JOURNAL OF BACTERIOLOGY, Feb. 1983, P. 813-8210021-9193/83/020813-09$02.00/0Copyright C 1983, American Society for Microbiology

Vol. 153, No. 2

Formation of Competent Bacillus subtilis CellstYOSHITO SADAIE* AND TSUNEO KADA

Department ofInduced Mutation, National Institute of Genetics, Mishima, Shizuoka-ken 411, Japan

Received 30 March 1982/Accepted 19 October 1982

The process of competent cell formation for transformation has been studiedwith early-stationary-phase (T1) cells ofBacillus subtilis which had been grown inan enriched Spizizen minimal medium and transferred to a second syntheticmedium. Rifampin, chloramphenicol, and tunicamycin were strong inhibitors ofcompetent cell formation, as well as vegetative growth. After formation, compe-tent cells were no longer sensitive to the above agents. Methicillin and an inhibitorof chromosomal replication, hydroxyphenylazouracil, did not inhibit the develop-ment of competence. A D-alanine-requiring mutant strain developed competenceeven in the absence of D-alanine in the second medium. A Tl-stage cultureshowed the activity of extracellular serine protease which is necessary forsporulation. Competent cell formation was completely blocked by 0.7 M ethanol,which is a specific inhibitor of early events during sporulation, including foresporeseptum formation. Competent cells were formed even in media which supportedsporulation. The development of competence was also studied with spoO mutantsat 10 different loci. Most spoO mutations repressed the development of compe-tence except for spoOC, spoOG, and spoOJ. These results suggest that competentcells are formed from early sporulating cells with the synthesis of cell wallmaterials and by factors whose genes are activated by the supply of nutrients. It issuggested that common steps are involved both in forespore septation and incompetent cell formation.

Transformation in Bacillus subtilis is mediatedby competent cells which are metabolically lessactive and smaller in cell size and have a lowerbuoyant density (for a review, see references 20and 33). Competent cells develop during earlystationary phase (6) or in a fresh medium towhich early-stationary-phase cells are trans-ferred (2), depending on the composition of themedium. The supernatant of competent culturesseems to contain factors which induce compe-tence in noncompetent cultures, and these areassumed to be autolysins. Although there is atheory (1) of mesosomal uptake of transformingDNA and there are some suggestions (29, 31)that the development of competent cells is relat-ed to the sporulation process because someasporogenous mutants showed poor compe-tence, questions still remain as to how thecompetent cells are formed and how the compe-tence-inducing factors function (20, 33).

In the present communication, we examinethe conditions necessary for the formation ofcompetent cells from early-stationary-phasecells and discuss the possibility that competentcells are formed during cell division in an earlystage of sporulation.

t Contribution no. 1466 from the National Institute ofGenetics, Mishima, Japan.

MATERIALS AND METHODS

Bacterial strains. The bacterial strains used aredescribed in Table 1. Strain NIG1121M (met) was usedas a recipient strain in most experiments. Strain 168T(thyAl thyBI) (25) was used as a donor for transforma-tion.

Transformation procedures. The competent cellswere prepared by the methods described by Dubnau etal. (10), which are essentially the same as thosedescribed originally by Anagnostopoulos and Spizizen(2). Incubation was at 37°C in all experiments. Anovernight culture of the recipient strain grown inPenassay broth (Difco Laboratories, Detroit, Mich.)was added as a 5% (vol/vol) inoculum into SPI medi-um, which contained Spizizen minimal salts-glucosesupplemented with 0.02% Casamino Acids (Difco),0.1% yeast extract (Difco) and 50 ,ug of amino acid perml. The cells were grown to the end of log phase andthen diluted 10-fold into fresh SPII medium, whichwas made by adding 0.5 mM CaCl2 and 2.5 mM MgCI2to SPI medium. The growth of cells was followed asdescribed before (26). A maximal competence wasobtained around 90 min after the dilution of the firstculture. A competent culture of 1 ml was treated with5.8 Fg of transforming DNA per ml for 30 min followedby DNase (50 ,ug/ml) treatment for 10 min. The num-ber of viable cells was scored on a broth-supplementedVogel-Bonner minimal salts-glucose medium (25) so-lidified with 1.5% Difco agar. Difco nutrient broth wasadded at a concentration of 1% (vol/vol). Amino acidwas supplied at a concentration of 20 >*/ml. The

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814 SADAIE AND KADA

TABLE 1. Bacterial strains

Straina Genotype Source ofspoo168T thyAl thyBIQB928 aroI906 purB33 dal-l trpC2NIG1121M metNIG1121 met hisNIG1131 met his spoOA34 PiggotNIG1132 met his spoOB136 PiggotNIG1133 met his spoOC9 PiggotNIG1134 met his spoOD8 PiggotNIG1135 met his spoOG14 PiggotNIG1136 met his spoOH17 PiggotNIG1137 met his spoOJ87 PiggotNIG1138 met his spoOK141 PiggotNIG1139 met his spoOE81 KawamuraNIG1140 met his spoOF221 Kawamura

a Strain QB928 (9) was obtained from H. Saitothrough Y. Fujita. Strain NIG1121M is a His' trans-formant of NIG1121 (26). All spoO strains are Leu+Spo- transformants of strain HA101 (met his leu) (22)with DNA from original spoO mutants. Original spoOstrains (23) are SL566 (spoOA34 phe-12 rif-2 tal-1),SL964 (spoOB136 metC3 tal-1), SL966 (spoOC9 metC3tal-1), SL225 (spoOD8 metC3 tal-1), SL221 (spoOG14metC3 tal-1), SL513 (spoOH17 rif-2 trpC2), SL670(spoOJ87 metC3 tal-1), SL741 (spoOK141 trpC2),SCR162 (spoOE81 trpC2 lys-3), and UOT274(spoOF221 metBS nonBl).

Vogel-Bonner minimal salts medium was used as adilution buffer.The competent cells were also prepared in the

synthetic medium of Schaeffer et al. (30). Schaef-fer's basal medium contained 1.05% K2HPO4,0.35% KH2PO4, 0.005% MgSO4 * 7H20, 0.0005%FeSO4 * 7H20, 0.005% CaC12, and 0.0005%MnCI2 * 4H20. The basal medium was supplementedwith appropriate carbon and nitrogen sources as de-scribed in the text.

Isolation of transforming DNA. DNA was purified bythe phenol method (28) from cells of strain 168T grownas described elsewhere (25), and its concentration wasdetermined by its absorbance at 260 nm.

Chemicals. Rifampin, chloramphenicol, and methi-cillin were purchased from Boehringer MannheimCorp., New York, N.Y., Sankyo, Tokyo, Japan, andBanyu Co. Ltd., Tokyo, Japan, respectively. Tunica-mycin and hydroxyphenylazouracil were kindly sup-plied by G. Tamura and N. Cozzarelli, respectively.1,10-Phenanthroline and phenylmethylsulfonyl fluo-ride were purchased from Wako, Tokyo, Japan, andSigma Chemical Co., St. Louis, Mo.

RESULTSDevelopment of competence in a synthetic me-

dium devised for transformation. The compe-tence of a culture expressed as the number ofMet+ transformants per recipient cell developedduring vegetative growth in SPI medium anddeclined toward the end of log phase (Fig. 1).The dilution of the culture at the end of log phasewith fresh SPII medium conferred an elevated

competence to the culture about 90 min afterdilution. The dilution of the culture later than 2 hafter the end of log phase was less effective fordeveloping competence (Table 2).

Therefore, with the medium employed in thepresent experiments, the development of com-petence was biphasic. Competent cells appearedduring log phase as well as after the transfer oflate-log-phase cells to SPII medium. The num-ber of viable cells did not increase immediatelyafter dilution in contrast to an immediate in-crease in turbidity. The eventual number oftransformants increased about 100 times in SPIImedium. Since competent cells are penicillinresistant (19) and nondividing, the increase intransformants must mean that new competentcells were being formed in SPII medium.

In the experiment described above, a compe-

Time (h)FIG. 1. Development of competence in SPI and

SPII medium. An overnight culture of NIG1121M inPenassay broth was inoculated in fresh SPI medium5% (vol/vol). At the end of the log phase, a portion ofthe culture was diluted 10-fold into fresh SPII medium.One milliliter of the culture was taken and incubatedwith DNA to examine competence. Symbols: 0, tur-bidity in SPI medium; 0, turbidity in SPII medium; A,Met' transformants in SPI medium; A, Met', trans-formants in SPII medium; O, CFUs in SPI medium; *,CFUs in SPII medium.

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COMPETENT CELL FORMATION OF B. SUBTILlS 815

TABLE 2. Dependence of competence developmenton the length of incubation of the cultures in SPI

mediumNo. of Frequency

Stagea Met+ trans- CFU per of transfor-formants ml (10') mation

per ml (103) (10-4)

To 260 32.0 8.1To.5 93.0 21.6 4.3T, 71.0 15.9 4.5T1.5 70.0 12.9 5.4T2 20.0 13.1 1.5T2.5 5.00 10.3 0.49

a The symbol T,, means n hours after the end of thelog phase. At the indicated time, NIG1121M cellsgrown in SPI medium were diluted 10-fold with freshSPII medium. Competence was measured after 90 minby adding DNA to the culture.

tent culture was incubated with DNA for 30 min.During such a long period, new competent cellsmay be formed or competent cells alreadyformed may lose competence, giving an errone-ous estimation of the number of competent cellsat a specific time. To examine the rate of in-crease of competent cells, DNA was added to acompetent culture for 3 min, followed by DNasetreatment for 2 min. The number of competentcells existing at the time of dilution decreasedwith further incubation (Fig. 2). Eventually thenumber of transformants began to increaseabout 45 min after dilution, roughly in accor-dance with the onset of viable cell division.

Blocking of competent cell formation by inhibi-tors ofRNA and protein or cell wall synthesis. Wenext examined conditions for the developmentof competence in SPII medium. To reduce theinitial number of competent cells already formedat the time of transfer, the culture was diluted 1h after the end of log phase when the compe-tence of the first culture had begun to decline.The inhibitors of DNA, RNA, and protein or

cell wall synthesis were used at concentrationsas low as possible. These drugs were added atthe time of dilution, and the increase of compe-tent cells was examined 60 min after dilution byadding DNA to the culture. In control experi-ments, the drugs were added with the transform-ing DNA 60 min after dilution, and the relativefrequency of transformation was compared un-der these two conditions (Table 3).

Hydroxylphenylazouracil (HPUra) (12, 17),which is a specific inhibitor ofDNA polymeraseIII of B. subtilis, was not effective at a concen-tration of 10 F.M (and even at 100 ,uM; data notshown). New competent cells were formed inthe presence of the inhibitors, whereas an in-crease in the number of the majority of noncom-petent cells was blocked. Therefore, the forma-

tion of competent cells does not requirechromosome replication, which is necessary forcell multiplication. This conclusion is supportedby the finding that the formation of competentcells was seen in thymine-requiring strains in theabsence of thymine (data not shown: 3).Rifampin (0.01 pLg/ml) and chloramphenicol

(1.0 Rg/ml), however, were very effective inhibi-tors and inhibited the formation of competentcells strongly when added at the beginning.These results indicate that de novo transcriptionand translation are required for competent cellformation as was suggested earlier (16). Compe-tent cell formation and the process of transfor-mation were not blocked by the drug added 60min after transfer.Tunicamycin (5 pLg/ml), an antibiotic which

inhibits the synthesis of cell wall materials (32,35), lipid-P-P-N-acetylmuramic acid-pentapep-tide, as well as teichoic acid (18), blocked com-petent cell formation. However, methicillin, asynthetic penicillin, an inhibitor of cross-linkingin peptidoglycan (4), was not inhibitory at aconcentration of 0.1 ,ug/ml, whereas the multipli-cation of noncompetent cells was blocked at thisconcentration. These results may suggest thatthe process of competent cell formation is de-pendent on the synthesis of wall materials butnot on the final process of the cross-linking ofpeptidoglycan.

o0 15 30 45 60 75 9(0Time (nn)

FIG. 2. Rate of development of competence. Onemilliliter of culture in SPII medium was incubated withtransforming DNA (11.6 ,ug) for 5 min followed byDNase (250 ,ug) treatment for 3 min. Met' transform-ants were scored. CFUs were scored just before theincubation of the culture with DNA. Symbols: A,Met' transformant; O, CFUs.

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TABLE 3. Inhibitory nature of antibiotics'Exp 1 Exp 2

Drug (concn) Time Met'/ml CFU/ml Fre- Ratio Met'/ml CFU/ml Fre- Ratio(102) (106) quency (0/60) (102) (106) quency (0/60)(10-4 06) (10-4) /0

HPUra 0 14 28 5.0 2.6 13 21 6.0 2.5(10 FM) 60 11 54 1.9 14 59 2.4

Rifampin 0 0.070 44 0.016 0.0053 1.0 95 0.11 0.017(0.01 ,ug/ml) 60 19 61 3.0 39 88 4.4

Chloramphenicol 0 0.090 50 0.018 0.023 0.50 52 0.096 0.044(1 ,ug/ml) 60 5.5 69 0.80 11 73 1.5

Tunicamycin 0 0.57 40 0.14 0.041 0.49 26 0.19 0.12(5 ,ug/ml) 60 19 56 3.4 15 78 1.9

Methicillin 0 3.5 22 1.6 2.6 2.8 16 1.7 1.8(0.1 jig/ml) 60 3.5 57 0.62 14 84 1.6

No drug 9.5 52 1.8 28 107 2.6No DNA 0.0050 71 0.00071 0.0050 148 0.00034

a At the T1 stage in SPI medium, NIG1121M cells were transferred to SPII medium. Drugs were added to theculture at 0 and 60 min. DNA was added at 60 min. After incubation for 30 min, DNA incorporation wasterminated by the addition of DNase. The cells were collected by centrifugation at 10,000 x g for 5 min, washedonce in SPII medium without drugs, and plated on selective agar to score Met' transformants. The number ofCFUs was also scored for each sample.

The inhibitory nature of these drugs was notdue to the release of DNase in the culturemedium. A supernatant of a competent culturedeveloped in the presence of inhibitor did notinactivate transforming DNA appreciably (datanot shown).Time of appearance of competent celis after

transfer of early-stationary-phase cells into SPIImedium. The accurate time of appearance ofcompetent cells in SPII medium was measuredwith the inhibitors, rifampin, chloramphenicol,and tunicamycin. The drugs were added at 10- to15-min intervals after the dilution of early-sta-tionary-phase cells, and the eventual formationof competent cells was examined at 90 min byadding transforming DNA. Rifampin was used ata higher concentration (1.0 ,ug/ml) than that usedfor the experiment described in Table 3, becauserifampin at a concentration of 0.01 ,ug/ml be-came ineffective during incubation for longerthan 60 min.These three drugs completely inhibited com-

petent cell formation until about 50 min aftertransfer (Fig. 3). Around 50 min, competent cellsbegan to appear at the time of the multiplicationof viable cells. Rifampin was toxic toward non-competent cells, and we could not accuratelydetermine when noncompetent cells began todivide. On the other hand, tunicamycin blockedthe multiplication of noncompetent cells withoutcell lysis and allowed us to determine when theybegan to divide. Thus, it is very probable thatcompetent cell formation is in some mannerrelated to cell division. Similar challenge experi-ments showed that HPUra (10 ,uM)-resistant cellmultiplication began around 60 min after trans-

fer, although HPUra did not block competentcell formation.Development of competence in a D-alaine-

requiring strain. D-Alanine is found in pentapep-tides linked to N-acetylmuramic acid in cellwalls (4), and D-alanine residues are required forcross-linking in the cell wall. We next examinedthe effect of D-alanine depletion in a D-alanine-requiring strain. Cells of the mutant strain weregrown to T1 phase in the presence of D-alanineand transferred to SPII medium with and with-out D-alanine. Competence developed even inthe absence of D-alanine, whereas the totalnumber of recipient cells decreased without D-alanine (Fig. 4a). To see whether or not compe-tent cells of the D-alanine-requiring strain wereformed with the synthesis of cell walls, tunica-mycin was added to the culture, and its effectwas examined. Tunicamycin completely inhibit-ed the formation of competent cells until 50 minafter transfer (Fig. 4b). This result indicates thatthe D-alanine-requiring strain needs cell wallsynthesis to produce competent cells even in theabsence of D-alanine in the medium. Theseresults suggest that competent cells do not re-quire the cross-linking of peptidoglycan in thesynthesis of cell wall as suggested by the experi-ment with methicillin or that the formation ofcompetent cells may be related to the presenceor absence of cell wall accessory materials otherthan peptidoglycan because D-alanine is alsofound in some accessory components and itsdepletion may affect their synthesis. Tunicamy-cin also blocks their synthesis. Actually, tei-choic acid (14) was found to act as a specificligand for B. subtilis cell wall lytic enzymes,

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oo*7 25

15 30 45 60 75 90Time (min)

FIG. 3. Time of appearance of competent cells ofNIG1121M in SPII medium. T1-stage cells in SPImedium were transferred to SPII medium. Inhibitorswere added at the time indicated. Competent cellswere examined by addding transforming DNA at 90min and incubated for 30 min followed by DNasetreatment for 10 min. Cells were washed once withSPII medium free of drug as described in Table 3,footnote a, and Met+ transformants and CFUs werescored. The number of transformants or CFUs wasnormalized by that of the sample which received nodrug. Symbols: A, Met+ transformants; O, CFUs; Rif,rifampin (1 ,ug/ml); CM, chloramphenicol (1 ,ug/ml);TM, tunicamycin (5 Rg/ml).

which are assumed to be competence-inducingfactors.

Blocking of competent cell formation at a suble-thal concentration of ethanol, a specific inhibitorof early events of sporulation. B. subtilis cellsform an asymmetric forespore septum after theend of log phase in a relatively poor mediumwhich supports maximal sporulation (for a re-view, see references 15, 23). The addition ofenough nutrient or the dilution of early sporulat-ing cells with fresh medium might make sporu-lating cells resume vegetative cell division. Theprocedures adopted in the present experiment

30 60Tinme(min)

FIG. 4. Development of competence in D-alanine-requiring strain QB928 in SPII medium with or withoutD-alanine. (a) Cells of D-alanine-requiring strainQB928 were grown to T1 stage in SPI medium supple-mented with D-alanine and washed once in SPII medi-um without D-alanine. They were then suspended inSPII medium with or without D-alanine. TransformingDNA was added to 1 ml of competent culture. DNasewas added after 30 min. Trp+ transformants werescored on selective agar without Difco broth. FreshSPII medium was used as a dilution buffer. Symbols:0, turbidity without D-alanine; 0, turbidity with D-alanine; A, Trp+ transformants without D-alanine; A,Trp+ transformants with D-alanine; C, CFUs withoutD-alanine; *, CFUs with D-alanine. (b) At the indicat-ed time, tunicamycin (5 ,ug/ml) was added to theculture in SPII medium without D-alanine. The devel-opment of competence was measured as described inthe legend to Fig. 3. The number of transformants wasnormalized to that of the sample which receivedtunicamycin at 90 min. Symbol: A, Trp+ transfor-mants without D-alanine.

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TABLE 4. Activities of extracellular proteasesaActivity (%)b

Inhibitorspo+ spoOA34

+ 1,10-Phenanthroline (1 mM) 53.5 2.93+ 1,10-Phenanthroline (1 mM) 3.22 1.04+ Phenylmethylsulfonyl

fluoride (2 mM)a Protease activity was mneasured by the methods

described by Dancer and Mandelstam (8). StrainsNIG1121 (spoO+) and NIG1131 (spoOA34) were grownto T, stage in SPI medium. Two milliliters of theculture supernatant were combined with 10 mg of hidepowder (Sigma) in 1 ml of imidazol buffer and incubat-ed at 37°C for 16 h with or without inhibitor. Thesample was filtered, and the extinction of the filtratewas measured at 595 nm. Klett values of Tl-phasecultures of spoO+ and spoOA34 strains were 135 and138, respectively.

b The activity was normalized with that of subtilisin(1 mg) (Sigma), which completely digested hide pow-der at 37°C for 20 min.

might make early sporulating cells resume newcell division. Because sporulation requires man-ganese (7, 34) at a relatively high concentrationand sporulation is arrested before forespore sep-tum formation in the absence of manganese (21),the effect of manganese on sporulation frequen-cy and competence development was examined.The addition of manganese even at 1 ,uM in-creased the sporulation frequency of the cells inSPI medium from 10-6 to 10-2 (data not shown),whereas competence development was stimulat-ed only to a small degree.The activity of serine protease, which is

thought to be a specific early-sporulation event(8) before forespore septum formation, was mea-sured in the culture of the T1 stage with whichwe examined the development of competence.The supernatant of the culture of the Spo+ strainshowed an activity of serine protease which was1,10-phenanthroline resistant and was inhibitedby phenylmethylsulfonyl fluoride (Table 4).1,10-Phenanthroline is an inhibitor of metallo-proteases, and serine protease is inhibited com-pletely by phenylmethylsulfonyl fluoride. On theother hand, the spoO strain did not produceextracellular serine protease to an appreciableamount. Thus, it is very probable to considerearly-stationary-phase cells in SPI medium asearly sporulating cells, possibly before foresporeseptum formation.

Ethanol at 0.7 M reduces the growth rate ofthe cells without any effects on the final yield ofcell density and blocks sporulation at stage 0,before forespore septum formation (5, 24). Fore-spore septum formation is specifically inhibitedby ethanol, whereas normal cell division re-mains intact.

We examined the effect of ethanol on theformation of competent cells during growth inSPI and SPII medium. As shown in Fig. 5, 0.7 Methanol completely inhibited the appearance ofcompetent cells during vegetative growth in SPImedium (data not shown) and in SPII medium.The addition of 0.7 M ethanol to the competentculture grown in ethanol-free medium, just be-fore the addition of transforming DNA, reducedthe eventual number of transformants to 17% ofthat obtained without ethanol addition. Cellswhich received DNA were resistant to ethanol.Thus, the transformation of already competentcells seemed to be affeced by ethanol or compe-tent cells seemed to be sensitive to ethanol.However, the complete absence of competencein an ethanol medium and the increasing appear-ance of competent cells in ethanol-free medium(Fig. 2) suggest that ethanol blocks the forma-tion of competent cells. When early-stationary-phase cells grown in SPI medium in the presence

'I X2

Time(nrni)FIG. 5. Inhibitory nature of ethanol on competence

development. Ethanol was added at a concentration of0.7 M. NIG1121M cells were grown in SPI mediumwith ethanol to T, stage and transferred to SPIImedium with or without ethanol. The development ofcompetence was examined as described in the text.Symbols: 0, turbidity with ethanol; 0, turbidity with-out ethanol; A, Met' transformants with ethanol; A,Met' transformants without ethanol; *, CFUs withethanol; O, CFUs without ethanol.

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8COMPETENT CELL FORMATION OF B. SUBTILIS 819

TABLE 5. Development of competence of NIG1121M in medium which supports sporulationaFre-

Met+ CFU/ quency

Mediumb Phase transfor-mml of trans- Probabilityc

(10-4)

Glucose NH4Cl Log 1.50 39.0 0.039 5 X 10-2T2.5d 1.83 4.40 0.42

Glucose Casamino Acids Log 1.70 27.1 0.063 3 X 10-5T2.5 1.60 8.60 0.19

Glucose Histidine Log 1.71 13.3 0.13 1.2T2.5 63.0 16.5 3.8

Glucose Glutamate Log 60.5 15.1 4.01 2.5 x 10-2_T2.5 49.0 1.75 28.0 25 x 10-2

Complexe Log <0.00500 13.4 0.0004 10-3T2.5 0.0500 27.0 0.0019

Synthetief Log 21.5 30.0 0.72T2.5 90.0 13.3 6.8

a An overnight culture in each medium was added as a 5% (vol/vol) inoculum into fresh medium. The cellswere grown to T1 phase and transferred to fresh medium. One milliliter of the culture was mixed with DNA toexamine competence.

b Schaeffer's basal medium (30) was supplemented with carbon and nitrogen sources. Glucose, 0.2%; NH4Cl,0.05%; Casamino Acids, 0.1%; histidine, 0.1%; and glutamate, 0.1%.

c The probability for a cell to become committed to sporulation calculated by Schaeffer et al. (30).d Cells of T1 phase were diluted 10-fold with fresh medium and incubated for 90 min.e A complex sporulation medium described by Schaeffer et al. (30).fA synthetic medium for transformation described by Dubnau et al. (10).

of ethanol were transferred to ethanol-free SPIImedium, competent cells developed, but theirformation was reduced and retarded. The etha-nol effect on the membrane is reversible (24).Thus, the process of competent cell formation isone of the early events during sporulation andrequires an intact membrane. We also examinedthe activity ofDNase which might be released into the medium from cells grown in the presenceof ethanol and could not detect any decrease ofthe activity of transforming DNA by incubationwith the supematant of the culture containingethanol (data not shown). The development ofcompetence in a D-alanine-requiring strain in theabsence of D-alanine described in Fig. 4 was alsoinhibited by ethanol (data not shown). Thus, theformation of competent cells of the D-alanine-requiring strain is also inherent in the earlysporulation stage.Development of competence in medium which is

favorable for sporulation. Generally, competentcells have been prepared in a specially devisedmedium which does not support optimal sporu-lation. On the other hand, sporulation occurs ina medium which does not support the develop-ment of competence. If competent cells areformed from early-phase sporulating cells by thesupply of enough nutrients to resume new cell

division, competent cells might be producedbest in the sporulation medium by transferringthe sporulating early-stage cells to fresh mediumor by growing a vegetative culture with a highprobability of sporulation. In 1965, Schaeffer etal. (30) proposed that the initiation of sporula-tion is under the control of catabolite repression.They examined the probability of sporulation inthe continuous culture which contained a mini-mal synthetic medium with various kinds ofcarbon and nitrogen sources.We examined the development of competence

for transformation in such a medium. Cells weregrown logarithmically in Schaeffer's basal medi-um (30) with various kinds of nitrogen andcarbon sources, transforming DNA was addedto the culture, and Met+ transformants werescored. The wild-type strain showed fairly goodcompetence for transformation in almost everysynthetic medium (Table 5). In the medium withhistidine as the sole nitrogen source, compe-tence was comparable to that developed in SPIand SPII medium. In this medium, sporulationbegins with an extremely high probability (30).Competence also appeared by diluting Tl-stagecells in fresh medium; this result again supportsthe idea that competent cells are formed fromcells at an early sporulation stage. In a complex

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TABLE 6. Frequency of transformation of spoOstrains in synthetic medium

Genotype MetF transfor- Frequency ofa Km'of recipi- mation (10-4) transformation (10-6)

ent main104

Wild 8.9 (1.0) 1.1 (1.0)spoOA 0.20 (0.022) <0.24 (<0.23)spoOB 0.047 (0.0053) <0.023 (<0.012)spoOC 3.9 (0.44) 0.36 (0.34)spoOD 0.031 (0.0035)spoOE 0.19 (0.021) 1.6 (1.5)spoOF 0.072 (0.0081) 0.0066 (0.0063)spoOG 5.1 (0.58) 4.1 (3.9)spoOH 0.12 (0.014) 0.14 (0.13)spoOJ 5.1 (0.57) 2.1 (2.0)spoOK 0.015 (0.0017) 0.010 (0.0096)Wild 1.7 (1.0)spoOD 0.043 (0.025)Wild 12 (1.0)spoOA 0.18 (0.015)a A competent culture (1 ml) was incubated with

plasmid DNA (5.7 ,ug) at 37°C for 2 h to allow theexpression of kanamycin resistance. The Kmr trans-formants were scored on minimal salts-glucose agarcontaining kanamycin (15 ,ug/ml) purchased from MeijiCo. Ltd (Tokyo, Japan). Plasmid DNA was purifiedfrom strain NIG1121 carrying pUB110 (27) as de-scribed by Gryczan et al. (13).

medium, the development of competence wasvery limited. Some factor(s) may function as adirect or indirect repressor for the expression offactor(s) required for the formation of competentcells.

Effect of spoO mutations on the development ofcompetence. It is generally accepted that mu-tants defective in an early stage of sporulationshow poor competence. However, their compe-tence was not examined systematically underthe same conditions (23). To see the effect ofspoO mutations on the development of compe-tence, we constructed 10 isogenic strains ofdifferent spoO loci (23) and examined the devel-opment of competence in SPII medium. Theresults shown in Table 6 revealed that most spoOmutations reduced the development of compe-tence except for spoOC, spoOG, and spoOJ. InspoOB, spoOD, spoOK, and spoOF strains, thedevelopment of competence was highly re-pressed. Thus, the development of competencedepends on some spoO gene products during anearly stage of sporulation. The poor competenceof these spoO mutants is not due to the deficien-cy of recombinational ability. Competence forplasmid pUB110 (13)-mediated transformationfrom Kms to Kmr was also poor for thesestrains, except for spoOE strain, which showedcompetence for plasmid DNA-mediated trans-formation. spoO strains used in the present studywere resistant to UV light and chemical muta-

gens (data not shown). Because rec strains of B.subtilis were sensitive to the above agents (25),spoO mutants are considered to be proficient inrecombinational ability.

DISCUSSIONWith the method employed in the present

study, competent cultures were induced fromcultures returning to vegetative growth from anearly sporulating (T1) stage, possibly beforeforespore septum formation. T, stage cells areheterogenous in their ability to acquire the com-petent state. A small fraction of T1 cells (whichmay have been committed to sporulation beyondsome critical point) can only be induced forcompetence, whereas the majority of T1 cellsregain vegetative growth and are not induced intheir ability to incorporate exogenous DNA.Therefore, the development of competence dur-ing vegetative growth is attributable to the smallfraction of cells which have started to sporulatebut fail to continue in the presence of sufficientnutrients in the medium.The formation of competent cells from T1-

stage cells requires cell wall synthesis, whichwas inhibited by tunicamycin but not by methi-cillin or D-alanine depletion, whereas vegetativegrowth was blocked by these inhibitors. As themultiplication of viable cells and the formationof competent cells concomitantly became resis-tant to inhibitors after about 50 min in the SPIImedium and some initiation mutants in celldivision were defective in the development ofcompetence (manuscript in preparation), it isvery probable that cell wall materials requiredfor competent cell formation are associated withcell division.The sporulation of B. subtilis begins with a

separation of the daughter chromosomes fol-lowed by the cessation of cell wall synthesis andthe formation of an asymmetric forespore sep-tum with less cross wall material between thetwo forespore membranes (15). Cell wall materi-als formed along the forespore membrane maybe digested away by some autolysin specific tosporulation (15). That a mutant strain defectivein more than 95% of total autolytic activity isstill competent for transformation and proficientin sporulation (11) may suggest some sporula-tion-specific autolysins involved in competentcell formation and forespore septum formation.As suggested earlier, the site of forespore sep-tum formation may be the site of the entry oftransforming DNA (37), although we have nofirm evidence in support of this hypothesis. Thesporulation-specific nature of competent cellformation was also supported by the experimentwith 0.7 M ethanol and some spoO mutationswhich blocked sporulation before forespore sep-tum formation and the development of compe-

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COMPETENT CELL FORMATION OF B. SUBTILIS 821

tence although we could not determine whetherthey block the development of T1-stage cells inSPI medium or the formation of competent cellsin SPII medium.The formation of competent cells also requires

de novo synthesis of RNA and protein. Thedevelopment of competence may be accompa-nied by the activation of intracellular recombina-tion ability involving the expression of a varietyof genes responsible for DNA metabolism otherthan cell wall synthesis. Some inducible func-tions responsible for mutagenesis or prophageinduction were found to operate in competentcells of B. subtilis (36).

ACKNOWLEDGMENTSWe thank H. Yoshikawa and R. H. Doi for reading the

manuscript critically and for many valuable comments. Weare grateful to G. Tamura and N. Cozzarelli for chemicals andH. Saito, P. J. Piggot, F. Kawamura, and Y. Fujita forbacterial strains.

This work was supported by a grant from the Ministry ofEducation of Japan.

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