[Reprinted from THE JOUILUAL OF EWERIXEKTAL MEIWXNE, October 1, 1959, Vol. 110, No. 4, pp. 571~5841 Printed in U.S.A. SIMULTANEOUS PRODUCTION OF TWO CAPSULAR POLYSAC- CHARIDES BY PNEUMOCOCCUS I. PROPERTIES OF A PKETJMOCOCCVS MAMFESTING BIKARY CAPSUL.4TION* Blr ROBERT AUSTRIAX, M.D., Ah3 HARRIET P. BERNHEIMER, PH.D. (From the Departmest of Medicine, Slale Uwiversity of New York College oj Medicine ot New York City, Brooklyn) PLATES 32 TO 35 (Received for publication, June `4, 1959) The concept that a pneumococcal cell may produce more than one capsular antigen is not new. The possible occurrence of the phenomenon was considered as early as 1928 by Griffith (l), who described, in his original report of the transformation reaction, a strain of pneumococcus iagglutinated specifically by antisera to two different pneumococcal types. In the same year, the serologic cross-reactivity between pneumococcus Type III ,snd a strain classified later as pneumococcus Type VIII was recognized (2, 3). It was shown subsequently by Goebel (4), however, that the immunologic relationship between these two capsular types resulted from the presence in their respective capsular poly- saccharides of a common chemical subgroup rather than from the production by each type of several discrete capsular antigens, one of which was common to both. Similar immunologic cross-reactivity has been demonstrated among a number of other pneumococcal types (j), but in no instance has it been shown conclusively that an organism of this species produces more than one molecular type of capsular polysaccharide. In 1953, Leidy, Hahn, and Alexander (6) reported on the production in vitro of new types of HemophilzGs irzfluensae obtained by transforming capsulated or non-capsulated organisms of this species with deoxyribonucleates (DNA) derived from bacterial cells of heterologous capsu'.ar type. Organisms reacting with each of two unitypic anticapsular sera were isolated and, although deiin- itive experiments are not described, it is possible that these cells were pro- ducing capsular polysaccharides of two molecular species. Analogous experi- ments performed with pneumococcus have led to the isolation of a variety of phenotypes manifesting binary capsulation. In th:ls and in subsequent reports, some of their biological, biochemical, and genetic properties will be set forth. * This investigation x-as supported in part by research grant E-1018(C2) from the In- stitute of Allergy and Infectious Diseases, Cnited States Public Health Service. 571 572 PRODUCTlOX OF C.kPSULAR POLTS.iCCR.iRIDES. I Materials and Methods Xonzenclalwe oj Pnezr~r~ococcal l'arianls.--In certain of the pneumococcal strains to be described, there is lack of complete correspondence between phenotype and genotype. For this reason, complexities arise in the description of these strains. For the present, only pheno- typic designations will be employed and terms descriptive of genotype will be introduced later. Fully capsulated phenotypes are indicated by the letter S followed by a Roman numeral designating capsular type; e.~., SIII is a capsulated strain of pneumococcus Type III. Be- cause of certain biochemical relationships to be described subsequently and for convenience, non-capsulated variants and intermediate capsular variants agglutinable by antibody to somatic antigens, including those described by Taylor (7) under the term SIII-1, will be designated by the expression S-x,, the subscript designating the capsular type from which the mutant strain was derived and the strain's designation; e.g., S-rrr, is Strain 4 of a non-capsu- lated phenotype derived from pneumococcus Type III. Phentotypes manilesting binary capsulation will be indicated by the letter S followed by the two Roman numerals descriptive . of the strain's capsular components separated by a hyphen; e.g., SI-III is a pneumococcus characterized by the production of both Type I and Type III capsular polysaccharides. S1rair.s of Pneztmococcus.-SI phenotypes-SVI, ID, 141S, IP: strains of the SI genotype isolated originally from human sources and carried in the laboratory for several years. SIII phenotype-111466: a strain of the SIII genotype carried in the laboratory for many years. S-r11 phenotypes- S-rrr., S-rrrl: strains of a non-capsulated variant of pneumococcus Type II (R36.1) which carry different mutated Type III genomes acquired by transformation with DNA from variants of Strain IIIA66. S-rrrl: the same non-capsulated mutant of pneumococcus Type II transformed with DN.4 of a variant of the Type III Strain SV3. (The three preceding strains are Ephrussi- Taylor's SIII-1, SIII-lb, and SIII-lc (8) and were made available to us by her and by Dr. Arnold Ravin.) S-rrrI: a non-capsulated variant of strain IIIA66. Preparation of DNA (Transforning Principles) and Techniqzbe of Transformation Re- acfionJ.--The methods used were those described by MacLeod and Krauss (9). Preparation of Anticapszdaar Sera.-Vaccines of unitypic pneumococci and of pneumo- cocci producing two capsular polysaccharides were prepared from cultures grown at 37oC. for 12 to 16 hours. After standing for 1 hour at room temperature following the addition of neutralized formalin to a final concentration of 1 per cent, the organisms were collected by centrifugation and resuspended in a volume of normal saline solution one-tenth that of the original culture. The suspension was heated at 63oC. for 30 minutes and stored at 4'C. Rabbits were immunized by the injection intravenously of 1 cc. of vaccine daily for 5 days and were bled on the 10th day following each course of vaccine. Precipilin Tests.-Precipitin tests were carried out in small test tubes. 0.2 cc. of each of several serial dilutions of antigen was layered over 0.2 cc. of serum. The tubes were incubated for 1 hour at 37oC. and the reactions read after the tubes had stood overnight at 4oC. Precipitin reactions in agar gels were carried out according to the technique of Ouchterlony as modified by Halbert (10). I'irzllewe and Prelection T&$.-Tests of virulence were performed by the injection intra- peritoneally into mice of the CFCV strain of 1 cc. of an appropriate dilution of a blood broth culture of the strain to be tested which had been incubated for 16 hours. Counts of viable bac- terial units were obtained by dilution and plating. In protection tests, mice were injected intraperitoneaily with 1 cc. of the serum to be tested 24 hours before being infected with pneumococci as in tests of vixlence. Control animals received normal rabbit serum. All tests R. AUSTRIAN ASD H. P. BERNHEIMER 573 were continued for 10 days and results were scored in terms of survival or death, The cause of death was determined by culture of the animal's blood at autopsy. In experiments in which the enzyme which hydrolyzes specifically Type III capsular polysaccharide' was employed, 2 mg. of enzyme were injected intraperitoneally. Tests of Phagocytosis by Human Polymorphonzdear Leukocytes.--The technique employed was one described previously (11). When the enzyme hydrolyzing Type III polysaccharide was used, it was present in a final concentration of 2 mg./cc. EXPERIMENTAL Phemtypes Manifesting Binary Capsdation.-When cells of pneumococcal Strain S-r1r, are transformed in the presence of DNA from any of the Type I pneumococcal strains listed above, organisms of two different phenotypes can be recovered: cells of the SI phenotype and cells of the SI-III phenotype. TABLE I Relationships of Pneumococcal Strains Participating in Transjormafion Reactions Citing Rise to Variants Producing F&o Capsular Polysaccharides Original capsular ty of strain manifesting the SI-II!?phenotype followrng transformation SI SII SIII SV _- Parent Type III strain of variant with S-III genome IIIA66 IIIA66 IIISV3 IIIA66 III.466 .- - Source of SI genome SVI SVI, ID, 141S, IP WI, ID SVI, ID, 141S, IP ID In similar fashion, 5X-111 strains may be obtained by transforming cells of lines S-I~I~, S-Irr, and S-IIIJ. From these experiments, it can be seen that mutant Type III cells, the capsular genomes of which were derived originally from two different Type III strains, IIIA66 and IIISV3, can be transformed to the S-111 phenotype with DNA from several SI strains. In addition, it is evident that non-capsulated cells derived originally from capsulated strains of different capsular types, i.e. Type II and Type III, may manifest binary capsu- lation when endowed with the proper genetic factors. It has been possible also to transform non-capsulated cells derived from Type I and from Type V pneu- mococci to the SI-III phenotype. The phenomenon of binary capsulation, therefore, may be expressed by cells derived from a variety of capsular types when they are endowed with the appropriate genetic factors derived from any of several Type I and mutant Type III strains (Table I). 1 The enzyme preparation x-as made available through the generosity of Dr. Alan W. Bemheimer, Department of Microbiology, New York University College of Medicine. 574 PRODUCTION OF CAPSULAR POLYS.ACCH.4RIDES. I SI-III cells were observed first in experiments in which strain S-rIr4 had been exposed to DNA from a capsulated Type I strain. In quellung prepara- tions,2 it was noted that the transformed cells showed capsular swelling when exposed either to Type I or to Type III antiserum. The size of the refractile capsular halo in Type III antiserum was approximately that seen about cells of the normally capsulated Type III phenotype but the refractile zone about cells in Type I antiserum was distinctly smaller than that about capsulated Type I cells and at times could be seen only with difficulty. Variation in the size of the two capsular components in relation to the phase of bacterial growth was not observed. When the cells manifesting binary capsulation were exposed to a mixture of Type I and Type III antisera, the capsular swelling was not recognizably greater than that which followed contact with Type III antiserum alone. In smears stained by the Gram technique, such cells showed no consistent variation in size or in shape which permitted their distinction from singly capsulated variants. SI-III cells isolated from the peritoneal cavity of the white mouse did show elongated forms often in short chains, morphologic charac- teristics not manifested by the organism when grown in vitro. .I On solid medium, SI-III cells give rise to clones morphologically indis- tinguishable from those formed by pneumococcus Type III, a not surprising finding in view of the significant amount of Type III polysaccharide formed by the SI-III cell. Eridence Establishing the Existence of the S-III Cell.-The observation that more than 90 per cent of cells from clones of the SI-III phenotype gave a positive quellung reaction in both Type I and Type III anticapsular serum provided strong presumptive evidence for the existence of a cell producing two capsular polysaccharides or one so constituted chemically as to possess the serologically reactive groups of Type I and of Type III polysaccharide. To distinguish between cells manifesting binary capsulation and mixtures of SI and SIII cells, several types of experiment were performed. Table II shows the results of growing SI, SIII, mixtures of SI and SIII cells and SI-III cells in broth containing either Type I or Type III anticapsular serum. Only those cells producing simultaneously two capsular polysaccharides are agglutinated by both unitypic antisera and yield cultures with limpid supernatant fluids in the presence of each antiserum in so called "thread tests." Similar evidence distinguishing cells with binary capsules from unitypic cells has been obtained from mouse protection tests. The evidence in Table III indicates that all animals infected with cells with binary capsules were protected by either unitypic capsular antiserum whereas none was protected by either 2 Capsular typing sera were made available through the courtesy of Dr. H. D. Piersma, Lederle Laboratories Division, American Cyanamid Company, and Dr. E. F. Roberts, Wyeth Laboratories, American Home Products Corporation. R. AUSTRIAN ASD H. P. BERKHEIMER 575 antiserum when infection was carried out with mixtures of Type I and Type III cells. Evidence for the Production of Two Capsular Polysaccharides by Z-III Cells.- SI-III cells grown by continuous passage in broth containing either Type I or Type III anticapsular serum give rise to mutant cells producing in amounts detectable by the quellung reaction only Type III or Type I capsular poly- saccharide. These mutants derived from SI-III cells suggest that separately variable factors controlling two capsular components are present in the SI-III cell but do not exclude the possibility that each mutant is associated with a qualitative variation in the composition of one of two polysaccharides. A second type of evidence is derived from the exposure of SI-III cells to the enzyme which hydrolyzes Type III capsular polysaccharide. After such treat- TABLE II .4ppearance of Supernatazt Fluid of Pneumococcal Cullzcres Containing Unitypi Anlicapsdar Serum Pneumococcal type or types in culture Serum An ti-Sl Anti-S111 SI L T SIII T L SI + 911 T T SI-III L L SI-III + SI L T SI-III + SIII T L Medium: neopeptone broth plus 10 per cent anticapsular rabbit serum; L = limpid supematant fluid. T = turbid supernatant fluid. ment, SI-III cells are no longer agglutinable by Type III anticapsular serum and they fail to give a quellung reaction with Type III antibody. Their aggluti- nability by Type I anticapsular serum remains unimpaired, however, and the treated cells give still a positive quellung reaction with Type I antibody. It has been shown that the action of the enzyme hydrolyzing Type III pneumo- coccal polysaccharide is more highly specific than antibody to Type III poly- saccharide. In addition, the action of this enzyme has been demonstrated recently to be that of an endoenzyme rather than that of an exoenzyme attack- ing the end-groups of a macromolecule (12). The experiments suggest strongly, therefore, that the SI-III cell produces normal Type III polysaccharide or a substance resembling it very closely and one that is probably distinct from the Type I component of the capsule. In precipitin reactions carried out in an agar gel by the modified technique of Ouchterlony (lo), the precipitates formed when Type III anticapsular 576 PRODUCTIOS OF C.APSUL.kR POLYS.XCCH.\RIDES. I antibody is allowed to react in the same plate with SI-III cells, SIII cells and Type III polysaccharide give rise to a line of continuity (Fig. 1); and, in like manner, a line of continuity is produced when the reactants are Type I anti- capsular antibody, SI-III cells and Type I polysaccharide (Fig. 2). Precipitin tests performed with partially purified polysaccharides from SI-III cells show that the Type III polysaccharide can be precipitated by Type III anticapsular serum without significant removal of the Type I com- TABLE III Proleclicc Ejecl of Type Z and Type ZZZ Antiserum in Mice Znfected with SZ-IZZ and with Mixtures of SZ and SIII Pnexmococci strain SI-III Amount of culture injected Serum injected cc. 10-E 10-s 10-s 10-G 10-7 10-s Anti-S1 Anti-SD1 Kormal None None Iione D/S' Viable units/cc. (undiluted culture): 4 X 10s Strains Amount of culture injected Serum injected D/S' cc. SI + SIX1 10-s Anti-S1 6/O 10-C Anti-S111 6/O 10-s Normal 6/O 10-s X'one 6/O lo-' Kane 6/O 10-8 None 4/z Viable units/cc. (undiluted culture) : 3 X 108 (equal numbers of SI + SIII) * D/S = died/survived. ponent of the capsule and conversely, specific precipitation of the Type I capsular component is not accompanied by removal of the Type III component from solution. A 200 cc. culture of SI-III cells was grown overnight afte: which time glucose was added to a final concentration of 1 per cent. During an additional 6 hours' incubation, the lactic acid formed was neutralized by the addition of 3 N NaOH. The cells were then removed by centrifugation and 1 volume of 93 per cent ethyl alcohol was added to the separated super- natant fluid. The suspension was stored overnight at 4oC. after which time, the precipitate formed was collected by centrifugation and dissolved in 10 cc. of normal salt solution. This solution served as the unfractionated capsular antigen. Aliquots thereof were allowed to R. XUSTRIXN AXD H. P. BERNHEIXER 577 react with appropriate amounts of Type I or Type III anticapsular rabbit serum or with normal rabbit serum. All volumes xere adjusted to give the same quantity of the original antigen in precipitin tests. The results are shown in Table IV. On the bases of the independent variability of its capsular components, of the susceptibility of the Type III capsular component to the enzyme hydro- TABLE IV Independent Precipitabilily oj Each Capsular Contponerzt of the SI-III Phenolype by Unilypic Serum Anti-S1 Anti-S111 Normal Serum - _- Anti-S1 Anti-S111 Normal - !- -. -- Serum Anti-S1 .4nti-SIII Piormal - Polysaccharide precipitated after treatment with normal rabbit serum Dilution of antigen 1:32 1 1:6+l 1:128 13256 1:512'1:1024'1:20481:40961:8192' C pp____-__p-- +++ ++ ++ + + 0 0 0 0 0 t+++ +++ ++ ++ + + , + f 0 0 0,o o/o Polysaccharide precipitated after treatment with type I anticapsular rabbit serum Dilution of antigen 1~32 1:6-l m-- It 0 t+++/ +++ O I0 I. - - ! 1:128 1:256 1:512 1:102~1:20481:4096~1:8192 C __- -p__--- 0 0 0 0 0 0 0 0 t++ ++ + + f 0 0 0 Polysaccharide precipitated after treatment with type III anticapsular rabbit ~erurn Dilution of antigen 1:32 ' 1:64 1:128! 1:2561 1:512 1:1024'1:2048 1:40961:8192 C -pp----- ++ + + f 0 0 0 0 0 0 0 ; 0 ; ; ; 0 lyzing Type III pneumococcal polysaccharide, of the serologic cross-reactivity of its cells with Type I and Type III polysacchsrides in precipitin tests in agar gels, and of the independent precipitability of its two capsular components, SI-III cells may be considered to produce two capsular polysaccharides. Phagocyiosis of SI-III Cells by Human Polymorphonuckar Leukocytes.-In Table V are shown the results of exposing SI-III cells to human polymorpho- 578 PRODUCTIOK OF C-\PSUL:lR POLTSlCCHARIDES. I nuclear leukocytes in a smooth-walled system. Several facts of interest are demonstrable in these experiments. First, the persistence of the antiphagocytic effect of the Type III capsular component is demonstrable even in the presence of Type I anticapsular serum. That the Type I antiserum is fully effective is shown in the last line of the table. Second, the relatively small antiphagocytic effect of the Type I capsular component becomes apparent when the SI-III cells are exposed to the enzyme depolymerizing Type III polysaccharide to- gether with normal rabbit serum. The efficacy of Type I antibody is definitely demonstrable in the presence of the same enzyme, however, by a further in- crease in phagocytosis. The Relative Importance of the Two Capsdar Components to the Virulence of 5'1-111 Pnezinzococci.-Table VI shows the effect of several reagents upon the TABLE V Phagocytosis of SI-III Pnezcntococci by Hwnan Polymorpkonuclear Leukocytes in the Presence of Various Sera and of the Enzyme Hydrolysing Type III Polysaccharide Strain SI-III SI Anti-S1 Anti-S111 Normal Anti-S1 Anti-S111 Normal Anti-S1 Per cent PMN con- Average No. of thing pneumococci, mococci per P IX gneu- Absent " " Present " " Absent 8 0.36 100 10.0 4 0.05 96 8.2 96 8.0 32 0.92 / 85 I 8.0 outcome of infection with SI-III cells in mice. It may be seen that each capsular polysaccharide contributes to the virulence of the infecting organism. Of interest is the role played by the Type I capsular component. Although the number of animals infected is small, it may be noted that the only animal infected with pneumococci manifesting binary capsulation and not receiving antiserum to survive was one treated with the enzyme depolymerizing Type III polysaccharide. Not shown in the table but noteworthy also is the fact that animals treated with a single injection of enzyme at the time of infection survived twice as long after inoculation as did untreated controls. Similar results have been observed in analogous experiments performed with cells of the binary capsular SIII-V phenotype. These observations are in accord with those derived from the study of phagocytosis of these strains. They suggest that the smaller Type I capsular component is able to protect the population of SI-III cells from complete elimination during the period that the enzyme is still hydrolyzing effectively the Type III capsular component but is unable by R. AUSTRIAK XX0 H. P. BERSHEILIER 579 itself to shift the balance of infection decisively in favor of the pneumococcus. Later, when the enzyme is no longer effective, regeneration of the Type III capsular component provides more effective protection against phagocytosis and permits rapid progression of the infection to a fatal outcome. Properties of Anfiserum Prepared zailh Vaccines of SI-III Pneumococci.-- Rabbits immunized with vaccines of pneumococci manifesting binary capsu- lation form antibodies to each of the capsular components of such cells. The relative amount of antibody to each of the capsular antigens may vary during the course of immunization. Antisera to SI-III cells cause capsular swelling of SI, SIII, SI-III, and SIII-V cells. Shown in Table VII is the protective effect TABLE VI E.ject of the Enzyme Hydrolyzing Type III Polysaccharide and of Unitypic Antisera otl the Strain SI-III SI SIII - -- - Amount of culture injected cc. 10-G 10-6 10-6 lo-6 10-e 10-1 lo-6 10-G 10-a Serum injected None Anti-S111 Anti-S111 Anti-S1 None " `< Anti-WI - .- - Enzyme injected D/S me. 2 2 None " " " 3/l 014 o/4 o/4 3/o 2/o 2 4/o 2 o/4 None o/4 \kble units/cc. (undiluted culture): SI-III = 2 X 108; SI = 2 X iv; SIII = 3 X 108. of such antiserum in mice infected with cells of each of the aforementioned phenotypes and with mixtures of SI and SIII cells. Recorded also in the table is the absence of protection against infection with pneumococcus Type V. Ana- logous results are observed in experiments concerned with the phagocytosis of pneumococci of several capsular types in the presence of Type I-III antiserum (Table 1'111). Precipitin tests in an agar gel (Fig. 3) disclose several facts. First, with Type I cells and Type I capsular polysaccharide, the anti-SI-III serum forms a continuous band of precipitate, and with Type III cells and Type III capsular polysaccharide a second and distinct band is formed. Second, these lines of precipitate are continuous with those formed by the antibody reacting with each of the capsular components of the SI-III cell. Third, there is no band of precipitate formed to indicate the presence of a single species of antibody Strain or strains SI-III SI SIII SI + SIII SIII-v SV SIII + sv SI-III SI SIII SI + SIII SIII-V sv SIII + sv SI-III SI SIII SI + SIII SIII-v sv SIII + sv Amount of culture injected CC. 10-e 10-e 10-G 10-e 10-G 10-C IO-6 10-G 10-e 10-C lo-6 10-C lo-6 10-C 10-7 lo-' lo-' lo-' lo-' lo-' lo-' Serum injected .-lnti-SI-III `I L` ii " " 1` Normal `I `I 8, 6` l` C` &Cone 1` `< " `i " " D/S O/j O/j O/j O/j O/j 5/O 5/O Viable units/cc. (undiluted culture): SI-III = 5 X 10'; SI = 3 X 10'; SIII = 2 X 10'; SI + SIII = 2 x 108; SIII-v = 2 x 10s; sv = 2 x 106; SIII + sv = 2 x 103. TABLE VIII Phagocytosis of Pneunmocci of Sewal Types by fIt~nza~z Polyvzorphonuclear Leukocytes i~z tire Presence of Type I-III Anticapsdar Rabbit Serum Strain SI-III SI SIII SIII-v sv s-111 SI SIII SIII-v sv ,- Serum Anti-SI-11 " " " " ?iormal L` " " " :I I I- PMIN COD taining pneumococci per cc-n1 8-l 60 92 72 0 1 Average No.`of 2neumococci per!PJiN 8.6 9.4 8.1 7.4 0 . 0 0 0 i 0 0 580 R. ATJSTRLlS AND H. P. BERNHEIXER 581 reacting with both capsular components or the presence of a capsular com- ponent reacting with antibody in a fashion different from that of unitypic cells. The results are consistent with those presented earlier which indicate that cells manifesting binary capsulation produce tmo species of capsular polysaccharide each of which is closely similar if not identical with that of the corresponding unitypic pneumococcus. In precipitin tests performed in tubes, Type I polysaccharide and Type III polysaccharide are each precipitated by antiserum prepared with a vaccine of TABLE IX Independed Precipilabilily of Capsular Antibodies from an Antiserum Prepared with a T*accine of SI-III Pna~ntococci Polysaccharide precipitated by anti-SI-III serum before absorption Dilution of antigen __. Polysaccharide precipitated by anti-5X-111 serum after absorption with SI cells Dilution of antigen Antigen Polysaccharide precipitated by anti-SI-III serum after absorption with SIII cells Dilution of antigen __- Antigen 1:16/ 1:3?! 1:6+! 1:128 ~______ SI SIII * Salice solution of purified Type I or Type III capsular palysaccharide in a concentration of 1 mg./cc. diluted as indicated. %-III cells. That two distinct anticapsular antibodies are present in such an antiserum can be demonstrated by the independent precipitability of each. \Vhen an antiserum prepared Cth a vaccine of SI-III cells is absorbed with either capsulated Type I or Type III cells, the corresponding anticapsular antibody is removed without significant alteration of the titer of antibody precipitating the other capsular component (Table IX). Analogous experiments in which precipitin reactions were carried out in an agar gel demonstrate the independent removal of one capsular antibody without alteration in the reaction of the other with appropriate antigens (Figs. 4 to 6). 582 PRODUCTION OF CAPSULAR POLTS.iCCH.ARIDES. I Properties of Deoxyribomcleates of P?leunzococci of the SI-III Phenotype.--To determine the nature of the genetic determinants of the capsular components of the SI-III cell, DKA was prepared from cultures of such cells in the usual manner. When cells of a non-capsulated pneumococcus (R36NC) derived originally from a strain of capsular Type II were exposed in the transforming system to DNA from SI-III cells, organisms of three different phenotypes were recovered: S- rrr, SI and SI-III. No SIII cells were observed. The results indicate that there has been no major alteration in either of the genetic units concerned with the formation of the two capsular components of the cell mani- festing binary capsulation during their residence within that cell and that each is able to engender expression of that character controlled by it when it is reintroduced as the sole genetic factor concerned with capsular formation into another pneumococcal cell. DISCUSSION Through the agency of the transformation reaction, it has been possible to isolate pneumococci manifesting binary capsulation of several phenotypes. Examination of cells of the SI-III phenotype in a number of experimental systems reveals that organisms of this type produce two distinct capsular polysaccharides which are closely similar if not identical to those produced by cells of the corresponding unitypic phenotypes. The evidence available suggests that the two types of polysaccharide are intermingled in the capsule in a random fashion. Study of cells of the SIII-V phenotype yields results of a similar nature. Binary capsulation in pneumococcus provides a new basis for serologic cross-reactivity in this species. Cells manifesting binary capsulation react with unitypic antiserum to each of their capsular components and stimulate the formation of antibodies which will react with cells bearing either of the capsular antigens produced by cells of the binary capsular phenotype. Whether or not pneumococci manifesting binary capsulation exist in nature remains an un- answered question. Examination by genetic and serologic techniques of several groups of pneumococcal strains the capsular antigens of which show cross- reactivity has failed to result in the recognition in any of the strains of separable capsular antigens. It would appear more likely that the serologic interrelation- ships of these strains are based upon the presence of common chemical sub- groups in their single capsular polysaccharides rather thanupon their production of multiple types of capsular polysaccharide. For this reason the nomenclature of pneumococcal types proposed by Eddy (13) seems preferable to that proposed by Kauffmann and Lund (14). These experiments with pneumococcus show many similarities to those performed with a. irzfEuenzae by Leidy, Hahn, and Alexander (6), and it ap- pears probable that binary capsulation may be manifested by organisms of the R. AUSTRIAN AND II. I'. BERNHEIXER 583 latter species. In each, hoxvever, it would appear to represent a product of the laboratory. This fact notwithstanding, binary capsulation provides a useful object for the study of a variety of biologic phenomena. Pneumococci manifesting binary capsulation have been produced through the agency of transformation reactions. Such cells produce two capsular poly- saccharides which are closely similar if not identical with those produced by corresponding singly capsulated strains. Binary capsulation in pneumococcus provides a new basis for serologic cross-reactivity in this species. BIBLIOGRAPHY 1. Griffith, F., Thesignificance of pneumococcal types, J. Byg., 1928, 27, 113. 2. Sugg, J. Y., Gaspari, E. L., Fleming, W. L., and Neil& J. M., Studies on im- munological relationships among the pneumococci. I. A virulent strain of pneumococcus which is immunologically related to, but not identical with typi- cal strains of Type III pneumococci, J. Exp. Med., 1928, 47, 917. 3. Harris, A. L., Sugg, J. Y. and Neill, J. M., Studies on immunological relation- ships among the pneumococci. II. A comparison of the antibody responses of mice and of rabbits to immunization with typical Type III pneumococci and to immunization with a related strain, J. Exp. Med., 1928,47, 933. 4. Goebel, W. F., Chemo-immunological studies on the soluble specific substance of pneumococcus. II. The chemical basis for the immunological relationship between the capsular polysaccharides of Types III and VIII pneumococcus, J. Biol. Ckem., 1935, 110, 391. 5. M@rch, E., Serological studies on the pneumococci, London, Humphrey Mil- ford, Oxford University Press, 1953. 6. Leidy, G., Hahn, E., and Alexander, H. E., In vitro production of new types of Hemophilus i~q`lz~ensae, J. Eap. Med., 1953, 97, 467. 7. Taylor, H. E., Additive effects of transforming agents from some variants of pneumococcus, J. Exp. Med., 1949, 89, 399. 8. Ephrussi-Taylor, H., Transformations allo&es du pneumocoque, Ext. Cell Research, 1931, 2, 589. 9. MacLeod, C. M., and Krauss, M. R., Stepxise intratype transformation of pneumococcus from R to S by way of a variant intermediate in capsular poly- saccharide production, J. Exp. Med., 1947, 86,439. 10. Halbert, S. P., Snick, L., and Sonn, C., The use of precipitin analysis in agar for the study of human streptococcal infections. II. Ouchterlony and Oakley technics, J. Esp. Med., 19%, 101, 557. 11. Austrian, R., Morphologic variation in pneumococcus. I. An analysis of the bases for morphologic variation in pneumococcus and description of a hitherto undefined morphologic variant, J. Exp. Med., 19j3,98, 21. 12. Torriani, A. ;2I., personal communication. 58-l PRODUCTIOS OE C.WSUI.-\I~ l'OI.~S:~CClI.~RID~S. I 13. Eddy, B. E., Nomenclature of pneumococcic types, P'uh. Hcullh Rep., 1941, 59, 449. 11. Kauffmann, F., and Lund, E., 1\Iemorandum on the nomenclature of the pneu- mococcus group, I~lcr~af. HUM. IM. Nomcirclalirre nrd Tarommy, 1951, 4.125. PLATE 32 FIG. 1. Reaction oi Type 111 anticnpsular antibody (.A) with Type III cells (B), Type I-III cells (C), ~tl Tl-pc IlL capwlnr l~olysnccharidc (D). FIG. 2. Reaction of Type I anticapsular antibody (.I) with Type I cells (B), Type I-III cells (C), and Tvlx I cal)sular p01>-saccll;kdc (D j. Tll .E JOLYRS.\L OF ESPERIMEST.\L 3KDICISE VOL. 110 PL.1TE 3