{Reprinted from THE JoURNAL OF EXPERIMENTAL Mepicing, April 1, 1949,
Vol. 89, No. 4, pp. 451-460]

ACQUISITION OF M PROTEIN BY PNEUMOCOCCI THROUGH
TRANSFORMATION REACTIONS*

By ROBERT AUSTRIAN, M.D., anp COLIN M. MacLEOD, M.D.

(From the Department of Microbiology, New York University College of Medicine,
New York)

(Received for publication, November 29, 1948)

Since the initial observations by Griffith (1) of the transformation of pneu-
mococcal capsular types in the mouse, considerable progress has been made in
elucidating the mechanism whereby this specific cellular alteration is effected.
The studies of Avery, MacLeod, and McCarty (2) have shown that acquisition
‘of the ability to produce a different capsular carbohydrate is dependent upon
the application to unencapsulated pneumococcal cells of highly polymerized
desoxyribonucleic acid. More recently, Boivin and his coworkers (3) have
shown that an analogous transformation can be induced in E. coli with surface
carbohydrate as the acquired character and have brought evidence that the
agent responsible for the change is desoxyribonucleic acid. Weil and Binder
(4) using culture filtrates, were able occasionally to produce type transforma-
tions of three strains of Shigella poradysenteriae but no information is available
concerning the chemical nature either of the principle inducing the transforma-
tion or of the acquired character. To the present time, therefore, no clear
evidence has been obtained of the transformation of bacterial cells with a pro-
tein as the acquired character.

In the present paper, transformation of pneumococci in vitro and in vivo
with the acquisition of a different M protein (5) is reported. In addition, the
independent variability of M protein and specific capsular polysaccharide is
described.

Materials and Methods

Transformation Reactions.—The techniques employed in preparing transforming extracts
and vaccines were those described by MacLeod and Krauss (6). Transforming extracts and
vaccines prepared from pneumococcus strains I-SVI and IIT-A66 were used. Transformation
reactions in vive were carried out by the method of Griffith (1). For transformation reactions
in vitro, the technique of Avery ef al. (2) was employed with the following modification. When
cultures of Dawson rough (7) variants on solid media were used as the source of inoculum, the
tubes containing the transforming system were inoculated by means of a straight needle which
had been touched to a peripheral portion of a colony. When pneumococci were grown in
liquid medium, the inoculum was one drop of a 10~ dilution of an 18 hour culture.

* This study was supported in part through the Commission on Acute Respiratory Dis-
eases, Army Epidemiological Board, Office of The Surgeon General, Department of the Army,
Washington, D.C.

451
452 ACQUISITION OF M PROTEIN THROUGH TRANSFORMATION

Strains of Griffith Rough and Dawson Rough Pneumococci.—Pneumococcus strain II-R36NC
(Griffith rough) and two derivatives of it were used as inocula in most of the experiments re-
ported. One derivative strain, II-R36N1, was obtained by transferring strain TI-R36NC
daily for 43 transfers in broth containing 1 per cent absorbed antiserum directed against the
M protein of the parent strain. Lancefield extracts of this strain, II-R36N14s, gave only faint
reactions with anti-M sera that reacted strongly with extracts of the parent strain, II-R36NC.
Strain II-R36N 143 retains the colonial appearance characteristic of Griffith rough pneumococci.
A Dawson rough variant of strain II-R36NC, selected according to Dawson’s method (7), was
also used. Plates of neopeptone-meat infusion agar containing 5 per cent defibrinated rabbit
blood were inoculated from a broth culture of strain II-R36NC with a fine wire in such a man-
ner that colonies were spaced at a distance of 0.5 to 1.0cm. The plates were incubated for 7
to 14 days at 37°C. After 5 days, rough, flat, fan-like excrescences similar to those described
by Dawson were noted at the periphery of some colonies. Portions of a number of these out-
growths were inoculated by means of a straight needle into tubes of neopeptone-meat infusion
broth and incubated overnight. Only those broth cultures showing growth characterized by
complete autoagglutination were used to inoculate a second series of plates. In Dawson’s
experience, cultures of the rough variants of pneumococci obtained by this process of selection
showed a strong tendency to revert spontaneously to the Griffith rough colonial form when
cultivated in nutrient broth (7). By repeated selection and inoculation from those colonies
showing the least capacity for reversion, however, strains of Dawson rough organisms were
obtained after 10 serial transfers which showed little or no tendency to revert spontaneously to
the Griffith rough form even after 5 or 6 rapid serial transfers in a highly nutrient liquid medium.
In Lancefield extracts of such relatively stable Dawson rough strains, the M protein charac-
teristic of the parent culture could still be demonstrated, but it appeared reduced in amount.
In view of Dawson’s observation that reversion of colonial type is favored by transfer in nu-
trient broth, inocula of the Dawson rough strain R36NDu, used in the present transformation
experiments, were obtained by touching a needle to the desired portion of the colony on solid
medium. This inoculum was then transferred directly to tubes containing the transforming
system.

In addition to the pneumococcal strains described above, in certain experiments another
Griffith rough variant, II-R36A, derived from the encapsulated type II strain II-D39S, was
used as inoculum.

In the designation of transformed strains the following set of conventions has been used:
the designation of the parent strain is followed by a dash, the letter M and an Arabic numeral
to indicate the type of M protein, and the letter S and a Roman numeral to indicate the type
of capsular carbohydrate possessed by the cell. For example, strain II-R36NC-M2’SIII is
a strain of pneumococcus derived from capsular type II possessing the type-specific protein
(M2’) of the parent type II cells and the capsular carbohydrate (SII1) of type III. Similarly,
strain II-R36NC-MISIII is one derived from capsular type II possessing the type-specific
protein (M1) of type I and the capsular carbohydrate of type IIT (SIID.

Preparation of Anti-M Sera, M Extracts, and Techniques of Precipitin and Agglutination
Tests. —The methods used were those described in the preceding paper (5).

EXPERIMENTAL

1. Transformation Reactions of Pneumococci with Acquisition of Capsular
Polysaccharide but without Acquisition of M Protein.— Because pneumococcus
strains II-R36NC and II-R36A had been shown previously to be susceptible to
transformation reactions involving the acquisition of capsular polysaccharides,
these two Griffith rough variants of pneumococcus II-D39S (capsular type IT)
R. AUSTRIAN AND C. M. MacLEOD 453

were selected for study. The results of iz vitro transformation of these two
strains to capsular type III and of strain II-R36A to an intermediate variant of
capsular type I are shown in Table I. Although capsular polysaccharides have
been acquired, the type-specific protein remains that of the parent strain,
demonstrating the independent variability of the two cellular components.
Strain II-R36A-M2’SI(int) is of further interest because of its low virulence
for mice. Pneumococci of this intermediate strain, possessing a very small,
but detectable capsule of type I polysaccharide, kill mice only when amounts of
16 hour culture as large as 1 X 107! to 1 X 10-* cc. are injected. In view of
the fact that this strain was derived from strain II-D39S which is fully virulent
for mice (M.L.D. = 1 X 10-8 cc. of a 16 hour broth culture) and because both

TABLE I

Antigenic Composition of Pneumococcus Strains II-R36N C and II-R36A and of Derivative
Strains Obtained by Transformation Reactions in Vitro

 

 

Source of
Strain transforming extract M protein Capsular SSS
strain

II-R36NC _ 2’ None
TI-R36NC-M2’SIII ITI-A66 2’ TI
TI-R36A _ 2’ None
TI-R36A-M2’SIII IIT-A66 2’ Til
TI-R36A-M2’SI(int) I-SVI 2’ I

 

strains possess the same M protein, the findings suggest that the M protein of
pneumococcus plays a minor rdle in the determination of virulence. This ob-
servation is in accord with the one noted in the preceding paper that anti-M
sera afford little or no protection against infection with homologous encapsu-
lated organisms.

2. Transformation in Vitro of Pneumococet with Concomitant Acquisition of M
Protein and of Capsular Carbohydrate.—In none of the experiments performed
in vitro with strains TI-R36NC or II-R36A was the acquisition of M protein
demonstrated. Inasmuch as these two strains possess M2’ protein, this finding
may not be altogether remarkable. Although transformation of pneumo-
cocci of one capsular type in the mucoid or encapsulated phase directly to the
mucoid phase of another capsular type has been reported (8), it seems probable
that the transformation occurred because of the appearance during growth of
unencapsulated mutants derived from the original mucoid strain. The cells
of these unencapsulated variants, lacking presumably the characters requisite
for the production of capsular polysaccharide of any type are susceptible to
transformation upon exposure in a suitable environment to desoxyribonucleic
acids from the homologous or heterologous types. When transformation
takes place, the altered cells again produce capsular carbohydrate, the type
being determined by the source of the nucleic acids.
454 ACQUISITION OF M PROTEIN THROUGH TRANSFORMATION

It was reasoned by analogy, therefore, that if it were possible to select cells
deficient in M protein, these cells might be capable of acquiring this cellular
component through the transformation reaction. To obtain cells deficient in
M protein, pneumococcus II-R36NC was grown in neopeptone broth con-
taining 1 per cent of a potent anti-M2’ protein rabbit serum which had been
absorbed with strains I-192R and III-A66R2 to remove species-specific anti-
bodies. Lancefield extracts of cells obtained from the sixteenth daily transfer
in antiserum broth and of cells from the same culture transformed to capsular
type III showed still the original M2’ protein. Extracts of cells from the
thirty-fourth transfer showed, however, an apparent reduction in the amount
of M2’ protein produced by the culture, though its presence was still detectable.
When cells from the forty-third transfer, strain II-R36N1,3, were tested in
transformation reactions with an extract of strain III-A66, colonies were ob-
tained on subculture which were of capsular type III. Of two colonies tested,
one gave rise to a strain possessing type 2’M protein. The cells from the
other colony, however, were found on subculture to possess type 3 M protein as
well as type IIISSS. The parent cell of this culture had undergone apparently,
a double transformation with acquisition of SSS III and M3 protein.

An alternative method for obtaining cells deficient in M protein was sought
by growing rough variants of pneumococcus II-R36NC according to the method
of Dawson described in the section on methods. Transformations to capsular
type III of Dawson rough variants from serial transfers 3, 5, and 8 showed no
concomitant acquisition of M3 protein. On the eleventh serial transfer of
the Dawson rough variant derived from strain II-R36NC, examination of
Lancefield extracts made from these cells suggested a diminution in the quan-
tity of M2’ protein produced by the culture. Cells from several colonies of
the eleventh passage, strain II-R36NDu, were tested in transformation reac-
tions with an extract made from pneumococcus III-A66. Following incuba-
tion at 37°C. for 24 hours, samples were plated on blood agar. Examination of
these platings revealed three colonial forms: mucoid, Griffith rough, and Daw-
son rough. Control cultures showed only the Dawson rough colonial phase
initially and after 6 serial subcultures. Griffith rough and mucoid colonies
from several cultures were selected and grown for the preparation of Lance-
field extracts. Extracts of cultures of most of the mucoid colonies (capsular
type III) showed M2’ protein. One mucoid colony, however, gave rise to a
culture possessing M3 protein in addition to the capsular polysaccharide of
type III. It is of interest that a Griffith rough strain recovered from the
same culture contained M2’ protein although it had undergone alteration in
colonial morphology from the Dawson rough form.

By two independent methods of selection, therefore, strains of pneumococcus
were obtained which are capable of acquiring through transformation reactions
R. AUSTRIAN AND C. M. MacLEOD 455

in vitro, a type specific M protein different from that possessed by the original
type II strain from which they were derived. The results of these experiments
are summarized schematically in Fig. 1 together with an antigenic analysis of
the strains concerned.

3. Transformation of Pneumococct in Vivo, with Concomitant Acquisition of
M Protein and of Capsular Polysaccharide.—Transformation of pneumococci
with acquisition of M protein was accomplished i# vivo by the method of
Griffith. Mice were inoculated subcutaneously with a live culture of the Grif-
fith rough strain, II-R36NC, together with a heat-killed vaccine of encapsulated
pneumococci prepared by the method of MacLeod and Krauss (6). In none
of the experiments were viable organisms detected in the transforming vac-
cines, either by mouse inoculation or by culture. Transformation of pneumo-
coccus IJ-R36NC to capsular type I was induced by a vaccine prepared from
pneumococcus J-SVI in eight of ten mice inoculated. Transformation to cap-
sular type III was effected with a vaccine of pneumococcus III-A66 twice in
ten attempts. In one instance of transformation to each capsular type, con-
comitant acquisition of M protein occurred. The strains in which the acquisi-
tion of two new characters took place differed distinctively in their colonial
morphologies from those of the strains from which the transforming vaccines
were made, whereas capsular transformations of pneumococcus IJ-R36NC
possessing either the M protein of that strain or the M protein of the transform-
ing vaccine were indistinguishable from each other in their colonial appearance.
In view of these morphological observations and the absence of viable organisms
from vaccines, there can be no doubt that a different M protein can be acquired
through transformation carried out in vive by the technique of Griffith.

4. Combination of Three Inheritable Characters within a Single Pneumococcal
Sirain.—To determine whether a strain of pneumococcus possessing the colonial
properties of one type and the M protein of a second type could be transformed
so that it now produced the capsular polysaccharide of a third type, the follow-
ing experiments were undertaken. Pneumococcus strain II-R36NC-MI1SI,
which had acquired M1 protein and SSSI through transformation ix vivo of
pneumococcus II-R36NC in the presence of a transforming vaccine of pneumo-
coccus I-SVI, was grown in 20 per cent antipneumococcus capsular type I
serum broth. A Griffith rough variant containing M1 protein was obtained
after eight transfers. This variant was then exposed in viiro to a transforming
extract of pneumococcus JII-A66, Platings of this culture revealed encapsu-
lated pneumococci which on analysis showed the basic colonial morphological
properties of strain II-R36NC, the type 1 M protein of strain I-SVI, and the
type III capsular polysaccharide of strain III-A66. It is possible, therefore,
by transforming reactions to combine three inheritable characters of three anti-
genically distinct pneumococci within a single cell. (Fig. 2.)
FIGURE 1

Methods of Selecting Rough Variants of Pneumococcus Strain II-D39S and of Directing the Acquisition of New S ‘pecific Capsilar Polysaccharide
and M Protein by Means of Transformation Reactions in Vitro

11-D39S
(M2’, SII)

Anti-SIT

serum

TI-R36NC
(M2’, S—)

Anti-M2’
serum, 43
transfers

|
IL-R36N 143
7 (M2'+, S—)
/
|
/
/

<

\

\
ON

Selection
of Dawson
rough mu-
tants, 11
transfers

II-R36NDu:
(M2/+, S—)

/

Transforming 7

extract IIT-A66 \

II-R36N13-M2’SIII
7 (M2’, SITD)

/

\

\._- IL-R36N14-M3SIII
(M3, SIII)

II-R36NDy1-M2’SIIL
7 (M2, SIT)

/

Transforming :
extract ITI-A66 \

NX TI-R36NDn-M3SH1
(M3, SHI)

Antigenic analysis of strains is contained in parentheses below strain designations. The type-specific protein is indicated by the letter M and

OSP

NOILVNYOASNVUL HONOUHL NIGLOUd W AO NOILISINOOV

the Arabic numeral following it, the capsular polysaccharide by the letter S and Roman numeral following it. Absence of the antigen is indi-
cated by —, a culture containing probably an increased proportion of deficient cells by +. Notations above and below the arrows indicate the
method used in selecting variants and the source of transforming extracts.
’ FIGURE 2 .
Combination of Inheritable Characters of Three Pneumococcal Strains within a Single Strain

tiSII Transformi
II-D 39S a II-R 36NC Se ILR36NC-MiSI. ——~-——>
(col. 2, M2’, SI) serum (col. 2, M2’, S—) vaccine 2 (col. 2, M1, ST)
th vive
ti-ST Transformi
—Antist nr 3encm1 = —Lfansforming _, eR 36NC-MISIII

serum — (col, 2, M1, S—) _e*tract IIT-A66 (col. 2, M1, SII)

The legends used are the same as those employed in Fig. 1. The retention of basic colonial morphological properties (autolytic and hemolytic)
of the parent strain in the ensuing transformations is indicated by the inclusion of the symbol col. 2 in the parentheses below the strain
designations.

dOZDVW “WO UNV NVIALSAV °H

LSP
458 ACQUISITION OF M PROTEIN THROUGH TRANSFORMATION

DISCUSSION

The studies described in this paper demonstrate that the M protein of pneu-
mococcus can be acquired through the transformation reaction and add a
new class of biological compounds, the presence and type specificity of which
are subject apparently to the contro! of highly polymerized desoxyribonucleic
acids. Transforming extracts are capable of causing the acquisition, not only
of the capsular polysaccharide of pneumococcus as described previously by
others, but also of the M protein and would appear capable also of inducing
alteration of colonial morphology from the Dawson rough to the Griffith rough
variant when the appropriate conditions exist. Inasmuch as it has been demon-
strated that pneumococcus capsular carbohydrate and M protein may vary
independently of one another, the results obtained suggest that transforming
extracts of encapsulated pneumococci contain a multiplicity of desoxyribonu-
cleic acids which control the specificities of the several cell characters described.

At the present time, transformation of pneumococci involving acquisition of
M protein cannot be carried out with as high a frequency as can that of the cap-
sujar polysaccharides. Although acquisition of a new M protein by pneumo-
coccus strain II-R36NC occurs occasionally in the mouse under the influence
of heat-killed transforming vaccines, acquisition of a new M protein by this
strain iz vitro has not been observed. It is possible, however, that cultures of
strain II-R36NC contain cells which are deficient in M protein but in such
small numbers that detection of transformation is unlikely unless strongly selec-
tive factors operate. In a sense, acquisition of a new M protein by pneumo-
coccus strain II-R36NC i vivo is analogous to the transformation in vitro of
pneumococci of one capsular type directly to another capsular type reported
by Dawson and Warbasse (8). It was their opinion that although unencapsu-
lated forms were not seen in the cultures, transformation took place probably
by way of these variants. If, however, pneumococcus II-R36NC is grown in
the presence of anti-M2’ serum or if Dawson rough variants are selected by
suitable cultural methods, strains are obtained by either technique which,
when extracted by hot acid solutions, yield less M protein than the parent
strain. It seems probable that the methods of selection employed have in-
creased the relative number of M protein-deficient cells in the culture and have
improved the probability of detection of cells with a newly acquired M protein
following exposure of the culture to transforming extracts. This hypothesis
would account for the presence of capsular transformations associated with
either the M protein of the cells used as transforming agents or that of the cul-
ture subjected to transformation. That only a fraction of the cells exposed to
transforming extract acquire a new M protein is not remarkable if it be recalled
that in all probability but a small percentage of M protein-containing, unen-
capsulated cells similarly treated undergo capsular type transformation.
R. AUSTRIAN AND C. M, MACLEOD 459

The experiments reported here suggest that acquisition of a class of proteins
by pneumococci is probably controlled by desoxyribonucleic acids. Of the
transformations reported previously, all analyzed chemically have concerned
the acquisition of polysaccharides. Although transformation involving the
surface carbohydrate of E. coli (8) and of the type-specific antigens of Sz.
paradysenteriae (4) have been accompanied by altered fermentative activities,
the alterations suggest the loss of enzymes rather than their acquisition.

The demonstration of the independent variability of capsular polysaccharide
and of M protein in transformed pneumococci is of interest in view of the obser-
vation in the preceding paper (5) that the same capsular polysaccharide may
be associated with different M proteins in nature, and that the same or very
similar proteins may occur with different capsular polysaccharides. An addi-
tional finding of interest is the fact that by the use of transformation reactions
it is possible to combine within a single pneumococcal cell characters derived
from three distinct pneumococcal strains. If it be assumed that the number
of type-specific M proteins and of distinctive colonial forms approximates the
number of known capsular types, then the possible number of antigenically dis-
tinct pneumococci is in excess of 500,000. The demonstration that it is possible
to effect such combinations of antigens in the laboratory and the observations
made upon naturally occurring strains suggest the desirability, whenever pos-
sible, of isolating chemically the reactive substances which enter into an anti-
genic analysis of bacteria in order to avoid or to elucidate “cross-reactions”
which may prove confusing. That such a procedure is useful is borne out by
the studies of the relations of the M and T antigens of group A @-hemolytic
streptococci (9).

SUMMARY

Acquisition by pneumococcal variants of M protein and of capsular polysac-
charide different from those present in the parent strain has been effected in
vitro by means of transforming reactions with extracts of heterologous encapsu-
lated pneumococci. Similar transformations have been accomplished in vivo
with heat-killed vaccines as the transforming agents.

Independent variation of pneumococcal capsular polysaccharide and M pro-
tein observed in nature can be brought about also in the laboratory. By means
of transforming reactions, it has been demonstrated that inheritable characters
of three distinct pneumococcal strains can be combined within a single strain.

It is suggested that acquisition of M protein through transformation reac-
tions occurs in cells deficient in that character.

BIBLIOGRAPHY

1, Griffith, F., 7. Hyg., Cambridge, Eng., 1928, 27, 113.
2. Avery, O. T., MacLeod, C. M., and McCarty, M., J. Exp. Med., 1944, 79, 137.
460

3.

cost An Oe

ACQUISITION OF M PROTEIN THROUGH TRANSFORMATION

Boivin, A., in Cold Spring Harbor Symposia on Quantitative Biology, Cold
Spring Harbor, Long Island Biological Association, 1947, 12, 7.

. Weil, A. J., and Binder, M., Proc. Soc. Exp. Biol. and Med., 1947, 66, 349.

. Austrian, R., and MacLeod, C. M., J. Exp. Med., 1949, 89, 439.

. MacLeod, C. M., and Krauss, M. R., J. Exp. Med., 1947, 86, 439,

. Dawson, M. H., J. Path. and Bact., 1934, 39, 323.

. Dawson, M. H., and Warbasse, A., Proc. Soc. Exp. Biol. and Med., 1931-32, 29,

149.

. Lancefield, R. C., and Stewart, W. A., J. Exp. Med., 1944, 79, 79. Watson, R.

F., and Lancefield, R. C., J. Exp. Med., 1944, 79, 89. Stewart, W. A., Lance-
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