JOURNAL OF BACTERIOLOGY, Nov., 1965Copyright ( 1965 American Society for Microbiology

Vol. 90, No. 5Printed in U.S.A.

Relationship Between the Competence Antigen andthe Competence-Activator Substance in

PneumococciALEXANDER TOMASZ AND SAMUEL M. BEISER

The Rockefeller Institute, and Department of Microbiology, Columbia University, New York, New York

Received for publication 14 June 1965

ABSTRACTTOMASZ, ALEXANDER (The Rockefeller Institute, New York,N.Y.), AND SAMUEL M.

BEISER. Relationship between the competence antigen and the competence-activatorsubstance in pneumococci. J. Bacteriol. 90:1226-1232. 1965.-Antisera prepared againstpneumococci in their competent phase inhibit deoxyribonucleic acid (DNA)-mediatedgenetic transformation as well as binding of radioactive DNA by the cells. The samesera do not inhibit transformation of competent Haemophilus influenzae and Bacillussubtilis cells, but transformation of a Streptococcus strain genetically related to pneumo-cocci is inhibited. The kinetics of immune inhibition of transformation resembles theinactivation of bacteriophage by phage-neutralizing antisera. The appearance of thecompetence antigen on the surface of pneumococci can be induced by the competence-activator substance. Antisera prepared against competent pneumococci can alsoinhibit the conversion of incompetent cells to competence by the competence-activatorsubstance. The possibility is considered that part of the new antigenic determinantappearing on the cell surface during competence may be the activator itself.

During their competent phase, pneumococcalcells possess a new antigenic determinant whichis absent from the same cells in the incompetentphysiological state (Nava, Galis, and Beiser,1963). Antisera prepared against competent cellsby injecting rabbits with formalin-killed pneu-mococcal cells could inhibit the transformationof competent pneumococci by deoxyribonucleicacid (DNA), whereas sera prepared against in-competent pneumococci had no inhibitory effecton the transformation.The rabbit sera prepared against both the

competent and the incompetent cells containedhigh titers of antibodies against the commonpneumococcal surface antigens; however, seraprepared against competent cells contained, inaddition, a minor component which could inhibitthe transformation of competent pneumococci.The nature of this inhibitory effect suggestedthat the new antigenic determinant of the com-petent cells is a chemical structure which is partof the cellular site involved in the first stages ofthe transformation process.

Recent investigations have shown that thecompetent physiological state in pneumococci is"induced" and controlled by a proteinlike cellproduct-the activator (Tomasz and Hotchkiss,1964)-which seems to be localized on the cell

surface (Tomasz, in preparation). It appeared ofsubstantial interest to investigate the relation-ship between the competence antigen and thepneumococcal activator.

MIATERIALS AND METHODSThe bacterial cultures used in this work were all

derivatives of the Avery R36A strain-a noncapsu-lated variant of pneumococcus type II (McCarty,Taylor, and Avery, 1946), subline R6. The methodsused for the preparation of DNA (Marmur, 1961),and for growing of the competent and incompetentcells, and the procedures used for testing the com-petence of the cells, have been described (Tomaszand Hotchkiss, 1964). The activation of incompe-tent cells to competence was terminated by theaddition of 0.1 ,ug/ml of subtilisin (Nagarse;Teikoku Chemicals, Osaka, Japan). Competentand incompetent cells were stored by freezing thecultures at a concentration of 5 X 107 cells permilliliter in medium containing 10% glycerol (Foxand Hotchkiss, 1957). Such frozen-cell prepara-tions were melted in an ice bath and then incu-bated, after dilution or transfer to fresh mediumby Millipore filtration, at 30 C for 10 to 15 minprior to use. This "resuscitation" after storage inthe frozen state was found essential for obtainingreproducible results.Assaying of the inhibitory titer of antisera was

routinely done by incubating antisera diluted in

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saline with 5 X 106 to 1 X 107 highly competentcells at 30 C for 30 min; the antiserum was dilutedto ineffective levels, and a saturating concentra-tion of DNA carrying the streptomycin-resistancemarker was added to the cell suspension. After 20min of incubation at 30 C, the transformationprocess was terminated by the addition of pan-creatic deoxyribonuclease (twice recrystallized;Worthington Biochemical Corp., Freehold, N.J.),and the number of transformants was determinedaccording to the usual procedure (Hotchkiss,1954).The 'y-globulin nature of the inhibitory serum

protein was tested by reacting samples of rabbitanti-competent pneumococcus sera (anti-co+sera) with excess goat anti-rabbit -y-globulin sera(kindly supplied by Curtis A. Williams, Jr.). Ascontrol for a possible inhibitory effect of the goatserum itself, rabbit sera prepared against incom-petent pneumococcus (anti-co- sera) were alsotreated with goat serum. After incubation of themixtures at 37 C for 1 hr followed by overnight inthe refrigerator, the precipitated rabbit y-globulinwas removed by centrifugation, and the titer ofthe inhibitory component was determined in thesupernatant fractions in the usual way.

In testing the interaction of antisera with DNA,0.1-ml samples of undiluted antiserum were mixedwith a series of concentrations of DNA in testtubes containing growth medium. After 30 minof incubation at 30 C, competent cells were added,and the inhibitory titer of the antiserum was de-termined. Cultures of a-hemolytic Streptococcusstrain D [the strain used by Bracco et al. (1957),kindly supplied by M. Krauss of C. M. McLeod'slaboratory] were grown in C-medium supple-mented with fresh yeast extract (Tomasz andHotchkiss, 1964). Pneumococcal activator wasprepared by the following procedure. Highly com-petent cells were suspended in saline buffered withpotassium phosphate buffer (0.01 M, pH 7.6) andcontaining 2-mercaptoethanol, and were heatedfor 10 min at 60 C to kill the cells and solubilizethe activator substance. The suspension was cen-trifuged in the cold at 10,000 X g, and the super-natant fraction was used. The method used for thepreparation of antisera was previously described(Nava, Galis, and Beiser, 1963). Three separatepreparations of anti-co+ and anti-co- sera wereused in the experiments. The sera prepared againstthe competent and incompetent pneumococci hadthe same titer with respect to antibodies againstthe common pneumococcal antigens. The antiserahad no detectable effect on the viability of highlycompetent or incompetent cultures.

Radioactive DNA was prepared from a uracil-requiring pneumococcal mutant resistant to strep-tomycin, which had been grown in C-medium(Tomasz and Hotchkiss, 1964) to which 0.1 sc/mlof uracil-R-C4 (specific activity, 20 to 30 #c/mmole;New England Nuclear Corp., Boston, Mass.) and5,ug/ml cold uracil had been added. The incorpora-tion of radioactive DNA into competent cellsduring transformation was determined as follows.

TABLE 1. Effect of pretreatment of the anti-co+sera with DNA

No. of transformants per ml after preincubation

DNA concn Without anti-co+ serum With anti-co+ serum

sgIml1.6 1.1 X 106 2.2 X 1051.0 0.98 X 10 2.2 X 1050.5 1.1 X 106 2.2 X 1050.16 0.9 X 106 2.2 X 106

The incubation of the competent cells with C14-DNA at 30 C was terminated by chilling the sus-pension on ice; after centrifugation in the cold,the cells were suspended in growth medium towhich 10 jug/ml of deoxyribonuclease were added,and the suspension was incubated at 37 C for 10min. After three washings with fresh medium, thecells were lysed by the addition of a few drops of5% deoxycholate solution and were plated onplanchets. Radioactivity was determined in awindowless gas-flow counter (Nuclear-ChicagoCorp., Des Plaines, Ill.).

RESULTS

Specificity of immune inhibition. The antibodynature of the inhibitory component of sera wasindicated by two features of this material: (i) thesubstance(s) which inhibited the transformationprocess was in the y-globulin fraction of thesera, and (ii) the production of this inhibitorycomponent in rabbits depended on injection withthe specific kind of cell. Sera taken from rabbitsprior to injection with formalin-killed competentcells did not have any inhibitory effect on trans-formation.The nature of the inhibitory serum component

was further investigated in the three experimentssummarized in Tables 1 and 2. Preincubation ofthe transforming DNA with a high concentrationof antiserum did not affect the inhibitory titerof the serum (Table 1), indicating that the inhi-bition was not due to an interaction with DNA.

Short pretreatment with papain (1 ,ug/ml,30 C, 15 min) destroyed the inhibitory compo-nent of the serum (Table 2). The data in Table 2also show that a considerable fraction of theinhibitory component(s) of the serum could beremoved by precipitation with goat anti-rabbit7y-globulin serum. The incomplete restoration ofthe uninhibited level of transformation was ap-parently due to the presence of some unspecifiedinhibitory component in the goat serum, since anidentical lowered level of transformation wasobtained in the control samples also (i.e., in thetubes with anti-co- sera mixed with goat anti-rabbit 7y-globulin).

Table 3 shows the results of experiments on

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TOMASZ AND BEISER

TABLE 2. Effect of pretreatments of the anti-co+sera with papain (experiment 1) and goat anti-

rabbit -y-globulin serum (experiment 2)

No. of transformantsExpt Pretreatment per ml

1 No anti-co+ serum added 2.5 X 104With anti-co+ serum 5 X 103With papain-treated anti- 3 X 104

co+ serum2 No antiserum added 7 X 104

With anti-co+ serum 30With anti-co- serum 7 X 104With anti-co+ serum after 1.5 X 103removal of y-globulin

With anti-co- serum after 1.2 X 103removal of 'y-globulin

TABLE 3. Species specificity of the antiserumprepared against competent pneumococci

No. of transformants per ml

Competent cells usedNo With anti- With anti-

antiserum co+ serum co- serum

Haemophilusinfluenzae ..... 8.8 X 105 1.0 X 106 1.1 X 106

ca-HemolyticStreptococcusstrain D (ofBracco. et al.,1957).......... 5.3 X 103 90 5.3 X 103

the species specificity of the immune inhibition.High concentrations of the anti-co+ sera causedno inhibition of the transformation of competentHaemophilus influenzae cells. (These experimentswere kindly performed by Grace Leidy, Colum-bia University.) On -the other hand, the trans-formation of competent cells of a Streptococcusstrain closely related to pneumococci, the ahemolytic Streptococcus strain D (Bracco et al.,1957), was inhibited by the anti-co+ serum.The anti-co+ serum had no effect on the trans-

formation of competent Bacillus subtilis 168-2(indole-, leucine-). (This experiment was per-formed in collaboration with David Dubnau,Albert Einstein College of Medicine.)A suspension of pneumococci activated to com-

petence by cell-free activator (about 108 cellsper milliliter) was distributed in 5-ml samples inthree tubes; 0.1 ml of anti-co+ serum was addedto the cells in tube A, 0.1 ml of anti-co- serumwas added to the cells in tube B; no antisera wereadded to tube C. After incubation at 30 C for20 min, 0.1 ml of C14-DNA was added to eachtube; the number of transformants and theamount of radioactivity bound by the cells was

determined after 20 min of incubation at 30 Caccording to the procedures described in Materi-als and Methods. Table 4 shows that the anti-co+serum inhibited the transformation as well as thebinding of radioactive DNA.

Kinetic characteristics of the immune inhibitionof transformation. Figure 1 shows the results oftypical antibody titration experiments. The anti-serum at various dilutions was allowed to reactwith 107 highly competent pneumococcal cells ina series of test tubes, and the inhibitory titer ofthe antiserum was determined according to theroutine procedure. It can be seen that a dilution

TABLE 4. Inhibition of the binding of radioactiveDNA by anti-co+ serum

No. of DNA-C14transformants bound

Preincubation of competent cells per ml per 5 ml

count/minWith anti-co+ serum ......... 4.3 X 105 100Without antiserum................2.4 X 107 2,715With anti-co- serum ......... 2.8 X 107 3,200

104

E

a)

c

0E0

0

6)

Ez

02

0

001 0 05 0.1

ml antiserum per ml medium

FIG. 1. Titration of anti-co+ serum with compe-tent cells. Open and closed circles indicate two sepa-rate experiments.

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VOL. 90,1965 ANTIGENIC CHANGE DURING ACTIVATION TO COMPETENCE

of the antiserum beyond 100-fold completely re- * Saturating concentrationmoved its inhibitory effect. of DNA, no antibody

Figure 2 shows the kinetics of the inhibition. A 105series of dilutions of the antisera were added totest tubes containing identical concentrations of Saturting concentration of DNA,competent pneumococci, and the mixtures were + 25 x diluted antibodyincubated at 30 C for various times. (The in- /herent instability of the competent state limitedthe maximal duration of these incubations to Eless than 60 min.) Samples of the cells were then.diluted 10-fold (to terminate the effect of anti- a 4body) into test tubes containing saturating con- c

0centrations of transforming DNA, and the num- EXber of transformants "surviving" the antibody o Subsoturating concentrationinhibition was determined. It can be seen that the c - DNA, no antibodyinactivation of the competent cells occurred by ° Subsaturating concentration of DNAa first-order process. However, at higher dilutions q + 25 x diluted antibodyof the antibody, the shape of the inactivationcurves revealed a more complex process. The use - 3of subsaturating DNA concentrations did not in- 3

crease the effectiveness of the anti-co+ serum(Fig. 3.)By use of the data obtained with higher con-

centrations of antibody, whefe the inhibition oappears to be a "single-hit" process, a K value of4 or (in some experiments) a maximum of 6 to 8was obtained, showing the relatively low titer of _ _ _ _ _ _ _of the antibody preparations. (The K value is 5 10 20 30defined as the specific rate of immune inhibition: Minutes

D T. FIG. 3. Rate of inactivation of competent cells byK = 2.3 - log anti-cot serum. For the determination of the number

t Tt of surviving transformants, saturating (0.2 pg/ml)and subsaturation (0.002 pg/ml) concentrations of

where T represents transformant, Ti indicates the transforming DNA were used.number of transformants at zero-time, and Tt

refers to the number of transformants at time t.AOs04 D is the final dilution of the antiserum.)S 0°03 In subsequent experiments, the effect of vary-10 \ tn> D 0.01 ing the concentration of the competent cells on_\\\gE\Fnone the extent of inhibition was determined. A mini-

mal inhibitory concentration of antiserum wasE incubated with increasing concentrations of com-

F\+\\\0if petent cells at 30 C for 30 min, and the extent ofo1 inhibition was determined by adding saturating

E concentrations of DNA and plotting the numberc of "surviving" transformants against the concen-

A \ \ tration of the competent cells. Figure 4 shows thatwo 3 \ \ the extent of inhibition was apparently independ-

3DQ10 ~ \ \ ent of the concentration of the cells: at all cellz concentrations used, a constant proportion of thez

cells was inhibited. This result is reminiscent ofA the behavior of phage-neutralizing antibodies

2 (Burnet, Keogh, and Lush, 1947). This pesudo0 20 30 40 50 50 first-order kinetics presumably means that the

Minutes number of antibody molecules per -"competenceFIG. 2. Rate -of inactivation of competent cells by site" is overwhelmingly high, even in the diluted

anti-co+ serum. sera and in the presence of high cell concentra-

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TOMASZ AND BEISER

TABLE 5. Adsorption of the inhibitory activity ofanti-co+ serum with co+ cells

No. of transform-Treatment ants per ml

No anti-co+....... 5.6 X 101Anti-co+....... 2 X 102Anti-co+ adsorbed with co+ cells. .. 2 X 104Anti-co+ adsorbed with co- cells. .. 6 X 102

tions, and that the number of competent sites percell is probably not large.The probability that the number of antibody

molecules per "competence site" is very high alsoprobably explains the difficulties encountered inattempts to adsorb the inhibitory activity fromanti-co+ sera with competent cells. Most adsorp-tion attempts were unsuccessful, but, occasionally,with very heavy suspensions of formalinized co+and co- cells, and with repeated adsorption, evi-dence for a specific decrease in the inhibitoryactivity of anti-co+ sera could be obtained(Table 5).Immune inhibition of the transformation of cells

activated to competence by purified activator prepa-rations. A 2-ml suspension of incompetent pneu-mococci (107 cells per milliliter) was treated with0.2 ml of cell-free activator preparation. Afterincubation at 30 C for 30 min, the activation ofthe cells was terminated by the addition of sub-tilisin (0.1 jug/ml). To a sample of the cell sus-pension, anti-co+ serum was added (0.1 ml to 1ml of suspension). The rate of inactivation of thecompetent cells is shown in Fig. 5. Figure 5 alsoshows the rate of inactivation of competent pneu-mococci which were allowed to develop compe-tence "spontaneously" during growth. It can beseen that the kinetics of immune inhibition weresimilar with the two kinds of competent cells.

Inhibition of the activation process by anti-co+sera. The activation process in which incompetentcells are converted to competence as a result ofthe interaction with the activator is inhibited bylow concentrations of proteolytic enzymes. Thesame enzymes have no effect on the transforma-tion process, in which competent cells react withDNA molecules (Tomasz, in preparation). Thisdifferent sensitivity to proteolytic enzymes pro-vides an operational distinction between the twoprocesses. Since, at the present time, at least,there is no way to assay the activation processindependently of transformation, the effect ofanti-co+ serum on the activation process couldonly be determined in an indirect way.Samples of an activator preparation of known

activity were added to two test tubes, A and B,

each containing equal concentrations of a stand-ardized incompetent cell suspension. In addition,tube B also received anti-co+ serum of knowntiter. Both tubes were incubated at 30 C for 30min, at which time 0.1 ,ug/ml of subtilisin wasadded to all tubes to destroy the activator and tostop the activation process. (A control experi-ment showed that subtilisin had no effect on theinhibitory power of the anti-co+ serum.)Two 0.1-ml samples of tube A, Al and A2, were

diluted 10-fold into fresh media; both Al and A2received saturating concentrations of transform-ing DNA, and A2, in addition, received anti-co+serum in the same concentration as was used intube B. A 0.1-ml sample from tube B was alsodiluted and treated with DNA. The three tubeswere incubated at 30 C, and the transformationprocess was terminated 30 min later by the addi-tion of deoxyribonuclease. The number of trans-formants was determined in the routine way(Table 6).The number of competent cells in both tubes

Al and A2 must be the same; however, the actualnumber of transformants observed in tube A2 isless, since, in this tube, a fraction of the compe-tent cells was prevented from reacting with DNAby the antiserum added with the DNA. The greatdecrease in the number of transformants in tube

0

Ea)

a

0

c-

0)-o

EDz

5x 10 vioble units per ml

FIG. 4. Effect of the concentration of competentcells on the extent of immune inhibition.

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B, as compared with tube A2, clearly reflects aninhibition of the activation process by the anti-co+ serum.

Since antiserum prepared against incompetentcells had no effect on the activation process (seeTable 6), the inhibition observed with the anti-co- serum seems to be caused by an interactionwith the activator rather than by the interactionwith some specific cellular site. This latter possi-bility was also made unlikely by experiments inwhich incompetent cells were allowed to reactwith anti-co+ serum in the absence of activatorfor 30 min. The antiserum was subsequently re-moved by washing the cells on a Millipore filter.Upon resuspension and addition of activator,

,Without antiserum

0

anti CO+ serum

)I

l0 20 30 40

Minutes of incubation

FIG. 5. Rate of inactivation of competent cells byanti-co+ serum. Symbols: X, competent cells whichdeveloped the competent state spontaneously duringgrowth; 0, cells activated to competence by cell-freeactivator preparation.

TABLE 6. Inhibition of the activation process byanti-co+ serum

No. of transfor-Tube Treatment mants per ml

Al No anti-co+ 1.5 X 105A2 Anti-co+ present during 6 X 104

transformationB Anti-co+ present during 3 X 102

activationAnti-co- present during 1.5 X 105activation and transfor-mation

these cells could be converted to competence,showing at least that an irreversible binding ofco+ antibody by incompetent cells does not occur.A similar washing procedure performed with com-petent cells which have reacted with anti-co+serum did not cause reversal of the immune in-hibition.An alternative explanation for the inhibition

of the activation process by anti-co+ serumwould be that cells in the process of conversionto competence may go through a "precompetent"state in which they are much more sensitive toantibody inhibition than are "fully" competentcells (i.e., cells which have completed the processof conversion to competence).

DIscussIoNThe greatly decreased capacity of competent

cells treated with anti-co+ serum to bind radio-active DNA indicates that the mechanism ofimmune inhibition of the transformation process isan interference of the antibodies with the combi-nation of DNA molecules with the cellular DNAbinding sites. The antigenic structure of thesesites seems to be specific for pneumococci, sincepneumococcal anti-co+ sera do not inhibit genetictransformation in other transformable species.The exception to this in the case of a-hemolyticStreptococcus strain D is particularly interesting,since the major pneumococcal surface antigen (R)seems to be absent from this streptococcus(Bracco et al., 1957). It is most reasonable tointerpret the inhibition of genetic transformationin this streptococcus by antisera prepared againstcompetent pneumococci as a further evidence forthe close relation between pneumococci and thisorganism. Other evidence for a close taxonomicrelation comes from successful interspecific trans-formation between the two species (Bracco et al.,1957) and from our recent finding that pneumo-coccal activator can activate the a hemolyticstreptococcal cells, and, in turn, the activator ofthis streptococcal strain can activate pneumococ-

0.1

o0.I

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a)

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TOMASZ AND BEISER

cus cells to competence (Tomasz, unpublisheddata).The specific "competence antigen" on the sur-

face of competent pneumococci appears to be pro-duced in the interaction of incompetent cells andthe activator. This conclusion may be inferredfrom the following findings: (i) incompetent cellsdo not possess the competence antigen (as judgedfrom their inability to induce the production ofanti-co+ antibodies in rabbits) and (ii) such in-competent cells do develop this antigen duringactivation to the competent state by purifiedactivator preparations (as judged by the fact thatthe transformation of competent cells produced inthis way is inhibited by anti-co+ sera).The finding that competent cells produced by

activation with cell-free activator preparationsand cells which have developed the competentstate during growth are inhibited with equal ef-ficiency by anti-co+ sera shows that the "compe-tence sites" produced in the activation process areidentical antigenically to those appearing on thecell surface during spontaneous development ofcompetence. This is a further addition to theevidence (Tomasz and Hotchkiss, 1964; Tomasz,in preparation) suggesting that the spontaneousdevelopment of competence in growing culturesis the result of an activation process by endoge-nously produced activator. It is not known at pres-ent whether the appearance of the new antigenicsites during activation is a result of an "unmask-ing" process or involves de novo synthesis of theantigen.The inhibition of the activation process by

anti-co+ sera "raises the possibility that one of thenew antigenic determinants on the competent

cell surface may be the activator itself. Certainobservations make this possibility a plausible one.It was shown earlier that the activator content ofpneumococcal cultures closely parallels the levelof competence (Tomasz and Hotchkiss, 1964).Furthermore, some recent experiments indicatethat the activator is localized in the cell envelope,from which it can be solubilized as a trypsin-sensitive material of considerable molecularweight. The antigenic activity of such activatorpreparations is presently being tested.

LITERATURE CITEDBRACCO, R. M., M. R. KRAUSS, A. S. ROE, AND

C. M. MACLEOD. 1957. Transformation reactionsbetween pneumococcus and three strains ofstreptococci. J. Exptl. Med. 106:247-259.

BURNET, F. M., E. V. KEOGH, AND D. LUSH. 1937.The immunological reactions of the filterableviruses. Australian J. Exptl. Biol. Med. Sci.16:231-236.

Fox, M. S., AND R. D. HOTCHKISS. 1957. Initiationof bacterial transformation. Nature 179:1322-1325.

MARMUR, J. 1961. A procedure for the isolation ofdeoxyribonucleic acid from micro-organisms. J.Mol. Biol. 3:208-218.

MCCARTY, M., H. E. TAYLOR, AND 0. T. AVERY.1946. Biochemical studies of environmentalfactors essential in transformation of pneumo-coccal types. Cold Spring Harbor Symp. Quant.Biol. 11:177-183.

NAVA, G., A. GALIS, AND S. M. BEISER. 1963. Bac-terial transformation: an antigen specific for'competent' pneumococci. Nature 197:903-904.

TOMASZ, A., AND R. D. HOTCHKISS. 1964. Regula-tion of the transformability of pneumococcalcultures by macromolecular cell products. Proc.Natl. Acad. Sci. U.S. 51:480-487.

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