BIOCHEMICAL STUDIES OF ENVIRONMENTAL FACTORS ESSENTIAL IN TRANSFORMATION OF PNEUMOCOCCAL TYPES MACLYN MCCARTY, HARRIETT E. TAYLOR,l AND 0. T. AVERY The phenomenon of transformation of pneu- mococcal types provides an outstanding example of the induction of specific and heritable modifications in microorganisms. Basically the phenomenon rep- resents the transformation of a nonencapsulated (R) variant derived from one specific type of Pneu- mococcus into encapsulated (S) cells of heterolo- gous specific type. By the technique employed at present, this is accomplished by growing the non- encapsulated cells in a special serum broth to which has been added the active fraction extracted from encapsulated pneumococci of a heterologous type. The production of a new polysaccharide capsule is induced in the R cells so that they acquire the type- specificity of the organisms from which the extract was obtained. The property of forming the new capsule is transmitted indefinitely to subsequent generations, and, in addition, the substance respon- sible for the induction of transformation is itself reduplicated in the transformed cells. It is thus ap parent that one is dealing with hereditary bacterial modifications, which are predictable and subject to direct experimental control. The chemical nature of the substance capable of bringing about this type of heritable change presents a problem of primary importance. Accumulated evi- dence based on the results of innumerable tests of the specificity and biological activity of various preparations, together with the data obtained by chemical, enzymatic, and serological analysis of the active material, has established beyond reasonable doubt that the active substance responsible for transformation is a specific nucleic acid of the des- oxyribose type (2, 9, 10). These results suggest that nucleic acids in general may be endowed with biologically specific properties not hitherto demon- strable. Results confirming this view have been pub- lished recently in a preliminary report by Boivin and his co-workers on the role of desoxyribonucleic acid in inducing a transformation analogous to the pneumococcal phenomenon, using encapsulated strains of E. coli (3). These investigators state that evidence obtained by chemical and enzymatic tech- niques shows that desoxyribonucleic acid and not the protein of the nucleoprotein molecule is re- sponsible for the specific effect. In so far as one can judge from this brief report, the results agree in principle with those obtained with Pneumococcus. In the historical development of the problem of 1 Work done in part as Fellow in the Natural Sciences of the National Research Council. transformation it is of interest to recall that Griffith (6), who originally described the phenomenon in viva, was unable to obtain positive results in vitro. The first successful demonstration of the reaction in the test tube was carried out by Dawson and Sia (5) in nutrient broth containing anti-R rabbit serum. From that time on, serum or serous fluid of one sort or another has always been used and has been shown to be an essential factor, since in its absence it is impossible to induce transformation. The function of serum is not merely one of enrich- ment of the culture medium, however, since the nu- trient broth itself contains adequate amounts of accessory growth factors required for initiation and maintenance of growth. It is evident, then, that serum provides essential environmental factors for pneumococcal transformation in vitro, and clarifi- cation of the role played by the serum is of fore- most importance in understanding the nature of the phenomenon. The following questions naturally arise: Why is the presence of serum or serous fluid in the medium essential? Why are some sera ca- pable of supporting transformation while others ut- terly fail? What components function as essential factors, how do they act, and what is the biochemi- cal nature of their action in respect to the cellular changes evoked by the specific pneumococcal nu- cleic acid? Although anti-R rabbit serum was used in the initial studies, in recent years this has been largely supplanted by human chest or abdominal fluids which occur as the result of a variety of pathologi- cal processes. These serous fluids invariably contain more or less R antibody, but they show marked variations in their ability to support transforma- tion, which are unrelated to the antibody titer. It has been observed, for example, that chest fluids accumulating as the result of mechanical factors, as in the case of cardiac decompensation, regularly have little or no activity, while fluids formed as the result of tuberculous or acute infectious processes are usually highly effective. This suggests that one of the essential serum components may be present only in very low concentration in normal sera, but may increase in the course of infectious disease. This empirical observation has served as a useful guide in selection of effective sera. The results of studies on the role of serum in the transforming reaction indicate that at least three essential constituents are involved. These are: (1) the R antibody, which causes agglutination of un- encapsulated R pneumococci; (2) a dialyzable con- 178 M. MCCARTY, H. E. TAYLOR, 0. T. AVERY stituent; and (3) a protein factor, in addition to the R antibody. Each of the three components is considered individually in the following discussion. The evidence for assuming that the serum factor depends on the collective action of the three com- ponents is summarized, together with a description of certain experiments designed to elucidate the function of each and the mechanism of their com- bined effect. THE ROLE OF R ANTIBODY All sera that have proved effective in supporting transformation have contained R agglutinins, but despite this fact it was difficult to be certain that these antibodies were essential in the reaction. For example, there seems to be no relation between the titer of R antibody and the efficacy of the serum in the transforming system, and indeed some of the most potent sera have the lowest anti-R titers. The results of recent experiments, to be described below, provide some indication of the role of R antibody. During growth in the serum broth used in the transformation system, the R ceils agglutinate as they divide, so that each cell of the inoculum ap- parently gives rise to a colony, which becomes visible to the naked eye after several hours of growth (Fig. 1, a). Subsequently these colonial ag- gregates become larger and settle to the bottom of the tube, leaving a clear supernatant (Fig. 1, b). ,.. .* . . . - u tl . , *** . ** , . . . . . . ' , ? o ?? ? *a b C FIG. 1. Growth of pneumococci in serum broth. (a) Six- hour growth of R pneumococci. (b) Eighteen-hour growth of R pneumococci. (c) Transformation; l&hour growth of R pneumococci in serum broth plus Type III transforming substance. This latter fact is useful in the technique of the $est, because newly formed S cells occurring as the result of transformation are not agglutinated by R antibody and grow diffusely throughout the tube (Fig. 1, c). Thus the appearance of the growth gives immediate presumptive evidence concerning the presence or absence of transformed pneumo- cocci. Colonial growth of R pneumococci somewhat an- alogous to that caused by anti-R results from the use of a semisolid medium (II). For example, in a viscous medium containing a low concentration of agar (0.2 yO) R pneumococci grow in loose aggre- gates not unlike those formed by antibody agglu- tination, The growth differs, however, in that the colonies do not settle to the bottom of the tube (Fig. 2, a). S pneumococci give a fluffy, cotton-ball colony, which is readily distinguished from the R colony (Fig. 2, b) , so that transformation in this type of semisolid medium can be recognized by the appearance of characteristic S outgrowths on the R colonies (Fig. 2, c). It has been found possible cl l;i C FIG. 2. Growth of pneumococci in semisolid medium. (a) Eighteen-hour growth of R pneumococci. (b) Eighteen-hour growth of S III pneumococci. (c) Transformation. to bring about transformation in an agar semisolid medium containing normal rabbit serum but wholly lacking in R antibody. Neither normal rabbit serum nor the semisolid medium were by themselves ca- pable of supporting transformation. This experi- ment suggests that the type of colonial growth pro- duced by anti-R is an important factor and that when this type of growth is simulated by other means the anti-R can be dispensed with. It is im- portant to note, however, that serum is required in the semisolid medium, although it is not essential that it contain R antibody. Although it cannot be stated with certainty why colonial growth is required, it is possible that local reducing conditions arising in the aggregated cells are of primary importance. This thesis is supported by the results of experiments in which the medium is placed in a shallow layer not exceeding l-2 mm. in depth. In the shallow layer, oxidizing conditions are promoted, and, even in the usual serum medium containing R antibody, manifest transformation does not occur. Attempts to reverse the effect of the shallow layer by the addition of reducing agents have not yielded consistent results, but on one oc- casion transformation was obtained in a group of flasks in which glutathione had been added to the usual serum medium. There is, then, some evidence that reducing conditions are essential in some phase of the transforming reaction. On the basis of the evidence available at present it would appear that R antibody serves the purpose of causing an essential colonial aggregation of R pneumococci, which in turn results in local con- ditions, possibly reducing in character, that are re- quired for transformation. #-' TRANSFORMATION OF PNELJMOCOCCAL TYPES 179 DIALYZABLE SERUM CONSTITUENT Early attempts at salt fractionation of serum fac- tor by the classical methods of protein chemistry yielded totally inactive fractions. Some light has been thrown on these results by the discovery that a dialyzable constituent of the serum is essential. When an active serum is dialyzed against physio- logical saline, there is a progressive decrease in its efficacy in the transforming system, and if dialysis is sufficiently prolonged the serum becomes completely inactive. Under these conditions, however, the R antibody is unimpaired, and no denaturation of protein is apparent. The period of dialysis required for complete in- activation varies for different sera from two days to three weeks. This suggests that the dialyzable substance may not be free in the serum but com- bined in loose linkage with a nondiffusible molecule. It is of interest, also, that sera are not inactivated by dialysis against distilled water, indicating that the salt ions may have some effect in displacing the dialyzable component from its linkage to the nondiffusible substance. The problem of the nature of the dialyzable con- stituent has been approached by a study of the reactivation of serum rendered inactive by dialysis. Two distinct types of reactivation, which appear to be of different mechanism, have been devised. In the first place, if inorganic phosphate in concentra- tions of 0.005 M or above is added to dialyzed serum and the mixture incubated l-2 hours, the phate mixture after treatment, as indicated in Table 1, and the serum medium was then tubed in 2.0-cc. amounts for the test of ability to support trans- formation. Type III transforming substance was added and the tubes inoculated with a diluted cul- ture of a susceptible R strain derived from Pneurno- coccus Type II. The tubes were incubated and the occurrence of transformation determined by the methods described in previous communications from this laboratory. The data given in Table 1 demonstrate that re- activation of dialyzed serum can be achieved with inorganic phosphate if sufficient time is allowed for interaction. A relatively slow reaction occurs, which may conceivably be enzymatic in character. In contrast to this procedure, it is possible to bring about immediate reactivation of dialyzed serum by the addition of such materials as unheated Neopep- tone or tryptic digest of casein. Further investiga- tion of substances causing this type of reactivation disclosed that inorganic pyrophosphate has a simi- lar effect and is not influenced by the presence of nutrient broth. The effects of pyrophosphate and Neopeptone, as compared with the effect of phos- phate, are illustrated in Table 2. Sodium pyrophosphate was added to dialyzed serum in a final concentration of M/150, Neopep- tone sterilized by filtration in a final concentration of 0.2%, and inorganic phosphate as in the previ- ous experiment. Nine volumes of nutrient broth were added immediately and the test for ability to TABLE 1. REACTIVATION OF DIALYZED SERUM BY INCUBATION WITH PHOSPHATE Period of Incubation Source of Serum Factor Additions before Addition Transforming Test with Type III of Nutrient Broth Transforming Substance and Strain R36A Quadruplicate Tubes ------ Dialyzed serum None 2 hrs. at 37" C. Dialyzed serum Phosphate 2 hrs. at 37" C. gyg?* R only s III R only R only s III s III Dialyzed serum Phosphate None R only Whole undialyzed serum None None R only s III s III E TYty E%i * S III indicates the occurrence of transformation as evidenced by the recovery of encapsulated cells of Pneumococcus Type III, while the term "R only" means that transformation did not take place, and only unencapsulated R variants were recovered. serum regains the ability to support transformation when added to broth in the usual concentration. The period of incubation of the serum with phos- phate is essential. The interaction between phos- phate and the serum appears to be prevented by the presence of nutrient broth, for if the latter is added at the same time as the phosphate, or after a short period of incubation, no reactivation is achieved. A protocol of an experiment demonstrating the reac- tivation of dialyzed serum by incubation with phos- phate is given in Table 1. The phosphate was added to dialyzed serum at a final concentration of M/150 in the form of Na,HPO,-KH,PO, buffer, pH 7.8. Nine volumes of broth were added to the serum or serum-phos- support transformation carried out as before. The fact that pyrophosphate and Neopeptone restore the activity of dialyzed serum under conditions that are ineffective when simple inorganic phosphate is used is apparent from the data presented in Table 2. Transformation is as prompt in the reactivated serum medium as in the system containing undia- lyzed serum. It is not known whether the effect of Neopeptone is dependent on the presence of inorganic pyro- phosphate or whether other substances, such as cer- tain organic phosphates, are perhaps also capable of reactivating dialyzed serum. A few organic phosphates, including adenosine triphosphate, py- ridoxal phosphate, glycerophosphate, and fructose 180 M. MCCARTY, H. E. TAYLOR, 0. T. AVERY diphosphate have been shown to be without effect. that some organ of the animal body contains the The difference in the action of phosphate and enzyme in much higher concentration than does pyrophosphate is emphasized in the case of globulin serum and would serve as a more favorable source fractions of active sera. These fractions have for for possible purification and identification of the the most part been prepared by half saturation enzyme. To test this assumption, a preliminary sur- with ammonium sulfate followed by dialysis of the vey was made of several rabbit organs by preparing redissolved precipitate to remove excess sulfate ion. simple saline extracts and testing them for the Globulin fractions so obtained are totally inactive presence of the third component. The procedure in the transforming system, and even prolonged used consisted of adding the extract to broth con- incubation with inorganic phosphate does not serve taining a small amount of concentrated rabbit R an- to reactivate them. On the other hand, immediate tibody plus unheated Neopeptone as a source of the reactivation results from the addition of either in- dialyzable constituent. The broth containing these TABLE 2. IMMEDIATE REACTIVATION OF DIALYZED SERUM BY PYROPHOSPHATE AND NEOPEPTONE Source of Serum Factor Additions Treatment I/ Transforming Test with Type III Transforming Substance and Strain R36A Quadruplicate Tubes Dialyzed serum Dialyzed serum Dialyzed serum Dialyzed serum Whole undialyzed serum None R only* R only R only R only Phosphate Nutrient broth R only Pyrophosphate added immediately s 111* h%`y : I";;" p&Y Neopeptone to all tubes. s III s III SIX SIII None s III s III s III s III * Symbols same as in Table 1. organic pyrophosphate or Neopeptone. It appears that salt fractionation removes an unknown sub- stance that is essential in the reaction by which phosphate effects reactivation but plays no part in the pyrophosphate reaction. It seems inescapable that phosphate has an im- portant function in the action of serum factor. Whether this effect is direct or indirect has not yet been conclusively determined. An example of an indirect action of phosphate in an enzymatic reac- tion has recently been provided by the work of Colowick and Price (4): who showed that phosphate brings about the reactivation of dialyzed prepara- tions of muscle hexokinase. In this case, the phos- phate is involved in a separate enzyme reaction which results in the release of guanine, an essential coenzyme for hexokinase activity. TEIE THIRD COMPONENT OF SERUM The activity of serum in supporting transforma- tion does not depend solely on R antibody and the dialyzable component. This is demonstrated by the fact that puriiied R antibody is not effective in the transforming system even when fortified by the addition of pyrophosphate or Neopeptone to pro- vide the dialyzable component. The third compo- nent of serum is a nondialyzable substance, which has not yet been sufficiently characterized to estab- lish its chemical identity. However, the results of fractionation procedures indicate that it is protein in nature. For the purpose of orienting further research, the possibility has been considered that the third factor may be an enzyme. If this is indeed the case, it seems highly probable on general grounds added components was tested for its ability to sup- port transformation. Positive results indicate that the organ extract has supplied the missing con- stituent, since as pointed out above, R antibody and dialyzable factor alone are unable to support trans- formation. Rabbit spleen proved to be a good source of the third component. To provide larger organs as source material, extracts of calf spleen and calf thymus were then tried, and it was found that thy- mus extracts were more active than those of any of the other organs tested. The apparent superiority of thymus extracts may be due in part to the almost complete absence of desoxyribonuclease, which in- activates the specific transforming substance and thus interferes with the test. Although these pre- liminary experiments have demonstrated that the third component is present in certain mammalian or- gans, little progress has been made in the isolation of this substance. Thymus extracts have proved to be exceedingly difficult to handle in fractionation attempts, because of the presence of mucoid ma- terial. Nevertheless, it is likely that a more favor- able source will be found so that purified prepara- tion of this component of the transformation sys- tem can be obtained and analyzed. THE OCCURRENCE OF AN R VARIANT NOT REQUIR- ING DIALYZABLE COMPONENT FOR TRANSFORMATION It has become increasingly apparent in recent years that bacterial populations, in common with populations of other living organisms, constantly undergo discontinuous variations, which must be presumed to be of the nature of genetic mutations. Inevitably, spontaneous variation of this sort be- TRANSFORMATION OF PNEUMOCOCCAL TYPES 181 comes involved in a problem like that of pneumo- coccal transformation, in which relatively large bacterial populations are employed. The importance of mutation, or, as it is commonly called by micro- biologists, dissociation, has been previously pointed out in connection with one aspect of this problem (2). The strain of R pneumococcus (R36A) derived from Type II which is used in the majority of trans- formation experiments in this laboratory gives rise to numerous variants recognizable in a general way by slight alterations in colony topography. Certain of these variants acquire significance by virtue of the fact that they are totally insusceptible to the effects of the transforming substance. In all, four distinct variants of the parent R strain have been isolated which are not responsive to the influence of the transformation substance. Despite the fact that the R strain is cultivated continuously under more or less uniform conditions, subject to the limitations imposed by the complexity of the medi- um required for growth, the incidence of this type of dissociation is variable. This is presumably due to undefined differences in environmental condi- tions, which result in changes in the selective properties for a given mutant. Another variant of strain R36A has been en- dountered, which is of special interest in connec- tion with the dialyzable component of serum fac- tor. This R variant, designated 6e, was originally isolated from a culture in a medium containing dialyzed serum, which appeared to be deficient for growth, since only a few of the R cells of the inocu- lum multiplied to form visible colonial aggregates. Strain 6e has been found to be not only susceptible in the transforming system, but completely inde- pendent of the presence of the dialyzable compo- nent of serum factor. Thus, it undergoes transforma- tion readily in systems that contain dialyzed serum or globulin fractions of serum, without the addi- tion of dialyzable component in the form of pyro- phosphate or Neopeptone. Strain 6e therefore be- haves in all respects as though it supplies its own dialyzable component or effects restoration of the serum by some analogous reaction. The protein factor is required, however, since transformation cannot be effected when only the R antibody is present in the system. Strain 6e is of potential value in further study of the serum factors. Attempts have been made to determine in what way it differs from the parent strain with respect to synthesis or release of a sub- stance replacing the dialyzable serum component. It has not been possible to show that larger amounts of such a substance accumulate either in the cells or in the supematant medium of cultures of the variant. EXPERIMENTS ON THE MECHANISM OF ACTION OF SERUM FACTOR A series of experiments, which were designed to provide a more intimate knowledge of the interac- tion between the specific transforming substance (pneumococcal desoxyribonucleic acid) and the susceptible pneumococcal cells, proved to have an important bearing on the problem of the role of serum factor. The customary procedure in demon- strating the phenomenon of transformation is to add the specific desoxyribonucleic acid to the serum medium and to inoculate with a susceptible strain of R pneumococcus. Transformation becomes ap- parent after 16 to 20 hours' incubation, but little is known of the course of events during this period of incubation. The purified enzyme, desoxyribonu- clease, which specifically inactivates the transform- ing substance (8, 9), has been used as a tool in an attempt to study certain phases of this problem. TABLE 3. THE USE 08 DESOXYRIBONUCLEASE IN DETERMINING THE TIUE REQUIXED FOB TEE &TAKE OF TXANSJORMING SUBSTANCE BY SUSCEPTIBLE CELLS Time of I Transformation Test Addition of Active enzyme; triplicate tubes I No enzyme; duplicate tubes 1 hour R only' R only R only s 111* s III -F-P- 2 hours R only R only Ronly S III s III ----- 3 hours R only R only R only s III s III ~~-~- 4 hours s III s III s III s III s III ----- 5 hours s III s III s III s III s III -~-_______ 6 hours s III s III s III s III s III o Symbols same as in Table 1. By adding desoxyribonuclease to the transform- ing system at various intervals after inoculation in a concentration known to cause almost immediate inactivation of the transforming substance, it is possible to determine the length of time required for the transforming substance to be taken up or "fixed" by the susceptible cells. An experiment of this type is illustrated in Table 3. An amount of Type III transforming substance representing 1000 minimal effective doses was added to each of 30 tubes containing 2.0 cc. of serum medium. The tubes were all inoculated with 0.05 cc. of a suitable dilution (containing approximately 1000 cells) of the susceptible R strain, R36A, and then incubated at 37O C. At hourly intervals groups of five tubes were treated in the following way. To three of the tubes were added 4.0 pg. of purified desoxyribonuclease in 0.2 cc. of nutrient broth con- taining 0.03 M MgSO, ; and to the remaining two tubes was added 0.2 cc. of the MgSO, broth with- out enzyme, as control on the effect of the &a- tion required to mix the enzyme thoroughly with the culture. The cultures were incubated overnight and the presence of transformation determined by 182 M. McCARTY, H. E. TAYLOR, 0. T. AVERY the usual procedure. The results are recorded in Table 3. It will be seen that the addition of desoxyribo- nuclease at any time up to four hours after inocula- tion interferes with the reaction so that transforma- tion does not occur, and it is therefore likely that throughout this period the transforming substance is readily accessible to the action of the enzyme. After four hours, on the other hand, the addition of desoxyribonuclease has no observable effect on the course of the reaction. This type of experiment was repeated with essentially the same results on sev- eral occasions and with different sera. Consequently, it appears that growth of the R cells in serum medium for 3 to 5 hours is required before the specific desoxyribonucleic acid is taken up by the cells and thus protected from enzymatic destruction. Further experiments have demonstrated that this does not depend solely on the increase in popula- tion (from the original inoculum of 1000 cells to an approximate l,OOO,OOO cells at 4 hours) and the consequent appearance of susceptible variants. Confirmation of the importance of the four-hour period of growth is provided by experiments in which the R cells are grown in serum medium in the absence of the specific transforming substance. After 4 to 5 hours' growth under these conditions the cells are so "sensitized" that, when they are transferred to a medium containing the transform- ing substance, the latter is taken up in as short a time as 1.5 minutes. That is, desoxyribonuclease has no effect on the outcome of the transforming test when added 15 minutes after the previously %ensi- tized" cells are brought into the presence of the specific desoxyribonucleic acid. If the transfer is made after shorter periods of growth in the serum medium-e.g., 2 to 3 hours-the "sensitization" has apparently not taken place and rapid fixation of the transforming substance cannot be demonstrated. Furthermore, if growth in the serum medium is prolonged 7-8 hours or more the "sensitization" is lost, indicating that the alteration in the cells is not permanent but is affected by further environ- mental changes that occur on continued growth. These experiments demonstrate that the events which occur in the first four hours of growth of R cells in the transforming system are independent of the presence of the specific transforming sub- stance. It must be concluded that growth under these conditions alters the cell in some way, or provides suitable environmental conditions, so that interaction between the cell and transforming sub- stance can take place. The relation of these experiments to the role of serum factor becomes apparent from the fact that a complete and active serum containing all the es- sential components muse be present in order to achieve "sensitization" of the cells during the four- hour period. Growth in the presence of purified R antibody, in serum inactivated by dialysis, or in globulin fractions of active sera, fails in each case to "sensitize" the cells. Thus the hypothesis is sug- gested that the major part played by serum in the transforming system is concerned with a modifica- tion of the R cell so that the specific transforming substance can be taken up. DISCUSSION It seems well established that the environmental conditions required for transformation of pneumo- coccal types in vitro depend on the combined action of several distinct factors. The present studies fall far short of the ultimate goal of determining the chemical nature and mode of action of the various factors involved. However, they provide a basis for and give direction to further research and make possible the formulation of a tentative hypothesis concerning the role played by serum in the trans- forming reaction. The experiments in which desoxyribonuclease was used to inactivate the transforming substance free in the reaction system have demonstrated that the specific desoxyribonucleic acid does not participate in the preliminary phase of "sensitization" which takes place during the first four hours. On the other hand, the three known components provided by serum or serous fluid are all required during this four-hour period. At present, the most reasonable interpretation of the available data is that the action of the serum factors during this early phase results in alterations at the surface of the R cells so that they are capable of taking up or adsorbing the specific transforming substance. The alternative in- terpretation that the serum provides a strongly selective environment for growth of R mutants sus- ceptible to transformation has been discarded on the basis of several important considerations. First, "sensitization" takes place in a relatively short time and is not dependent to any marked degree on the size of the original inoculum or on the total population. During the last 30 minutes of the four- hour period the number of "sensitized" cells in- creases from zero to at least 5000, representing ap- proximately 0.5% of the total population. The final and most important evidence against the assump- tion that selection of a mutant is involved depends on the fact that the state of "sensitization" is tem- porary and readily lost. Cells that have been "sensi- tized" by growth for 4 hours in serum medium can be deprived of their acquired "sensitization" by re- peated washing with nutrient broth or simply by allowing them to grow an additional 2 to 4 hours in the serum medium. If it is assumed, then, that the action of the en- vironmental factors is exerted at the surface of the R cells, the problem resolves itself into one of de- termining the nature of the alteration of the cell surface and the way in which the serum components bring it about. Perhaps the most attractive hy- pothesis is that the reaction is enzymatic in char- TRANSFORMATION OF PNEUMOCOCCAL TYPES acter and is dependent upon the action of an WI- identified enzyme, represented by the third compo- nent of the serum, on certain specific groupings at the cell surface. It is well established that profound but temporary alterations in the peripheral mosaic of living bacterial cells can be brought about by enzymatic action. For example, the bacterial en- zyme that hydrolyzes the capsular polysaccharide of Pneumococcus Type III is capable of removing the capsule from viable Type III organisms (1). The functions of capsule formation and polysaG charide synthesis are not impaired, however, and in the absence of the active enzyme, descendants of cells so treated again have all the characteristics of normal Type III cells. A similar phenomenon has been described in the case of hemolytic strepto- cocci, from which one of the surface antigens, the M protein, can be removed by the use of proteolytic enzymes without affecting the viability of the bac- teria (7). Therefore, in the case of the transforming reaction, it is not unreasonable to suppose that less extensive reversible alterations at specific sites of the surface of pneumococcal cells can result from enzymatic action, and that these alterations make possible the adsorption or penetration of the specific desoxyribonucleic acid. In terms of the enzyme hypothesis, the occur- rence of a dialyzable constituent is referable to a dissociable cofactor. The reactivation of dialyzed serum by incubation with phosphate has its counter- part in the work of Colowick and Price (4) in which the hexokinase activity of dialyzed muscle extracts is restored by incubation with phosphate. In this latter case, the release of a cofactor, guanine, by a secondary enzymatic reaction involving ribonucleic acid is responsible for reactivation. However, in the restoration of the transforming activity of dialyzed serum the immediate effect of pyrophosphate sug- gests that a different type of reaction is involved than in the case of hexokinase. The function of the essential colonial aggrega- tion of the R cells resulting from the action of R antibody is more difficult to interpret from the point of view of the enzyme hypothesis. However, bacterial aggregation may have an important rela- tion to the period of growth required for "sensitiza- tion" of the cells, since at four hours the indi- vidual aggregates are just beginning to reach a size (about 1000 cells) which could provide special local conditions. It is conceivable that these local environmental changes, reducing or otherwise, pro- vide essential conditions for the action of the en- zyme. Thus, it is not necessary to make the un- likely assumption that the preliminary four-hour period is required because of the slow action of the hypothetical enzyme. It scarcely need be reiterated that these con- siderations are tentative in character and subject to modification in the light of future results. How- ever, the various assumptions discussed above pro- vide the basis for a useful working hypothesis, the validity of which can be further tested experi- mentally. The results of the present studies leave little doubt that the contribution of serum to the transformation system is complex and dependent on at least three components: the R antibody, an ad- ditional protein component, and a dialyzable fac- tor which may be combined with the protein com- ponent as it occurs naturally. Furthermore, evidence is presented which strongly suggests that the role of serum is concerned with alteration of the surface of the R cells so that the specific desoxyribonucleic acid is taken up or adsorbed. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. REFERENCES AVERY, 0. T. and DUBOS, R. The protective action of a specific enzyme against Type III Pneumococcus infec- tion in mice. J. Exp. Med. 54: 73-89. 1931. AVERY, 0. T., MACLEOD, C. M., and MCCARTY, M. Studies on the chemical nature of the substance inducing transformation of pneumococcal types. Induction of transformation by a desoxyribonucleic acid fraction iso- lated from Pneumococcus Type III. J. Exp. 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