[Reprinted from THE JouRNAL OF EXPERIMENTAL MEDICINE, July 1, 1953,
Vol. 98, No. 1, pp. 35-40]
Printed in U.S. A,

MORPHOLOGIC VARIATION IN PNEUMOCOCCUS

II. Controt oF PNeumococcaL MorpHoLtocy THROUGH TRANSFORMATION
ReEactions*

By ROBERT AUSTRIAN, M.D.

(From the Biological Division, Department of Medicine, The Johns Hopkins University
School of Medicine, Baltimore)

(Received for publication, March 7, 1953)

‘In the preceding paper (1), a hitherto undefined morphologic variant of
pneumococcus has been described and its relation to the three previously
recognized morphologic variants of this species set forth. The data presented
revealed, as had earlier studies (2, 3), that two of the principle factors influ-
encing morphologic patterns in pneumococcus are the mode of cellular sepa-
ration after division and the production of type-specific capsular polysac-
charide. Both these pneumococcal characters have been shown to be subject
to control through transformation reactions (4, 5) and in addition, to be in-
herited independently of one another in such reactions (6, 7). If these findings
are applicable generally, it should be possible to alter predictably any com-
petent pneumococcal morphologic variant through transformation reactions
with the transforming principles of any of the other morphologic variants.
The present experiments are concerned with an examination of this problem.

Materials and Methods

1. Nomenclature —The nomenclature used to describe the morphologic variants of pneu-
mococcus is that employed in the preceding paper (1).

2. Preparation of Transforming Principles and Techniques of Transformation Reactions.—
Transforming principles (T.P.) were prepared according to the method of MacLeod and
Krauss (8). In all experiments save those concerned with the possible exchange of desoxyribose
nucleic acids controlling production of type-specific capsular polysaccharide, the technique
for carrying out the transformation reaction was that used by these two authors. 0.1 cc. of a
10~* dilution of an 18 hour blood broth culture of the strain to be transformed was inoculated
into 2 cc. of charcoal-absorbed neopeptone broth containing 5 per cent human pleural fluid
and 0.1 cc. of the appropriate transforming principles. Cultures containing no T.P. were pre-
pared as controls. After 24 hours’ incubation at 37°C., a loopful of each culture was streaked
on the surface of a neopeptone agar plate containing 5 per cent defibrinated rabbit’s blood. In
experiments yielding both autoagglutinable and diffusely growing variants, both supernatant
fluid and sediment from the transformed and control cultures were inoculated on the surface

* This work was supported by a grant-in-aid from the National Institutes of Health, Public
Health Service, Federal Security Agency.

 

35
36 MORPHOLOGIC VARIATION IN PNEUMOCOCCUS. II

of solid media. The plates were incubated for 24 hours and examined then for preliminary
identification of the several morphologic variants under a colony microscope at a magnifica-
tion of 30 diameters. Individual clones were then isolated and the variants identified further
by the use of the Gram stain and of quellung and agglutination reactions. In addition, to test
for the presence of the factors controlling mode of cellular separation after division in capsu-
lated forms, transforming principles were obtained from the strain in question in many in-
stances. Recovery of filamentous non-capsulated (fi/+ S—) and of non-filamentous capsulated
(fl— S++) forms following growth of a non-filamentous non-capsulated (fl— S—) form in
the presence of T.P. obtained from a putatively filamentous capsulated (fi!+ S-+) variant
demonstrated conclusively the genetic structureof the latter. Similarly, isolation of non-filamen-
tous non-capsulated (f/— S—) and of filamentous capsulated (fl+ S++) variants following
the growth of a filamentous non-capsulated (fl-+ S—) strain in the presence of T.P. from a
putatively non-filamentous capsulated (fi/— S+) strain established the genetic constitution
of the latter.

In experiments concerned with the possible exchange of desoxyribose nucleic acids con-

‘ trolling the production of type-specific capsular polysaccharide, a modified technique of
transformation (9) was employed. The capsulated strain to be tested was inoculated in the
manner described above into tubes containing T.P. from a pneumococcus of heterologous
capsular type and into tubes without T.P. which served as controls. As the medium contained
agglutinins against non-capsulated pneumococcal] variants but lacked agglutinins directed
against type-specific capsular antigens, an environment inimical to the selection of non-
capsulated variants was maintained. After incubation of the cultures for 24 hours at 37°C.,
10 gamma of crystalline desoxyribonuclease (Worthington Biochemical Sales Co., lot D333)
in 0.2 cc. of charcoal-absorbed broth were introduced into each tube and the cultures were re-
incubated for 1 hour. Appropriate controls showed this time to be adequate for destruction
of the biological activity of the T.P. Thereafter, 1.6 cc. of each culture was transferred to a
tube containing 4 cc. of neopeptone broth to which 10 per cent anticapsular rabbit serum
directed against the inoculated strain had been added. The cultures were incubated overnight
and the several morphologic variants were recovered and identified by the same cultural,
serologic, and biologic procedures described above.

3. Strains of Pneumococcus.—The strains employed were those described in the preceding
paper. One additional strain of pneumococcus capsular type I, strain IF (f— SI), and its
morphologic variants were also used. This strain, isolated in Denmark in 1943, and designated
I 519/43 41° X 300 by Dr. Erna Mgrch-Lund (10), was kindly sent by her from the State
Serum Institute, Copenhagen.

EXPERIMENTAL

In Table I are shown the pneumococcal morphologic variants which might
be expected to result from the interaction of the several variants with the
transforming principles of heterologous forms. As indicated in the experimen-
tal data which follow, it has been possible to isolate eighteen of the twenty
predicted recombinants. Because combination of the filamentous form with
various capsular types led to the more ready identification of the several
morphologic variants on blood agar plates, several strains of pneumococcus
were used to facilitate the experiments.

Transformation of the Filamentous Non-Capsulated (fl-+ S—) Variant.—
When strain JID39S (fl+ S—) was grown in the presence of the T.P. of
strain IID398 (sil— S—), non-filamentous non-capsulated (fJ— S—) variants

were recovered. This transformation was described originally by Taylor (6).
ROBERT AUSTRIAN 37

Growth of strain IF (f//4+- S—) in the presence of the T.P. of strain ID
(il+ ST) resulted in the isolation of the filamentous capsulated strain IF
(fl+- Slir).

Transformation of the filamentous non-capsulated strain IID39S (fi+- S—)
with the T.P. of the non-filamentous capsulated pneumococcus VIIEB (fl—
SVIII) yielded three new morphologic variants: a filamentous capsulated
strain (f/+ SVI Isr), a non-filamentous non-capsulated strain (fil—ir S—),
and a non-filamentous capsulated strain (fil—tr SVIIIir). The presence of
the two genetic factors controlling non-filamentous growth and production of
type VIII capsular polysaccharide in the doubly transformed, non-filamentous

TABLE I

Morphologic Variants of Pneumococcus Arising from the Interaction of Cells of One Morphologic
Variant with the Transforming Principles of an Heterologous Variant

 

 

 

 

 

Strain
T.P. 1
flt S- fl— S— flt+ SY | fil- SY
flt S— — flttr S— — ftir S¥
fl- S- fil—ir S— _ fil—ir SY | _—
|
filt SX ji+ SXir fil— SXtr fil+ SXtr | fil— SXtr
fl+tr S— | fltir SY
(fl--ar SXtr) | (fl--tr SXtr)
| |
fil— SX fil+ SXtr fil— SXtr | filt+ SXtr fil— SXtr
fl-ir S— : fil-tr SY
jil-—tr SXtr | fl— SX |

 

 

jul-+, filamentous; f/—, non-filamentous; SX, SY, capsulated organisms of different cap-
sular type; S—, non-capsulated.
Symbols in parentheses indicate predicted forms not isolated.

capsulated (fii— S-+) variant was demonstrated through the effect of T.P.
prepared from it upon a filamentous non-capsulated strain (fi/-+ S—), result-
ing in the recovery of non-filamentous non-capsulated (fJ— S—) and fila-
mentous capsulated (ji/+- S+-) forms.

Transformation of the Non-Filamentous Non-Capsulated (fil— S—) Variant.
—Growth of the non-filamentous non-capsulated variant, I1D39S (fi— S—),
in a transforming system containing the T.P. of strain IID39S (fl+ S—)
yielded filamentous non-capsulated (fil+- S—) forms as demonstrated first by
Taylor (5).

Transformation of strain IID39S (fl— S—) in the presence of the T.P. of
the filamentous capsulated strain ID (ji/-+- SI), resulted in the recovery of
two new morphologic variants: a filamentous non-capsulated strain (fil+ér
S—) and a non-filamentous capsulated strain (fl— Slr). The doubly trans-
formed strain, (fil+-ir SIér), was not recovered in this experiment. This fail-
38 MORPHOLOGIC VARIATION IN PNEUMOCOCCUS. II

ure may have resulted from the lack of selective factors favoring the growth
of filamentous forms. Unlike non-filamentous variants which grow diffusely
in liquid media, filamentous forms manifest varying degrees of autoaggluti-
nability and are therefore not favored selectively as are the former in cultures
containing both types of cells. As the probability of occurrence of a double
transformation is in all likelihood the product of the individual probabilities
of the two distinct transformations involved in the reaction, a selective factor
may be necessary to facilitate its detection. The absence of such a selective
factor from the experimental system may account for the failure to recognize
doubly transformed cells.

Interaction of the non-filamentous non-capsulated strain, ITD39S (f7—
S—), with the T.P. of the non-filamentous capsulated strain, IIIA66 (fil—
STIT), resulted in the recovery of non-filamentous capsulated type III pneu-
" mococci. This reaction is the classical example of pneumococcal capsular type
transformation, discovered originally by Griffith (4) and elucidated by Avery,
MacLeod, and McCarty (11).

Transformation of the Filamentous Capsulated (fil+- S+-) Veriant.—In these
experiments, the filamentous capsulated strain, IID39S (f/-+ SVIIUr), was
used because of its morphologic properties detectable in the gross. When
grown in the presence of T.P. from the strain IF (f7— S—), transformation
of strain IID39S Gil+ SVIIUr) to the non-filamentous capsulated variant
(fil—tr SVIIIir) occurred. Although growth in liquid media favors selection of
non-filamentous mutants arising spontaneously from filamentous forms, de-
tection of such mutants requires usually several transfers in liquid media. In
the controls employed in this experiment, no non-filamentous forms were
observed. Two other aspects of this experiment merit comment. First, trans-
formation took place in the absence of antibody capable of agglutinating the
strain undergoing transformation and second a character of a capsulated
pneumococcus appeared capable of transformation without evident loss of
capsulation.

The possible transformation of pneumococcal capsular type through the
mediation of desoxyribose nucleic acid exchange reactions has been discussed
in a previous preliminary report (9). Although definitive proof of exchange
reactions involving this pneumococcal character is lacking, study of reactions
between capsulated strains of pneumococcus is not without interest. Experi-
ment has shown that when the filamentous capsulated strain IID39S (fl-+
SVIII¢r) is transformed in the presence of the T.P. of a filamentous capsu-
lated strain of heterologous type, e.g. strain ID (i/-+ SI), according to the
modified technique described previously, filamentous capsulated type I cells
may be recovered from the system. Whether or not this reaction is mediated
through the development of filamentous non-capsulated forms which acquire
the new capsular type cannot be ascertained with certainty.

Employing the modified technique referred to above, transformation of the
ROBERT AUSTRIAN 39

filamentous capsulated variant, IID39S (fl+ SVIIIir), with the T.P. of the
non-flamentous capsulated strain, IITA66 (f/— SIID, of heterologous cap-
sular type, has resulted in the recovery of three new pneumococcal strains:
a non-filamentous capsulated type VIII strain (il—¢ SVIIIir), a filamentous
capsulated type HII strain (fi/+ SIII#r), both of which represent single trans-
formations and a doubly transformed strain (fJ—fr SIIIir). In this experi-
ment, recovery of the doubly transformed variant is favored selectively by its
properties of diffuse growth and by the absence of agglutinating antibody.

Transformation of the Non-Filamenious Capsulated (fl— S+-) Variant.—
In this series of experiments, the non-filamentous capsulated variants, ITD39S
(fl— SIT) and IID39S (f/— SVIIsr), were used. Growth of the latter strain
in the presence of T.P. from the filamentous non-capsulated strain, IID39S
(fl4+- S—), resulted in the isolation of a filamentous capsulated pneumococcus
(fil+ S++). The reaction was carried out in an environment lacking agelu-
tinins for the diffusely growing strain undergoing transformation demon-
strating again that transformation of diffusely growing pneumococci may be
recognized if selective factors favor detection of the transformed cells. In this
instance, transformation of a character of a capsulated pneumococcus oc-
curred under conditions unfavorable to the selection of spontaneous muta-
tions to the filamentous variant recovered from the transforming system. The
result supports the view that capsulated pneumococci may participate in
transformation reactions without loss of capsulation.

With the use of the modified technique of transformation, the non-filamen-
tous capsulated strain, IID39S (fiJ—- STI), was transformed with T.P. from
the filamentous capsulated strain, D398 (fil+ STIIir). Although the doubly
transformed variant was not recovered, a filamentous capsulated type II
strain, (fil-++ir STI) and a non-filamentous capsulated type III strain (fl—
SIITér) were isolated. Lack of factors favoring selection of the doubly trans-
formed variant, (fi/-+¢r SIII#r) militated probably against its detection.

The possible exchange of capsular type has been demonstrated also through
the interaction of the cells of a non-filamentous capsulated variant with the
T.P. of a second non-filamentous capsulated variant of heterologous capsular
type in the modified transformation reaction. Strain IID39S (fl— STLI¢r)
was recovered following growth of strain IID39S (fJ— SVIIIir) in the pres-
ence of T.P. from strain IITIA66 (fil— STII). This reaction has been described
previously in detail (9).

DISCUSSION
Studies with selective cultural techniques have indicated previously that
morphologic variation in pneumococcus is determined chiefly by the mode of
cellular separation after division and by the ability or inability of the cell to
produce capsular polysaccharide. In addition, it has been shown that the
genetic factors governing these two characters of pneumococcus are almost
40 MORPHOLOGIC VARIATION IN PNEUMOCOCCUS. II

certainly desoxyribose nucleic acids which are inherited independently of one
another. If these observations are correct, it should be possible to predict the
results of transformation reactions involving competent pneumococci of one
morphologic variant and the transforming principles obtained from cells of
a different morphologic variant in a fashion analogous to the prediction of re-
combinations of independent characters in sexual forms. Experimental study
of the predicted reactions has resulted in the isolation of all but two of the
morphologic variants expected to result from such interactions. The unde-
tected variants were those least likely to be found on theoretical grounds,
representing double transformations arising in the absence of any selective
factor to favor their discovery following interaction of non-filamentous non-
capsulated (f/-- S—) and non-filamentous capsulated (fJ— SX) strains with
the transforming principles of a filamentous capsulated (fil+- SY) strain of
heterologous capsular type.

These data reveal also that transformation of diffusely growing pneumo-
cocci may take place in the absence of agglutinating antibody and that such
transformations may include alteration of mode of cellular separation after
division and acquisition of new capsular type as well as the acquisition of
resistance to penicillin by diffusely growing cells demonstrated previously by
Hotchkiss (12). The results show also that capsulated pneumococci may par-
ticipate in transformation reactions involving characters other than the cap-
sule of the pneumococcal cell.

SUMMARY

The interaction of each of the four morphologic variants of pneumococcus
with the transforming principles of the heterologous morphologic variants of
the same species has been studied and has resulted in the isolation of eighteen
of the twenty variants predicted to arise through such interactions. The
analogy of the findings to the recombination of independently heritable char-
acters in sexually reproducing forms has been noted.

BIBLIOGRAPHY

. Austrian, R., J. Exp. Med., 1953, 98, 21.

. Griffith, F., Great Britain Rep. Pub. Health and Med. Subj., Ministry of Health,
No. 18, 1923, 1.

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

. Griffith, F., J. Hyg., 1928, 27, 113.

. Taylor, H. E., Compt. rend. Acad. sc., 1949, 228, 1258.

. Taylor, H. E., J. Exp. Med., 1949, 89, 399.

. Austrian, R., and MacLeod, C. M., J. Exp. Med., 1949, 89, 451.

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

. Austrian, R., Bull. Johns Hopkins Hosp., 1952, 90, 170.

10. Mgrch-Lund, E., Acta path. et micrebiol. scand., 1949, 26, 709.

11. Avery, O. T., MacLeod, C. M., and McCarty, M., J. Exp. Med., 1944, 79, 137.

12. Hotchkiss, R. D., Cold Spring Harbor Symp. Quant. Biol., 1951, 16, 457.

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