Metabolism of Resistant Mutants of Streptococcus faecalis
II. Incorporation of Exogenous Purines*t
M. EARI.BALIS,VALIAHYLIN, M. KATHARINE
COULTAS
ANDDORRISJ. HUTCHISON
(Laboratories of the Sloan-Kettering Institute, New York, N.Y.)
Several agents which inhibit neoplastic growth
in man and in experimental animals appear to
exert their effects at least partially by interference
with nucleic acid purine metabolism. The clinical
usefulness of such compounds is severely limited
by the fact that, after a period of treatment, re
sistance develops to the agent employed. The
availability of mutant strains of Streptococcus
faecalis1 resistant to the action of certain agents
(13) has presented an avenue of approach to the
mechanism of the development of such resistance
and to the problem of the action of these agents.
This work represents an attempt to compare
the information derived from tracer studies with
that which is known about the growth require
ments of these bacteria. The organisms were grown
in the presence of labeled purines, and the conver
sion of these compounds to nucleic acid adenine
and guanine was measured.
MATERIALS
AND METHODS
Reagents.—Theadenine-8-C1* and the guanine-8-C14 were
synthesized, as has been previously described (2, 8). The
xanthine-8-C14 and hypoxanthine-8-C14 were purchased from
the Southern Research Institute. The adenosine-8-Cu and
guanosine-8-C14were prepared by Dr. B. Lowy and synthesized
from adenine-8-C" and guanine-8-C14 (10).
* A preliminary report of this work has been presented (4).
t This investigation was supported by funds from the Na
tional Cancer Institute, National Institutes of Health, Public
Health Service (Grant No. Cy 3190, C-2699), and from the
U.S. Atomic Energy Commission (Contract No. AT(8001)810).
1The strains studied have been designated as follows:
SF/O = parent (ATCC No. 8043).
SF/MP = resistant to 6-mercaptopurine.
SF/MP/A = double mutant, resistant to 6-MP and Amethopterin.
SF/MPcc = resistant to 6-MP, isolated independently of
SF/MP.
SF/DAP = resistant to 2,6-diaminopurine.
SF/A = resistant to A-methopterin.
SF/A/O = partial revert of SF/A.
SF/A/MP = double mutant, resistant to A-methopterin
and 6-MP.
Received for publication September 19, 1957.
Bacteria and media.—Theisolation and maintenance of the
strains of S. faecalis have been described elsewhere (11, 13).
The standardized inocula used in these experiments were
grown and prepared according to the following regimen: Two
successive transfers were made into 5 ml. of liquid medium
identical with that on which the culture is carried. The third
transfer with saline-washed inoculum was then made into 5 ml.
of an unlabeled equivalent of the medium which was to be used
in the labeled experiment. The inoculum used was one which
gave approximately 8 X IO5cells/ml. In each experiment 400
ml. of a folie acid (1 m^g/ml) or thymine (1 ;ug/ml) supple
mented purine and pyrimidine-free medium (F-PP) was further
supplemented with the labeled purine at a concentration of
3.7 X 10-»
M.This medium was sterilized at 121°
C. for 15 min
utes; if the labeled compound was heat-labile it was omitted
from the medium, sterilized by filtration through an ultrafine
sintered glass filter, and added aseptically to the cooled me
dium. Just prior to inoculation, a 12-ml. aliquant was removed
aseptically from each flask to serve as a titration blank. The
flasks were then inoculated so that there were approximately
8 X IO5cells/ml and incubated at 35°C. for 18 hours. At the
termination of the incubation period, 19 ml. was removed from
each flask, and the rest of the cells were harvested by centrifugation. A 5-ml. aliquot was handled aseptically, since it was to
be used as the inoculum for a series of controls to determine the
resistance and purine requirement for the mutants after the
above growth conditions had been carried out. A 2-ml. sample
was used to determine the amount of growth by turbidity
measurement on a Coleman Junior Spectrophotometer, and a
12-ml. sample was used for titer determination.
Controls on A-methopterin and 6-mercaptopurine resist
ance and purine requirements were made on each culture for
each experiment, with the use of the saline-washed suspension,
prepared from the 5-ml. aliquot removed from the experimen
tal flasks, as seed. The basal medium for the resistance controls
was the one supplemented with PGA at 1 mMg/ml, while the
response to adenine, guanine, hypoxanthine, and xanthine was
determined in this medium and also in one supplemented with
thymine at 1 fig/ml rather than with the PGA.
The controls were set up in 13 X 100-mm. tubes which con
tained 2 ml. of medium; these tubes were sterilized at 121°
C.
for 8 minutes and, after being cooled, were inoculated with a
saline-washed suspension diluted so that each inoculated tube
contained 8 X IO6 cells/ml. Turbidity measurements were
made after incubation for 18 hours at 35°C.
Isolation of nucleic acid purines.—Theharvested cells were
washed successively with cold trichloroacetic acid, alcohol, and
ether. The washed cells were hydrolyzed at 37°C. for 24 hours
with 1 ml. of l N sodium hydroxide per 180 mg. of bacteria
(16). The degraded pentosenucleic acid (PNA) was separated
from the deoxypentosenucleic acid (DNA) by acidification
with HC1, followed by the addition of 1.5 volumes of ethanol.
220
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1958 American Association for Cancer Research.
BALIS et al.—Metabolism of S. faecalis Mutants.
The DNA was collected by centrifugation. The individual
purines were isolated, as has been previously described (7), by
hydrolysis of the nucleic acids, precipitation of silver purines,
regeneration of the purines as hydrochlorides, and separation
of the individual purines by paper chromatography. The radio
activities were determined as has been previously described
(6). Infinitely thin films on aluminum planchéiswere measured
in a Geiger-Müllerflow counter, with helium-isobutane gas.
All the planchéiscontained sufficient radioactivily lo result in
counts of at leasl twice Ihe background, and Ihe aclivities
were determined to within slandard errors of less lhan 5 per
cent (15) except where noted. Aclivilies are reported as relalive
specific activity (RSA), defined as
ESA
counts/min/^mole isolated compound
X 100.
counls/min/Mmole supplemenl
RESULTS
The capacity of the eight strains1 to use adenine
as a precursor of the nucleic acid adenine and
guanine was determined (Table 1). With the ex-
II
221
cursor and the amount of DNA purines which
were derived from the same source.
The capacity of these eight mutants to utilize
exogenously supplied adenosine was determined
(Table 1), and the results were similar to those
obtained with the free base, with the exception of
those found with SF/MP, which used exogenous
adenosine even more poorly than it did exogenous
adenine for the synthesis of nucleic acid adenine.
The degree of conversion of the adenine moiety to
guanine was about the same when the free base or
when the ribosides were used.
A similar series of experiments was carried out
with guanine and guanosine used as precursors.
The results of these studies are shown in Table 2.
Again it can be seen that all the mutants except
SF/MP could convert the exogenous purine and
the exogenously supplied riboside to both the
TABLE 1
UTILIZATION
OFEXOGENOUS
ADENINEANDADENOSINE
IN FOLICACID-CONTAINING
MEDIA
STHAIN
EXP. 1: ADENINE*
Adenine
Guanine
PNA
DNA
PNA
DNA
Exp.
RSAt
SF/0
SF/MP
SF/MP/A
SF/MPcc
SF/DAP
SF/A
SF/A/O
SF/A/MP
48
1»
SO
SS
45
58
47
47
36
80
37
84
SS
41
80
2: ADENOSINEf
Adenine
PNA
DNA
Guanine
PNA
DNA
RSAt
44
4
51
38
40
65
47
50
50
5
41
SO
9
31
31
56
47
40
41
40
54
43
34
11
26
31
37
31
26
28
29
6
28
29
47
41
86
36
28
4
26
27
35
28
27
28
* Experimenl 1 medium contained 3.7 X 10 6 moles/liter of adenine-8-C14.
t Experiment 2 medium contained 3.7 X 10~6moles/liler of adenosine-8-C14.
__.
_ counls/min/Atmole
iVSA —: -.
isolated
compound
:
:
-
counls/mm/^mole
supplemenl
X
100.
ception of SF/MP, all were able to synthesize a nucleic acid purines. SF-/MP was again inefficient
considerable portion of both of their nucleic acid in the conversion of the purine from the supplied
purines from adenine. This one exception was able compound to adenine, and again it can be seen
to derive its polynucleotide adenine from the exog that the mutant SF/A converted the exogenously
enously supplied adenine to about one-half the supplied purine to nucleic acid purines to a greater
extent that the other mutants were able to do, but extent than did the other strains. In the experi
it was extremely inefficient in converting this exog ments listed in Table 2, it is evident that there was
enous precursor to guanine. This is consistent no significant difference in the incorporation of the
with the fact that all the strains except SF/MP
exogenously supplied purine into the PNA as
were able to grow in a folie acid-free medium with compared with that into the DNA.
adenine as the sole source of purines. Mutant
The capacity of these eight strains to use
SF/A incorporated the exogenous purine to a xanthine as a precursor of the two nucleic acid
greater extent than did the other strains; this is purines was determined, and the results are pre
consistent with the growth studies which would sented in Table 3. Here again, there was no exten
seem to indicate that this mutant is less efficient sive difference in the relative amount of the purine
at synthesis de novo than are the other strains. It that was converted into the PNA as compared
will further be noted that there is no significant with that into the DNA. In contrast to the results
difference between the relative amount of PNA obtained when adenine and guanine were the pre
purines that have been synthesized from this pre- cursors, mutant SF/MP was able to use xanthine,
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222
Vol. 18, February, 1958
Cancer Research
particularly for guanine synthesis, to about the
same degree as did the wild strain or the other
mutants. Xanthine, in contrast to guanine and
adenine, was used to a greater extent for the syn
thesis of nucleic acid guanine than it was for the
synthesis of nucleic acid adenine by all strains
which were studied. The capacity of these micro
organisms to utilize xanthine in a folie acid-free
for several strains. With the exception of the mu
tant which is resistant to 2,6-diaminopurine, about
three-fourths of the adenine was synthesized from
the exogenously supplied adenine, and SF/DAP
made only hah' its adenine from this source. All
the mutants synthesized more of their nucleic acid
guanine from the exogenously supplied adenine
than did the mutant SF/DAP. In Table 3, results
TABLE 2
UTILIZATION
OFEXOGENOUS
GUANINEANDGUANOSINE
IN FOLICACID-CONTAINING
MEDIA
EXP. 3*
STRAIN
Adenine
PNA
DNA
HSAt
ÕÕ4
41
12
2
SO
SF/O
EXP. 4t
Guanine
PNA
DNA
SF/MP
1
30
SF/MP/A
45
39
:u
40
SF/MPcc
35
49
38
40
SF/DAP
38
50
SF/A
66
33
SF/A/O
45
43
SF/A/MP
38
41
* Experiment 3 medium contained 3.7 X
t Experiment 4 medium contained 3.7 X
Adenine
PNA
DNA
Guanine
PNA
DNA
RSAÃŽ
43
38
l
18
28
42
47
41
62
32
40
42
82
52
4(î
37
«7
34
4*
52
38
4(¡
38
40
Vf
10 ' moles/liter of guanine-8-C14.
10~5moles/liter of guanosine-8-C14.
34
40
2
SS
S«
41
39
31
68
38
48
42
43
Ì
See footnote t Table 1.
TABLE 3
UTILIZATION
OFEXOGENOUS
PURINES
EXP. 5*
6 + DAPf
7 + DAPÃŽ
8}
(A-8-C»)AdeninePNARSA34393834fil606685512972807166637867646694503976727254777505658158827874DNARSA487682Guani
(X-8-C»)AdeninePNADNAGuaninePNADNAEXP.
(X-8-C»)AdeninePNA2816302145482935GUIDNA
(H-8-C»)AdeninePNA214235250412932DNA234103923252
STRAIN
PNA27182724343823RSA#4348484045684748nineDNAEXP.
SF/O
SF/MP
SF/MP/A
SF/MPcc
SF/DAP
SF/A
SF/A/O
SF/A/MP
The following abbreviations have been used: X, xanthine; DAP, 2,6-diaminopurine; A, adenine and H, hypoxanthine.
* Experiment 5 medium contained 3.7 X 10~*moles/liter of xanthine-8-C" and 1 m/ig/ml of folie acid.
t Experiment 6 medium contained 3.7 X 10~6 moles/liter each of xanthine-8-C1* and of 2,6-diaminopurine, and 8 X 10~*
moles/liter of thymine.
ÃŽ
Experiment 7 medium contained 3.7 X 10~6 moles/liter each of adenine-8-C" and of 2,6-diaminopurine, and 8 X 10~6
moles/liter of thymine.
§Experiment 8 medium contained 3.7 X 10~5moles/liter of hypoxanthine-8-C" and 1 m/xg/ml of folie acid.
# See footnote J, Table 1.
medium, but in the presence of 2,6-diaminopurine,
was also determined and is shown in Table 3. With
the exception of mutant SF/MPcc, all the strains
studied synthesized a larger fraction of the nucleic
acid purines from xanthine than they did from the
2,6-diaminopurine. Indeed, mutant SF/MP syn
thesized only a very small amount of its adenine
and guanine from the 2,6-diaminopurine. The
utilization of adenine in a folie acid-free medium
containing 2,6-diaminopurine was also determined
are also shown from an experiment in which the
capacity of the organisms to use hypoxanthine as
a purine precursor was determined. It can be seen
that the mutant SF/MP used hypoxanthine ex
tremely poorly, and it synthesized just as little
nucleic acid adenine as it did nucleic acid guanine
from this precursor. Most of the mutants, with the
exception of SF/MP, used hypoxanthine more
readily than did the parent strain, SF/O. In gen
eral, the mutants synthesized a little more nucleic
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BALISet al.—Metabolism of S. faecalis Mutants. II
223
this compound. These observations are consistent
with the postulated mechanism of action for 6-MP
(5) which proposes that 6-MP, in the course of be
coming inhibitory, is converted to a nucleotide de
rivative that antagonizes the interconversion of
DISCUSSION
the natural purine ribotides involved in the syn
Several strains of S. faecalis have been studied, thesis of nucleic acid purines. If we imagine that
strains which are resistant to purine antagonists
the organism cannot use the exogenous adenine,
or to substances implicated in the synthesis of guanine, or hypoxanthine, because it lacks en
purines, and yet with the exception of the one zymes necessary for the conversion of these exog
mutant, SF/MP, there is no definitive change in enous purines to the biologically active nucleothe pattern of purine utilization that would in any tides, and if we further postulate that the enzymes
way explain the resistance of these bacteria to which are responsible for this conversion are also
the ones which convert 6-MP to the nucleotide
these particular inhibitors. The one mutant,
SF/MP, shows that it lacks the ability to convert (the true inhibitor), then it can be seen that the
exogenous adenine or exogenous guanine into the absence of these enzymes would result both in the
corresponding nucleic acid purine, and thus it is inability of the organism to use adenine, hypoxan
logical that this organism should be unable to grow thine, or guanine as a purine source for growth and
in a folie acid-deficient medium with adenine or in the organism's being resistant to 6-MP. It was
further noted that the mutant did not interconguanine as a source of purines for growth. How
ever, it is not immediately apparent how loss of vert exogenous adenine and guanine. If the bac
synthetic ability should result in resistance to an terium possesses very limited ability to convert
exogenous adenine and guanine to the active ribo
antagonist.
The double mutants are particularly enigmatic. tides, then in effect only trace amounts are avail
A mutant which was isolated from SF/MP on the able to the cell. Such a situation was studied with
basis of A-methopterin resistance was able to Escherichia coli, and it was found that small con
convert adenine from the medium into nucleic acid centrations of adenine or guanine resulted in a
guanine and could convert guanine from the me very limited interconversion of the exogenous pre
dium into nucleic acid adenine; this ability is lack cursor, though higher concentrations resulted in
ing in the parent SF/MP. If the inability of the extensive interconversion (14).
mutant SF/MP to utilize exogenously supplied
In view of the fact that neither adenine nor
adenine for this synthesis of nucleic acid guanine guanine is extensively converted to the other by
is fundamentally associated with the mutant's re the mutant SF/MP and the fact that this organ
sistance to the action of 6-MP, it is difficult to ism is able to convert xanthine into both nucleic
understand how the biochemical changes con acid adenine and nucleic acid guanine, this or
comitant with the second mutation could restore ganism offers evidence again of the fact that there
the synthetic ability and yet not restore sensitivity must be more than one pathway leading from
to the action of 6-MP. In essence, the conversion adenine to guanine. It has also been noted that
of the mutant SF/MP into SF/MP/A has resulted this organism, when grown on suboptimal amounts
in the resistance to 6-MP being manifested in a of xanthine in a folie acid-free medium, is stimu
manner more similar to that observed with SF/ lated in its growth by the addition of mixtures of
MPcc, and perhaps this is merely evidence of a adenine and guanine. The bacterium derives a suf
very close relationship between the two routes to ficient quantity of nucleic acid purines from these
resistance—one exhibited by SF/MP and the two (adenine and guanine) together to spare the
other by SF/MPcc. Certainly, the data presented requirement for xanthine. Thus, it appears that
have shown the complexity of the mechanisms adenine and guanine in combination should be
that lead to the development of resistance to anti- able to supply all the nucleic acid purines re
metabolites, and they have shown an interrelation
quired for growth; yet no growth results (13). This
ship between these actions.
is another example of a situation in which a quan
The mutant SF/MP, is, for all practical pur
titative reduction in the ability of an organism to
poses, unable to utilize exogenously supplied hy carry out a necessary biological process has resulted
poxanthine as a precursor of nucleic acid purines. in a qualitative alteration in growth response. If
This mutant can synthesize nucleic acid purines the organism cannot make a quantity of nucleic
de novo. Therefore, it must be able to utilize ino- acid purines above some threshold level, there is
sinic acid, and the mutation cannot involve any not a reduction in growth proportional to the re
metabolic steps subsequent to the formation of duction in this synthetic process, but no growth at
acid adenine than guanine from this precursor.
When hypoxanthine was used as a precursor, there
was definitely less utilization of the precursor for
DNA synthesis than for PNA synthesis.
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224
Cancer Research
all. A similar situation was observed with a purineless mutant of Aerobacter aerogenes (1), which was
able to synthesize nucleic acid purines from 2,6diaminopurine but was able to do so only at a level
of about one-third that of the parent; as a result
of this quantitative reduction in ability to use 2,6diaminopurine, there was no growth whatsoever
on this substrate.
The mutant SF/A, when studied with regard to
its folie acid requirements, was found to have a
lower requirement for this growth factor in the
presence of purines than did the parent strain for
optimal growth (13). However, the growth was
less extensive in the absence of purines, and under
these conditions the organism had a much higher
requirement for folie acid than did the parent.
These findings are made clearer in view of the in
corporation studies. This mutant utilizes the exogenously supplied purine to a greater extent than
do the other strains which were studied. This we
have interpreted to mean that the organism has a
lessened de novosynthetic ability as compared with
its parent, and, hence, in the medium which is free
of purines more folie acid is required to give the
optimal amount of growth than is required with
those organisms which are more capable of carry
ing out the de novo synthesis of purines. Further
more, this organism has apparently compensated
to some extent for this lessened de novo path by a
greater ability to utilize exogenously supplied
purines. This shift in metabolic pattern is remi
niscent of that which was indicated for an A-methopterin-resistant leukemia in which the capacity
to synthesize nucleic acid purines and thymine
from formate appeared to be reduced (3). The
ability of this organism to survive in the presence
of A-methopterin is perhaps most consistent with
the concept that this inhibitor acts, to some extent
at least, by causing unbalanced growth (3, 9).
Therefore, a reduction in the rate of synthesis of
nucleic acid purines via the affected routes would
make the organism less susceptible to the in
hibitor.
The second mutant that is resistant to the ac
tion of 6-MP, SF/MPcc, possesses no definite
metabolic trait, discernible by the methods of in
vestigation used, which would indicate in any way
the mechanism by which this organism has become
resistant to the action of 6-MP. The only differ
ence that this organism manifested was a lessened
utilization of xanthine in the presence of 2,6-diaminopurine. In view of the fact that, under the
conditions of this experiment, there was no folie
acid present, the lessened utilization of xanthine is
equivalent to an increased utilization of 2,6-diaminopurine. Other than that, the organism uti
Vol. 18, February, 1958
lized the purines which were studied in a manner
quite similar to the other strains and to the parent
strain.
Two different mutants have been studied which
arose in response to what appeared to be identical
treatment. These two organisms have different
folie acid requirements (12) and are distinct indi
viduals in response to exogenous purines. They are
one example of the variety of alterations which
can lead to the same end—resistance to an in
hibitor. It is undoubtedly true that many tissues
in response to a variety of agents will develop re
sistance in several independent fashions.
SUMMARY
Several strains of S. faecalis which are resistant
to agents which suppress the growth of some tu
mors have been studied with respect to their
ability to utilize certain purines and purine ribosides for nucleic acid synthesis. In those instances
in which more than one mutant resistant to the
same agents has been obtained, the mutants dif
fered in other respects. This was most strikingly
demonstrated by two strains resistant to 6-mercaptopurine, which differed in their growth re
quirements and in their ability to utilize various
purines for nucleic acid synthesis.
ACKNOWLEDGMENTS
The authors wish to thank Dr. George Bosworth Brown for
his continued interest and very helpful discussions of the prob
lem.
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Metabolism of Resistant Mutants of Streptococcus faecalis: II.
Incorporation of Exogenous Purines
M. Earl Balis, Valia Hylin, M. Katharine Coultas, et al.
Cancer Res 1958;18:220-225.
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