APPEARANCE OF STREPTOMYCIN RESISTANCE FOLLOWING THE
UPTAKE OF TRANSFORMING DEOXYRIBONUCLEIC
ACID IN PNEUMOCOCCUS

By Dr. HARRIETT EPHRUSSI-TAYLOR*

Laboratoire de Genetique Physiologique du C.N.R.S., Gif-sur-Yvette, Seine-et-Oise

N bacterial transformation, the experimenter is

able to examine the physiological action of normal
hereditary determinants, introduced into cells in the
form of DNA, by examining the events leading to
the manifestation of the newly acquired hereditary
trait. Thus far, such studies are limited in number!?)?.
In ono, the appearance of resistance to streptomycin
was followed in populations of pneumococci which
had been made to fix DNA from a streptomycin-
resistant donor strain. The conclusion reached was
that the resistant phonotype appears as a discrete
change from sensitive to resistant: no stages of
partial resistance could be recognized. It was found,
furthermore, that the probability of a cell becoming
resistant was normally distributed over a_ time-
interval ranging from about 15 min to 90 min follow-
ing penetration of the streptomycin-resistance gene,
even though DNA fixation had been limited to a 5-
min period. The most probable moment for a cell
to become resistant was about 60 min following
uptake of DNA. The interpretation of these results
at the time of their publication was rendered difficult
owing to the absence of information concerning the
mechanism of streptomycin-resistance and the types
of syntheses involved in its establishment. Later,
a brief description was made of experiments showing
that the discrete event, described here, is not in
fact the development of typical streptomycin-
resistance, but, after all, only an intermediate stage
in its development’. Experiments documenting this
contention are presented here, in conjunction with a
hypothesis concerning the mode of action of the
streptomycin-resistance gene. The hypothesis is a
development of the recently published theory of Spotts
and Stanier* concerning the mode of action of strepto-
mycin and the nature of streptomycin-resistance.
Since it may open some interesting new approaches
to the study of gene action, and, in particular, to
the question of the relationship between genes
and ribosomes, the publication of these experi-

* Present address: Developmental Biology Center, Western Reserve
University. Cleveland, Ohio.

ments, and the accompanying hypothesis on the
mode of action of the streptomycin-gene, seems
worth while.

(1) The experimenial demonstration of the appearance
of resistance. All investigations of the appearance of
antibiotic resistance following uptake of transforming
DNA use a single basic procedure. Following a
period of DNA fixation which is sharply limited by the
destruction of unabsorbed DNA with DNase, the cells
are diluted into fresh medium and incubated at
37° C. At intervals, samples are withdrawn and
plated in agar containing the antibiotic. The number
of cells able to give rise to a colony in the presence of
the antibiotic are thus scored. Fig. 1, curve a, shows
how pneumococci transforming for streptomycin-
resistance develop this ability. This curve is typical
of those obtained by Fox! and Schacffor’ as well as by
me. The number of cells able to give rise to colonies
in streptomycin-agar rises rapidly from’ about the
15th min following DNA fixation. A shoulder is
observed at about 80-90 min, following which the
numbers increase again, but at a slower, exponential
rate characteristic of the overall population increase
of the growing culture. Various experiments’ have
shown that: (1) at 90 min, virtually every cell which
fixed a transforming molecule is able to form &
colony in the presence of streptomycin; (2) that the
increase observed after 90 min is due to the formation
of genetically transformed daughter cells. In fact,
it is established that thé transmission of an acquired
gone to both daughter cells may begin as early as the
second generation after DNA fixation’, that is, at
about 45 min. It may, however, begin only at 4
third or fourth generation in some cells’. The early
transmission of an acquired gene is, however, not
reflected immediately in the numbers of strepte-
mycin-resistant colony-forming units observed, owing
to the tendency of sister cells to remain attached
after cell division. Differences in the degree to which @
shoulder is observed at 80-90 min are almost certainly
due to differences in the extent of chain formation 10
different media.
November 24, 1962

No. 4856

 

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No, of streptomy cin-resistiunt colonies
-
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60 120 180
Min after contact with DNA

Fig. 1. Evolution of the numbers of transformed cells able to form

colonies in streptomycin agar. A, Transformation culture growing

in absence of streptomycin ; B, streptomycin has been added 90

min after DNA uptake; C. streptomycin has been added 135 min
after DNA uptake

 

If, in the agar medium used to score the resistant
cells, the concentration of streptomycin is insufficient
1o arrest rapid growth of the recipient strain,
residual metabolism in the presence of streptomycin
will cause some transformants to complete the change
from sensitive to resistant on the agar plate’. Thus,
the transformed phenotype will seem to appear earlier
at concentrations of streptomycin below a critical
level, and the precocity of the appearance of the
phenotype will appear to be a function of streptomycin
concentration. ‘Therefore, in order to avoid under-
estimation of the time required for resistance to de-
velop it is necessary to challenge the cells in agar
containing streptomycin at a concentration yielding
a maximally selective effect. In the experiments
reported here, it was found that the rate of appearance
of streptomycin-resistant cells is the same at all
concentrations of streptomycin equal to or greater
than 200 pg/ml. The majority of experiments were
performed at 200 g/ml., but in some the concentra-
tions were higher. ;

(2) Limitations of the procedure for demonstrating
resistance. ‘The foregoing procedure for determining
the appearance of resistance, adequate at first sight,
in fact, leaves one parameter unexplored. The
method reveals only when a transforming cell can
form a colony at a maximally selective concentration
of streptomycin. It does not tell us whether the
transformant does so at once, or whether its growth
and division is temporarily suspended by the chal-

lenge. The streptomycin-resistant donor strain is
completely indifferent to streptomycin at the con-
contrations used: Is the nowly resistant cell,

which yields a colony, also really indifferent to
streptomycin ? ;

(3) Two steps in development of resistance. To test
this question, instead of challenging the transformants
in streptomycin agar, a small amount of strepto-
mycin was added to the cells in liquid medium, at
a time when resistance is generally presumed to be
complete. Following the addition of streptomycin,

NATURE

749

platings in agar were performed in order to determine
the evolution of the numbers of resistant cells. . Fig. 1
shows the results of one such experiment, in which
cells which had fixed DNA for 2 min were diluted
100-fold into fresh medium and the culture divided
into three portions: (a) no streptomycin is present
in the liquid culture and platings are made directly
into streptomycin agar; (b) 50 ug/ml. of streptomycin
were added after 90 min of growth, and platings
made into streptomycin agar (at 200 ug/ml.);. and.
(c) 50 ug/ml, of streptomycin were added after 135 min
of growth, and platings are made in streptomycin
agar. If, as is generally believed, all transformants
have achieved the synthesis of the streptomycin-
resistant phenotype by 90 min, there should be no
difference in the numbers of streptomycin-resistant
cells present in these three liquid cultures. This is,
however, clearly not the case. The increase in the
numbers of resistant cells present in the cultures
receiving a small amount of streptomycin is almost
immediately arrested by the antibiotic. Thus, the
immediate replication of the newly formed resistant
cells is blocked by as little as 50 ug/ml. of streptomy-
cin. Yet these cells are able to form colonies in agar
containing 200 ug/ml. or more. Other experiments
showed that, in fact, streptomycin transformants
become completely indifferent to streptomycin only
after some 150-180 min have elapsed following DNA
fixation.

Two explanations of these observations can be
offered: (1) that resistance develops in two steps.
First, the bacteria are altered so that streptomycin is
no longer bacteriocidal, and seeondly, they become
completely indifferent to streptomycin. If this explana-
tion is to be retained, it must be assumed also that
cells can pass from the first state to the second in the
presence of streptomycin. (2) That the cells which
survive the streptomycin challenge are not genetically
transformed. For example, the acquired factor can
be supposed to be not yet a part of the linear array of
genes of the bacterial chromosome, but transmitted
via an extra-chromosomal mechanism. Streptomycin
could then be supposed to block the extra-chromo-
somal mechanism so that the majority of the
daughter cells produced in its presence would bo
streptomycin-sensitive and therefore die. This would
be analogous to the situation found in the induction
of ‘petites’ by acriflavine acting on yeast®. The
eventual formation of a colony in streptomycin-agar
would reflect a shift from the extra-chromosomal stato
to a chromosomal state, achioved through recombina-
tion at one of the numerous cell divisions which the
mother cell could make.

Results of a number of types of experiments invalid-
ate the second hypothesis. One critical argument
against it is the fact that when a cell acquires a
DNA particle, recombination does ensue very shortly
thereafter?*. Further, it is reported that when a
particle of transforming DNA is genetically marked at
several points, so that it is able to give rise to several
types of different, recognizable recombinants, one
observes that a unique recombinant type is formed
from a single absorbed particle, most if not all of the
time!®, Were the acquired particle transmitted at the
outset by an extra-chromosomal mechanism prior
to recombination, this result could not be observed.
The first hypothesis is, therefore, to be retained in
considering why streptomycin arrests the multiplica-
tion of resistant cells newly formed by transformation.

Accordingly, in order to explain the results exempli-
fied by Fig. 1, we can assume that even though from
750

90 min on, every cell which acquired the streptomy-
cin-resistance gene can form a colony at high con-
centrations of streptomycin they do so only after a
considerable period of arrested growth. In other
words, at this stage of phenotypic transformation,
streptomycin is a bacteriostatic substance from the
effects of which the transforming cell can eventually
escape. This characterizes what we shall call stage 1
in the development of resistance, while complete
indifference characterizes stage 2, the definitive state.

Another type of experiment confirms this point
of view, and, in addition, informs us of further
characteristics of stage 1. Following a challenge of
500 ug/ml. of streptomycin for a 30-min period at
37° ©, surviving transformed cells are washed on a
membrane filter to eliminate unbound streptomycin.
transferred to fresh medium by washing them off
the membrane, and their growth followed by
plating samples at intervals, in two different media:
agar with and without streptomycin. In the experi-
ment shown in Fig 2, the resistant transformants
were selected 60, 110 and 180 min after DNA fixation
as described.

One may note the following features of the curves
in Fig. 2. (1) There is a small lag in the onset of
replication of the resistant cells selected 180 min after
DNA fixation which is not observed when strepto-
mycin is simply added at this time to a transforming
culture and left there. The lag observed in Fig. 2 is
almost certainly caused by the vigorous aeration of
the cells during washing on the membrane filter
(Pneumococet are microaerophilic). (2) Growth of
streptomycin-resistant cells selected 60 and 110 min
after DNA fixation is severely retarded, even though

NATURE

 

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500 -~
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7} & e
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75° i60' 210°
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1 2 3 4 5 6
Hours of incubation
Fig 2. Onset of division of streptomycin-resistant transformants

in the absence of extracellular streptomycin. The resistant

transformants were selected: A, 60 min; B, 110 min; C, 180 min

after DNA uptake by treating the transforming population with

500 wg/ml. of streptomycin for 30 min. Survivors were collected

on a ‘Millipore’ membrane, washed, resuspended in medium, and

their growth followed: x, on plates containing no streptomycin
and: @ on 200 ug/ml. of streptomycin

November 24, 1962 voi. 196
the antibiotic has been removed. This means that
either the cells have fixed enough streptomycin so
that the extracellular concentration of the antibiotic
is no longer critical, or that the 30-min treatment has
inflicted a finite damage which is not subject to
reversal by the removal of streptomycin. The latter
seems more likely since the amount of streptomycin
bound to bacteria is very small"). (3) No sensitive cells
survive the selection at 500 ug/ml., and few or no
sensitive progeny are formed by stage 1 resistant
cells.

By extrapolating the exponential slopes of the
curves of Fig. 2, one can calculate from curve c¢ the
delay caused by aeration, and from curves a and b the
delay caused by the combined factors of aeration and
streptomycin-inflicted damage. Correcting for the
delay caused by aeration, one finds that the cells in a
required 135 min of incubation to resume exponential
increase, while tho cells in 6 required 85 min. The
difference between these two times is 50 min, which
is the same as the difference in the incubation times
of the two cultures prior to the streptomycin chal-
lenge. In other words, the time required for definitive
resistance to develop is constant, and independent
of the moment of application of the streptomycin
challenge. Thus, cells which are at stage 1 in the
development of resistance, and which may have
arrived at this stage at very different moments, are a
homogeneous poptlation in so far as their attainment
of definitive resistance is concerned. With respect to
definitive resistance, primary” transformants are
apparently no different from their second, third or
even fourth generation daughters.

In the experiment of Fig. 1, 50 ug/ml. of strepto-
mycin was added to the liquid culture, while in the
experiment of Fig. 2, 500 ug/ml. were added. Both
concentrations arrested the multiplication of the
resistant transformants. Irrespective of whether the
damage to the cells was inflicted by 50 or 500 ug/ml.
and of whether the streptomycin was left in contact
with the survivors, the moment of onset of inerease
of the streptomycin-resistant cells was at about 180
min. This again suggests that streptomycin inflicts
finite damage on stage 1 transformants, and that their
recovery is independent of the external concentration
of streptomycin. To show this more clearly, an
experiment was performed in which fluctuations in
the numbers of streptomycin-resistant colony-forming
units was followed in a control and two streptomycin-
containing cultures. The latter received 50 and 500
ug/ml. of streptomycin, respectively. 60 min after
DNA fixation. Fig. 3 shows the results of such an
experiment. It can be seen that the time required for
stage 1 resistant transformants to resume division
after the addition of streptomycin is approximately
the same, irrespective of the external streptomycin
concentration. Hence, the conversion of a stage |
resistant transformant into a definitely resistant cell
is essentially independent of streptomycin concentra-
tion. Further, the damage inflicted on the stage |
cells must be finite and independent of streptomycin
concentration, within the limits explored.

The most striking feature of the way in which
definitive resistance develops in a transforming
population is that cells destined to transform, and
their immediate progeny. show this resistance at the
same time, that is. about 180 min after fixation of
transforming DNA. Yet some of these cells are the
original transformants and some are their first,
November 24, 1962

 

 

 

 

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rig. 3. Evolution of the numbers of streptomycin-resistant cells

ina transforming culture x in the absence of streptomycin; in the

prsenee of @ 50 zg/ml.;O, 500 ug/ml. of streptomycin added 60

nin after DNA uptake. Some of the trregularities in the curves
are probably due to synchrony of division.

sseoud or later generation progeny. Therefore, as
transforming cells grow and divide, they must produce
daughters which are similar to themselves not only
venotypieally but also with respect to the degree to
which they have developed definitive phenotypic
resistance. Since genetic integration usually occurs
at one of the first two or three divisions following DNA
linution, it is difficult to imagine that this phenotypic
uniformity of transformants and their progeny is
established by the process of genetic integration itself.
On the other hand, DNA fixation has occurred during
a very short interval. Its penetration into the cell
vould very well be the event which initiates the
process of phenotypic transformation.

if this were the case, the following mechanism of
the development of definitive resistance can be
advanced. On peonctration of the DNA, the strepto-
inyein + gene immediately induces the formation of
the system which confers resistance. Since the com-
picte phenotype is manifested only some 180 min after
DNA fixation, we can suppose that resistance results
from the synthesis of a very large number of specific
macromolecules. As cell division proceeds, both the
generating system initiated by the acquired gene and
the specific macromolecules which it determines are
‘listributed more or less equally between sister cells.
In those cells where the resistance gene is fixed
permanently by genetic recombination, the resistance
gene will also be transmitted. It is only in these cells
that the generating system will be stable enough for
final resistance to be manifested. What. then, is
Stage 1 resistance? As shown by Fox, it is a discrete
change which occurs on an average after about 60
min have elapsed following DNA-uptake, and which
shows a normal but fairly wide distribution with
Fespoct to the moment it occurs!. As shown by the

NATURE

751

foregoing experiments, it is a change which enables
a cell to survive a challenge of maximally selective
amounts of streptomycin, and to escape from a strong
bacteriostatic effect of the antibiotic. Further, the
rate at which a stage | resistant escapes is independ-
ent of the external streptomycin concentration in the
growth medium.

A suitable explanation of stage 1 resistance was not
evident so long as theories of the nature of strepto-
mycin resistance were based on supposing the resist-
ant cell impermeable to streptomycin. Even with
the publication of a theory! to the effect that, in the
presence of streptomycin, sensitive bacteria synthesize
an abnormal membrane constituent which results in
disruption of transport mechanisms, an explanation
of stagel resistance did not seem possible. Supposing
that, at the onset, the acquired resistance gene were
to confer on the cell the capacity to form normal
membrane substance in the presence of streptomycin.
at early stages the cell membrane could be at best a
mosaic, for the old membrane and membrane-forming
system should still be present in the cell. It is
hard to see how a mosaic membrane could confer on
cells an immunity to the lethal effects of streptomycin.

The recent hypothesis of Spotts and Stanier*
provides, on the other hand, an explanation of the
nature of stage 1 resistance. According to these
authors, streptomycin attacks the ribosomes of
sensitive cells, causing their disruption. Resistant
cells, according to the theory, contain ribosomes
which do not combine with streptomycin, and are.
therefore, resistant to its action. There is, indeed.
some direct evidence in favour of this view!*. In the
light of this hypothesis, stage 1 resistance can be inter-
preted as resulting from the synthesis of adequate
numbers of streptomycin-resistant ribosomes so that
at least one copy of each of the different messenger
RNA’s of the cell which are necessary for the continua-
tion of vital specific functions could be housed in
streptomycin-resistant ribosomes. Bacteriostasis
would ensue. however, at this stage owing to the
destruction of residual streptomycin-sensitive ribo-
somes, which could still represent the majority of
the ribosomes of the cell. Stage 1 resistant cells
would recover their ability to divide as soon as the
streptomycin-resistant ribosome population were built
up to a level compatible with normal growth and
division. Recovery-rate would be independent of
the amount of streptomycin in the system. for
recovery would result from the function of surviving
streptomycin-resistant ribosomes.

The ribosome hypothesis is particularly satisfying
beeause it explams why stage 1 resistance appears
after an interval which is normally distributed over
a fairly wide time-range.

There are presumably many different messenger
RNA’s determining vital functions which must be
housed, and the probability that any one cell possesses
one streptomycin-resistant ribosome-messenger RNA
particle of each type would be expected to be dis-
tributed in this way, assuming random association
of the RNA with ribosomes. Further, the ribosome
hypothesis of streptomycin action can explain why all
transforming cells and their progeny show definitive
resistance at about the same time: the existing ribo-
somes would be shared at each division.

However, it should be mentioned that there is one
fact concerning the action of streptomycin which
the Spotts and Stanier theory does not explain. This
is the observation that if chloramphenicol and
streptomycin are added simultaneously to sensitive
752

cells, streptomycin has no lethal action. It appears,
thus, that the lethal effects of streptomycin are
the consequence of protein synthesis. It has
been proposed that streptomycin does not enter
cells unless a special permease is synthesized,
following contact of cells with streptomycin, and this
could account for the protective effect of chloramphen-
icol. Since so many of the biological actions of
streptomycin can be explained by the theory of
Spotts and Stanier, including the very particular
way in which resistance develops following trans-
formation, one is inclined to conclude that only a
minor modification of it may be necessary in order to
explain why chloramphenicol eliminates the bacterio-
cidal effect of streptomycin.

This work was supported by a grant from the
Rockefeller Foundation to the C.N.R.S.

NATURE

VoL. 198

November 24, 1962

Fox, M.S8., J. Gen. Phys., 42, 737 (1958).
2 Task Ss 8. F., and Hotchkiss, R. D., Biochim. Biophys. Acta, 45, 155
3 Epheass Payor, H., in Microbial Genetics, 132 (Camb. Univ. Pregg,

‘Spotts, C, R., and Stanier, R., Nature, 192, 633 (1961).

* Schaeffer, P., Ann. Inat. Pasteur, P1, 323 (1956).

* Hotchkiss, R. D., in Chemical Basis of Heredity, 321 (Johns Hopking
Univ. Press, 1956),

7 Ephrussi-Taylor, H., In Recent Progress in Microbielogy, 51,
{Almaqvist and Wiksells, Stockholm, 1958).

*Ephrussi, B., and Hottinguer, H., Cold Spring Harbor Symp. on
Quant. Biol., 16. 75 (1951).

* Ephrussi-Taylor, H., J. de Chimie- Physique, 68, 1090 (1961). Fox,

, Nature, 187, 1006 (1960). Voll, M. J., and Goodgal, 8. H..

Bre "U.S. Nag. Acad. Sci., 47, 503 (1961).

Ravin, A. W., Exp. Cell Res,, 7, 58 (1954).

‘| Hancock, R., Biochem. J., 78, 7P (1961),

as Anand ltya, Davis, B. D., and Armitage, A. K., Nature, 185, 29

13 Speyer, J. F.. Lengyel, P., and Basilio, C., Proc. U.S. Nat, Acad. Sci.,
48, 684 (1962).
\* Hurwitz, C., and Rosato, C., Biochim. Biophys, Acta, 41, 162 (1960),