JOURNAL OF BACTERIOLOGYVol. 87, No. 4, p. 867-875 April, 1964Copyright © 1964 American Society for Microbiology

Printed in U.S.A.

PENICILLIN- RESISTANCE OF COMPETENT CELLS INDEOXYRIBONUCLEIC ACID TRANSFORMATION

OF BACILLUS SUBTILIS

E. W. NESTERDepartment of Microbiology, School of Mledicine, University of Washington, Seattle, Washington

Received for publication 29 November 1963

ABSTRACT

NESTER, E. W. (University of Washington,Sieattle). Penicillin resistance of conmpetent cells indeoxyribonucleic acid transfornmation of Bacillussubtilis. J. Bacteriol. 87:867-875. 1964.-Trans-formants are resistant to penicillin killing forseveral hours after deoxyribonucleic acid (DNA)addition. The present study indicates that thisresistance is a consequence of such cells still re-maining competent and is not the result of anyinteraction of donor DNA with the recipient cell.The following data support this conclusion: (i)the frequency of transformationi can be increasedfive- to tenfold if penicillin acts on a competentculture prior to DNA addition; (ii) the percentageof competent cells in such a penicillin-treatedculture calculated on the basis of a random co-incidence of DNA molecules entering the same cellincreases some 25-fold over that of a penicillin-nontreated population; (iii) the kinetics of peni-cillin killing of a recipient cultuire are identicalwhether or not transforming DNA has been added;(iv) the extent of killing by penicillin is related tothe level of competence of the recil)ient culture;and (v) the kinetics of appearance and disappear-ance of competence in a poptulation as well as inindividual cells indicate that a cell imay remaincompetent for 3 to 4 hr.

The physiological basis for competence, i.e.,the ability of a cell to take u) and integrate highmolecular weight deoxyribonucleic acid (DNA)into its genome, is poorly understood. As a rule,recipient cultures of Bacillus subtilis, Pneniiococ-c(Us, and Haemzophilus become maximally com-

Ipetent under conditions in which the cells arenot rapidly multiplying (Anagnostopoulos andSpizizen, 1961; Young and Spizizen, 1961; Mc-Carthy, Taylor, and Avery, 1946; Alexander andLeidy, 1953; Goodgal and Herriott, 1961; Stuy,1962). A competent cell must undergo the fol-lowing interactions with the transforming DNA.First, the cell must take utp D'NA into a deoxy-

ribonuclease-insensitive state; secondly, it mustintegrate the 1)NA into its genome. Apparently,the inability to take up DNA is the most commoncause of incompetence, because a constant rela-tionship between uptake of P'32-labeled DNA andtransformation frequency has been noted undera variety of conditions in three transformationsystems: B. subtilis, pneumococcus, and Hae-mophilus (Fox, 1957; Lerman and Tolmach, 1957;Goodgal and Herriott, 1957; Young and Spizizen,1961). This uptake appears to be an energy-re-quiring process (Stuy, 1962) involving probablyboth a specific enzyme(s) and receptor sites onthe cell surface. The receptors probably containprotein because their synthesis is inhibited bychloramphenicol (Fox and Hotchkiss, 1957; Stuy,1962) and they appear to be antigenic in Pneu-mococcus (Nava, Galis, and Beiser, 1963). Theinability of a cell to take up DNA may reflect theabsence of such receptors or enzyme(s). Fewdata are available on factors which influenceintegration.

In a previous paper (Nester and Stocker, 1963),we reported that transformed cells of B. subtilisare penicillin resistant for several hours followingtransformation, although over 90 %7 of the popula-tion is killed. This investigation was initiated todetermine whether the penicillin resistance is afeature of the competent cell or is a result of thecompetent cell's interaction writh transformingDNA.

MATERIALS AND MXIETHODS

Transformation proceduire. The detailed pro-tocol for preparing competent cells, the subse-quent treatment of the culture with penicillin,and the techniques of biological assay were de-scribed (Nester and Lederberg, 1961; Nester andStocker, 1963) as follows. The recipient cultureswere grown for 4 hr at 37 C in a glucose-saltsmedium supplemented with casein hydrolysateand L-tryptophan. The culture was then diluted

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J. BACTrE1RIOL.

1:10 into a second medium (CHT-10) andincubated at 32 C. The culture reached its peakof competence between 120 and 150 min afterdilution, and D1NA was added at various timesduring this secontl incubation period. D)NAprel)arations weere always used at saturatinglevels (3 to 10 jig/mil of recipient culture followedby 20 ,ug/ml of deoxyribonuclease) unless other-wise noted. In some experiments, cells were

collected on a 0.45-, -Millipore filter disc, washedNvitl 10 ml of minimal medium, and resuspendedin appropriate medlium. This procedure did notsignificantly alter the overall competence of theculture, because the same frequency of trans-formants resulted whether DNA was added be-fore or after filtering. Unless otherwise noted,penicillin was used at 200 units per ml and peni-cillinase at 400 units per ml. Reversion controlswere included with each experiment.

Bacterial strains. Strain 168 (try2-), the usualrecil)ient strain, re(luires indole or tryptophanfor growth. Strain SB210 (try2-) is a poorlytransformable strain derived as a spontaneousmutation from 168. Strain SB1 carries the un-

linke(d markers Ihis, and try2. The prototrophicstrain used as a source of DNA was SB19 or

SB455. The pedigrees of these strains have beendescribed (Nester, Schafer, and Lederberg, 1963).

RESULTS

E;ffect of penicillint treatment prior to DAVAaddition on the frequiency of transformants. To

distinguish between the two alternativ es forpenicillin resistance, namely, that competent cellsipso facto are penicillin resistant, or thlat theinteraction of transforrrming DNA w-ith a com-

petent cell confers penicillin resistaice, the effectof addin,g penicillin I)efore the addition of D)NAon the frequency of transformiants was stu(lied.Penicillin wvas added at dlifferent timnes to samplesof the competent cultures ani(l after 60 iruin wsasdestroYed by )enicillinase. T'lhei culture was thenfiltered an(d the resuspended cells exl)soCe(d to1)N-A. Table I pjresents the results of three suchexl)eriments. In each case, the freqtlency oftransformants is five- to ternfoldl greater tlhani itwas in a similar culture not treatedl with peniciil-lin. These (lata suggest that the competent cellsare penicillin-resistatnt )ior to 1)DNA atdlition.However, it should be ntoted that tlhere is also a

several-fold decrea.e irn the absolute numbher oftransformants in th( n)enici llin-treated tullt ures.The explanation for this find(ling is not cleain

Kinetics of penlicillin killing of a noiflpetentcultore. 'Th}e kinetics (of penicillin killingo of a

competent cultuire ill the labsence of transform-ing DNA.. are show n in Fig. 1. There is ilnitiallyan exlponential killing, followed by a ogreatlyreduced or even colnm)lete cessation, followed byfurther killing. An exl)onential rate of killinggenerally ceases after 90 to 95%/1t of the popllulationhas been killedl. This poiint most likely indicatesthe time when thle surviving population (con1sistsprimarily of peanicillin-rsistant.auI presumably

TABLE 1. Effect of penicillin on competence*

Time of Viable cells per ml Transformants per ml t'erfrent Fof i

Expt addiofpn transiormat____ transfornm.tion(min) frequency

-Pen Xl°7 + Pen X16 -Pen X 104 + Pen Xl -14 Pen + Pen

1 90 2. 9 3.0 1.4 1.3 0.048 0.44 9.

150 6.4 2.8 2.6 (.88 0.040 0.31

2 90 2.3 1.4 5.4 1 0.2.3 1.1 4.8150 2.8 1.4 7.6 1].9 0.27 1.3 4.8

3 210 11 3.1 3.5 0.82 0.033: 0.26 7.9

* Strain 168 cells were prepared for competence by the usual procedure; 90 irin after dilutioni inltoCHT-10, the cultuies were diluted 1:5 into fresh CHT-10 and penicillin added at the indicatedl tiunies(measured from the original dilution into CHT-10). Penicillinase and deoxyriboniuclease were a(ddedand the cultures filtered 60 min after penicillin addition. The cells were resus)ernded in fresh CHT-10,DNA was added for 10 min, and the cultures were assayed for try+ transforiiiants. Both the treated anduntreated cultures were handled the same, except for the addition of penicillin. Control experimlentsindicated approximately a 70cc recovery of cells from the M\illipore filter.

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PENICILLIN RESISTANCE OF B. SUBTILIS

still-competent cells. If the kinetics of penicillinkilling reflect the competence of the culture, thenthe surviving population of cells should be pro-portionally more competent than a comparablep)opulation not treated with penicillin (Table 1).Furtlher, the pronounced decline in killingoccuriing after the majority of cells have beenkilled should be independent of DNA addition,an(d the survival level after the exponentialphase slhould be related to the competency of theculture; a highly competent culture should resultin a hiigher survival level than a less competentculture.

IPropoortion of competent cells in a recipientpopuiilation after penicillin treatment. The cellp)o)ulation surviving penicillin treatment con-tains a higher proportion of competent cells thandloes a l)opulation vithouit such treatment (Table1). This observation was verified and extended inthe following way. 'T'he fraction of competentcells inl a l)opulation (loubly auxotrophic for twouinliniked markers can he estimated by relatingthe frequency of double transformants observedto the number predieted from the frequency ofsinigle transformants; the frequency of doubletransformants should be a product of the fre-quency of single transformants if the two eventsare independent. Under our conditions of trans-formation, the frequency of double transformantsis generally far greater than is calculated fromthe frequency of single transformants (Nester andStocker, 1963), unlike the situation in Hae-mophiluis (Goodgal and Herriott, 1961) and Pneu-mococcus (Ephrussi-Taylor, 1959).The ability of penicillin to increase the pro-

portioni of competent cells in a population isshown in Table 2. The penicillin survivors aretransformed at a frequency 20-fold higher tohisi+ and try + than are the untreated cells. Inaddition, the fraction of competent cells in thepopulation without penicillin treatment is lessthan 3%, whereas 50%/,, of the penicillin survivorsare calculated to he competent. As discussedpreviously (Nester andl Stocker, 1963), thesecalculations are based on certain assumptions,some of which may not be valid. NI!evertheless,the fact that penicillin treatment results in anincrease in the frequency of transformants whichis closely paralleled by the calculated increase incoml)etence lends support to the validity of thecalculation and verifies that the population ofpenicillin survivors is greatly enriched for com-

petent cells.

109.

-iZ.(ni

-4tA-i.jt-ki.QjIR

VIABLE CELLSNO PENICILLIN

VIABLE CELLS+ PENICILLIN

O 60' 120' 180'240300'TIME AFTER PENICILLIN ADDITION (MIN)

FIG. 1. Penicillin killing of competent recipientcultures. Strain 168 was prepared for competence;120 min after dilution into CHT-10, 200 units perml of penicillin were added and incubation con-tinued. At the indicated timnes, samlples were removed,diluted into penicillinase, and assayed for viablecells.

Level of competence, DNA addition, and peni-cillin killing. The kinetics of killing of two cul-tures of strain 168 of different levels of com-petence which were treated with penicillin areshown in Fig. 2. There is a significant differencein the level of survivors in the two cultures afterthe initial exponential killing. Thus, after approx-imately 90% of the cells of the more competentculture had been killed, further killing ceased fora period of time. However, in the less competentculture, more than 99% of the cells were killedbefore any decrease in the killing rate was de-tectable. Furthermore, the kinetics of killing wereidentical, whether or not DNA had been added.A similar experiment was performed in whichthe penicillin resistance of 168 wvas comparedwith that of SB210 (try2-) which, under optimalconditions, is only about 1 .000 as transformableas 168 (Fig. 3). In this case, there was even amore pronounced difference in the level of sur-vivors.

Kinetics of attaining and maintaining compe-tence. Although all of the data presented are

consistent with the hypothesis that competent

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TABLE 2. Competence of culture with and without penicillin treatment*

Transformants per ml Viable cells Per cent Per centTreatment his+ X 103 try+ X 103 kis+ry+ X 102 X 107 transformation competent

+ Penicillin 471 194 82 2.2 1.5 51- Penicillin 70 37 17 6.8 0.08 2.5

* SB1, hisf-try2- (unlinked), was prepared for competence by the described procedure (total volumeof culture, 100 ml); 120 min after dilution into CHT-10, 1,000 units per ml of penicillin were added, andincubation was continued for an additional 120 min. The culture was centrifuged, washed once, resus-pended in Ho the original volume, and treated with prototropic DNA (14 ,ug/ml) for 30 min. An identicalculture was taken through the same procedure except for penicillin treatment. After penicillin treat-ment, 8.5% of the original cells survived. The transformation frequency with 14 ,Ag of DNA for 30 minat 120 min after dilution into CHT-10 was 0.7%.The per cent of competence was calculated from the following formula:

% competence =\

cells ipso facto are l)enicillin-resistant, they donot answer the question of whether there is an

exact concordance between penicillini resistanceand competence of a cell. If the gain and loss ofcompetence coincides with the gain and loss ofpenicillin resistance, a cell must remain com-

petent for 3 to 4 hr-the duration of penicillinresistance of transformants (Nester and Stocker,1963). However, a cell might lose its competenceor proceed through cycles of competence andnoncompetence and still remain penicillin-re-sistant. Alternatively, a cell might becomepenicillin-sensitive and still remain competent.Although the exact temporal relationship betweencompetence and penicillin resistance is not easilydetermined, because penicillin requires a periodof time to act, it is possible to gain data bearingon the above possibilities by relating the kineticsof attaining and maintaining competence in a

culture to the kinetics of penicillin killing oftransformants. The duration of competence incells was studied in two ways: first, by deter-mining the kinetics of achieving and maintainingcompetence in the whole culture and, second, bydetermining the length of time an individual cellremains competent. The competence of the totalpopulation was evaluated by assaying the num-

ber of transformants after exposure to DNA forincreasing periods of time, as well as after a

10-min contact with DNA added at variousintervals (Fig. 4). The former assay measures allcompetent cells as they arise in the culture; thelatter measures the competence of the culture

+ \ ( try2+ \

cells /viable cells X10)his,+tryX+viable cells

only during the particular 10-min period theI)NA and cells are in contact.

Tlnhe number of transformants increases withcontinued exposure to DNA. After 60-min ex-posure, additional contact with the DNA evokesno further transformants. Likewise, the numberof transformants from a 10-min contact withDNA increases and reaches its peak at approxi-mately the same time. This number remainsrelatively constant for the next several hours. Theobservation that at 150 min a 10-min pulse ofDNA yields a lower transformation frequencythan does continued exposure would not bepredicted if the number of competent cells in theculture had reached its maximum and if com-petent cells maintained comipetence for severalhours. However, these data may simply meanthat a competent cell cannot be saturated withDNA in 10 min but continues to take up mole-cules upon more prolonged exposure.The lack of any significant increase in the total

number of transformants arising in the cultureafter 150 min (even though the cells are multi-plying and in contact with DNA), coupled withthe constancy in the number of transformants inthe culture at any one time after 150 min (meas-ured by the 10-min pulse), can be explained bythe hypothesis that no cells in the populationbecome competent after 150 min and that anycompetent cells assayed after this time reflectthe continued competence of those cells whichbecame competent before 150 min. Anotherinterpretation which we consider less likely is

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that a balance is reached between dying andl -9 X newly arising transformants. If this latter ex-

planation is correct, the constancy of competentcells observed at any one time by the 10-min

4 s contact with DNA would then result from a lossAP- in competence of some cells, counterbalanced by

Xi < '/a gain in competence of other cells. This would beit {; lo8 ,#Jf / similar to the situation described for the kineticsQt~,'/ of the appearance of competent cells in Pneumo-

d:¢' { coccus (Hotchkiss, 1954). Accordingly, the dura-I ,8/ tion of competence in individual cells was de-

- COMPEJENT termined. SB1 (hisj- try2, unlinked) was exposed---LESS COMPETENT to his,- try2+ I)NA. After removal of this DANA

IOT*and after various periods of incubation, the

07 \+2 culture was exposed to hisl try2- DNA; theculture was scored for hisi+ try2-, his7- try2+,and his,+ try2+ transformants. The level of

-4 <random coincidence from the two loci enteringthe same cell was estimated by- adding the his,-

106 < try2+and his1+ try2 DNA lpreparations simul--4< 106- \taneously (Table 3). A significant point of these

data is that the number of double transformants- +DNA arising when the his+ marker is added at 0 timeDNA is approximately the same as when it is added 3

| v hr later. Because double transformants can onlvresult when the try± and his+ markers enter the

105 8 same cell, by random coincidence, every doubletransformant must have been competent duringthe time it picked up the first marker (try+) andduring the time it took up the second marker(his+). It is conceivable that these cells have notremained competent during the entire interval

G +DNA between the addition of the first and secondl0o4 "markers. However, the ratio of double trans-

- DNA formants to the product of the single transform-ants obtained when the two different DNApreparations were added simultaneously com-pared to that obtained when they were added 3hr apart suggests that the cell continually main-

103-_I

tains its competence. If competence were gained)' 60' 120' 160' 240' and lost, even though it involves the same try+TIME AFTER PENICILLIN ADDITION transformants, the ratio of double to single

(MIN) transformants would be greater when the cellswGere exposed to the two DNA l)reparationsFIG. 2. Kinetics of penicillin killingy of highly simult1aneously The mo.st econlomical interpareta-

competent and poorly competent cultutres. Competent tioistatetheysame cllnminainteopetecstrain 168 cells were prepared by the usutal regimen(highly competent) and by a procedure in which the over a period of 3 to 4 hr.initial 4-hr incubation period wvas reduiced to 2 hr(poorly competent). DNA was added 90 win after and assayed for try+ transforntants and viable cellsdilution into CHT-10, antd deoxyribonnclease at as indicated. Without penicillin, thetransfor7mation118 min. At 120 min, the cdtuires were dilutted into frequency of the more comtpetent cdtiture assayed atprewarmed CHT-10 containting 200 units per mnl of 120 mmin was 0.3%c, and the poorlty comn,petent cuilturepenicillin where indicated. Sam7tples were removed was 0.03c%o.

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210 to sustain its competence for several hours, the

o9N penicillin effect of chloramphenicol and amino acid starva-/168 No penicillin tion on a competent population was determined

./ ,' (Fig. 5). It is clear that the addition of chloram-phenicol or the removal of a required amino acid

./ / (tryptophan) results in a loss of competence. We/8j Xconclude that, although a competent cell is not.-,,* multiplying and is not synthesizing proteins

specified by the donor DNA, it nevertheless must

e-'- carry out some protein synthesis to remain com-

07 petent.

DISCUSSIONThe results reported in this paper strongly

168 + Penicillin suggest that in the B. subtilis transformation106- A *. Viable Ceils system a competent cell is penicillin-resistant;

when competence is lost, the cells again becomesensitive to killing by this antibiotic. The rele-vant facts which lead to this conclusion are thefollowing. Transformants are totally resistant to

I j5_ -___ 168-Trcnsformants-r\

No penicillin 6

I06t K 210-+ Penicillinq 41 \*~ Viable Cel Is

S: 'Continuous exposure-,/to DNA.

10 f0 2 1 0- Transformrntns

No penicillin

/0L DNA Contoct 10

I09-/ - 4/~~~~~~~~~~~I10

0 60 120 180 240Time Af ter Penicillin Addition (Min.)

FIG. 3. Kinetics of penicillin killing of strains 0-.*168 and SB210. The experimental procedure of Fig. 70 120 180 290 300 350 4102 was employed. Time of Incubation in CHT-10(Min.)

FIG. 4. Kinetics of appearance of competence.

Effect of chloramphenicol and amino acid Competent 168 cells were prepared as usual; 60 minstarvation on competence. From present and after dilution into CHT-10, the culture was dilutedpreviousdata(Neterane 1:5 into fresh CJHT-10 and divided into two lots-aPrevioUS data (Nester and Stocker, 1963), we flask, and a series of tubes. Prototrophic DNA

eranvismionthecompalet cellyias autnonmltiingb (S plg/ml) was added to the flask, and samples wereorganism incapable of carrying out certain bio- removed at the indicated times for transformantsynthetic functions. To determine whether the assay. DNA was added to the tubes for 10 min, andcompetent cell requires any biosynthetic activity the culture was assayed.

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TABLE 3. Duration of competence of individual cells*

Time after Transformants per ml Mixed donor DNAaddition of first hi~r~added at 0 timemarker (m)in his+ (X 104) X104) (X 102) (his+) (try+) X 10-4 his+ try X 10-4try+(X10)his+try+(X 102) (hs+) (try+)(his+) (try+

0 110 125 387 2.8 2.660 181 90 707 4.3 1.8120 180 79 533 3.8 2.7180 138 86 297 2.5 2.1240 71 76 90 1.7 2.9

* SB1, hisctry2- (unlinked), was prepared for competence by the usual regimen; 120 min after dilu-tion into CHT-10, 2,ug/ml of hisr-try2+ DNA were added, followed 20 min later by 8 Ag/ml of deoxyribo-nuclease. After an additional 10 min of incubation, the culture was filtered through a 0.45-,u Milliporefilter disc, resuspended in fresh CHT-10, and samples dispensed into tubes. This is 0 time. At the in-dicated times thereafter, 168 DNA, hisj+trys- (1 ug/ml) was added for 20 min followed by deoxyribo-nuclease. The removal of the hisI-try2+ DNA was verified by adding 0.1 ml of the resuspended cellsexposed to try2+ DNA to competent try2- cells. No appreciable number of try2+ transformants resulted

the killing effect of penicillin for several hoursafter DNA and deoxyribonuclease addition(Nester and Stocker, 1963). This resistance doesnot depend on the addition of transforming DNA,because the kinetics of penicillin killing areindependent of DNA addition, but is related tothe level of competence of the culture. Further,the addition and subsequent removal of penicillinprior to DNA addition consistently results in afive- to tenfold increase in the frequency oftransformants. Finally, the data indicate thatcells remain competent for approximately thesame time span in which transformants areresistant to penicillin action. Because the ma-jority of the recipient population is killed bypenicillin in our experiments, only a smallminority of the recipient population is pre-sumably competent. This conclusion was reachedindependently by the finding that the number ofdouble transformants is far greater than would bepredicted on a random entrance of DNA mole-cules into the same cell. Indeed, the calculatedproportion of competent cells is roughly equiva-lent to the number of cells in the populationwhich possess a marked but transient resistanceto penicillin. This comparison is probably onlyfortuitous, in view of the several simplifyingassumptions on which the calculation is basedas well as the unlcertainties of the activity ofpenicillin required for cell death.Although there are several possible explana-

tions why competent cells are penicillin-resistant,the most reasonable interpretation is that thecompetent cell is not growing and multiplying.The following facts support this contention.

10:

K

c1zK;

I 9Od 1 5('MTIME OF STARVATION OR CM TREATMENT (MIN)

FIG. 5. Effect of starvation and chloramphenicolon competence. Strain 168 was prepared for com-petence as usual; 120 min after dilution into CHT-10, the culture was diluted 1:5 into fresh CHT-10.At 170 min, the culture was filtered and resuspendedin CH; 0.9-ml samples were dispensed into tubescontaining tryptophan (20,ug/ml) or CM (10IOg) asindicated. At the indicated times, a saturating levelof SB19-DNA was added for 5 min and the cultureassayed for transformants. Chloramphenicol did notdecrease the viable-cell count. When the CM andDNA were added together, the CM had no effect on

the yield of transformants after incubation periodsup to 180 min.

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Penicillin acts only on growing bacteria (Hobby,Meyer, and Chaffee, 1942). The number oftransformants does not increase for severalhours after DNA and deoxyribonuclease addition(Nester and Stocker, 1963). The number of

competent cells in a recipient culture remainsrelatively constant for several houirs. A similarsituation may exist in lHaeitiophiliis, because thecoml)etence of a culture can be prolonged if thecells are placed under poor nutritional conditions(Goodgal and Herriott, 1961).One question which the above interpretation

cannot answer is why penicillin treatment ofcompetent cells results in a decrease in thenumber of transformants in most experiments.One possible explanation is that anyV potentially-com)etent or "slightly comletent" cell is killedbefore it becomes fully competent. A second pos-sible interpretation is that some portions of thecell surface, perhaps associated with the D)NAreceptors, are not, totally inert during the periodof competence but are being synthesized. Peni-cillin apparently does not kill these cells (asevidenced by the total resistance of transform-ants), but may prevent the -ynthesis of cell-wallrecel)tors or enzymes required for the irreversibleuptake of the DNA. Sublethal effects of lenicillinon the synthesis of cell-wall components were

recently described (AIichael, 'Massel, and Perkins,1963). A similar explanation might hold in partfor the inhibitory effect that chloramphenicoland amino acid starvation have on the mainte-nance of competence. The rapid loss of compe-

tence under these conditions again suggests thatthere must be biosynthesi.s, including presumablyprotein sy-nthesis, occurring in the nongrowingcoml)etent cells. Altenbern (1963) recently notedthat chloramlhenicol inhibited the reversion ofsI)herolplasts of Proteuis to an osmotically re-

sistant form, although a net synthesis of proteincould not be measured. If the competent, cell isnot growing, any biosynthesis may- result in onlya turnover and not a net synthesis of cellularcomlponents.The lpresent studies have not directly deter-

mine(l at what stage of competence, irreversibleul)take, or subsequent, integration into the re-

cipient genome the l)enicillin-resistant state ofthe recil)ient cell is involved. However, the work

of other investigators suggests the irreversibleul)take of the DNA. Young and Spizizen (1961)obs;erved that, under the usual conditions oftransform-ation, a change in competence l)roduced

by shortening the time of incubation of therecipient culture prior to D)NA addition resultedin a decreased uptake of P32-labeled DNA. Thisdecrease wvas directly )roportional to the de-creased transformation frequency. Further, theyobserved that a genetically incompetent culture,characterized as nonsporulating, did not irre-versiblv take up labeled DNA. The culture SB210appl)ears to sporulate very poorly.The relationship of this nonmultiplying state

to a cultural state of the l)opulation is unclear.The data suggest that a minority of the recipientcells go through a lparticular stage in the growthof the culture which is characterized by anextraordinarily long period (several hours) of non-multiplication (competent cells) under environ-mental conditions in which the majority of thepopulation (noncompetent cells) have a genera-tion time of approximately 45 min. B3ecausenonsporulating cells do not transform well, it isconceivable that this stage of nonmultiplicationis a p)reslportilation stage in the life of the or-ganism, as lpreviously suggested by Young andSpizizen (1961). Experiments are currently inprogress to test this hypothesis.

ACKNOWLEDGMIENT

This study was supported in part by grantRG 9848-01 from the U.S. Public Health Serv-ice, and by the State of W1ashington Fund for13iological and MIedical Research.

LITERATURE CITED

ALEXA\kNDER, H. E., ANI) GJ. LEIDY. 1953. IndUctionof streptomycin resistance in sensitive Hemno-philus inflnenzae by extracts containingdeoxwribonucleic acid from resistant Hemo-philuis influenzae. J. Exptl. Med. 97:17-31.

ALTENBERN, R. 1963. Reversion of L fornms andspheroplasts of Paroteus niirabilis. J. Bacteriol.85:269-272.

ANAGNOSTOPOULOS, C., AND J. SPIZIZEN. 1961. Re-quirements for transformation in Bacillussubtilis. J. Bacteriol. 81:741-746.

EPIIRUSSI-TAYLOR, H. 1959. The mechanism ofdeoxyribonucleic acid-induced transforma-tions. Intern. Congr. Microbiol. 7th, Stock-holm, 1958, p. 51-68.

Fox, AI. 1957. Deoxyribonucleic acid (DNA)incorporation by transforiimed bacteria. Bio-chiml. Biophys. Acta 26:83-85.

Fox, MI., AND R. HOTCIIKISS. 1957. Initiation ofbacterial transformations. Nature 179:1322-1325.

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GOODGAL, S., AND R. HERRIOTT. 1957. Studies ontransformation of Hemophilus influenzae, p.336-340. In W. D. McElroy and B. Glass [ed.],Symposium on the chemical basis of heredity.Johns Hopkins Press, Baltimore.

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