com-plex media at concentrations far in excess of theamounts contributed by the salt. The chloramphenicolisomers (obtained from Parke, Davis & Co., Detroit,Mich., through the courtesy of H. M. Crooks, Jr.)consisted of the first three of the four compoundswhose structural formulas are shown in Fig. 1. Thesample of bacitracin (lot 80163-3) was obtained fromCommercial Solvents Corp., Terre Haute, Ind.,through the courtesy of M. C. Bachman. Radioactiveleucine was purchased from Schwarz Bio Research,Inc., Orangeburg, N.Y.

RESULTS

Induction by Mn+2 of sporulation in B.megaterium, and ofbacitracin and spore formationin B. licheniformis. The dose-response curves ofspore formation and bacitracin production toaddition of Mn2 to cultures in the maximalstationary phase are shown in Fig. 2 and 3. ForB. megaterium, the time of addition of the metallicion was 14 hr; for B. licheniformis, 8 hr. To ob-tain maximal sporulation and efficient productionof bacitracin, the pH of 14-hr B. megateriumcultures was changed from 6.6 to 7.3, and of8-hr B. licheniformis cultures from 7.8 to 7.3.Also presented in Fig. 3 are the quantities of

bacitracin formed when Mn+2was added to cul-tures of B. licheniformis at the time of inoculation.The reason for the decrease in ability to formthe peptide by cells of cultures deprived of Mn+2during the logarithmic phase of growth is notknown. The decrease was not a function of a

,08 L

102 L

I-

0.0! .1 1.0 10

CONCENTRATION OF MN 2 ( X 106 M)

FIG. 2. Number of total viable organisms (0) and ofviable spores (0) of Bacillus megaterium 24 hr afterthe addition to 14-hr cultures of various concentrationsofMn4'. The 14-hr cultures, which contained 4.2 X 107total viable cells and 1CP viable spores per milliliter, wereadjusted to pH 7.3 prior to addition of Mn'2.

100

90

soI \'80

60-70

60

30

20

100.7 .0 2.0 l0 20 l00 200

CONCENTRATION OF MN+2 (x 10 M)

FIG. 3. Quantity of bacitracin in supernatant fluidsof cultures of Bacillus licheniformis to which variousconcentrations of Mn+2 had been added at the time ofinoculation (0) and at 8 hr after inoculation (0). Datawere obtained 24 hr after Mn+2 was added. At a concen-tration of 0.06 X 10-6 m Mn+2, a slight amount ofbacitracin was detected; at 0.02 X 10-' m Mn", nobacitracin was found.

change in cell numbers, inasmuch as neither thegeneration time of 40 min nor the total cell yieldwas altered by the delay in addition of the metallicion. When Mn+2 was supplied at the time ofinoculation, bacitracin was first detected in 8-hrcultures (i.e., 2 hr after exponential growth hadceased); when the metallic ion was added tomaximal stationary-phase cultures, the peptidewas first detected within 4 hr.The minimal quantity of Mn+2 needed for

sporulation of B. licheniformis was identical tothat required for bacitracin production. As withthe latter, the amount of sporulation was lesswhen the metallic ion was added after exponentialgrowth had occurred than if added at the time ofinoculation. To learn whether the cell density hasan effect on the quantity of Mn+2 required forsecondary biosynthetic processes, the normalpostlogarithmic concentration of 109 viable cellsper milliliter was increased 10-fold at the sametime as the addition of the metallic ion. Theamount of Mn+2 needed for maximal sporulationand bacitracin production was identical to thatrequired in systems in which a cell density of109 per milliliter was used.

852 APPL. MICRO'BIOL.

-i10 7M.

crL'i

U)2 0cn

z<(D(r0

10 5L'i

(a-

LI I0 4

0

a:wm2D I0 3z

on August 17, 2019 by guest

http://aem.asm

.org/D

ownloaded from

VOL. 14, 1966 CHLORAMPHENICOL ACTION ON BIOSYNTHESIS IN BACILLUS

Suppression of sporulation by chloramphenicolisomers. Each of the three isomers tested was ableto prevent sporulation of B. megaterium in con-centrations less than those required to preventgrowth or to kill vegetative cells. Data for theD(-)-threo and L(+)-erythro isomers are sum-marized in Fig. 4; the D(-)-erythro form pos-sessed about half of the antisporogenic, bacterio-static, and bactericidal activity of the L(+)-erythro compound. Amounts of added Mn+2as high as 5 X 104 M were unable to reverse theeffects of any of the three isomers. Figure 5 showsthat the D( -)-threo isomer could prevent sporula-tion only if added within a period of 4 to 5 hrafter the introduction of 5 X 10-6 M Mn+2; thiswas true also for each of the other two isomers.

Similarly, with B. licheniformis, the D(-)-threoand L(+)-erythro isomers prevented sporulationat concentrations that were without effect onviability (Table 1). However, the effective con-centration of the former compound was 10-foldhigher for this organism than for B. megaterium,whereas that of the latter isomer was essentiallyidentical for both species. The difference in activ-ity of the D(-)-threo compound did not resultfrom the difference in media used for the two testorganisms; when B. megaterium was grown in

100 -

50

20

0

Z 5.00

Z 2.0LJ

z0V- ,

1.0 t

0.5 C

<,a z

o )o DrAllwL 0 <n

0 m U< U)

Ss W

5

tz

zo o0

x

41

41-D

D(-)-THREO ISOMER L (+)-ERYTHRO ISOMER

FIG. 4. Concentrations of D(-)-threo and L(+)-erythro isomers ofchloramphenicol required to suppresssporulation, prevent growth, destroy viability, and sup-press protein synthesis of Bacillus megaterium. Dosesbelow arrows had no effect; those above arrows hadcomplete effect.

ID8 I

MN21 ADDED A °

_.A7I CAP ADDED AT 14 51617 HRS

a 14 15 16 17 IS 9 20 21 22 23 24 25 26

ar.F or r,11 THODr {nlAGE OF CULTIURE (HOURS):

FIG. 5. Number oftotal viable organisms (0) and ofviable spores (a) of Bacillus megaterium-at varioustimes after inoculation of 4 X 105 vegetative cells permilliliter. At 14 hr, 5 X 10-6 m Mn+2 was added, andthe pH was adjusted to 7.3. Addition of 1O-' M D(-)-threo isomer of chloramphenicol (CAP) at 14, 15, 16,or 17 hr completely suppressed spore formation; addi-tion at 20 hr or later hadno action on sporulation; effectofaddition at 14 through 19 hr is indicated on thefigure.Cell viability was not altered in any ofthe samples.

nutrient broth, spore formation was prevented bythe same low concentration effective in the syn-thetic medium.

Effect of chloramphenicol isomers on culturallongevity and on protein synthesis of B. mega-terium. The decline in number of living organismsin control cultures and in cultures supplementedwith Mn+ plus sub-bactericidal concentrations ofeither the D(-)-threo or theL( +)-erythro isomersis shown in Fig. 6. It may be noted that culturesretained viability only when provided with Mn+2in the absence of the antibiotic isomers. Longevityof the cultures appeared to be associated, in part,with sporulation; however, not all of the viableorganisms in the Mn+2-supplemented culturesformed spores. The rate of killing of vegetativecells by a bactericidal concentration of the D(-)-threo isomer (10-fold higher than the quantityused for the experiments summarized in Fig. 6)is shown in Fig. 7. The dose-response curves tothe D(-)-threo and L(+)-erythro isomers withrespect to suppression of protein synthesis aregiven in Fig. 8. The activity of the latter isomerwas approximately 3% of that of the D( -)-threocompound. In a cell-free system of E. coli, theisomer was found to have about 12% of theactivity of chloramphenicol (13).

Effect of chloramphenicol isomers on formationofbacitracin by B. licheniformis. Each of the threeisomers tested was able to prevent formation ofbacitracin at concentrations less than those re-quired to kill vegetative cells (Table 1). Complete

853

-j 107mEr

a.w(n 1062z.2Q)cr0 105

-iuim

> 104U-0

crcoui2 103:3z

u)

ZwJ.O11:XL

U.0

z0U)U)wcr(L0.

0)

on August 17, 2019 by guest

http://aem.asm

.org/D

ownloaded from

WEINBERG AND TONNIS

TABLE 1. Concentrations of chloramphenicol and its isomersa required to prevent growth, bacitracinformation, and sporulation, and to kill vegetative cells of Bacillus licheniformisb

Bacteriostatic activity"c Suppression of Suppression of Bactericidal activityd1Concn of I, II, or III bacitracin formation" sporulationd

(X 10-5 1)

I II III I II III I II III I II lII

zero 0 0 0 0 0 0 0 0 0 0 0 03 0 0 0 0 0 0 0 0 0 0 0 06 + 0 0 + 0 0 0 0 0 0 0 010 ++ 0 0 ++ 0 0 ++ 0 0 0 0 020 ++ 0 0 ++ + 0 ++ 0 0 0 0 030 ++ + 0 ++ ++ + ++ 0 0 0 0 060 ++ ++ 0 ++ ++ ++ ++ ++ 0 0 0 0100 ++ ++ + ++ ++ ++ ++ ++ 0 + 0 0

a Symbols used for isomers: I, D(-)-threo; II, L(+)-erythro; III, D(-)-erythro.b Results expressed as: 0, no effect; +, partial effect; ++, complete effect.c Isomer added with 2 X 10- M Mn+2 at time of inoculation.d Isomer added with 2 X 10-6 M Mn+2 to 8-hr cultures.

z

a O

_I Jm

so 90 100 110 <

>-Ilo 20 > E40 50 60 70

AGE OF CULTURE (HOURS)

FIG. 6. Number of total viable organisms of Bacillusmegaterium at various times after addition to 14-hr cul-tures (adjusted to pH 7.3) of 5 X 10-6 m Mn2 (0),S X 10-6 M Mn+ plus 10-' M D(-)-threo isomer (A),or 5 X 10-6 M Mn+2 plus 20 X 10-5 M L(+)-erythroisomer (A). Control (neither Mn+2 nor isomers), El.Sporulation (0) occurred only in cultures in which Mn+2was the sole addition; the number ofspores in all othercultures remained at < 102.

suppression of bacitracin formation without in-terference with sporulation was obtained by theaddition of a concentration of either 30 x 1O-5 Mof the L(+)-erythro isomer or 60 x 10-5 M ofthe D(-)-erythro isomer. Quantities of addedMn+2 as high as 50 X 10-' M were unable toreverse the action of either chloramphenicol orits isomers.The D(-)-threo isomer could prevent bacitra-

cin formation only when added within a periodof 2 hr after the introduction of 5 X 10-6 MMn+2 (Table 2); this was true also for each of theother isomers. Chloramphenicol and its isomershad no effect on bacitracin production when

103 L14 5 16 17 I8 19 20 21 22 23 24 25 26 27 28 29

AGE OF CULTURE (HOURS)

FIG. 7. Number ofviable organisms ofBacillus mega-terium at various times after addition of 10-4 M D(-)-threo isomer ofchloramphenicol to 14-hr cultures. Con-trol, 0; chloramphenicol, Li.

added to 8-hr cultures that had been suppliedwith Mn+2 either at the time of inoculation -orduring exponential growth. In a previous study(3), exposure of cultures of strain A-5 ofB. licheniformis to high concentrations of chlor-amphenicol or its isomers resulted in apparentreduction of the compounds to the correspondingaryl amines with subsequent lysis of the cells.In the present study with strain 10716, neither the

854 APPL. MICROBIOL.

108

2aL-

V.

2"I 107..i

q-7

m

5 106

cr

z

-i2xL'iCL

v

cn

z

on August 17, 2019 by guest

http://aem.asm

.org/D

ownloaded from

VOL. 14, 1966 CHLORAMPHENICOL ACTION ON BIOSYNTHESIS IN BACILLUS

L (+)-ERYTHROISOMER

loo r"I

zI}::

J-

I

DD(-)-THREOcISOMER

225 -

z

L'i

.0 3.3 10 33 loo 330

CONCENTRATION X 10i5 M)

FIG. 8. Suppression of protein synthesis of Bacillusmegaterium by various concentrations of the D(-)-threo and L(+)-erythro isonters of chloramphenicol.Values were obtained after 30 min of exposure of cellsto C'4-leucine; control samples averaged 1,786 countsper min.

TABLE 2. Quantity ofbacitracin produced by Bacilluslicheniformis after addition ofMn+2 or chloram-phenicol, or both, to maximal stationary-phasecultures

Age of culture when Quantity of bacitracin (j.g/ml) in(hr) culture supernatant fluid at

Mn+2 Chloram-(2 X phenicol 8 to 11 12 hr 13 hr 14 hr 15 hr

10'6M) (lOX hr1O'5M)

8 0 0 0 0 0- 8 0 0 0 0 0

8 _ 0 8 25 50 588 8-10 0 0 0 0 08 11 0 0 12 31 42

change in color indicative of aryl amine formationnor cell lysis was observed.

DIscussIoN

The results obtained in this study provide evi-dence that the ability of chloramphenicol andits isomers to prevent sporulation parallels theextent of their ability to inhibit protein synthesis.Furthermore, effective antisporogenic concentra-tions for each of the three isomers are less thanthe quantities needed to prevent growth and tokill vegetative cells. In contrast to a previousobservation (3) that the DL-erythro isomers havestrong antisporogenic action, we found that theorder and relative proportion of activity ofchloramphenicol and its isomers are similar tothe antibiotic potency of the compounds for

species of Bacillus and, as reported by otherworkers (12), for E. coli.

In addition to suppression of sporulation, sub-bacteriostatic concentrations of chloramphenicoland its isomers interfere with maintenance ofMn+2-dependent cultural longevity. Apparently,the antibiotics suppress the synthesis of a specificprotein(s) that is required for viability of cells inmaximal stationary-phase cultures. Excess quanti-ties of Mn2 are unable to neutralize any of theactivities of chloramphenicol or its isomers.

In the present study, a concentration of 10-5 Mchloramphenicol completely inhibited sporulationof B. megaterium, but suppressed total proteinsynthesis by only 58%. The formation of anenzyme that catalyzes the synthesis of L(+)-N-succinyl glutamic acid by presporulating cells ofB. megateinum is suppressed by 12 X 105 Mchloramphenicol (1). Presporulating cells of B.cereus are unable to synthesize acetoacetyl co-enzyme A reductase (8) and total protein (16)in the presence of 3 X 10-5 M and 33 X 10-5 Mchloramphenicol, respectively. In cells of B. sub-tilis, 15 x 1O M chloramphenicol suppressesprotein synthesis and spore coat formation (14).As was true in the present study, the antibioticbegins to lose effectiveness when added to culturesin which heat-resistant spores have begun to ap-pear (16).When sufficient Mn+2 is available in cultures of

growing cells, so that enzymes required for theassembly of bacitracin can be preformed, chlor-amphenicol added to postlogarithmic-phase cul-tures cannot interfere with synthesis of thepeptide. The present finding is in agreement withprevious reports (3, 6). Such enzymes might in-clude racemases needed for synthesis of the fouramino acids present in bacitracin in the D con-figuration. However, when Mn+2 is withheld fromthe cells, one or more enzymes are presumablyabsent, since no detectable bacitracin is syn-thesized. Addition of Mn+2 to maximal sta-tionary-phase cells results in appearance of thepeptide within 4 hr. When either chloramphenicolor its isomers are supplied within 2 hr after addi-tion of the metallic ion, bacitracin is notproduced; apparently, synthesis of either theracemases or the peptide assembly enzymes issuppressed. Predictably, the order of activity ofthe compounds is D(-)-threo > L(+)-erythro> D(-)-erythro.

ACKNOWLEDGMENTSThis investigation was supported by Public Health

Service grants AI-06268-01 and AL-06268-02 from theNational Institute of Alergy and Infectious Diseases.The experiments on protein synthesis were per-

formed in the laboratory of T. D. Brock with the

855

0.33

on August 17, 2019 by guest

http://aem.asm

.org/D

ownloaded from

856 WEINBERG

assistance of Pat Holleman. The technical assistanceof Dorothy L. Grisso, Carol L. Conrad, and Rita M.Norquest is gratefully acknowledged.

LITERATURE CITED1. AUBERT, J. P., J. MILLET, E. PINEAU, AND G.

MILHAUD. 1961. L'acide n-succinyl-L(+)gluta-mique chez Bacillus megaterium en voie desporulation. Biochim. Biophys. Acta 51:529-537.

2. BERNLOHR, R. W., AND G. D. NOVELLI. 1960.Some characteristics of bacitracin productionby Bacillus licheniformis. Arch. Biochem. Bio-phys. 87:232-238.

3. BERNLOHR, R. W., AND G. D. NOVELLI. 1963.Bacitracin biosynthesis and spore formation:the physiological role of an antibiotic. Arch.Biochem. Biophys. 103:94-104.

4. BROCK, T. D. 1961. Chloramphenicol. Bacteriol.Rev. 25:32-48.

5. BROCK, T. D. 1962. Inhibition of endotrophicsporulation by antibiotics. Nature 195:309.

6. CORNELL, N., AND J. E. SNOKE. 1964. Biosynthesisof bacitracin and protein. Biochim. Biophys.Acta 91:533-536.

7. HAHN, F. 1961. Inhibition of protein synthesis byantibiotics. Antimicrobial Agents Ann. 1960,p. 310-319.

8. KOMINEK, L. A., AND H. 0. HALVORSON. 1965.Metabolism of poly-fl-hydroxybutyrate andacetoin in Bacillus cereus. J. Bacteriol. 90:1251-1259.

9. LEONARD, C. G. 1961. Inhibition of protein syn-thesis by antibiotics-discussion. AntimicrobialAgents Ann. 1960, p. 325-326.

10. LEONARD, C. G., R. D. HOUSEWRIGHT, AND C. B.THORNE. 1958. Effects of some metallic ions onglutamyl polypeptide synthesis by Bacillus sub-tilis. J. Bacteriol. 76:499-503.

AND TONNIS APPL. MIcROBIOL.

11. MACH, B., E. REICH, AND E. L. TATUM. 1963.Separation of the biosynthesis of the antibioticpolypeptide tyrocidine from protein biosyn-thesis. Proc. Natl. Acad. Sci. U.S. 50:175-181.

12. MAXWLL, R. E., AND V. S. NICKEL. 1954. Theantibacterial activity of the isomers of chilor-amphenicol. Antibiot. Chemotherapy 4:289-295.

13. RENDI, R., AND S. OCHOA. 1962. Effect of chlor-amphenicol on protein synthesis in cell-freepreparations of Escherichia coli. J. Biol. Chem.237:3711-3713.

14. RYTER, A., AND J. SZULMASTER. 1965. Action duchloramphenicol sur la sporogenese de B. sub-tilis. Ann. Inst. Pasteur 108:640-651.

15. SLEPECKY, R., AND J. W. FOSTER. 1959. Altera-tions in metal content of spores of Bacillusmegaterium and the effect on some spore prop-erties. J. Bacteriol. 78:117-123.

16. VJNTER, V. 1963. Spores of microorganisms.Chloramphenicol-sensitive and penicillin-re-sistant incorporation of 14C-diaminopimelicacid into sporulating cells of Bacillus cereus.Experientia 19:307-308.

17. WEINBERG, E. D. 1955. The effect of Mn+ andantimicrobial drugs on sporulation of Bacillussubtilis in nutrient broth. J. Bacteriol. 70:289-296.

18. WEINBERG, E. D. 1964. Manganese requirementfor sporulation and other secondary biosyn-thetic processes of Bacillus. Appl. Microbiol.12:436-441.

19. WEINBERG, E. D. 1965. Microbiological methodfor estimation of stability constants of baci-tracin complexes of zinc, cadmium, and manga-nese. Antimicrobial Agents and Chemo-therapy-1964, p. 120-125.

on August 17, 2019 by guest

http://aem.asm

.org/D

ownloaded from