JOURNAL OF BACTERIOLOGY, Jan. 1970, p. 188-195Copyright © 1970 American Society for Microbiology

Vol. 101, No. 1Printed In U.S.A.

Incorporation of 5-Diazouracil-2-14C into RibonucleicAcids of Escherichia coli During Division

Inhibition'EDWARD PREVIC AND GEORGE FISTER

Department oJ Microbiology, College of Medicine, University of Florida, Gainesville, Florida 32601

Received for publication 25 August 1969

Radioactive diazouracil (DZU-2-14C) was rapidly incorporated into acid-insolublematerial of Escherichia coli B. Isolated ribonucleic acid contained essentially allof the incorporated label, and this was solubilized by ribonucleases but not by de-oxyribonuclease. A maximum of 45 to 50% of added label was incorporated atdivision-inhibitory and subinhibitory concentrations. Incorporation levels andfilament-forming capacity were concomitantly depressed by preincubation of DZUin various medium components. The lower levels of incorporation brought aboutby preincubation were apparently related to an inherent instability or high reactivityof the DZU. The limit on incorporation of fresh DZU suggests the presence ofgeometrical isomers in the original DZU. The nature of these isomers and the re-activity of DZU are discussed in relation to its use in future cell division studies.

When 5-diazouracil (DZU) is added at ap-propriate concentrations to randomly-dividingcultures of Escherichia coli B in balanced growth,the more mature cells in a population completetheir division cycle (4). All cell division thenceases for some time, but the mass of the cul-ture continues to increase at a nearly normalrate. Previc and Richardson (4) have shown thatthe rates of protein, ribonucleic acid (RNA),and deoxyribonucleic acid (DNA) synthesis arenot grossly altered in the resultant filaments. Thisobservation of selective inhibition of cell divisionby DZU prompted an investigation of its meta-bolic fate. At least superficially, it appeared tobe a simple compound, and its chemistry in cellswas expected to give direct information about thedivision process. In the work reported here, amajor portion of added radioactive DZU (DZU-2-14C) is found in RNA, but at least 50% of theradioactive label usually remains in a solubleform. The failure to get complete incorporationis examined in relation to (i) nonhomogeneouscomposition of the original DZU, (ii) instabilityof the compound in the culture medium, or(iii) partial conversion to a nonincorporableform by the bacteria. It is shown that the firstand second are significant problems resultingfrom the possibilities for geometrical isomerism

' Part of this study was submitted to the University of Floridaby George Fister in partial fulfillment of the requirements for thePh.D. degree.

in DZU and from the inherent reactivity ofdiazonium compounds. These complicationsare discussed along with the prospects for theuse of DZU in further cell-division studies.

MATERIALS AND METHODSOrganisms. E. coli B (ATCC 11303) was grown in a

defined mineral salts-glucose medium (MSG) at 37 Cas described by Previc and Richardson (4).

Chemicals. DZU-2-14C (specific activity, 0.757 c/mole) and nonradioactive DZU were synthesized byNew England Nuclear Corp., Boston, Mass., bydiazotization of 5-aminouracil. The 5-aminouracilwas dissolved in 1 N HCl and cooled to -5 C in anice-salt bath. The proper amount of NaNO2 wasdissolved in water, cooled, and added slowly to thereaction flask. A precipitate was observed, and stirringwas continued for 1 hr at -5 C. The product wasfiltered and washed with ice water until free of chlo-ride. The product was then dried over P205. The DZU-2-'4C and nonradioactive DZU synthesized by thisprocedure had matching infrared spectra. Two possi-ble forms of the product, a hydrate and an anhyd-ride, are shown in Fig. 1. These proposed forms andthe synthetic procedure have been described (1).Elementary analyses on the nonradioactive DZUwere done by Peninsular Chemical Research, Inc.,Gainesville, Fla. The composition agreed well with thehydrate: DZU-anhydride (per cent calculated): C,34.8; H, 1.5; N, 40.6; DZU-hydrate (per cent calcu-lated): C, 30.8; H, 2.6; N, 35.9; synthetic DZU (percent found, duplicate analyses): C, 29.9, 30.0; H, 2.7,2.7; N, 35.0, 35.0.

Incorporation of radioactively labeled compound. At

188

on March 2, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

intervals, 2.0-ml samples were removed from culturestreated with DZU-2-14C. These samples were addedto 2.0 ml of cold 10% trichloroacetic acid, thoroughlymixed on a Vortex mixer, and allowed to stand in anice bath for at least 30 min. Then, 2.0 ml of the mixturewas filtered onto membrane filters (Millipore Corp.,Bedford, Mass., 25 mm in diameter, 0.45Mm pore size)and washed on the filters with 4 ml of 5% cold trichlo-roacetic acid and 6 ml of cold water. The filters werepulled dry by suction, thoroughly dried at 60 C for10 min, and then placed in scintillation vials with 10ml of scintillation fluid and counted in a PackardTri-Carb scintillation spectrometer (Packard Instru-ment Co., Inc., Downers Grove, Ill.). The countingfluid was 60 g of naphthalene, 4 g of 2,5-diphenyl-oxazole, and 200 mg of 1 ,4-bis-[2-(4-methyl-5-phenyloxazolyl)]-benzene (Packard Instrument Com-pany, Inc.) dissolved in 100 ml of methanol and 20ml of ethylene glycol, with sufficient p-dioxane tomake a total volume of 1 liter. Liquid samples, e.g.,total culture, filtrates, washes, etc., were counted bydissolving 0.1-ml samples directly in the scintillationfluid.

Preparation of crude cytoplasmic extracts. Normalor filamented bacteria were harvested by rapidlymixing 100 ml of culture with 40 ml of mineral saltssolution frozen at -18 C. Unless otherwise indicated,all subsequent handling was at 0 to 4 C. The cellswere pelleted by centrifugation for 9 min in a ServallGSA rotor accelerated to 13,000 X g and immediatelydecelerated. The cells were washed twice by resuspen-sion in 0.01 M sodium acetate (pH 5.1) followed by 7min in a Servall SS-34 rotor accelerated to 37,000 Xg and immediately decelerated. The cells were finallysuspended to a density of about 2 X 109 cells/ml in0.01 M sodium acetate containing, where indicated, 2tg of polyvinylsulfate potassium salt (PVS; K & KLaboratories, Inc., Jamaica, N.Y.) per ml, and dis-rupted in a French pressure cell (American Instru-ment Co., Inc., Silver Spring, Md.) at 18,000 lb/in .

Destruction of cellularity was at least 98% whenequated to loss of viable, colony-forming units (4).The broken-cell suspension was sedimented for 30min at 37,000 X g in the Servall SS-34 to pellet wall-membrane debris and unbroken cells.

Preparation of RNA. A modification of the Scherrerand Darnell (6) phenol extraction method was usedto isolate RNA. Crude suspensions of broken cells orsupematant fluids were mixed with sodium dodecylsulfate to a concentration of 0.5%. The samples werethen extracted with an equal volume of water-satu-rated phenol at 60 C for 3 min. The mixture wasquickly cooled to room temperature and the layerswere separated by gentle centrifugation. The aqueouslayer was re-extracted with phenol. The phenol layers,including insoluble interface material, were combinedand extracted with an equivalent volume of acetatebuffer. All aqueous layers were combined and ex-tracted with diethyl ether to remove residual phenol.The RNA was precipitated by addition of two vol-umes of 95% ethyl alcohol and overnight storage at4 C. The precipitate was centrifuged at top speed in theServall SS-34, redissolved in acetate buffer, repre-cipitated with ethyl alcohol, centrifuged, drained,

189

No ANHYDRIDE

0

11 NN'1-CNs ,-1N N Co

N H

11C

HN' "C-N=NOHI

O=C CHNH

HYDRATEFIG. 1. Structures of DZU from diazotizationi of

5-aminouracil, as proposed by Johnson, Baudisch, andHoffman (1).

washed with cold absolute ethyl alcohol, air-dried, andthen dried in a vacuum desiccator. When dissolved in 0.1M sodium phosphate buffer (pH 7.0), the 260 to 280nm absorbancy ratio for this material was 2.00 fromboth control and filamented bacteria. This indicatedno significant contamination with polypeptides con-taining aromatic amino acids.

Enzymes. Pancreatic ribonuclease was obtainedfrom Calbiochem, Los Angeles, Calif.; ribonucleaseT, from Schwarz BioResearch, Inc., Orangeburg,N.Y.; RNA phosphodiesterase from Miles Labora-tories, Elkhart, Ind.; and ribonuclease-free deoxy-ribonuclease I from Mann Research Laboratories,New York, N.Y.

RESULTS

Incorporation of DZU-2-14C into E. coli B. Asubinhibitory dose of 0.1 g.g of DZU-2-14C perml was rapidly incorporated into the trichloro-acetic acid-insoluble material of E. coli B (Fig. 2).In this and duplicate experiments, the incorpora-tion reproducibly approached a limit of about45 to 50% of the available counts. A filamentingconcentration (4) of 0.5 Mig DZU-2-14C per ml attwo different specific radioactivity levels alsowas incorporated to a limit of around 45 to 50%(Fig. 2). This showed that the incomplete in-corporation at the lower concentration was notlimited by the number of available reactive sitesin the cells. In fact, the cells retained the capacityto rapidly convert DZU to a trichloroaceticacid-insoluble form for at least 140 min after aninitial exposure (Fig. 3). A culture of E. coli B inbalanced growth in MSG at 37 C at an optical

VOL. 101, 1970 DIAZOURACIL-2-14C IN RNA OF E. COLI

on March 2, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

stantial portion of the original dose was not beingincorporated.Slow incorporation of depleted DZU. The

residual DZU label in the medium after the32- _ ~ usual 90-min treatment was not readily incor-porated by untreated cells. E. coli B at an opticaldensity of 0.150 in MSG at 37 C was permitted toincorporate DZU-2-'4C for 90 min from an14- initial concentration of 0.5 ,ug/ml. The uptake

E was 46%, leaving 0.27 ,ug/ml in the medium. Theculture was filtered on a membrane ifiter (Milli-ffi8- Z - pore Corp., 0.45 gm pore size) at 90 min, and 100

C) tml of the filtrate at 37 C was inoculated with 20ml of a culture of E. coli B in balanced growth in

E J _ * MSG at 37 C to give an optical density of 0.145.o4- r -Incorporation was followed in the usual way ono Z Sv this culture (Fig. 4). A control was provided by_ filtering a culture as described above immediately2

l *Atafter addition of DZU-2-14C and inoculating thefiltrate with fresh cells. Figure 4 shows that theincorporation pattern for the control was similarto that for straightforward addition of DZU, as

,- oStin Fig. 2, with 47% uptake. Cell division in thecontrol was inhibited completely after the usualresidual increase in numbers (4). The depletedDZU in the 90-min filtrate was incorporated to

0 30o 60 90 2 only 10% of the available dose, and with a slowMinutes initial rate (Fig. 4). With this depleted DZU, the

-14C into E. coli B calculated concentration was 0.225 ,ug/ml uponFIG. 2. Incorporation of DZU_2cuaton and ther was veyhteosralin MSG at 37 C, as material insoluble in cold 5% inoculation, and there was very little observable

trichloroacetic acid. Symbols: 0, 0.1 ;tg/ml, specific filamentation (<5%). An initial level of 0.225activity 0.757 mc/mmole; *, 0.5 jug/ml, specific Ag of fresh DZU per ml was adequate to filamentactivity 0.151 mc/mmole; *, 0.5 &g/ml, specificactivity 0.757 mc/mmole;O, residual activity, filtratesfrom*.

25density of 0.150 was treated with 0.45 jAg ofDZU-2-12C per ml. At zero-time, and in 10-minm

0

intervals, 25-ml portions of the culture were re- 20moved to clean tubes with the simultaneous Xaddition of 0.05 Mg of DZU-2-14C per ml. From i5tthe time of transfer, four 5-min samples of 2 ml Ieach were taken from the subcultures for meas- Yurement of 14C in trichloroacetic acid-insol- p 10-uble material. The 0.45 jig of DZU-2-12C perml was a filamenting dose, and the total of 0.50Ag/ml after addition of DZU-2-14C did not s-inhibit mass growth. Since the DZU-2-12Coriginally added to the master culture wasgradually being depleted by incorporation, the 0 20 40 60 80 100 120 140added DZU-2-14C was diluted less in successive Minutessamples and its incorporation rate increased ac- FIG. 3. Incorporation ofDZU-2-14C (0.5,.g/ml,cordingly, up to the 80-min sample (Fig. 3). The specific activity 0.757 mc/mmole) into E. coli B in MSGrate decreased slightly to the end of the experi- at 37 C, previously exposed to nonradioactive DZUrante ireandgeteogtoidcttht(0.45 Ag/ml) for various time periods. Incorporation wasment; it remained great enough to indicate that i("into cold 5% trichloroacetic acid-insoluble material.the growing filaments retained their capacity to The initial point for each curve corresponds to theconvert to insoluble form some portion of newly length of treatment time with nonradioactive DZUadded DZU during a time span in which a sub- prior to addition ofDZU-2-14C.

190 PREVIC AND FISTER J. BACrRIOL.

on March 2, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

DIAZOURACIL-2-'4C IN RNA OF E. COLI

30 to 50% of a population of E. coli B underotherwise similar conditions.

Alteration of DZU during chromatography andby medium components. Results of the type de-scribed above suggested that there were morethan one molecular species in the DZU sample.The mode of synthesis and the elementary analy-sis indicated a uniform molecular composition(see Materials and Methods). The radioactiveand nonradioactive DZU were chromatographedseparately and together on Whatman no. 3 filterpaper. The filter sheets were cut into small stripswhich were eluted to provide fractions for deter-mination of radioactivity and ultraviolet absorp-tion. Isopropanol-HCl-water [65:16.7:20 (8)]elution gave a single substance from freshly dis-solved DZU samples, but the eluate compoundwas tentatively identified as 5-hydroxyuracil ac-cording to RF and to ultraviolet spectrum (seeDiscussion). Elution by quinoline-collidine-water [1:2:1 (8)] gave a salmon-orange spot thattailed badly on the paper and was thought to be areaction product of the DZU with the quinoline.These furtive attempts at chromatographic analy-sis suggested an unstable or highly reactive char-acter for DZU. The likelihood was examinedthat DZU might have been rapidly converted toa nonincorporable form in the culture medium.Solutions of the various salts components of themedium, with and without glucose, adjusted topH 7.0, were incubated at 37 C for 90 min with0.5 ,ug of DZU-2-'4C per ml. The missing compo-nents were then added to complete the medium,and 5-ml inoculum suspensions of E. coli B wereadded to 100 ml of each preincubation mediumto an optical density of 0.150 (450 nm). Incor-poration was observed and was found to bemaximum by 90 min in each case. Table 1 showsthe maximum per cent incorporated. Glucose ap-parently had a protective effect on the subsequentincorporability of the DZU, permitting normalrates of incorporation. The capacity to inducefilaments was also preserved in the presence ofglucose. The loss of incorporability was associ-ated with a loss of filament-forming capacity, asdetermined by microscopic observation of thevariously treated systems. The changes that themedium components induced in the DZU werenot necessarily the source of any of the 50% inac-tive component initially observed. If the latterhad been the product of a competition betweenincorporation and degradation, preincubationin complete medium should have resulted in asignificant depression in incorporability belowthe 45 to 50% usually observed. The protectiveeffect of glucose, although potentially interestingfrom the chemical point of view, did not suggestany direct approach to the division problem andwas not pursued.

E

0

C.)

E06I.

0 20 40 60 80Minutes

FIG. 4. Incorporation of depleted DZU. E. coli B inMSG at 37 C was treated with 0.5 ;Ag ofDZU-2-14C perml, 0.757 c/mole. Filtrates of the treated culture, takenat 0 and 90 min, were inoculated with fresh cells, andincorporation was determined in cold 5% trichloro-acetic acid-insoluble material. Up to 47% of the 14Cwas incorporable byfresh cells after 0 min depletion (0,and up to 10% after 90 min depletion, *).

TABLE 1. Incorporation of DZU-2-14C intotrichloroacetic acid-insoluble material ofE. coli B growing in mineral salts-glucose(MSG) medium at 37 C after 90 min ofpreincubation of the DZU at 37 C invarious components ofMSG medium

DZU incorporated afterpreincubationb (per cent

of max)Salt solutiona .-

With glucose gWitcosuet

KH2PO4/K2HPO4 -Mg++ 41 14Sodium citrate-Mg.....-M 24 16(NH4)2SO4 - Mg .......... 42 25Mineral salts complete +Mg+.................... 45 26

a All solutions were adjusted to pH 7.b Specific activity of DZU, 0.757 c/mole.

DZU in cytoplasmic macromolecules. Fromthe preceding evidence, DZU incorporation anddivision inhibition appeared to be concomitant.It was thus relevant to examine the nature of theincorporated DZU. E. coli B in MSG at 37 Cwas permitted to form filaments for 90 min with0.5 ;Ag of DZU-2-14C per ml; the filaments wereharvested and disrupted in a French pressure cell

191VOL. IOI, 1970

on March 2, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

192PREVIC AND FISTER

TABLE 2. Solubilization of DZU-2-14C fromcytoplasmic fractions of E. coli

Per cent 14C release intodialysatea

Treatment Extract afterNative treatment atextract 100 C for

5 min

None+ PVSb ................. 0 0- PVS.................. 18.1 3.4

Pancreatic ribonuclease (1mg/ml)

+ PVS ................. 0 0- PVS ............... 19.4 41.2

a Dialyzed against nine volumes of 0.1 M sodiumphosphate buffer (pH 7) at 37 C.

b Polyvinylsulfate potassium salt.

as previously described. After centrifugation ofwall-membrane debris, the supernatant fluid(cytoplasmic fraction) contained 95 to 96% ofthe radioactivity that had been retained by washedwhole cells. The 4% in the pellet could be re-leased by resuspension and resedimentation ofthe wall pellet. Hence, it was probably cytoplas-mic material that had been mechanically oc-cluded. Cytoplasmic fractions were prepared inthe absence and in the presence of PVS. Of eachtype, four 1-ml samples were taken and two wereheated at 100 C for 5 min. One heated and oneunheated sample were dialyzed without furthertreatment. Pancreatic ribonuclease (1 mg/ml)was added to one heated and one unheated samplebefore dialysis. All samples were dialyzed against0.1 M sodium phosphate buffer (pH 7.0) satu-rated with chloroform. Dialysis was at 37 C for24 hr. In all cases, the solubilization of radioac-tivity, i.e., appearance of "4C in the dialysate, hadreached its maximal level before 24 hr. The percent "4C released, measured at 24 hr, is shown inTable 2 for the various cases. Solubilization bypancreatic ribonuclease was effectively blockedby PVS. It was assumed that endogenous ribonu-clease, which was largely heat denaturable, ac-counted for solubilization in the absence of addedenzymes. The failure of exogenous ribonucleaseto completely release the radioactive label fromcrude cytoplasm might have been explained bythe presence of label in a nonsusceptible form ofRNA, as in ribosomal particles. Alternativelythe remainder of the label might have been inmacromolecules other than ribonucleic acids.To resolve this question, RNA was isolated andpurified to determine whether it contained all ofthe DZU-2-14C label and whether the 14C in the

purified RNA was completely and specificallysusceptible to solubilization by ribonuclease. E.coli B was filamented for 90 min with 0.5 mg ofDZU-2-'4C per ml and was harvested as before.The resuspended cells, after addition of 2.0 ,ugof PVS per ml, were disrupted, and the RNA wasisolated in the manner previously described. Table3 summarizes the isolation of RNA containingDZU-2-14C. Recoveries of radioactivity in puri-fied RNA were 95 to 105% of those in the cyto-plasmic fractions. A typical experiment yielded35 mg of purified RNA-(DZU-2-14C), specificactivity 1.46 X 105 counts/min per mg, from1,500 ml of culture at an optical density of 0.60.The RNA-(DZU-2-14C) was dissolved in 0.1 Msodium phosphate (pH 7.5) and 1.0-ml samples,untreated or treated with various enzymes, weredialyzed against 9.0 ml of the same buffer for 24hr at 37 C. Dialysates were assayed periodicallyfor radioactivity. In Table 4, the per cent of 14Csolubilized is shown after 24 hr of incubation. Re-lease of counts is also shown at 24 hr and atselected earlier times. Pancreatic ribonucleasealmost completely solubilized label from RNA-(DZU-2-14C), and was inhibited by 2 MAg of PVSper ml. Deoxyribonuclease did not permit therelease of DZU-radioactivity from the purifiedRNA.

DISCUSSION

The capacity of DZU to induce the formationof filaments by rod-shaped bacteria (3) is reason-ably selective. Under appropriate conditions,the rates of mass increase and protein, RNA,and DNA synthesis are not grossly affected, andthe growth rate can be nearly normal for longperiods in the absence of cell division (4). Theseobservations and the possibility of relating theactivity of the inhibitor to its metabolic fate gavepromise that DZU might become an unusuallyuseful tool in the study of bacterial cell division.We have described the incorporation of radioac-tively-labeled DZU into trichloroacetic acid-insoluble components of E. coli B. When addedover a range of concentrations (0.1 to 0.5 ,ug/ml),only about 50% of the DZU-2-14C was incor-porated, but essentially all that becomes insolublecan be found in ribonucleic acids. Under certainconditions, such as preincubation in mediumwithout bacteria, a conversion occurs in the DZUthat leads to loss of incorporability and a con-comitant loss of filament-forming capacity. DZU,acting as an analogue of a normal base, mightlead to defective messenger RNA (mRNA),with consequent errors in the translation topolypeptides, but this should apply to its incor-poration into all types of mRNA. If there are

192 J. BACTERIOL.

on March 2, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

DIAZOURACIL-2-14C IN RNA OF E. COLI

TABLE 3. Incorporationi of DZU-2-14C into ribonucleic acids of E. coli B after treatment for 90 min inmineral salts-glucose medium at 37 C

Determination Medium No. of counts/ Total vol (ml) Total counts/min Recoverymin per ml tavo(m)Ttlcut/m Roer

Growth A, total culturea 2,942 102 30 X 104 100%Harvest B-1, washed cells, resuspended 13,238 10.2b 13.5 X 104 45%

B-2, medium plus washings 14,440 103 14.8 X 104 49%

Disruption C, French cell effluate 10,090 13.4b 13.5 X 104 100% of B-1

Fractionation D-1, cytoplasmic fractionc 8,656 15.0 13.0 X 104 D-1 + D-3 =99% B-1

D-2, resuspended wall-debris 1,056 5.0 0.53 X 104D-3, 2nd supernatant fluid 657 5.0 0.33 X 104D-4, 2nd wall-debris pelletd 320 5.0 0.16 X 104 1% B-1

Extraction E-1, phenol phasee 120 17.0 0.20 X 104 2% B-1E-2, aqueous phasee 9,703 13.0 13.0 X 104 96% B-1

Precipitation F-1, aqueous-ethylalcohol su- <10 25.0pernatant fluidf

F-2, redissolved, purified 14,140 10.0 14.0 X 104 104% B-IRNA

a Culture radioactivity and volume after chilling with frozen buffer.bIncreased French cell effluate volume includes washes.¢ After centrifugation of French cell effluate.d From recentrifugation of resuspended wall-debris.eAfter reextraction and pooling of extracts.f Combined supernatant fluids from 1st and 2nd precipitation of RNA by ethyl alcohol.

TABLE 4. Solubilizationz of radioactive label from RNA-(DZU-2-14C) purified as summarized in Table 3

No. of counts/min per ml in dialysate at of initial dsC at 24braTreatment

2 hr 6 hr 12 hr 24 hr Soluble Insoluble

None............. 62 91 383 665 2.9 93.2

Pancreatic ribonuclease (0.1 mg/ml)....... 6924 17,448 21,106 22,032 96.6 12.5

Deoxyribonuclease (0.1 mg/ml) ........... 310 776 1,518 2,762 12.1 91.9

a Solutions of 1 ml (5.2 mg/ml, specific activity 4.4 X 104 counts/min per mg) were dialyzed against 9ml of 0.1 M phosphate buffer (pH 7.5) at 37 C.

division-specific types of mRNA, it is difficult toimagine that these could have an extraordinarysusceptibility to DZU that could account for theselective action of that agent against cell division.Although incorporation of label into RNA

and inhibition of cell division seem related, noevidence was presented here that the radioactivelabel was retained in a moiety with the unalteredmolecular composition of DZU. Studies are inprogress to define the nature of the labeled portionin isolated RNA. One problem is that the DZUrepresents only 1 to 2% of the total base contentof the RNA, with the usual filamenting doses.Another problem is the potential instability of

diazonium compounds. Preliminary results indi-cate that at least a portion of the labeled materialmay have lost the nitrogen of the diazonium groupin the course of the treatments. If this is the case,and if the loss occurred in pathways subsequentto accumulation of the compound into intra-cellular pools, an examination of intermediatesin the conversion of DZU leading to RNA mayyet reveal its significance to the cell-divisionmechanism.The choice of DZU, because of its selective

division-inhibitory action, was balanced againstthe probable chemical instability that mightcomplicate an understanding of its mode of ac-

193VOL. 101,9 1970

on March 2, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

PREVIC AND FISTER

tion. Elementary analysis on the solid compound,as isolated in the synthetic procedure, suggestedthat it was a hydrated form with a configurationas in Fig. 1. Compounds of this type, termeddiazotic acids or diazohydroxides, are usuallystable in solution only above pH 9 as their alkalimetal salts. At neutral or acid pH, they convertto diazonium ions and become unstable or highlyreactive (6). Hypothetical reactions for diazo-uracil are shown in Fig. 5. The formation of azodyes with tyrosyl residues (Fig. 5b) or with otheramino acids has been ruled out by the appear-ance in RNA of the 14C label from DZU. Sinceintact DZU has not been identified in the labeled

HO HO + N HONN ' N2 OHHDl,

I 11~~~~~~~~~I Hg20 1IcCHN CHHC0' ~N HO'CN"' (a) H6' N

HC,,CH

/(b)

H,O O,H

NO;C ,C,N=N,C ;Ct11 11 1

CC4,CH HCs ,CHHO N ~ C

N "I0+ 11HCN vN

(C) OH

HC11

N C- ,N=N a

IIHO '4N"

9H

I 11 IHOC>+ CH OH

,NI/ syn-

HO,N

OH 11N'CN N

I 11HOCK CH

N/I/ OH

9H OHN

N C c<N /OH I 1'NN !~~~,-

CN

HOC CH

'Z~'N"'- anti- HOC CH

FIG. 6. Geometrical isomers ofDZU (hydrate). Allconfigurations are co-planar with the page.

portion of the RNA, the likelihood of hydrolysiswith loss of nitrogen (Fig. 5a) must be considered.In this case 5-hydroxy-uracil would be a product,and its importance to the cell division inhibitionmust, therefore, be assessed. Direct reaction ofDZU with a pyrimidine (Fig. 5c) is not excluded,since there has been no confirmation that theobserved incorporation is by substitution for anormal base. These problems will be examinedin a subsequent publication.

In addition to easy chemical convertibility, theoriginal preparation might have been a mixtureof geometrical isomers of the same molecularspecies. The formulae of Fig. 1 are over-simpli-fied. If there is a hydroxyl group covalentlybound to a diazo-N, then the orbitals of the dia-zonium N = N group would be of the wr-bondtype and would (i) be planar, (ii) be coplanarwith the uracil ring when the latter is in an aro-matic (lactim) form, and (iii) have syn- and anti-forms (2). A variety of configurations is thuspossible (Fig. 6). The coplanarity tends to re-strict free rotation of the N-C bond so the diazo-hydroxide has "up" and "down" configurationsfor each of the syn- and anti- forms. Attempts arebeing made to determine the significance, if any,that geometrical ambiguity may have on thebiological reactivity of DZU. For example,active transport might be selective for a par-ticular geometric form.The implications of the structural chemistry of

DZU are interesting, but they serve at the presentstage to complicate our evaluation of it in theinhibition of bacterial cell division. The originalidea was that its chemical involvement in cellmetabolism could hopefully expose its mechanismof action as a filamenting agent. This may yetbecome true, but not as simply as anticipated. Itsselectivity against cell division makes it still at-tractive as a tool in studying the process. Forexample, a population of filaments with the samemass growth rate versus time as a normallydividing culture should have a characteristic rateof cell envelope formation relative to its mass.The composition of the cell wall-membrane com-plex, and its cross-linking patterns, should alsobe characteristic. It may be more meaningful tostudy these parameters in DZU filaments thanin systems in which filamentation is accompaniedby gross aberrations in envelope polymerizations(7). Studies are in progress to compare cell wallcomposition in normal and DZU-filamentedcells to see whether there are any significant dif-ferences.

ACKNOWLEDGMENTSWe thank E. C. Bates for technical assistance.This investigation was supported by Public Health Service

research grant A1-07541 from the National Institute of Allergy

FiG. 5. Hypothetical reactions of diazouracil basedon the charged diazonium ion intermediate. (a) Hydroly-sis to 5-hydroxyuracil. (b) Formation of azo-dye-typebonds with tyrosyl residue in a polypeptide. (c) Forma-tion of azo-dye-type bonds with uracil residue in anucleic acid.

194 J. BACTERIOL.

on March 2, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

DIAZOURACIL-2-14C IN RNA OF E. COLI

and Infectious Diseases and by a stipend to G. Fister from train-Ing grant STI AI-0128-09 from the same agency.

LITERATURE CITED

1. Johnson, T. B., 0. Baudisch, and A. Hoffman. 1931. Forma-tion of diazouracil anhydride from aminouracil. Berichte64B :2629-2631.

2. Karrer, P. 1950. Organic chemistry, 4th English ed. ElsevierPublishing Company, Inc., Amsterdam.

3. Loveless, L. E., E. Spoerl, and T. H. Weisman. 1954. Asurvey of effects of chemicals on division and growth ofyeast and Escherichia coli. J. Bacteriol. 68:637-644.

4. Previc, E., and S. Richardson. 1969. Growth-physiological

195

changes in Escherichia coli and other bacteria during divi-sion inhibition by 5-diazouracil. J. Bacteriol. 97:416-425.

5. Roberts, J. D., and M. C. Caserio. 1964. Basic principles oforganic chemistry. W. A. Benjamin, Inc., New York.

6. Scherrer, K., and J. Darnell. 1962. Sedimentation character-istics of rapidly labeled RNA from HeLa cells. Biochem.Biophys. Res. Commun. 7:486-490.

7. Weinbaum, G., and S. Okuda. 1968. Inhibition of envelopepolymerizations in filamentous Escherichia coil B. J. Biol.Chem. 243:4358-4363.

8. Wyatt, G. R. 1955. Separation of nucleic acid components bychromatography on filter paper, p. 243-265. In E. Chargaffand J. N. Davidson (ed.), The nucleic acids, vol. I. Aca-demic Press Inc., New York.

VOL. 101, 1970

on March 2, 2021 by guest

http://jb.asm.org/

Dow

nloaded from