Published November 1, 1931
I N V I T R O T R A N S F O R M A T I O N OF P N E U M O C O C C A L T Y P E S
II. T ~ E N A T U R E OF THE FACTOR RESPONSIBLE FOR THE
TRANSFORMATION OF PNEUMOCOCCAL TYPES
BY RICHARD H. P. SIA,* M.D., AND MARTIN H. DAWSON, M.D.
(From the Department of Medicine, The Presbyterian Hospital, and the College of
Physicians and Surgeons, Columbia University, New York)
(Received for publication, July 10, 1931)
The Nature of R Cultures Most Suitable for Transformation of Type
by in Vitro Methods
As originally pointed out by Griffith (2), and subsequently confirmed by one of the authors (3), there are varying degrees of constancy
or stabiRty of the R variant of pneumococcus. In previous work on
transformation of type by in vivo procedures it was found t h a t the
nature of the R culture employed materially affected the results (4, 5).
Thus, in the in vivo procedure, it was shown that an R culture which possessed
only a slight degree of R stability showed a tendency to revert to the S form of the
original type. However, R cultures which were more definitely stabilized in the R
form showed less tendency to revert to the S form of the original type and were
more readily transformed into S forms of heterologous types. R cultures which
were still more definitely stabilized in the R form were more "resistant" and could
be converted into S forms of either homologous or heterologous types only with
considerable difficulty.
* On leave of absence from the Peiping Union Medical College, Peiping, China.
701
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In the accompanying communication (1) a method of inducing transformation of pneumococcal types entirely by in vitro procedures is
reported. In the present study the conditions under which transformation of pneumococcal types can be effected by in vitro procedures
have been further analyzed and the factor responsible for transformation has been investigated.
Published November 1, 1931
TRANSFORMATION O1e PNEUMOCOCCAL TYPES.
702
II
In the in vitro experiments R cultures of varying degrees of stability were
employed. The degree of R stability was determined by one of the following
methods: (1) the number of transfers necessary to effect reversion in 10 per cent
anti-R serum broth (3, 6); (2) the amount of living R culture necessary to induce
reversion when injected subcutaneously in white mice; (3) the ease with which
transformation of type could be effected by in vivo procedures.
The results obtained in in vitro experiments in which various R cultures were
employed may be presented in brief as follows: (1) transformation of type was
most readily effected with those R cultures which possessed only slight degrees of R
stability; (2) transformation of type was not effected with certain cultures which
were more definitely Stabilized in the R form; (3) under the conditions employed,
the R cultures, in the presence of heterologous S vaccines, showed little tendency
to revert to S forms of the original type.
T h e significance of t h e s e o b s e r v a t i o n s will b e r e f e r r e d to in a s u b -
TABLE I
The Effect of Growing S Organisms in Media Containing R Vaccine
S culture, 1 drop 10 ~ dilution; 0.5 cc. blood broth; 0.1 cc. normal rabbit serum;
1 R vaccine, equivalent Of 100 cc. of original culture.
Type of culture
Used for inoculation
"
IS
Colonies on plates streaked
24 hours
48 hours
72 hours
96 hours
I S only
I S only
I S only
I S only
II "
It "
"
II "
"
II "
"
II "
"
iII "
III "
"
III "
"
III "
"
III "
"
A t t e m p t s to Convert S F o r m s into R F o r m s by Growth i n M e d i a
C o n t a i n i n g R Vaccines
A s d e t a i l e d in t h e p r e c e d i n g e x p e r i m e n t s , t h e g r o w t h of R f o r m s of
p n e u m o c o c c i , u n d e r s u i t a b l e c o n d i t i o n s , in m e d i a c o n t a i n i n g S v a c icines of h e t e r o l o g o u s t y p e , f r e q u e n t l y r e s u l t e d i n t h e t r a n s f o r m a t i o n
o f : t h e R f o r m s i n t o S: f o r m s o f ~t h e t y p e of t h e v a c c i n e . T h e effect of
g r o w i n g S o r g a n i s m s in m e d i a c o n t a i n i n g R v a c c i n e s w a s n e x t
determined.
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sequent communication.
Published November 1, 1931
RICHARD H. P. SIA AND MARTIN H. DAWSON
703
Type I S, Type II S and Type III S organisms were grown in media to which
were added relatively large amounts of a 1 R vaccine. Very small seedings o5
the S cultures were employed and plates were streaked from the cultures every 24
hours for 4 days. Normal rabbit serum was used in the culture media instead of
anti-R serum. The results of the experiment appear in Table I.
F r o m the d a t a presented in Table I it appears t h a t S organisms are
capable of living and multiplying in the presence of large a m o u n t s of
R vaccine. U n d e r the conditions employed the S forms showed no
t e n d e n c y t o r e v e r t to the R form. N o a t t e m p t was made to determine
the effect of serial transplants of S organisms in media containing R
vaccines.
In previous experiments it was found t h a t the smallest a m o u n t of
an S vaccine effective in inducing transformation of type was t h a t
representing the bacteria from 0.1 cc. of the original S culture. I t
therefore appeared improbable t h a t either the s u p e r n a t a n t material
or the filtrate of an S vaccine would prove effective in inducing the
change. However, in order to analyze further the conditions responsible for transformation, experiments were done using the supern a t a n t and the filtrate obtained from an S vaccine.
A III S vaccine was prepared in the usual way and divided into two equal lots.
One lot was centrifuged at high speed for 1 hour. At the end of this time the
supernatant material was removed and used in transformation experiments.
The other lot of vaccine was filtered through a Berkefeld candle and the filtrate
similarly employed in transformation experiments. Relatively large amounts of
the supematant material and the filtrate were employed. Transformation of type
was not obtained in any instance.
T h e experiment was repeated m a n y times with m a n y variations in
technique b u t the results were uniformly negative.
In other experiments a t t e m p t s were made to effect transformation
of t y p e b y the use of purified specific soluble substance of T y p e I I I S
pneumococcus 1 (7). All such experiments were unsuccessful.
1 We are indebted to Dr. M. Heidelberger of the ColIege of Physicians and
Surgeons, Columbia University, for placing material at our disposal for these
experiments.
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Attempts to Effect Transformation of Type by the Use of (1) the Supernatant Fluid from an S Vaccine, (2) the Filtrate of an S Vaccine,
(3) _Purified Specific Soluble Substance
Published November 1, 1931
704
TRANSFORMATION
OF PINIEU~fOCOCCAL TYPES.
II
F u r t h e r experiments in which the filtrate of an actively growing S
culture was employed also yielded negative results.
Attempts to Effect Transformation of Type by Extracts of S Organisms
I n all successful t r a n s f o r m a t i o n experiments carried o u t u p to this
point a suspension of S cells, killed b y heating, was employed. E x p e r i m e n t s were next devised in an a t t e m p t to effect t r a n s f o r m a t i o n
b y the use of cell-free extracts of S organisms. A convenient m e t h o d
of preparing such extracts a p p e a r e d to be b y the m e t h o d of freezing
a n d thawing. T h e following experiment was accordingly arranged.
Table II.
T h e d a t a p r e s e n t e d in T a b l e I I show t h a t a I I I S vaccine, h e a t e d
for 30 m i n u t e s at 60°C., was effective in inducing t r a n s f o r m a t i o n of
type. T h e same vaccine, when subjected to the procedure of freezing
a n d thawing t w e n t y - t h r e e times, was likewise effective in inducing
t r a n s f o r m a t i o n . I t is again emphasized, however, t h a t the freezing
a n d thawing procedure failed to b r e a k u p the previously h e a t e d cells.
T h e filtrate from the heated, frozen a n d thawed m a t e r i a l was not
effective in inducing transformation. Likewise the m a t e r i a l which
was first frozen and thawed, and subsequently h e a t e d at 60°C., failed
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The bacteria obtained by centrifugation from 1200 cc. of a I I I S culture were
taken up in 12 cc. of plain broth and divided into two equal lots of 6 cc. each,
designated as Lots A and B. Lot A was heated for 30 minutes at 60°C. At the
end of this period 1.5 cc. was withdrawn for experimental and control purposes.
The remainder, 4.5 cc., was frozen andthawed twenty-three times in an attempt
to break up the ceils. It was found, however, that bacterial cells which had
previously been subjected to heating for 30 minutes at 60° were very resistant to
disruption by this method. The resulting material still showed an abundance of
well formed cells when stained by Gram. 0.5 cc. of the heated, frozen and thawed
material was withdrawn for experimental purposes. The remaining 4.0 cc. of
this material was centrifuged and the supernatant filtered through a Berkefeld
candJe. The resulting filtrate was used in transformation experiments. Lot B,
which had not been subjected to heating, was frozen and thawed twenty4hree
times. At the end of this time, smears showed only Gram-negative detritus and
no intact cells. 1.5 cc. of this material was withdrawn and heated for 30 minutes
at 60°C. This material was used for control and experimental purposes. The
remainder, 4.5 cc., of the frozen and thawed material was centrifuged and filtered
through a Berkefeld candle. The resulting filtrate was employed in transformation experiments. The remaining details and results of this experiment appear in
Published November 1, 1931
RICHARD
H. P. SIA A N D
MARTIN
H. D A W S O N
705
t o i n d u c e t r a n s f o r m a t i o n of t y p e . F i n a l l y , t h e f i l t r a t e of t h i s m a t e r i a l
w a s also i n e f f e c t i v e . T h e s e r e s u l t s m a y b e s e t d o w n in t a b u l a r f o r m
as follows:
Suspension of S Cells
1.
2.
3.
4.
5.
Heated 30 rain. at 60°C . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Heated 30 rain. at 60°C., frozen and thawed . . . . . . . . . . .
Heated 30 rnin. at 60~C., frozen and thawed, filtered . . . . .
Frozen and thawed, heated 30 rain. at 60°C . . . . . . . . . . .
Frozen and thawed, heated 30 rain. at 60°C., filtered . . . . .
Effective
Effective
Not effective
Not effective
Not effective
TABLE II
Attempts to Effect Transformation of Type by the Use of Cell-Free Extracts of S
Organisms
Colonieson platesstreaked
Method of preparingvaccines
and vaccine"extracts"
Heated 30 min. 60°
Heated 30 rain. 60 °, frozen
and thawed 23 times
Heated 30 rain. 60°, frozen
and thawed 23 times
centrifuged and filtered
Frozen and thawed 23
times, then heated 30
mira 60°
Frozen and thawed 23 times,
then heated 30 min. 60°,
filtered
24 hours
I Numerous
III S
2
48 hours
72 hours
96 hours
Nearly all
III S
Nearly all
III S
Nearly all
III S
R only
R only
R only
3
R only
6
8
C~
C~
I n i n t e r p r e t i n g t h e r e s u l t s of t h i s e x p e r i m e n t a t t e n t i o n is d r a w n
t o t h e f o l l o w i n g p o i n t s . I n t h e first p l a c e , t r a n s f o r m a t i o n of t y p e
w a s e f f e c t e d o n l y w i t h t h o s e f r a c t i o n s w h i c h c o n t a i n e d f o r m e d cell
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2 R culture, 1 drop 10 4 dilution; 0.5 co. blood broth; 0.1 co. anti-R serum;
I I I S vaccine and vaccine "extracts" as indicated.
Published November 1, 1931
706
TRANSFORMATION
OF PNEUMOCOCCAL
TYPES.
II
elements. I n the second place, the process of freezing and thawing was
carried out under aerobic conditions. I t therefore a p p e a r e d possible
t h a t the factor responsible for t r a n s f o r m a t i o n m i g h t h a v e been
destroyed b y oxidation during the manipulations.
I n the next experiment an a t t e m p t was m a d e to p r e v e n t oxidation
b y carrying out all procedures u n d e r anaerobic conditions.
T h e results of this experiment m a y be t a b u l a t e d as follows:
Suspension of S Cells
1. Heated 30 min. at 60°C . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effective
2. Heated 30 min. at 60°C., frozen and thawed anaerobically . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effective
3. Heated 30 min. at 60°C., frozen and thawed anaerobically, centrifuged, supernatant material . . . . . . . . . Not effective
4. Frozen and thawed anaerobically, heated 30 min. at
60°C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Not effective
5. Frozen and thawed, anaerobically, heated 30 rain. at
60°C., centrifuged, supernatant material . . . . . . . . . . . . . Not effective
T h e additional points to be n o t e d in this e x p e r i m e n t are: (1) the
s u p e r n a t a n t m a t e r i a l from heat-killed, frozen a n d thawed bacterial
suspension of S cells was not effective; (2) a suspension of S cells,
frozen a n d thawed anaerobically and subsequently h e a t e d for 30
m i n u t e s a t 60°C., was not effective, This experiment suggested t h a t
the failure to induce t r a n s f o r m a t i o n w a s due to the existence of unfavorable conditions created during the disruption of living bacterial
cells.
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The bacteria obtained by centrifuging a culture of I I I S organisms were divided
into two lots, A and B. Lot A was heated for 20 minutes at 60°C. A portion
was then withdrawn for test and control purposes. The remainder was frozen
and thawed twenty times under a heavy layer of oil. A portion of this material
was withdrawn for experimental purposes and the remainder was centrifuged at
high speed for 30 minutes. At the end of this time the cell-free supernatant
material was pipetted off and similarly used for experimental purposes. Lot B
was frozen and thawed twenty times under a heavy layer of oil and then heated
for 20 minutes at 60°C. A portion was withdrawn for test purposes. The
remainder was centrifuged at high speed for 30 minutes. The clear supernatant
fluid was pipetted off and used in tests.
Published November 1, 1931
707
RICHARD H. P. SIA AND MARTIN H. DAWSON
The Effect of (1) Bacterial Peroxide, (2) the pH in Cultures from Which
Vaccines Are Prepared for Transformation Purposes
In the experiments carried out up to this point the observation had
been made that S vaccines prepared from different lots of culture
varied greatly in their capacity to induce transformation. It was
noted that vaccines made from old and autolyzing cultures were quite
ineffective. Moreover the procedure of subjecting the culture to a
preliminary heating for 10 minutes at 60° before centrifugation
TABLE III
TI~¢ Effect of (1) Bacterial Peroxide, (2) ptt in Cultures from Which Vaccines Are
Made for TransformaHon Purposes
Ageof
pH of Bacterial
culture I culture peroxide
Colonies on plates streaked
2iholui
48hours
72 hours
hrs.
16
7.7
24
7.3
40
7.3
48
7.3
-}--{-
64
7 2
-t-++
R only
Over half I I I S
Numerous " "
R only
" "
Numerous I I I S
R only
"
Numerous I I I S
Nearly all " "
Several
" "
Numerous " "
Nearly all " "
R only
"
appeared to be of value in preserving the necessary factor (4). An
attempt was made to verify these observations in the following way.
A culture of I I I S pneumococcus in plain broth was incubated at 37°C. Samples
of the culture were withdrawn at the following intervals,--16, 24, 40, 48 and 64
hours. After centrifugation the bacteria obtained from these samples were
heated for 20 minutes at 60 °. The supernatant material was tested for the
presence of bacterial peroxide and its p H determined. The different lots of
vaccine were then used in transformation experiments. The results are presented
in Table I I I .
From the data presented in Table I I I it is evident that vaccines
prepared from cultures of S organisms are not effective in inducing
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2 R culture, 1 drop 10 -6 dilution; 0.5 cc. blood broth; 0.1 cc. anti-R serum;
I I I S vaccine, equivalent of 50 cc. of original culture, prepared as indicated.
Published November 1, 1931
708
TRANSFOILMATION
OF P N E U M O C O C C A L
TYPES.
II
transformation of type if bacterial peroxide is present in demonstrable
amounts in the cultures. Within the limits of the experiment the
pH range did not materially affect the results. The experiment was
repeated and entirely similar results were obtained. This finding
offered a solution to many hitherto unexplained results and suggested
that the principle responsible for transformation of type was possibly
susceptible to oxidizing influences. In subsequent attempts to isolate
the principle responsible for transformation of type the significance
of this observation was recognized. T h e s e experiments are being
continued.
DISCUSSION
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The study of the conditions under which transformation of pneumococcal types may be effected by in vitro procedures has led to a
clearer understanding of the mechanism of the transformation process.
It has been shown that the nature of the R culture selected for
-transformation purposes materially affected the results obtained.
Thus, R cultures which possessed only slight degrees of R stability
were found to be the most suitable for transformation purposes. An
adequate explanation of this finding cannot be offered until further
knowledge is available as to the factors which determine the degree of
stability possessed by various R cultures.
In considering the nature of the mechanism by which transformation of type is effected two possibilities present themselves: either a
latent attribute of the R cell may be stimulated by its association with
the S vaccine, or the R organisms may acquire a new property from
the vaccine. The former conception involves the assumption that all
R pneumococci possess at all times the latent capacity of elaborating
any one of the known varieties of specific polysaccharides associated
with S organisms. The latter hypothesis suggests the possibility
that, at times, certain attributes of bacteria may be transferred from
organisms of one type to those of another type of the same species.
In the course of the present investigation no evidence has been obtained to support either hypothesis but attention is directed to certain
characteristics of the property, possessed by S vaccines, which is
responsible for transformation of type.
Published November 1, 1931
RICHARD H. P. SIA AND MARTIN H. DAWSON
709
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In the previous paper it was shown that the factor responsible for
transformation was heat-labile, being destroyed at temperatures
above 80°C. It therefore cannot be related to the specific soluble
substance of pneumococcus for this has been shown to resist temperatures of 100°C. (7). Attempts to effect transformation by the
use of purified specific soluble substance were uniformly unsuccessful.
The experiments described, in which attempts were made to induce
transformation by cell-free extracts of S vaccines, throw further light
on the nature of the property responsible for transformation of type.
These experiments suggested that the essential factor was destroyed
by the bacterial enzymes released during disruption of living bacterial
cells. Additional evidence in support of this view was obtained in the
experiments in which it was shown that vaccines prepared from old
cultures in which bacterial peroxide was present in demonstrable
amounts were ineffective in transformation.
In these two characteristics, thermolability and susceptibility to the
action of enzymes liberated in old broth cultures, the property responsible for transformation bears a striking resemblance to the
"antigenic specific substance" described by Day (8). Day showed that
the antigenic specific substance of pneumococcus contains two groups,
(1) a stable portion (ordinary specific substance), which reacts with
immune serum, (2) an unstable, antigenic, portion, which provokes
the formation of antibody. The first of these groups is resistant to
heating at 100°C. and to the action of bacterial enzymes. The second
group is destroyed by heating at 100°C. and by bacterial enzymes. It
therefore appears possible that the factor contained in an S vaccine
responsible for transformation of type is closely related to, if not
identical with, the "antigenic specific substance" of pneumococcus.
The nature of this antigenic group is conjectural. In many respects it
resembles the enzymes of pneumococcus studied by Avery and Cullen
(9). Further work is required to determine its true nature and precise properties.
The significance of the results reported in this and the preceding communication in the field of bacteriology and epidemiology is largely a
matter of speculation. Further investigation is required to determine
whether similar transformations of type occur under natural or disease
conditions both in pneumococci and other bacterial species.
Published November 1, 1931
710
TRANSFOR~[ATION OF PNEU~OCOCCAL
SUMMARY
TYPES.
II
AND CONCLUSIONS
1.
2.
3.
4.
5.
6.
7.
8.
9.
BIBLIOGRAPHY
Dawson, M. H., and Sia, R. H. P., J. Exp. Med., 1931, 54, 681.
Griffith, F., Rep. Pub. Health and Med. Subj., Ministry of Health, No. 18,
1923, 1.
Dawson, M. H., J. Exp. Med., 1928,47,577.
Dawson, IV[.H., J. Exp. Med., 1930, 51, 99.
Dawson, M. H., J. Exp. Med., 1930, 51,123.
Dawson, M. H., and Avery, O. T., Proc. Soc. Exp. Biol. and Med., 1927,24, 943.
Heidelberger, M., and Avery, O. T., J. Exp. ]fled., 1923, 38, 73; 1924, 40, 301.
Day, H. B., Brit. J. Exp. Path., 1930, 11,164.
Avery, O. T., and Cullen, G. E., Y. Exp. Med., 1920, 32, 547, 571,583.
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I. Further observations on the conditions under which transformation of pneumococcal types may be induced by in vitro procedures
are presented.
2. R cultures possessing only slight degrees of R stability are most
suitable for transformation purposes b y in vitro procedures.
3. Vaccines prepared from cultures subjected to the action of
bacterial enzymes liberated (a) in old broth cultures, (b) during mechanical disruption of young bacterial cells, are not effective in inducing
transformation of type.
4. The property of an S vaccine responsible for transformation of
type is not related to the specific soluble substance of pneumococcus.
5. Attempts to effect transformation of type by the use of cell-free
extracts of pneumococcus have so far proved unsuccessful.