JOURNAL OF BACTERIOLOGY, Dec. 1973, p. 1398-1404Copyright i 1973 American Society for Microbiology

Vol. 116, No. 3Printed in U.S.A.

Inhibition of Deoxyribonucleic Acid Synthesisand Bud Formation by Nalidixic Acid in

Hyphomicrobium neptuniumRONALD M. WEINER AND MARCIA A. BLACKMAN

Department of Microbiology, University of Maryland, College Park, Maryland 20742

Received for publication 14 August 1973

The relationship between chromosome replication and morphogenesis in thebudding bacterium Hyphomicrobium neptunium has been investigated. Nali-dixic acid was found to completely inhibit deoxyribonucleic acid synthesis, butnot ribonucleic acid synthesis. The antibiotic was bacteriostatic to the organismfor the initial 5 h of exposure; thereafter it was bacteriocidal. Observation ofinhibited cultures revealed cells that had produced abnormally long stalks, butno buds. These results indicate that bud formation is coupled to chromosomereplication in H. neptunium. They do not exclude the possibilities that cross wallformation and bud separation may also be coupled to chromosome replication.

Hyphomicrobium neptunium is a procaryotewith a multiphasic developmental cycle (Fig.1). A swarmer cell (Fig. 1A), about 0.5 gtm indiameter, loses its flagellum (Fig. 1B) andbecomes a pear-shaped cell (Fig. 1C) that isabout 1.0 um long. A stalk, about 0.2 ,um wide,appears from the pointed end of this cell andnormally assumes a length of from 1.0 to 3.0 gm(Fig. 1D). From the tip of the growing stalk, abud emerges (Fig. 1E). The bud forms a septum(Fig. 1G), produces a flagellum distal to thestalk attachment site (Fig. 1F), and separatesfrom the parent. The stalked cell continues toproduce buds while the progenies reinitiate thelife cycle (12).Toward understanding the regulation of this

cycle at the level of chromosome replication, weinvestigated the applicability of using the de-oxyribonucleic acid (DNA) inhibitor, nalidixicacid. This antibiotic specifically inhibits DNAsynthesis in some gram-positive and gram-negative bacteria (3, 9), whereas in Caulobactercrescentus, it stops ribonucleic acid (RNA)synthesis and only partially inhibits DNA syn-thesis (5).

Additionally, in Escherichia coli, DNA syn-thesis is coupled to cell division (2, 10); whereasin some strains of Bacillus subtilis, recentevidence indicates that it is not (6).We show that H. neptunium is sensitive to

nalidixic acid, that it quickly blocks DNA butnot RNA synthesis, and that multiplication isdependent upon chromosome replication in thisorganism.

A portion of this work partially fulfills therequirements of M. Blackman for the M.S.degree from the University of Maryland, CollegePark, and was presented at the Annual Meetingof the American Society for Microbiology,Miami Beach, Fla., 6 to 11 May 1973.

MATERIALS AND METHODSBacteria and growth conditions. H. neptunium

was obtained from the American Type Culture Collec-tion (ATCC 15444). Cells were grown at 36 C in250-ml culture flasks in a New Brunswick gyratoryshaker set at 220 rpm. Culture broths were preparedby adding 11.22 g of marine broth (Difco 2216) to1,000 ml of distilled water. To prepare solid mediumfor plate counts and maintenance slants, agar (Difco)was also added to a concentration of 1.5%.

Metabolic inhibitor. Nalidixic acid was kindlysupplied by S. Archer of the Sterling-Winthrop Re-search Institute, Rensselaer, N.Y. Stock solutionswere prepared by dissolving 1 g of the antibiotic in 100ml of 0.1 N NaOH. During the tracer and growthexperiments, 1.0 ml of this stock solution was addedper 100 ml of marine broth to a final concentration of100 ug of nalidixic acid/ml. This addition to theculture medium did not raise the pH more than 0.03U.Measurements of cell growth and division. Stan-

dard plate counts on marine agar were made to titerviable cells. Dilutions were prepared in distilled wateror physiological saline. The cells remained fully viablefor more than 1 h in either diluent. In a culture in thelogarithmic phase of growth, we observed approxi-mately 2% of the cells in rosette formation, and someof these were disbursed by optimal mixing on aVortex mixer (Super-Mixer, Lab-Line Instruments,Inc., Melrose Park., Ill.) for 1 min just prior to plating.

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EFFECT OF NALIDIXIC ACID ON H. NEPTUNIUM

Total cell titers were ascertained on a Petroff-Haus-ser counting chamber in a Zeiss I photomicroscope(Baltimore Instrument Co., Baltimore, Md.), by usinga plan 16/0.35 objective lens in conjunction with aphase 3 condenser setting. Under these conditions,stalks were visible while a broad field was observed.

Turbidity was measured in a spectrophotometer(Spectronic 20, Bausch & Lomb, Inc., Rochester,N.Y.) at 540 or 600 nm by using uninoculated mediumas a blank.Assay of RNA and DNA synthesis. Initially,

thymidine-2-14C (International Chemical and Nu-clear Corp., [ICN] Irvine, Calif., 59.6 mCi/mmol) wasused to label DNA, and uracil-2-14C (ICN, 50.8mCi/mmol) was used to label RNA; however, thesetracers were incorporated at relatively low ratesinitially and not at all after 4 h. Therefore, adenine-8-"4C (ICN, 50 mCi/mmol) was used to label both RNAand DNA during the nalidixic acid inhibition experi-ments.To study the uptake of the various radioactive

precursors into macromolecules, a modification of themembrane filtration and fractionation technique de-scribed by Roodyn and Mandel (14) was employed. Tomeasure incorporation of the labeled purine into RNAand DNA, 2-ml samples of the culture were placed atintervals in equal volumes of 10% solutions of trichlo-roacetic acid and kept at 0 C for 30 min. Theacid-treated samples were then passed through mem-brane filters (Millipore Corp., HAWP, 0.45-am poresize, 47-mm diameter). Each filter was thoroughlywashed with 15 ml of a cold 1% trichloroacetic acidsolution and suspended in toluene scintillation fluid(15 ml/vial) containing 1.0% 2,5-diphenyloxazole and0.05% 1, 4-bis-2-(5-phenyloxazolyl)-benzene (PackardInstrument Co., Inc.). Radioactivity was determinedin a Unilux II scintillation counter (Nuclear-ChicagoCorp., Des Plaines, Ill.).To measure incorporation into the DNA fraction,

additional 2-ml samples of the culture were placed intest tubes containing 0.2 ml of a 5.5 N NaOH solution,and the mixtures were incubated at 37 C for 15 h.After this period, the suspensions were placed in anice bath and acidified with 0.2 ml of 6 N HCl and 2 mlof 10% trichloroacetic acid. The suspensions werefiltered, and the radioactivity was determined aspreviously described. Measurements of incorporationof the tracer into the RNA fraction were obtained bysubtracting the counts of the sample treated with theNaOH from those treated solely with trichloroaceticacid. The efficiency of counting ranged from 60 to77%.

Light microscopy. The cultures were negativelystained with nigrosin that was prepared according tothe methods of Dorner (7). A 0.5-ml portion of theculture was mixed with 0.5 ml of the stain, and themixture was smeared across a slide. The preparationswere observed and photographed in a Zeiss I photomi-croscope which was equipped with x 12.5 oculars, 1.25optovar, and a planachromat x 100/1.25 numericalaperture objective lens. Kodak Tri-X Pan film wasused for photography.

Electron microscopy. Cultures were washed andconcentrated by centrifugation, and the cells weresuspended in distilled water. One drop of the bacterial

AB0

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F EFIG. 1. Life cycle of Hyphomicrobium neptunium.

Morphogenic stages are identified and described inthe text.

suspensions was placed on a 200-mesh, Athene-type,copper grid covered with a Formvar film. This wasfollowed by negative staining by adding 1.0% (wt/vol)aqueous potassium phosphotungstate (pH 7.0). Theexcess was immediately removed by touching filterpaper to the droplet edge. The preparations wereallowed to dry and were then examined and photo-graphed in an RCA EMU-3F electron microscope.

RESULTSEffect of nalidixic acid on DNA and RNA

synthesis. Within 1 min after exposure to 100Ag of nalidixic acid/ml, the incorporation oflabeled adenine into the macromolecular frac-tion of DNA of H. neptunium was totallyinhibited. This inhibition was followed and wasfound to be complete for at least one observeddoubling time of 1.9 h (Fig. 2). The antibiotichad no effect upon the rate of incorporation ofthe purine into RNA, per unit of cell mass, overthe same period of time. These data are consist-ent with our observations and micrographsshowing continued cell growth in the presence ofthe antibiotic. We conclude that nalidixic acidinhibits DNA synthesis and that its action isspecific, immediate, and complete in H.neptunium.

Effects of nalidixic acid on growth, divi-sion, and viability. In logarithmically growingbatch cultures, the time between cell divisionsof the heterogeneous population averaged 115min, although observations of individual cellsimbedded in agar revealed the doubling time forstalked cells to be shorter than the doublingtime for swarmer cells (manuscript in prepara-tion).

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WEINER AND BLACKMAN

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FIG. 2. Effect of nalidixic acid on RNA and DNAsynthesis in H. neptunium. Cells in the logarithmicphase of growth were added to marine broth contain-ing 14C-adenine (final concentration, 0.01 4Ci/ml).The initial cell number was 2 x 108/ml. The culturewas sampled for incorporation for 1.3 generations andsplit, one sample being transferred to a flask contain-ing nalidixic acid and the other serving as a control.Results are shown as a differential plot of incorpo-rated radioactivity versus relative increase in turbid-ity. Details of optical density measurements andtracer assays are described in the text. RNA synthe-sis, control (0); DNA synthesis, control (0); RNAsynthesis, 100 ug of nalidixic acid/ml (A); DNA syn-

thesis, 100 ,g of nalidixic acid/ml (A).

The addition of nalidixic acid immediatelyinhibited cell division of most of the population(Fig. 3A, B). The small increase in cell titer,shortly after the addition of nalidixic acid, can

be attributed to the division of those cells thathad already triggered, though not completed,cell division just prior to being exposed to theantibiotic (1, 10). Nalidixic acid was bacterio-static to H. neptunium during the first 5 h ofexposure. During this time, the effects could bereversed by dilution. Thereafter, the inhibitorwas bacteriocidal (Fig. 3A).The culture to which nalidixic acid was added

at 12 h into the experiment had a more rapidkill rate than the culture that was exposed tothe inhibitor immediately. We believe that thisis a reflection of different rates of growth of therespective cultures at the time of addition of the

inhibitor (the former culture being in middle loggrowth and the latter in late log growth). Thisdoes not contradict earlier findings that nali-dixic acid is lethal only to growing populationsof E. coli (8) and B. subtilis (3) in which thedegradation of DNA and loss of viability arecorrelative. Moreover, it has been reported thatthe DNA is sequentially degraded, at the repli-cation fork, from the most recently replicatedDNA to the older DNA (13). This is consistentwith our observations that the most rapidlydividing H. neptunium, presumably with moreexposed replication forks, are most quicklykilled in the presence of the antibiotic.

Figure 3C shows that the optical densities ofthe inhibited cultures increase about fourfold.Similarly, our observations and micrographs ofnalidixic acid-treated cultures show a largeincrease in cell mass.

Effects of nalidixic acid on morphology.Growth studies showed that in the presence of100 tsg of nalidixic acid/ml the cells failed tomultiply. If one or more events in the progres-sion of the morphogenic cycle were coupled to,and dependent on, continuing chromosome rep-lication, then further development would havebeen halted at that (or those) stage(s) and,indeed, normal division could not have con-tinued. If only one morphogenic block wereexerted, then all cells in a batch culture con-taining the inhibitor would progress to andremain frozen at the stage of developmentproximal to the block. Therefore, to identify theinhibited stage(s) of development, observationsand micrographs of cultures were made. Loga-rithmically growing control cultures of H.neptunium contained cells in all stages of devel-opment, including swarmers, stalked cells, andbudding forms (Fig. 4A). Cells exposed to theinhibitor developed atypically. One of the morebizarre forms is shown in Fig. 4B. The cell, itselfelongated, had produced stalks at both poles.These events were uncommon occurrencesamong nalidixic acid-treated populations. Inother aspects, however, namely severe stalkelongation and the absence of bud formation,the cell was representative of at least 90% of theorganisms in a culture exposed to the antibioticfor 5 h (Fig. 4C).To see whether nalidixic acid promoted more

subtle morphological aberrations and to observethe fine structure of inhibited cells, electronmicrographs were prepared. Cells from a controlculture are shown in Fig. 5A. Stalks are 1 to 2,gm long and 0.2 um in diameter. Where thestalked cells formed buds, the progenies wereseparated from the parent by septa. On theother hand, the inhibited cell (Fig. 5B) pro-

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EFFECT OF NALIDIXIC ACID ON H. NEPTUNIUM

loll duced a stalk 17-,um long, that remained 0.2 gmA in diameter. The cell had not formed a bud.

However, an area where the stalk began to10'° _ swell, constrict, and then swell again was appar-

* ent. The tip of the stalk was also swollen,although no septum formed. We observed simi-lar morphologies among approximately 20% of

10 _ the nalidixic acid-treated population. The re-> / v mainder of the inhibited cells produced uni-z O form, though elongated, stalks and, likewise,

ios108 _ K _ did not form buds.

) - DISCUSSIONLLJ 17 X In bacteria like E. coli, which have life cycles'0 consisting primarily of cell elongation followed> ^ >> by binary fission, there is ample evidence (2, 3,

10, 11) that cell division can be dependent upon06 _ chromosome replication. Less is known about

similar controls governing various develop-mental stages of procaryotes with more elabo-rate life cycles (5, 15, 16).

1 0'°-B We report here that the morphogenesis of H.010 B _ neptunium (Fig. 1), from stages A through D, is

not directly dependent upon chromosome repli-cation. We found that progression to stage E is

N.w IO9 _ coupled to DNA replication. The possibility of aco/ 0 0 similar coupling of the events necessary to

recycle cell type E to D (plus A) awaits furtherZ clarification.

I ,Y The action of nalidixic acid enabled us toA/̂ ^ ^ observe control of morphogenesis at the level of

chromosome replication. It was ideally suited to107 , _ this experimental design in that it specifically

o inhibited DNA synthesis of H. neptunium, yetdid not impair protein, RNA synthesis, or cell

I I I I viability for at least one generation time. There-0.7 - _ fore, cells in a batch culture were able to

C proceed through successive morphogenic stages.06-

Where continued development was coupled to0.6 _ / _ DNA replication, however, the cycle termi-

EE nated, and cells were frozen at the stage proxi-c 0.5 - / _ nmal to the block. As a side benefit, the dis-o covery that the effects of nalidixic acid can be(0 reversed for up to 5 h permits us to make use>- 0.4 - - of the antibiotic in establishing synchronousH l .D*--o populations.z In the presence of nalidixic acid, swarmera 0.3_ _

_

I/ FIG. 3. Effects of nalidixic acid on growth, divi-O. 0.2 sion, and viability of H. neptunium. Culture condi-H ° ~ /, _ tions and titering methods are described in the text.O \ /)6 Three flasks were inoculated with a culture in the late0' logarithmic phase of growth to an initial titer of0. _ vvv _ approximately 1 x 107 cells/ml. One vessel served as a

A Uf^^ ^ , .control (0), a second contained the inhibitor (A), andthe third (0) received the inhibitor (-) 12 h into the

102 experiment. Results are shown as changes in (A)0501520 25 viable titer, (B) total cell number (from microscopic

TIME IN HOURS counts), and (C) turbidity versus time.

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WEINER AND BLACKMAN

FIG. 4. Bright-field micrographs of negatively stained H. neptunium: (A) control culture in the logarithmicphase of growth, (B) single cell exposed to nalidixic acid for 5 h, and (C) cells exposed to nalidixic acid for 5 h.Methods of cultivation, staining, and photography are described in the text. Bars represent 2 Mm.

cells lose their flagellum, become pear-shaped,and produce stalks. In most instances, the cellproper does not elongate nor does the stalkwiden beyond its normal dimension of 0.2 Am.However, occasionally, inhibited cells do formstalks with an interior area of swelling andconstriction (Fig. 5B). Infrequently, too, the tipof the stalk becomes swollen (Fig. 5B) as

though, perhaps, bud formation is initiatedthough not completed. The causes of the aberra-tions are unclear. Electron micrographs of thin-sectioned cells are expected to reveal whethersuch formations contain DNA, poly-,8-hydroxy-butyrate granules, and/or unique membraneconfiguration.

In control cultures in the logarithmic phase of

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FIG. 5. Electron micrographs of negatively stained H. neptunium: (A) control culture in the logarithmicphase of growth, and (B) single cell exposed to nalidixic acid for 5 h. Methods of cultivation, staining, and pho-tography are described in the text. Bars represent 1 pm.

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WEINER AND BLACKMAN

growth, 25% of the population consists of cells inthe process of budding. In contrast, when nali-dixic acid is added to such a culture, after 4 habout 5% of the population is comprised of cellswith buds. This is consistent with our findingsof a slight increase in cell titer within 20 minafter the addition of the inhibitor (Fig. 3).Therefore, we feel that it is unlikely that truebuds form when chromosome replication isinhibited.The data are insufficient to determine the

dependencies of cross wall formation and budseparation on continuing chromosome replica-tion. Assuming that bud formation is inhibitedby nalidixic acid, then about 80% of the cellsthat had formed buds, but had not yet divided atthe moment the inhibitor was added, did dividewith DNA replication blocked. Still, it is possi-ble that the two events are coupled to chromo-some replication. If so, the cells that underwentdivision in the inhibitor's presence might havepreviously completed sufficient chromosomereplication necessary to trigger cell division (1,5, 10). If this hypothesis is accurate, then thehigh percentage of inhibited cells with budsthat release progeny is indicative of a possiblecorrelation between bud formation and the com-pletion, or near completion, of a round ofchromosome replication.

Alternatively, it is conceivable that cross wallformation and bud separation are not coupledto chromosome replication in H. neptunium.The low numbers of replication-inhibited cellsthat retain their buds may not be viable.To examine these possibilities, it would be

valuable to develop a technique for synchroniz-ing populations of H. neptunium. It would thenbe possible to apply the inhibitor at sequen-tially timed intervals of development. A clocklinking DNA replication to morphogenic eventscould then be constructed (4).

ACKNOWLEDGMENTS

We thank Zigfridas Vaituzis for taking the electron micro-graphs. We appreciate his assistance and helpful discussion.

This work was supported in part by a General Research

Board Award and a Biomedical Sciences Support Grant toR.M.W.

LITERATURE CITED

1. Clark, D. J. 1968. Regulation of deoxyribonucleic acidreplication and cell division in Escherichia coli B/r. J.Bacteriol. 96:1214-1224.

2. Clark, D. J. 1968. The regulation of DNA replication andcell division in E. coli B/r. Cold Spring Harbor Symp.Quant. Biol. 33:823-838.

3. Cook, T. M., K. G. Brown, J. V. Boyle, and W. A. Goss.1966. Bacteriocidal action of nalidixic acid on Bacillussubtilis. J. Bacteriol. 92:1510-1514.

4. Degnen, S. T., and A. Newton. 1972. Chromosomereplication during development in Caulobactercrescentus. J. Mol. Biol. 64:671-680.

5. Degnen, S. T., and A. Newton. 1972. Dependence of celldivision on the completion of chromosome replicationin Caulobacter crescentus. J. Bacteriol. 110:852-856.

6. Donachie, W. D., D. T. M. Martin, and K. J. Begg. 1971.Independence of cell division and DNA replication inBacillus subtilis. Nature N. Biol. 231:274-276.

7. Dorner, W. C. 1930. The negative staining of bacteria.Stain Technol. 5:25-27.

8. Goss, W. A., W. H. Deitz, and T. M. Cook. 1964.Mechanism of action of nalidixic acid on Escherichiacoli. J. Bacteriol. 88:1112-1118.

9. Goss, W. A., W. H. Deitz, and T. M. Cook. 1965.Mechanism of action of nalidixic acid on Escherichiacoli. II. Inhibition of deoxyribonucleic acid synthesis. J.Bacteriol. 89:1068-1074.

10. Helmstetter, C. E., and 0. Pierucci. 1968. Cell divisionduring inhibition of deoxyribonucleic acid synthesis inEscherichia coli. J. Bacteriol. 95:1627-1633.

11. Jones, N. C., and W. D. Donachie. 1973. Chromosomereplication, transcription and control of cell division inEscherichia coli. Nature N. Biol. 243:100-103.

12. Leifson, E. 1964. Hyphomicrobium neptunium sp. n.Antonie van Leeuwenhoek. J. Microbiol. Serol.30:249-256.

13. Ramareddy, G., and H. Reiter. 1969. Specific loss ofnewly replicated deoxyribonucleic acid in nalidixicacid-treated Bacillus subtilis 168. J. Bacteriol.100:724-729.

14. Roodyn, D. B., and H. G. Mandel. 1960. A simplemembrane fractionation method for determining thedistribution of radioactivity in chemical fractions ofBacillus cereus. Biochim. Biophys. Acta 41:80-88.

15. Steinberg, W., and H. 0. Halvorson. 1968. Timing ofenzyme synthesis during outgrowth of spores of Bacil-lus cereus. II. Relationship between ordered enzymesynthesis and deoxyribonucleic acid replication. J.Bacteriol. 95:479-489.

16. Zusman, D., and E. Rosenberg. 1968. Deoxyribonucleicacid synthesis during microcyst germination in Myx-ococcus xanthus. J. Bacteriol. 96:981-986.

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