JOURNAL OF BACTERIOLOGY, Sept. 1967, p. 562-570Copyright © 1967 American Society for Microbiology

Development of Competence in the Bacillussubtilis Transformation System

KENNETH F. BOTT AND GARY A. WILSON

Department of Microbiology, University of Chicago, Chicago, Illinois

Received for publication 7 June 1967

Competence in Bacillus subtilis, assayed by the ability of cells to be transformedwith bacterial deoxyribonucleic acid (DNA) or transfected by phage DNA, hasbeen shown to occur in a single semisynthetic medium with peak activity occurring3 hr after the cessation of logarithmic growth. No step-down conditions or culturemanipulations were necessary for routine transfection of 1 % of the population. Theresults demonstrate that bacteriophage DNA is a valid assay for studying thedevelopment of competence in B. subtilis. Predictions of workers using transformingbacterial DNA, who have suggested that competence in B. subtilis is associated with aspecific phase of growth, are substantiated. The peak of competence is not affectedby marked differences in the rate of growth during the logarithmic phase. The effecton development of competence by this procedure of some components (includingcasein hydrolysate, tryptophan, and histidine) which were routinely included in thetransformation medium by other investigators has been determined by use of infec-tious phage DNA as an assay. We have demonstrated that tryptophan, as well ashistidine, increases the transformation frequency-even in strains which do not haveauxotrophic demands for these components. Glutamic acid and alanine depressoptimal levels of transfection.

Competence refers to the ability of a bacterialcell to bind irreversibly deoxyribonucleic acid(DNA) of high molecular weight in such a mannerthat it becomes resistant to deoxyribonuclease;it is the first major step in the transformationprocess. Later steps involve integration or re-

combination with the recipient genome, which isfollowed by replication, segregation, and ex-

pression of the new genetic information. Althoughcompetence can be subdivided into finer stages(reversible and irreversible), there appears to beno specificity in this step which enables a bac-terial cell to discriminate against heterologousDNA (6, 11, 13). It is generally agreed that theability to develop competence is genetically con-trolled-possibly by specifying the nature ofreceptor sites on the cell surface (13, 26).

Specific studies of the competence process havebeen hampered by difficulties in procedure, whichrequire that recombination and expression of thetransforming DNA occur before the transform-ants can be recognized. These are especiallycritical in evaluating the state of competence inBacillus subtilis, since only a relatively small per-centage of the recipient population (1 to 4%) hasever been transformed for any single marker, eventhough the whole population has the geneticcapability to become competent.

Several workers have noted the appearance ofcompetent cells of B. subtilis near the end of ex-

ponential growth in a minimal medium (althoughnot in complex medium) and the inability of someasporogenic mutants to be transformed. Theyhave suggested that this reflects an associationwith the process of sporulation (17, 20, 24, 25).However, the data are not yet sufficient to estab-lish a quantitative association (13).

Spizizen and co-workers (2, 25) described anefficient method of obtaining competent cultureswhich involves a "step-down" to a growth limit-ing medium after 4 or 5 hr of normal growth.When competence is induced by this method, onecannot be certain whether it is associated withnormal growth or whether it is a type of unbal-anced growth that requires "step-down" condi-tions for its most efficient expression.

In this report, we present evidence from studieson the uptake of infectious phage DNA that theappearance of competence can be identified witha specific stage of the growth cycle. In addition,we show that there is little, if any, difference inthe development of competence for phage DNAor bacterial DNA, since conditions which altercompetence development for the bacterial DNAsystem have similar effects when competence isassayed by use of infectious phage DNA. The

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level of competence obtained under our condi-tions of growth is usually 1%, and is reproduciblewithin a twofold range for several of the com-monly used recipient strains. Other strains havebeen transfected at the same stage of growth withsimilar reproducibility, but at lower frequencies.The results suggest that subsequent modificationswhich can shorten the doubling time of the loga-rithmic culture, in the absence of inhibitorycomponents, may cause the period of competenceto be shorter by virtue of its ability to "synchro-nize" the events which occur in stationary phase.

Several aspects of the technique are significant.First, this more thoroughly defined regimen forobtaining competence enchances the likelihoodthat normal strains, and mutants with abnormalgrowth characteristics, can be optimally trans-formed (or transfected). Second, it provides as-surance that each transformation experiment isbeing performed when the strain is maximallycompetent, and it allows unambiguous interpreta-tion of the effects of environmental or metabolicalterations. The use of an infectious phage DNAassay enables one to estimate the competence foruptake of biologically active DNA apparentlywithout the subsequent requirement of integra-tion.

MATERIALS AND METHODS

Definitions and abbreviations. "Synchrony" refersto the degree of uniformity with which events occurin the stationary phase, not to simultaneous divisionof cells, as is the conventional use. "Transfection" isthe process by which a cell produces bacteriophageafter uptake of DNA isolated from bacteriophageparticles. The DNA which brings about this processis referred to as "infectious DNA." "Transfectants"are cells which have been induced to produce phageby this technique.The abbreviations used are as follows: ind, indole

(tryptophan); thiy, thymine; met, methionine; ilv,isoleucine; leu, leucine; uvr, the ability to repairlesions induced by ultraviolet light. A superscriptminus following one of these abbreviations refers tothe inability of a cell to grow in a synthetic mediumlacking this component, or in the case of uvr the in-ability to repair the damage induced by ultravioletlight. A superscript plus following the abbreviationindicates the ability of a cell to grow in a syntheticmedium lacking this component.

Bacterial anid phage cultures. B. subtilis strain 168i,id- thy- was obtained from F. Rothman. Strains168 uvr-, 168 uvr-thy-, and the 168T+ prototroph fordonor DNA were obtained from B. Strauss. The 168uvr- thy- is a transformant of 168 int thy- which isdefective in its ability to repair the damage inducedby ultraviolet light. The acridine orange inducedmutants (166 AO and 26 AO) were derived from 168ind thy- (4). They are known to be defective intheir ability to sporulate, and they grow at a slowerrate than is normal on the conventional minimal

salts medium of Spizizen (2). B. subtilis Mu8u5ul(leu- ilv- met-, abbreviated here as Ul) was ob-tained from N. Sueoka.

Bacteriophage 029, its host for production ofphage lysates (HW+), and a streptomycin-resistantderivative of it, HW+Sr, were obtained from B.Reilly. Both of these strains require tryptophan forgrowth.DNA. DNA of bacteriophage +29 was obtained

by extracting the purified, concentrated phage (3)twice with phenol-saturated buffer, according to theprocedure of Reilly and Spizizen (10). Reagent-grade phenol was distilled before use. Bacterial DNAwas extracted from early stationary-phase cultures of168T+ grown in Difco Antibiotic Assay medium byuse of a modification of the method of Saito andMiura (12) in which the phenol was removed bydialysis against several changes of buffer containing0.1 M phosphate (pH 7.4) and 1 M NaCl. Before use,all preparations of DNA were equilibrated to, andstored in, standard saline citrate (0.15 M NaCl +0.015 M trisodium citrate, pH 7).

Growth of recipient cultures. Cultures were grownby use of a single medium which was identical to thegrowth medium of Anagnostopoulos and Spizizen(2) except for the addition of extra magnesium to thelevel which they routinely used in their transforma-tion medium (i.e., 0.072% magnesium instead of0.01%). Our medium had the following composition:0.5% glucose; 1.4% K2HPO4; 0.6% KH2PO4; 0.072%anhydrous MgSO4; 0.2% (NH4)2SO4; 0.19%Nacitrate-2H20; 0.02% acid-hydrolyzed casein (Nutri-tional Biochemicals Corp., Cleveland, Ohio); 50,ug/mloftryptophan and 50 ,g/ml of histidine; plus 50 ,ug/mlof any other amino acid required by the auxotrophicrecipient. The medium was prepared completely(except for magnesium) in glass-distilled water at a10-fold concentration, filter-sterilized by use of a pre-washed 0.45-,u membrane filter (Millipore Corp.,Bedford, Mass.), dispensed into sterile tubes in 10-mlamounts, and then frozen until use. Just before use,a tube was thawed, and the contents were added to85 ml of sterile glass-distilled water in a I-literErlenmeyer flask with 5.0 ml of 0.12 M MgSO4.

Experiments were begun with an overnight culture(37 C) of the recipient in Antibiotic Assay mediumwhich had been inoculated with a loopful of growthfrom a Difco Tryptose Blood Agar Base plate. Nodifference was noted between overnight culturesgrown 12, 16, 20, or 24 hr, although 12 to 14 hr wasmost routinely used. The overnight culture was cen-trifuged, resuspended in an equal volume of minimalsalts solution, and added to the growth medium togive an optical density at 500 m,u of 0.1 (approxi-mately 2 X 107 colony formers/ml) with a Bausch &Lomb Spectronic-20 colorimeter. Usually, 4.5 ml wasrequired. Growth was monitored by following thechanges in optical density at 500 m,. As noted byYoung and Spizizen (25), plates of the recipientculture which were 3 to 4 days old gave rise to cul-tures having higher transformation frequencies thanfreshly subcultured growth. In fact, the best resultswere obtained with cultures which had been kept atroom temperature for 2 to 3 weeks.

563VOL. 94, 1967

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Transformation. The transformation mixture con-tained 0.1 ml of DNA (6 to 10 ,ug) and 0.9 ml ofcompetent bacteria in a 150-mm test tube. The mix-ture was rapidly agitated at a 450 angle on a recipro-cal shaker[1Li-inch (2.9-cm) stroke, 450 strokes/min] at 37 C. After 30 min, 100 ,g (0.1 ml) of deoxy-ribonuclease was added, and the shaking was con-tinued for 15 min. The necessary dilutions were thenprepared with the use of a twice-concentrated solution(21.5 g/liter) of Davis Minimal medium withoutglucose (Difco) as diluent. Hereafter, this will bereferred to as Davis salts. If bacterial DNA (from168T+) was used as donor, 0.1 ml of dilutions from thetransformation mixture was plated on the minimalmedium of Spizizen (2), which contained all essentialamino acids for growth of the auxotroph, except theone being tested by transformation. When phageDNA was used, the plating was by the soft agartechnique of Adams (1) with the use of TryptoseBlood Agar Base plates and the peptone-NaCl soft-agar overlay of Reilly and Spizizen (10). A 0.1-mlamount of the transfection mixture at the desireddilution was added to 2.5 ml of warm soft agar; then250 ,ug (0.1 ml) of streptomycin and bacterial strainHW+Sr as an indicator were added. The use ofHW+Sr as indicator has several functions. It providesthe necessary lawn of indicator at high dilutions ofthe transfection mixture. It is not transfectable byDNA isolated from bacteriophage 029 (or trans-formable by bacterial DNA), and, as such, is adouble check that any infectious centers must havearisen from phage synthesis in competent cells. Thepresence of streptomycin inhibits development in thelawn of recipient colonies which might produce abacteriocin that could be confused with plaques (B.Reilly, personal communication).

Controls for both the phage transfection and bac-terial transforming activity were performed by incu-bating the DNA with 100 jg of deoxyribonuclease inthe presence of 0.0012 M magnesium sulfate for 15min before addition of the competent cells.

Transformation frequency refers to the number oftransformants or transfectants per milliliter times 100,divided by viable cells per milliliter (determined onTryptose Blood Agar Base medium at the time DNAwas added).

RESULTSUnder conditions of growth in the medium de-

scribed, the peak of competence has always beendetected at one specific point in the stationaryphase of growth. We have used the terminologyof Schaeffer et al. (14) to designate the beginningof stationary phase (To) as the point at which theoptical density curve departs from the straightline of logarithmic growth. Competence is maxi-mal 3 hr later, T3 (Fig. 1). The appearance of thispeak has been verified using as recipients strainsUt, 168 uvr-, 168 ind- thy-, and derivatives ofthem. Recent experiments suggest that the peak ofcompetence can be shifted away from T3 (in bothdirections) by changing some of the compo-

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with DNA from bacteriophage k29 and 168T+. At theindicated intervals after To, 0.9 ml of the bacterialculture (UJ) was withdrawn and added to 0.1 ml ofthe phage DNA. Another 0.9 ml was mixed with 0.1ml ofDNA from 168T+ and isoleucine+ transformantsrecovered on minimal medium plus leucine and methio-nine. A third sample was diluted appropriately andplated on complex medium to determine total cellviability. Transformation procedures were as describedin Materials and Methods. The turbidimetric doublingtime during the log phase was- 48 min. (Closed circlesrepresent optical density; open circles, isoleucine+transformants; starred circles, phage DNA transfect-ants).

nents of the growth medium. These alterations canresult in a decreased efficiency of competence.

Figure 1 illustrates that maximal competenceis attained simultaneously for bacterial DNA(ilv+ transformants) and infectious 429 DNA.This confirms the observations of Reilly andSpizizen (10) that the peak of transfection coin-cides with that for transformation with the use ofa different bacterial marker, and substantiates theusefulness of phage DNA to assay competence.We have consistently observed the peak of trans-fection to be sharper than that for transformationunder our experimental conditions. This differ-ence is not apparent in the data of Reilly and

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Spizizen (10). We suggest that it may reflect adiscrimination between the two types of DNA.Whether the broadness of the transformationpeak can be attributed to size heterogeneity of thebacterial DNA, or to some other difference, hasnot yet been determined. However, we have notedthat the number of transfectants is increased bymagnesium to a slightly greater extent than thebacterial transformants (unpublished data).Whether or not the two observations are relatedis also unknown.

Experiments illustrated in Fig. 2 have verifiedthat competence is a transitory stage in the growthof the population, which always occurs 3 hr afterTo and is not dependent upon the initial growthrate of the culture. Part A illustrates that when thelogarithmic growth rate is reduced by some nu-trient limitation (in this case, phosphate) thepeak is still at T3. Parts B and C show the develop-ment of competence in slow-growing mutantsthat had been induced by acridine orange (4).The 168 uvr- thyj strain in part D is very poorlytransformable by conventional methods. Thisgraph indicates that one reason is probably itsslower growth rate, since the 4 hr plus 90 minregimen would not give it time to reach the properpoint in the stationary phase. These results aresubstantiated by experiments of Okubo andRomig (9), who showed that another slow-grow-ing mutant of B. subtilis (MC-1) was made morehighly competent by conventional methods witha prolonged growth period. During the experi-ment illustrated by part E, the culture growth wasunusually rapid for reasons not understood; thismay have affected the "synchrony" of stationary-phase activities in such a manner that more of thecells became competent at the same time. One cansee that the peak of transfection was unusuallysharp under these conditions. Despite the varia-tions in growth rate or the strains used here (Fig.2), the peak of competence was always detectedat or near T3.

Effects of tryptophan and histidine. We havetested some of the constituents of the growthmedium to define their importance, and to verifythat their effect on competence for phage DNAwas the same as that for bacterial DNA. Table 1shows the effects of withholding tryptophan orhistidine from the growth medium of strain Ul,an auxotroph which does not require either ofthese amino acids. Deprivation of either of thesecomponents did not shift the peak of competenceaway from T3. It can be seen that both tryptophanand histidine are essential for maximal compe-tence, although a low level of transformationdoes occur in their absence. Although it appearsfrom these data that omission of tryptophan andhistidine stimulates a high cell viability while de-

creasing competence, the effect is not real; sub-sequent experiments have indicated that the finalpopulation size is not significantly affected by thisconcentration of tryptophan or histidine. Simi-larly, the growth curves have never varied in amanner that would suggest that these aminoacids were inhibiting growth. The importance ofhistidine was not unexpected, since Anagnosto-poulos and Spizizen (2) indicated that one of thefunctions of casein hydrolysate was to supplyhistidine. They proposed that it acted as a chelat-ing agent for divalent cations and noted that itseffects could be mimicked by a, a-bipyridyl. Sinceno alternative explanation has been proposed forthe effects of histidine, we attempted to test thetheory of chelation more directly. The completegrowth medium was extracted with dithizone ac-cording to the procedure of v. Hofsten (7). Thisextraction method has been shown to removecopper and other heavy metal ions to at least alevel of 0.5 X 10- 6 M. In this experiment, 100 mlof complete medium containing tryphophan andhistidine, without magnesium, was shaken withan equal volume of carbon tetrachloride contain-ing 6 mg of dithizone per liter. The extractionwas repeated five times (no further color changeof the solvent layer); then excess solvent wasevaporated with a stream of filtered air. TheMgSO4 was prepared as a concentrated solutionin deionized water and diluted appropriately intothe extracted medium. The medium was filter-sterilized by use of a prewashed membrane filterof 0.45 ,u porosity. The control consisted of asimilar portion of medium which was extractedthe same number of times with carbon tetrachlo-ride having no dithizone. Magnesium sulfatefrom the same solution was then added to thismedium; evaporation of carbon tetrachloride andsterilization procedures were the same.

If ions inhibitory to DNA uptake were beingcomplexed by histidine in the medium, we wouldhave expected the dithizone treatment to increasethe transfection frequency due to removal of theseinhibitors. This effect was not noted, although thegrowth rate and total amount of growth on thetwo media were identical, indicating that growth-supporting ability had not been altered. The di-thizone treatment reduced the number of trans-fectants to 11% of the value obtained in its ab-sence (i.e., 2.0 X 106 transfectants/ml with CCl4alone; 2.2 x 105/ml with CC14 plus dithizone).Essentially the same results were obtained whenthe medium was extracted with chloroform con-taining the dithizone. These results may be in-terpreted to mean that the inhibitory componentwhich is being complexed by histidine is not onlya heavy metal ion, as suggested by Anagnos-topoulos and Spizizen (2), but some other com-

565VOL. 94,7 1967

BOTT AND WILSON

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FIG. 2. Transfection ofstrains which grow at rates different from those ofthe typical recipient cultures. At eachindicatedinterval, 0.1 mlof 429DNA wasaddedto 0.9ml of culture according to procedure described in Materialsand Methods (0, transfectants; 0, optical density). (A) Normal Ul recipient growing in a minimal mediwn likethat described in Materials and Methods except that the phosphate component was provided by 0.5 g ofK2HPO4.FinalpH was adjusted to 7.0 with I N HCI. The turbidimetric doublingtime was 72 min. (B) Strain 166 AO growingin the minimal medium described in Materials and Methods. Turbidimetric doubling time, 58.5 min. (C) Strain26 AO in minimal medium. Turbidimetric doubling time, 72.5 min. (D) Strain 168 uvr, thy, in minimal mediumTurbidimetric doubling time, 114 min. (E) Strain Ul (a normal recipient) in minimal medium. For unknown rea-sons, this culture grew unusually fast. The turbidimetric doubling time of 37 min illustrated here is to be com-pared with the normal pattern illustrated in Fig. 1.

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COMPETENCE IN B. SUBTILIS

TABLE 1. Efrects of witholdintg certain conistituentsfrom the growth medium of Ul on the trans-

fection at the peak of competence (T3)

Transfor-Nutrient removed Transfectants Viable mation

from complete mediuma /ml cells/ml fre-quency

None 8.7 X 105 1.3 X 108 .65

Tryptophan 3.3 X 105 3.9 X 108 .084

Histidine 1.4 X 105 3.3 X 108 .042

Histidine and trypto-phan .. ............ 2.2 X 104 3.7 X 108 .006

Casein hydrolysate.....11.8 X 105 5.6 X 10' .32

a The minimal medium described in Materials and Methodswas complete except for the total removal of those componentsindicated in the left column. All values are those observed at

Ts, the peak of competence.

ponent of the casein hydrolysate, or a by-productof cell growth. Actually, we favor the formeralternative because of reports from variouslaboratories that the particular batch of caseinhydrolysate is extremely important in achievinggood transformation frequencies. Dithizonetreatment might not be expected to permit thesame degree of competence in both systems. Ametabolic by-product would be more likely toinhibit transfection in our system, since it is notdiluted out as it could be if a step-down procedurewere employed.The requirement of tryptophan for maximal

competence of a strain which does not requiretryptophan for growth was unexpected and is stillnot understood, but may reflect a function oftryptophan that has not yet been recognized.

Effect of glutamic acid and alanine on trans-fection. The acridine orange-induced mutants ofB. subtilis are restored to the parental growthrate by the presence of 2 mg of glutamic acid perml (unpublished data). Attempts to transfectthese strains first used a medium which had beenmodified to contain 2 g of glutamic acid per liter.Under these conditions, none of the mutants was

ever transfected. Subsequent control experimentswith 168 uvr- and Ul showed that normal levelsof competence were not produced in the presenceof glutamic acid. In fact, 50 ,g/ml was shown toreduce markedly the frequency of transfection.The results of these experiments are shown inTable 2. It does not appear that this inhibitionis due to a general stimulation of growth. At50 ,g/ml, little or no effect on growth rate wasdetected in the 168 uvr- and Ul recipients.The peak of maximal competence, although low-ered, was still at T3. The presence of 2 mg ofglutamic acid per ml did not affect the logarithmicgrowth rate of these recipients (unlike its effecton the acridine orange-induced mutants), but

TABLE 2. Effects of glutamic acid oni transfectionat the peak of competence

Concn Trans-of glu- Transfec- Vliable forma-

Recipient strain tamic tants/ml cells/mi fore

quency

mg/mlU1 ........... 0 2.9 X 106 4.07 X 108 .73U1 ........... 0.05 2.1 X 105 5.49 X 108 .038Ul........... 2 7.6 X 103 5.23 X 108 .0015

168 uvr......... 0 9.4 X 105 1.7 X 108 .57168 uvr- (lowMg) ......... 2 4.0 X 102 1.1 X 108 .00038

168uvr-. .........2 8.0 X 102 2.2 X 108 .00037

26AO......... 0 8.5 X 104 4.0 X 108 .02126 AO.........1 2 40 4.0 X 108 .000010

a Final concentration of neutralized glutamic acid solutionwhich was added aseptically to minimal growth medium at thebeginning of growth.

b Final magnesium sulfate concentration was 100 pg/mlinstead of the usual 720 ,g/ml.

it did slightly extend the logarithmic growthphase. Periodic samples of this culture testedfor competence indicated that glutamic acid wasindeed inhibitory and did not act to shift the posi-tion of the competence peak to a later hour.Alanine was also shown to be inhibitory when, insimilar experiments, it was added to the caseinhydrolysate medium. It, too, slightly increasedthe population size, and caused a decreasedtransfection frequency at the peak of competence(T3). In one experiment of this type, 50 ,ug/ml ofadded alanine reduced the number of trans-fectants per ml, with strain Ul, from 1.1 x106 (in the culture lacking alanine) to 4.5 x 105in the culture grown in the presence of alanine.These results suggest that several components ofcasein hydrolysate are acting to keep the levelof competence low, whereas other componentsmay be stimulatory.

DISCUSSIONThese experiments have illustrated that, when

B. subtilis is cultured in a single growth mediumcontaining 0.072% magnesium, a peak of com-petence appears 3 hr after the cessation of loga-rithmic growth (T3). The increased populationsize which accompanies growth in a singlemedium provides a larger number of competentcells than can be obtained by the "step-down"method previously used. The levels of competenceobtained by this procedure are as good as orbetter than we were able to achieve using themethod of Anagnostopoulos and Spizizen (2);results were also more reproducible. A directcomparison of the two methods is difficult, since

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BOT-r AND WILSON

in our hands the 4 hr plus 90 min regimen hasbeen highly variable even when used in duplicateor triplicate experiments on the same day. Thefrequency of competence with Spizizen's methodyielded from 0.05 to 0.5% in the majority of ourtrials. It does appear that under the conditionsdescribed (24) the maximal frequency of com-petence (1 to 4%) that has been obtained by themethod of Spizizen and co-workers makes thatmethod slightly more efficient, but we have neverbeen able to achieve quite that level of com-petence in many attempts to duplicate theirefficiency. Perhaps some slight difference, such asrecipient strain, amount of inoculum, the waterused to make up the medium, or shaker speed,would be enough to affect the final level of com-petence. We believe that these variables can nowall be tested systematically by use of the methodoutlined in this paper.Three different recipient strains have been

transfected by our procedure at a frequency ofapproximately 1% (within a factor of 2) for morethan 25 transfection experiments. All of thecultures examined were shown to develop a peakat T3. It is of interest that those cultures whichgrow at a rapid rate [similar to, or faster than, theconventionally used recipients (Fig. 2)] seem tohave the sharpest peak of transfection. It istempting to suggest that these conditions maybe providing a better opportunity for a more"synchronous" stationary phase in which a largerpercentage of the population would reach com-petence at the observed peak .A peak of compe-tence is still detected at T3 when the growthrate is reduced because of phosphate limitationor because of ancillary growth requirements(Fig. 2). The appearance of the same pattern ofcompetence, despite an altered growth rate inlogarithmic phase, suggests that metabolic activitydue to these alterations has little effect on timingof the development of competence.Our studies have illustrated that tryptophan

and histidine are both necessary for maximalcompetence under the conditions described,even in a strain (Ul) which does not requirethem to satisfy auxotrophic deficiencies. Morerecent experiments (unpublished data) have re-vealed that the peak of competence can also beobserved at T3 when cells are grown in the ab-sence of casein hydrolysate, tryptophan, andhistidine, i.e., a completely synthetic medium.In the absence of all of these supplements, themaximal frequency is roughly 30% of the valueobtained in their presence. These conditions alsoprolong the duration of logarithmic growth.Taken together, these results suggest that com-petence is indeed associated with a specificaspect of stationary phase; it appears that some

constituents of casein hydrolysate are stimu-latory, but under the conditions outlined abovethey must function in the presence of inhibitorysubstances which are also present. Certainly,a detailed analysis of the casein hydrolysatecomponent is essential before further experimentscan be effectively evaluated.Another observation from the current studies

is that the addition of some components doesalter the overall growth rate (as reflected byturbidimetric doubling time), but not competence.These results suggest that experiments of someworkers who have noted stimulatory or inhibitoryeffects of certain constituents on competencedevelopment may in fact have served only to alterthe growth rate sufficiently to make the routine4 hr plus 90 min regimen slightly out of phasewith the peak of competence. That is, an evalua-tion based on transformation at a single pointmay have resulted in submaximal levels and thusintroduced some inaccuracy into their interpre-tations.

Special note should be made of the observationsthat glutamic acid and alanine inhibit transfec-tion. This is consistent with the following. (i)No reports have appeared in the literature describ-ing transformation of an alanine-less or glutamic-less locus; furthermore, Spizizen et al. (18) re-ported that a group of their mutants requiringglutamate are defective in competence. (ii)Aconitase-less mutants which can only grow inthe presence of glutamic acid are not transform-able (19). (iii) Thorne and Stull (21) found thatglutamic acid inhibits transformation of B. licheni-formis. (iv) Several investigators (8, 15, 16)have suggested that alanine or glutamic acidmight alter the maximal number of transformantsin the B. subtilis system using bacterial DNA.(v) In the past, it has been well known to thoseworking routinely with the B. subtilis transfor-mation system that certain batches of caseinhydrolysate support the development of goodcompetence, whereas other batches from the samemanufacturer are completely useless. Attempts toreplace this constituent with yeast extract usuallylower transformation frequency (18).Glutamic acid and alanine are major compo-

nents of the mucopeptide chain in cell walls of atransformable strain of B. subtilis (22, 23, 27).Young (23) has proposed that hydrolysis of thiscross-linked peptide could permit expansion ofthe rigid cell wall. We suggest that, in the ab-sence of excessive glutamic acid or alanine (orboth), such an expansion might occur and beenough to accommodate the binding of DNAat a pertinent site on the surface of the cyto-plasmic membrane. If an excess of these twoamino acids were provided, then competence

568 J. BACTERIOL.

COMPETENCE IN B. SUBTILIS

might be inhibited by a strengthening of the cellwall.

Because of the timing of the appearance ofcompetent cells, it is not surprising that sugges-tions of an association between competence andsporulation have been made (16, 20). However,the observations that competence will not de-velop in medium which is conducive to sporula-tion, and that transformed cells do not sporulatebefore giving rise to transformants [the mediumusually employed does not have the necessarycofactors for sporulation (24)] indicate thatcompetence probably represents a stage beforethe irreversible commitment to sporulation.We would expect it to appear after the inductionof tricarboxylic acid cycle activity (5), sinceSpizizen et al. (18) have suggested that a-picolinicacid (an inhibitor of tricarboxylic acid activity)inhibits the development of competence.We suggest that the use of infectious phage

4)29 DNA to assay competence by this methodpermits more precision in the study of exogenousfactors affecting competence than some othermethods, as well as being potentially useful forstudies on competence per se. Some of the spe-cific advantages include the following: The entire429 genome is small (approximately the samesize as the average piece of bacterial DNA whichis isolated, 1.0 x 107 daltons versus 6 X 106 to1 X 107 for bacterial DNA), thereby providing adiminished chance of shearing during isolationand handling. This phage DNA is easy to isolateand has been shown by Reilly and Spizizen (10)to give a linear response of infectivity with in-creasing DNA concentration. It is also likely thatphage DNA does not have to integrate or recom-bine with the recipient genome to be expressed(although this fact has not been verified experi-mentally). This would eliminate the differentialprobability of integration or variation due tomarker reversion which has been associated withspecific bacterial markers. It would also facilitatethe study of competence in strains having noauxotrophic requirements. If integration is notnecessary, phage DNA should serve as a directassay of every competent cell in the populationand thus act as a much better indicator of com-petence than bacterial DNA. In this respect, weshould point out that the level of competenceobtained with phage DNA should be greater thanthat indicated by use of any single bacterialmarker, where selection of one class of trans-formants is a prerequisite for recording com-petence. In the phage system, all of the DNAmolecules which have successfully entered thecells should be scored as transfectants. As seenin Fig. 1, this has not been observed in our ex-periments. It also does not appear to be evident

in the experiments of Reilly and Spizizen (10).We cannot presently offer an explanation for thisdiscrepancy. One possibility is that some form ofDNA restriction results, since the phage aregrown on a host which is different from thetransformable cells, and is assayed on a straindifferent from the competent cells. Anotherfinding which may bear significance in this in-terpretation is that of Reilly and Spizizen, whodemonstrated that although the number oftransfectants with 4)29 DNA is directly propor-tional to the DNA concentration, an average of10,000 to 20,000 phage equivalents is necessaryfor the expression of each plaque. This may re-flect some form ofDNA degradation within com-petent cells, since the data of Anderson et al.(3), which we have confirmed (unpublisheddata), indicates that the isolated 429 DNA ishomogenous in size (5.8 u) and does not showthat a substantial portion of the molecules aresheared during isolation.

ACKNOWLEDGMENTS

We gratefully acknowledge the technical assistanceof Sheila Prachand and the suggestions of B. Straussduring preparation of the manuscript. This researchwas supported by grant GB 4291 from the NationalScience Foundation, by institutional grant 41-G fromthe American Cancer Society to the University ofChicago, and by Public Health Service Training Grant2-TO1-GM-0603.

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