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STUDIES
ON
METABOLISM
CONTROLLING
OF
MECHANISMS
VIRUS-INFECTED
IN
BACTERIA
THE
1
SEYMOUR S. COHEN
The Children's Hospital of Philadelphia (Department of Pediatrics), and the
School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
The work which I wish to present today extends
the observations reported six years ago (Cohen,
1947) at the Cold Spring Harbor Symposium on
Nucleic Acids and Nucleoproteins. Most of my
studies since then represent an effort to test certain hypotheses formulated at that time concerning
the control of the metabolism of E. coli, strain B,
infected by T2 bacteriophage. In an effort to
strengthen my biological equipment for the studies
ahead, I had the good fortune to spend some
months of 1947 and 1948 in the laboratory of M.
Andre Lwoff at the Pasteur Institute, where problems of bacterial variability, involving studies of
enzymatic adaptation and mutation, were being actively pursued. The practical advantages accruing
from the experimental use of genetic and environmental variability as a tool in chemical virology
will be apparent in this paper, and I take great
pleasure in presenting this work at this session
under the chairmanship of Dr. Lwoff. His laboratory has been a major institution of higher learning for workers from all over the world.
While still on the subject of the advantages of
biological equipment for the chemist, I would comment that, with the exception of work on bacterial
viruses and their host cells, the study of the preparation and properties of host cells in other fields of
virology has been sorely neglected. Indeed, it cannot be anticipated that chemical studies on the multiplication of plant and animal viruses can be seriously developed as long as the experimenter is constrained to the use of a biologically heterogeneous
assortment of cells made available by the exigencies of embryological development, rather than by
the requirements of the investigator. This follows
from the fact that the most valuable technique until
now in chemical virology has involved the analysis
of events in a population of infected cells in which
the proportion of uninfected cells is very low.
By means of this technique it was possible to
show that cells infected with T2 continued to
respire and synthesize nucleic acid and protein, but
no longer appeared to synthesize polymeric substances characteristic of structural constituents of
the bacteria (Cohen, 1947). The polymers produced after infection could largely be isolated in
virus under certain favorable conditions. This apparent redirection of the products of synthesis was
particularly dramatic in the case of phosphorus
1 The work described in the paper was conducted u n d ~
a grant from the CommonwealthFund.
utilization and nucleic acid synthesis. In growing
bacteria about 80 per cent of the cell P is nucleic
acid P and of this ribose nucleic acid (RNA) P is
three to five times that of deoxyribose nucleic acid
(DNA) P. After infection the P which would have
normally gone into RNA is now routed into DNA
while the RNA does not increase in amount. 2
It was proposed (Cohen, 1948a) that there was
a common precursor to both the ribose phosphate
of RNA and the deoxyribose phosphate of DNA,
and that an inhibition of the formation of this
ribose phosphate could account for the availability
of more P for deoxyribose phosphate and DNA. A
key portion of the hypothesis stated that the inability to make RNA, an important component of
metabolically active structures, might limit the synthesis of more of these structures. We see no reason at present to change this particular formulation. Therefore, we undertook to determine the
path of formation of ribose phosphate in E. cell,
and to see the effect of virus infection upon
this metabolic system. Although these working
hypotheses will be seen to be incomplete and perhaps somewhat misleading, they permitted a type
of experimentation which eventually tested and
even supported some of these ideas.
PATHS OF PENTOSE AND DEOXYPENTOSE
FORMATION
In the past six years, a schema of carbohydrate
metabolism in E. coli has been elaborated in my
laboratory~ as presented in Figure 1. It was shown
initially by the use of yeast enzymes that pentose
phosphates including ribose-5-phosphate were produced following the oxidative decarboxylation of
2 Hershey (1953b) has recently pointed to the existence
of an acid-soluble, alkali-soluble component, distinguish.
able from the bulk of the RNA, which has a rapid turnover in the infected cells as measured with pa2. Although
he calls this metabolically active fraction RNA he has not
demonstrated the existence of p82 in all or indeed any of
the nucleotides of RNA, a procedure which recent experi.
ments (Davidson and Smellie, 1952) would suggest to be
important. It is especially critical to know if cytosine
ribose nucleotides are synthesizedin virus infected cells. It
is conceivable that this interesting material may prove
to be an intermediate in the synthesis of DNA, a role excluded for the bulk of the RNA (Cohen, 1947; Manson,
1953).
8My collaborators in these studies of carbohydrate
metabolism have included Dr. D. B. McNair Scott, and
Miss Mary Lanning, Mrs. Ruth Raft, and Mrs. Lorraine
Roth.
[221]
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222
SEYMOUR S. COHEN
FIGURE 1.
glucose
ATP
;
glucose-6-phosphate.
"fructose 1, 6-diphosphate
'I[ TPN
6-phospho~luconolactone
ATP
gluconate ~
~ H20
6-phosphogluconate
TPN
ATP
ribulose ~
ribulose.5.phosphate
,triose phosphate
D-arabinose
D-ribose ~
CH3CHO
ATP
ribose-5-phosphate
deoxyribose-5-phosphate
rihose-l-phosphate
deoxyribose.l-phosphate
+tt8P04
[ base
rihose nueleoside
1
+H~P04 ] [ base
deoxyrihose nucleoside
1
RNA
DNA
F~cuRr- l. Paths of ribose and deoxyribose phosphateformation in E. coli.
6.phosphogluconate (Cohen and Scott, 1950). The
existence of ribose among the reaction products
was demonstrated chromatographically (Scott and
Cohen, 1951), and by means of a manometric
assay (Cohen and Raft, 1951) using specifically
adapted bacteria, which in this instance were
ribose-utilizing mutants of strain B. Another pentose phosphate was found among the reaction products in largest amount, and this substance was subsequently identified as ribulose-5-phosphate (Horecker, Smyrniotis and Seegmiller, 1951). Shortly
after these findings, the enzymatic formation of deoxyribose-5-phosphate from glyceraldehyde-3-phosphate and acetaldehyde was demonstrated (Racker,
1951). The reactions linking ribulose-5-phosphate
and triose phosphate form an area of active study
in several laboratories at the present time. These
reactions also have been demonstrated in E. coll
and this organism also possesses the enzymes to
convert glucose to glucose-6-phosphate and the
latter to phosphogluconate or to triose phosphate
via the Embden-Meyerhof scheme. Thus E. coli has
at least two alternative paths of glucose-6-phosphate
utilization. At the present time, there is no reason
to believe that other major pathways of gtucose-6phosphate utilization exist. The organism does not
form the ketohexonie acids and under most conditions of growth the enzymes for the anaerobic
cleavage of phosphogluconate (Entner and Doudoroff, 1952) are present in very slight amounts
(Scott, unpub.).
These reactions had been studied in cell-free extracts, but in order to approach these systems in
the intact bacteria, it is necessary to supply unphosphorylated substrates. The ability of strain B
or appropriate mutants to metabolize various carbohydrates is largely adaptive, and in order to
demonstrate the insertion of substrates into the
pathways in intact cells, it becomes necessary to
clarify the steps involved. We were able to demonstrate the existence of the reactions:
a. gluconate + ATP
~ 6-phosphogluconate
q- ADP, in the presence of an adaptive gluconokinase (Cohen, 1951a).
b. D-ribose q- ATP
> D-ribose phosphate qADP, in the presence of an adaptive ribokinase
(Cohen, Scott and Lanning, 1951).
c. D-arabinose ~ D-ribulose, in the presence of
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METABOLISM OF VIRUS INFECTED BACTERIA
adaptive pentose isomerase (Cohen, 1953).
d. D-ribulose -4- ATP
.~ D.-ribulose phosphate + ADP, in the presence of ribulokinase
(Lanning and Cohen, unpub.).
Thus, as shown in Figure 1, these substrates may
be inserted at various levels of the phosphogluconate pathway and would appear to traverse this
path alone, since, under conditions of growth and
infection, the conversion of glucose-6-phosphate to
6-phosphogluconate appears essentially irreversible
in contrast to most of the other steps presented.
This was demonstrated by means of gluconate-l-C 14
in experiments in which the isotope content of the
CO2 liberated was found to be 85 to 95 per cent
of the isotope in the gluconate fed. Thus, the C1
was converted Io CO2 in the conversion of phosphogluconate to ribulose-5-phosphate, whereas formation of glucose-6-phosphate and degradation to
triose via the Embden-Meyerhof scheme would
have resulted in the conservation of isotope in the
methyl carbon of pyruvate (Cohen, 1951b).
Test of the role of the phosphogluconate pathway
by the same technique during growth of E. coli on
glucose-l-C 14 revealed that only a part of the glucose, estimated to be in the range of 16 to 38 per
cent, was handled in this way (Cohen, 1951b),
whereas the remainder was presumably degraded
via the Embden-Meyerhof scheme.
Under conditions of virus infection when RNA
synthesis was inhibited and replaced by stimulated
DNA synthesis, the utilization of the phosphogluconate path was reduced markedly, although not eliminated, thereby suggesting that the ribose of RNA
is synthesized predominantly via this path. Although the rate of glucose utilization or of respiration was not significantly affected by infection, the
isotope experiment revealed that the relative balance of the two paths was affected, and that the
common precursor, glucose-6-phosphate, plays a
pivotal role in this phenomenon.
The pattern revealed by the isotope experiments
has been checked in two ways. First, it has been
shown that E. coli contains sufficient of the dehydrogenases of the phosphogluconate pathway to
permit the utilization of 40 per cent of the glucose
by this route (Scott and Cohen, 1953a and b).
Second, analysis of the ribose of RNA of bacteria
grown on glucose-l-C14 reveals that most of the
ribose came from a pathway such as the phosphogluconate pathway in which the Ca of glucose was
lost. However, the isotope content of the ribose
was sufficiently high as to indicate a partial formation from fragments derived from the EmbdenMeyerhof scheme (Lanning and Cohen, 1952).
THE INDIRECT EFFECT OF INFECTION ON
CARBOHYDRATEMETABOLISM
How does virus change the balance of the pathway? Are we merely shifting equilibria by removing deoxyribose phosphate in virus DNA? If so,
RNA synthesis should proceed under conditions of
223
infection in which DNA synthesis is inhibited, as
in infection with ghosts or irradiated phage. However, RNA synthesis is inhibited under all conditions of infection, and although this hypothesis of
the linking of equilibrated reactions can explain
the greater availability of P for increased DNA
synthesis, it does not explain the apparently complete inhibition of RNA synthesis.
The possible inhibition of the enzymes of the
phosphogluconate pathway has been tested in the
iollowing way:
Strain B or the appropriate mutants, adapted to
substrates which must be handled via the phosphogluconate pathway, were infected with T2r+ or T4r
in the presence of these substrates as the sole carbon source. These substrates included gluconate,
ribose, D-arabinose, and the purine ribosides. The
production of DNA and virus on these substrates
indicated the operation of all the known steps of
carbohydrate metabolism leading to ribose nucleoside formation (Cohen and Roth, 1953). Isotope
experiments with glucose-l-C 14 under conditions of
infection had indicated that glucose-6-phosphate
dehydrogenase and 6-phosphogluconate dehydrogenase were still operative (Cohen, 1951b). We
have concluded, therefore, that the lack of RNA
synthesis is not due to an effect of virus on known
steps of ribose phosphate formation and utilization.
The prevention of this synthesis could affect carbohydrate metabolism in the manner described, but
this would appear to be an indirect effect. It is
concluded that the control of RNA synthesis is
effected at other levels of metabolism.
THE BASE COMPOSITIONOF THE T-EVEN PHAGES
AND HYDROXYMETI-IYLCYTOSINE
A clue as to the critical level of metabolic control was discovered in the course of quite another
approach to the phages. It appeared of interest
from several points of view to compare the constitution of the DNA of the three mutant pairs of
related phages with which we have worked for
several years, namely, T2r + and T2r, T4r+ and
T4r, and T6r+ and T6r.
a. The rigorous chemical description of the
phages had been neglected. It could be anticipated
that satisfactory interpretation of physiological and
genetic data would not be possible without these
data.
b. In view of the hypothesis that variations of
the structure of DNA may be responsible for
genetic differences, organisms such as viruses with
small numbers of DNA molecules may provide material in which changes of DNA composition may
be detectable.
A collaboration was developed with Dr. G. R.
Wyatt of the Department of Agriculture of Canada
to study this problem. Earlier efforts to characterize the bases had failed to account for more than
70 per cent of the bases of the phages or to find
cytosine (Marshak, 1951). It is obvious that in-
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SEYMOUR S. COHEN
224
complete recovery of the bases does not permit
the report of the absence of a constituent, but
merely suggested the inadequacy of a method, in
this case the use of concentrated perchloric acid in
hydrolysis. However, it was equatly evident that
the phage nucleic acid possessed certain peculiarities warranting more careful study. As a result of
the trial of many hydrolytic procedures, the previously missing base came to light. It proved not to
be cytosine, but a compound whose spectrophotometric properties and chromatographic properties
suggested that it was a derivative of cytosine
(Wyatt and Cohen, 1953a). At our suggestion, 5hydroxymethyl cytosine was synthesized by Dr.
Charles Miller of Sharp and Dohme, Inc. and the
synthetic material was compared with the natural
compound isolated in crystalline form from T6r+
phage. The comparison and elementary analyses
became available within one week, and the natural
compound proved to be 5-hydroxymethyl cytosine
(HMC) (Wyatt and Cohen, 1952). Dr. Miller has
also kindly made available 5-hydroxymethyl uracil
(HMU).
HMC is labile in perchloric acid and in other
acids under various oxidizing conditions. A method
has now been devised in which, in analysis of
phage or of phage nucleic acid, molar base recovery approaches 100 per cent of the moles of P. The
details of the method are in press (Wyatt and
Cohen, 1953b) as are analyses of the T-even phages
and some other viruses.
In Table 1 are presented some of these analyses
on intact phages. The mean ratio of estimated
ba,~es/P is improved to 0.99 when the material
analyzed is isolated phage DNA. It can be seen
that the base compositions of the six phages are
substantially identical, within the experimental errors of the analyses. However, the conclusion can
not be drawn from these analyses that the nucleic
acids of these phages do not differ since position
isomerism without change in composition is possible. Furthermore, a real difference of one per
cent in the content of a particular base, which is
not a significant difference by current analytical
methods, would reflect a difference of hundreds of
residues.
In the course of the analyses, no significant
amounts of cytosine were found in these viruses,
TABLE1. DNA BASECOMPOSITIONOF PHAGEST2, T4,
A N D T6
Mean Moles/100 MolesEstimatedTotal Bases
Hydroxymethyl Total
Virus Adenine Thymine Guanine Cytosine Bases/P
T2r+
T2r
T4r+
T4r
T6r+
T6r
32.0
32.3
32.3
32.2
32.5
32.3
33.3
33.4
33.1
33.5
33.5
33.4
18.0
17.6
18.3
18.0
17.8
17.7
16.8
16.7
16.3
16.3
16.3
16.6
0.99
0.95
0.96
0.94
0.99
0.88
and it was estimated that the maximal content of
cytosine in T6r+, for instance, was 0.2 per cent of
its content of HMC. Conversely, analysis of the
bacterial host and of its DNA did not detect HMC
and the maximal HMC contents of these ~aterials
were estimated to be 0.2 and 0.6 per cent respectively of their cytosine content. It should be noted
that cytosine is a constituent of both the RNA and
DNA of E. coll. Analyses of the DNA of various
strains of E. coli have revealed the four bases,
adenine, thymine, guanine, and cytosine to be in
approximately equimolecular proportions (Gandelman, Zameuhof and Chargaff, 1952). 5-methyl
cytosine has not yet been found in any microorganism or virus.
Thus a new pyrimidine base has been found in
several viruses which has not yet been found in the
bacterial host or its nucleic acids. It is conceivable
that HMC might be present in trace amounts in E.
coli, but the substance has not yet been demonstrated. If HMC is not a product of normal bacterial activity, this would be the first instance of a
low molecular building block in a virus different
from those of ;is host. In any case, the absence of
cytosine in the virus and the shifting to HMC synthesis suggests that the metabolic systems involving
these compounds may account for the inhibition of
RNA synthesis, and now to extend the problem to
its proper scope, for the inhibition of host DNA
synthesis, as well. If an infected cell were compelled
to synthesize HMC and were nnable to make or
utilize cytosine such a cell would be unable to synthesize host nucleic acid, and might synthesize materials linked to HMC such as virus nucleic acid
and virus proteins. The case of the lost kingdom
and the horseshoe nail is herein postulated in modem dress.
If the hypothesis were true it would be expected
that virus infections in which host nucleic acids
continue to be synthesized, as in lysogenic systems,
would not involve the synthesis of HMC. This prediction has been confirmed by Simonovitch and
Smith (personal communication) at least with respect to the absence of HMC in temperate phages.
What situation prevails with other phages of the
T series? Analysis of T7 and T54 has not revealed
detectable amounts of HMC in these viruses. Of the
odd numbered phages of the T set, nucleic acid
metabolism has only been studied in T7 and T3
infected cells (Putnam et al., 1952; Pardee and
Williams, 1953). Careful examination of the data
presented in these papers reveal significant incorporation of paz into the RNA fraction in the first
instance, and pentose increment in the second. The
significance of these data has not yet been evaluated. We conclude that the data on the T-odd
viruses can not yet be considered an argument
against our hypothesis.
4 We are indebted to Dr. Kozloff and Dr. Lark, respectively, for these preparations.
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METABOLISM OF VIRUS INFECTED BACTERIA
HMC has not been found in any other DNA so
far examined for this substance. These include
mammalian DNA and some animal virus preparations, including a polyhedral virus, vaccinia virus,
and meningo-pneumonitis virus, which all contain
the usual DNA bases, including cytosine. The latter virus is of particular interest since it is a member of the psittacosis group of viruses, in the multiplication of which folic acid has been found to play
an important role.
ORIGIN OF THE RING STRUCTURE OF
HYDROXYMETHYL CYTOSINE
Weed and I have published data indicating the
transfer of the pyrimidine nucleotides of host DNA
to become pyrimidine nucleotides of virus DNA
(Weed and Cohen, 1951). In the course of that
work, one of the pyrimidine nucleotides of virus
DNA was stated to be deoxycytidylic acid. The incorrect identification of this material arose in the
following manner: DNA was extracted with hot trichloroacetic acid from various tissues or organisms
from which RNA had been previously removed by
one of various methods. RNA was not present when
the extraction was made from isolated virus. The
DNA extract was concentrated with N HC1 and
hydrolyzed in the acid solution. The hydrolysate
was chromatogrammed on papers using a tertiary
butyl alcohol--HC1 mixture (Smith and Markham,
1950), and two bands were obtained containing the
pyrimidine nucleotides. Following the hydrolysis
of thymus DNA, for instance, the band containing
the cytosine nucleotide has little other base. The
band containing thymine nucleotide is also fairly
homogeneous. Nevertheless, it has been customary
to purify the nucleotides further by adsorption and
elution with an acid of appropriate strength on an
anion exchange resin (Weed and Wilson, 1951).
In the experiments with virus small amounts of the
nucleotides were available, and were separated on
paper only. Although the band in the thymidylic
nucleotide region obtained in this way from a virus
DNA hydrolysate is substantially free of bases
other than thymine, the band in the "cytidylic" region has now been found to contain two components of approximately equal amount separable
on the resin. The first, eluted with 0.01 N HC1,
contains HMC as the sole base, and has an absorption maximum at 282 to 283 m~. The second,
eluted with 0.01 N HC1, contains equimolecular
amounts of HMC and thymine and has an absorption maximum of 274 rag. The mixture isolated
from paper had a maximum of 278 m~, or that of
deoxycytidylic acid. It is evident that it is neces.
sarv to separate these components on both paper
and resin in experiments in which the pyrimidines
of host DNA are labeled by growth of the bacteria
in labeled orotic acid.
These experiments have now been repeated in
this way in collaboration with Dr. Weed of the
Army Medical School. Bacteria were grown to 3 X
225
10s per cc in a mineral medium containing glucose
and orotic acid-2-C14, generously given us by Dr.
D. W. Wilson. The cells were centrifuged and
washed. One half was precipitated in two per cent
perchloric acid, washed and dried. The remainder
was resuspended at the same concentration in the
medium free of orotic acid, and infected at a six
fold multiplicity of T6r+ in the presence of 50 7
tryptophan per cc. The culture was aerated until
lysis and virus was isolated following two cycles of
differential centrifugation. The pyrimidine nucleotides of host DNA and virus DNA were isolated as
described above and their isotope contents were
estimated. The results are presented in Table 2,
TABLE 2. T h E PYRIMIDINE NUCLEOTIDES OF THE D N A o r
BACTERIA GROWN IN RADIOACTIVE OROTIC ACID AND
OF VIRUS PRODUCED IN THE LABELED HOST
Base
Host Nucleotides
Cts./Min./gMol.
Thymine
Cytosine
123
160
Virus Nucleotides
Base
Cts./Min./gMol.
Thymine
HMC
24, 25.4, 26.8
24
and show that the ring structure of a pyrimidine
can be converted to the ring of HMC. The extensive
dilution of the pyrimidine ring derived from host
in virus HMC and thymine is to be expected in
view of the extensive net synthesis of DNA in this
system.
However, whether the DNA base, cytosine, is an.
obligate intermediate of HMC is in no way answered by these experiments. When the DNA was:
analyzed from virus produced in ceils infected in
the presence of radioactive orotic acid, the radioactivities of HMC and thymine were essentially
similar suggesting that, despite the extensive dilution of the orotic acid by considerable de nova
synthesis of the pyrimidine ring, the two bases
might have a common precursor not far removed
from the end products of the reaction sequences.
ON THE ORIGIN OF THE HYDROXYMETHYL GROUP
The fl-carhou of serine is known to he a source
of one carbon fragments such as the methyl group
of thymine and the C2 and Cs of purines. Formate
has also been found to be a source of these atoms
in the nucleic acid bases in many organisms. However, the oxidation level at which these atoms are
added to the molecule has not yet been determined.
Serine is considered to be cleaved to glycin and a
moiety containing the fl-carbon at the oxidation
level of formaldehyde or that of the hydroxymethyl group. We, therefore, undertook to see if
the fl-carbon of serine could become the carbon
atom of the hydroxymethyl group of HMC.
These experiments 5 were facilitated by the fact
that the C2 of the pyrimidine ring, unlike that of
5 We are indebted to Dr. J. Stekol for a sample of DL~
serine-3-C14.
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SEYMOUR S. COHEN
226
TABLE 3. THE LABELtNC OF THE DNA BASES OF BACTERIA GROWN IN SERINE-3-C14, AND OF VIRUS PRODUCED IN THESE
BACTERIA IN THE PRESENCE AND ABSENCE OF LABELED SERINE5
Bacteria
Substance
Adenine
Guanine
Thymine
Cytosine
Cts./Min.//zMol.
2730
2840
1450
157
T6r+ Grown in Absence of
Serine-3-C14
T6r+ Grown in Presence of
Serine-3-C14
Substance Cts./Min./gMol.
Substance
Adenine
Guanine
Thymine
HMC
Adenine
Guanine
Thymine
HMC
the purine ring, is not derived from this type of
one-carbon precursor. Therefore, it could be anticipated that growth of E. coli in the presence of
serine-3-C14 would give rise to bases in the DNA
in which the purines, thymine, and cytosine should
contain two, one, and 0 labeled atoms respectively.
The isolation of the bases was carried out by
hydrolysis of the DNA fractions of the bacteria in
formic acid and chromatography on paper in isopropanol-HCl (Wyatt, 1951). To free the bases
from possibly contaminating amino acids, the individual bands were eluted, adsorbed on an anion
exchange resin and eluted, and rechromatographed
on paper using butanol-HzO. Typical analyses are
presented in Table 3, as are analyses of the bases
of T6r+ reproduced in the same bacteria in the
presence or absence of radioactive serine. Three
experiments have yielded consistent results.
It can be seen that the ratios of radioactivity in
purines, thymine, and cytosine of bacterial DNA
are 2, 1, and 0.1. In virus produced in labeled bacteria in the absence of labeled serine, the dilution
of thymine was somewhat greater than that accounted for by net DNA synthesis, whereas that of
the others was not far from the expected dilution
to about one-eighth the initial activity. The low
activity of the tIMC implies that the methyl group
of thymine is not converted in large amounts, if at
all, to hCH2OH, and that the --CHzOH in this
experiment was derived from essentially unlabeled
precursors, rather than serine or hydroxymethyl
donors stored in the host. This also implies that the
cytosine of host DNA can supply the pyrimidine
ring for HMC.
Virus grown in the presence of serine-3-C14 contained highly active HMC, which in one experiment
not presented in the table slightly exceeded the
activity of thymine. Since the ring structure of
cytosine formed in the presence of serine contains
small amounts of C14, and HMC, whose ring is derived from cytosine or cytosine precursor, contains
large amounts of C14, it is concluded that the /3carbon of serine is a potentially important precursor of the hydroxymethyl group. We have not
studied other possible precursors of this carbon
atom.
310
502
55
23
Cts./Min.//zMol.
1920
2090
952
700
THE ATTEMPTED ISOLATIONOF THE HMC
DEOXYRIBOSIDE FOLLOWINGENZYMATIC
DEGRADATION
In Figure 2 are presented the structures of the
pyrimidines with which we are concerned as well
as some of their possible metabolic relationships.
Three of these, cytosine, uracil, and thymine or 5methyl uracil have been found in the host. HMC
and thymine are known in the virus. 5-methyl cytosine and 5-hydroxymethyl uracil have not been
found in either. Very little is known of the metabolism of these compounds, and one must be prepared to cope with the possibility that their major
metabolic routes involve the deoxyribosides.
Methods for the isolation of deoxyribosides generally begin with the enzymatic degradation of
DNA by deoxyribonuclease followed by dephosphorylation with a suitable phosphatase. We have
developed this procedure using pancreatic deoxyribonuclease followed by purified intestinal alkaline
phosphatase for small amounts of thymus DNA,
cuhninating in the separation of the deoxyribosides by paper chromatography in butanol-NH4OH.
Following elution of the bands with H.~O, we have
been able to obtain solutions of deoxycytidine and
thymidine which possess satisfactory spectrophotometric characteristics and are free of contamination
by the other pyrimidine deoxyriboside, as determined with thymineless or cytosine-uracilless mutants of E. coll. Under short treatments with our
sample of alkaline phosphatase, it is possible to
obtain deoxyguanosine and deoxyadenosine. With
N~C-NH~
I
I
N=C-NH z
CH, O
o<1 IICH
RN--CN
CYTOSINE
N=C-OH
III
URACIL
.o:cI C-C.,OH
It
N~C-NH 2
1
I
? ,o:~ I-CH~
RN--CH
RN--C
5-HYDROXYMETHYL CYTOSINE 5-METHYL CYTOSINE
II c.,o
O,C C H
RN-CH
I I
N=C-OH
I I
-O=C C-CH2OH
Ill
RN-CH
~'
5"HYDROXYMETHYL URACIL
N=C-OH
II
I !
RN--C
:O~C C-CH~,
THYMINE
FmURE2. Structures and possible metabolic relations of
pyrimidines. R -" a hydrogenatom or a glycosylmoiety.
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METABOLISM OF VIRUS INFECTED BACTERIA
prolonged treatment, an impurity of adenosine
deaminase produces hypoxanthine deoxyriboside
which is less readily separable from the deoxy.
guanosine. Crystalline thymidine was also prepared
by Dr. D. B. McNair Scott (Brady, 1942 and personal communication).
Polymeric virus DNA, prepared by the urea
method (Cohen, 1947), was subjected to the same
procedure. Whereas the P of thymus DNA was
liberated to the extent of 92 to 98 per cent, various
preparations of ~:irus DNA liberated only 65 to 82
per cent of their P after prolonged treatment.
Chromatography of the digest revealed large
amounts of thymidine which was further purified
and crystallized by Dr. Scott. It had the same
melting point and spectrophotometric properties as
the thymidine of thymus DNA. No band containing the HMC nucleoside was detectable. A band
containing guanine and hypoxanthine in the position characteristic of their nucleosides was found.
This band has also been found on occasion to
contain very small amounts of HMC and thymine.
Hydrolysis of the purine nucleosides from this
band in 0.01 N HC1 and paper chromatography in
a butanol-ethanol-H20 mixture (4:1:5), revealed a
deoxypentose having the Rr value of deoxvpentose
derived from deoxyribosides of thymus DNA.
The bulk of the HMC was found in the nucleotide fraction non-mobile in butanol-NH4OH. This
material was eluted in water and showed a broad
spectrum whose maximum was shifted from 260 mg
to 265 mg. Hydrolysis of the enzymaticaUy resistant non-mobile nucleotides derived from three
preparations of T6r+-DNA had the compositions
presented in Table 4. In each case, the concentraTABLE 4. BASE COMPOSITION OF ENZYME-REsISTANT
NON-MOBILE NUCLEOTIOES
%P
UnhydroPreparation lyzed Adenine Thymine
Intact T6r+
DNA
A
B
C
-25
33
37
32.5
19.1
26.5
28.0
Guanine
HMC
17.8
16.6
12.1
11.9
16.3
47.1
35.5
34.6
33.5
17.3
24.6
25.5
tion of HMC in an enzyme-resistant fraction may
be observed. It is tempting to suppose that the
survival of injected virus DNA into a bacterium
containing enzymes capable of degrading host DNA
to the nucleoside level is associated with the existence of HMC in enzyme-resistant units. One might
therefore, imagine that survivability, specificity,
and the parasitic function may all be conferred on
,. virus DNA in the replacement of cytosine by HMC.
ISOLATION AND PROPERTIES OF
DEOXYRIBOSIDE
HMC
Several procedures involving acid hydrolysis of
virus DNA were then employed. Hydrolysis in N
227
HC1 was followed by separation of the nucleotides
on paper as described previously. The HMC nucleotides were then hydrolyzed with phosphatase and
the HMC deoxyriboside was separated in butanolNH4OH. The product so obtained is spectrophotometrically satisfactory, but is low in over-all yield.
Direct chromatography in butanol-NH~OH of the
product of acid hydrolysis and treatment by phosphatase yieIds a material slightly contaminated by
guanine.
The procedure of choice involved hydrolysis of
20 mg. DNA per cc. 1.5 N HC104 at 100 ~ for one
hour. Neutralization to pH 7.5 with KOH and centrifugation following chilling removed most of the
KC104 and guanine. The pH was adjusted to 8.8
with NH4OH and the Mg++ concentration was adjusted to 0.25 per cent. Alkaline phosphatase was
added (30 units per cc.) and the mixture incubated
for three hours at 37 ~ The hydrolysate was centrifuged to remove MgNH4P04, concentrated, and
chromatographed on paper in hutanol-HzO. Three
bands were discernible. The first, at Rt <0.1 contained about 10 per cent of the HMC of the preparation as the free base. The second, at R~ .13 to .17
when applied in small amounts on paper, and somewhat more diffuse in concentrated solutions, contained the HMC deoxyriboside and a slight contaminant of guanine ( < 5% of the preparation).
The third, at R~ .45 to .55 contained both adenine
and thymidine.
The band containing the HMC deoxyriboside has
been further purified by paper chromatography at
about 25 ~ in ethanol-acetate pH 3.5 (Cohen and
Scott, 1950), in which both guanine (Rt .36), and
free HMC (Rt .47) may be separated from the
deoxyriboside (Rf .57). The HMC deoxyriboside
has its maximum absorption at 282 m/~ in 0.1 N
HC1 in contrast to that of HMC at 278 to 279 rag.
The compound isolated in this way is devoid of
phosphorus. The maximum of the deoxyriboside is
the same at pH 7 and 13 in contrast to free HMC.
Owing to the small amount of deoxyriboside
available it has been used until now for metabolic
experiments, and has not been consumed in various
elementary analyses. As a consequence of this, the
molecular extinction coefficient for the deoxyriboside has not been determined, and the values obtained by Fox and Shugar (1952) for deoxycytidine have been adopted as a close approximation.
As an additional difficulty, it may be noted that
following storage of frozen solutions for a month,
30 to 40 per cent liberation of free HMC from the
deoxyriboside was revealed.
In the initial chromatography in butanol-H20 in
three of five isolations the HMC deoxyriboside
band has been split. When the contents of each of
the split bands were rechromatographed in dilute
solution in butanol-H20 and ethanol-acetate, they
were then apparently identical. They possessed the
same spectrophotometric properties at pH 1, 7,
Downloaded from symposium.cshlp.org on May 8, 2016 - Published by Cold Spring Harbor Laboratory Press
SEYMOUR S. COHEN
228
pH
.80
.70
and 13, and in tests to be discussed below appeared
essentially identical with respect to enzymatic deamination and microbiological assay. The nature
of this phenomennon is not clear at present. In
Figures 3A and 3B are presented spectra of the
deoxyribosides of cytosine and HMC as prepared
by these methods. It is a very difficult matter to
reduce end absorption below 245 m/~, when ~eparation procedures are confined to paper chromatography. Nevertheless, it would appear that an effect
of the hydroxymethyl group becomes spectrophotometrically visible at pH 13.
07
x
13
THE UTILIZATION OF PYRIMIDINE DERIVATIVESe
>-
tz
b.I
0 240 250 2s
2"}0 28o 26o 36o
m~
45-
pl-I
9
I
07
x 13
.35-
>-
z
ILl
t:3
.10-
'~
.05
o
36o
m~
Fzcu•E 3. (a) Spectra of deoxycytidine isolated from
thymus nucleic acid, as described in text, (b) Spectra of
deoxyriboside of HMC isolated from T6r-I" nucleic acid, as
described in text.
It appeared of interest to know whether 5-hydroxymethyl derivatives of the pyrimidines were
used by various microorganisms. Pyrimidine-requiring mutants of E. coli are rare, and those available at the present time possess numerous complicating quirks, as we shall see. The organisms, besides strain B, on which we have concentrated include a uracil or cytosine-requiring mutant of strain
W, We- obtained from Dr. B. Davis, and a thyminerequiring strain of strain 15, 15T- obtained from
Dr. J. Gots. These requirements may be replaced
by the pyrimidine nucleosides. We- was grown
overnight in a mineral medium containing 0.7 mg
glucose, and 4 -/cytosine per ml. Growth was limited to ca 7 )< 10 s cells per cc. Aliquots were then
inoculated to the medium containing glucose and
test pyrimidine or deoxyribose at 5 • 107 to 10s
cells per cc and growth was followed turbidimetrically over a six-hour interval. The compounds permitting growth and multiplication are presented in
Table 5. The mass doubling time was about 20 per
cent faster with uracil than with cytosine.
Similar studies were done with strain 15,~-. In
overnight growth about 0.4 ,/thymine was limiting
at about 8 X 10s cells per cc in mineral medium
containing one mg glucose per cc. The tests of the
ability of a compound to support growth were run
on cells grown in the glucose-thymine medium
which were inoculated at about 10 s per cc of glucose-mineral medium. In the growth of strain 15Ton thymine, uracil or uracil derivatives accumulated and were excreted in the medium, as determined by support of the growth of We-.
HMC, HMU, or the deoxyribosides of these compounds at 5 ~, base per ml did not significantly
affect the growth rate of B nor DNA synthesis of
B infected with T2 when the bacterial concentration
was at 5 X 107 to 3 X l0 s per cc.
Dr. Helen Skeggs of Sharp and Dohme, Inc. has
tested preparations of the HMC deoxyriboside in
microbiological assay with Lactobacillus bi~dus,.
Thermobacterium acidophilus R 26, and Lactobacib
lus leishrnaniL Since these organisms react nonspecifically to the deoxyribosides, the test is presum6 Miss Hazel Barner was a most active collaborator in
this phase of the work.
Downloaded from symposium.cshlp.org on May 8, 2016 - Published by Cold Spring Harbor Laboratory Press
METABOLISM OF VIRUS INFECTED B~4CTERIA
o
50'
+ HMC
x 0
rr 3 0
HMC
DEOXYRIBOStDE
DEOXYRiBOStDE
~,,
Ld
13-
o
o
o
< 20
Z
o
,-, I0
o
g
3o
6'o -g
io
MINUTES
FIGURE4. The lack of effect of HMC deoxyriboside on the
DNA content of T2r+-infected E. coli, W strain.
ably a measure of the availability of deoxyribose
phosphate rather than the utilizability of the base.
All three organisms grow equally well on HMC deoxyriboside, cytosine deoxyriboside, and thymidine.
However, the deoxyriboside of HMU was unable to
support the growth of L. bifidus, the only organism
of the three tested on this compound.
PHAGE INFECTION OF STRAIN W
It has been. observed that W strains of E. coli
will adsorb T2 and T6 , but do not support their
multiplication. The possibility arose that W was an
HMC-requiring strain. W was grown to 3 X 10s
cells per cc in glucose-mineral medium, and infected with T2r+ at a virus to cell ratio of 4.6 in the
absence and presence of HMC and HMC deoxyriboside. No significant differences were noted in these
cultures. As can be seen in Figure 4, the DNA content of the culture rapidly fell about one-half, a
frequently observed phenomenon with this organism after infection. No evidence has yet been obtained to account for the block to the multiplication
of T-even phage in this strain. It may be noted
that W has been reported effective in the hydroxymethylation of ketovaline to ketopantoic acid (Maas
and Vogel, 1953).
PHAGE INFECTION OF SULFANILAMIDE-INHIBITED
STRAIN B
It had been reported that sulfanilamide-inhibited
B could support the growth of T1, T3, and T7 in
the presence of the metabolites, methionine, xanthine, valine, and thymine, but not the growth of
T2, T4, and T6 (Rutten, Winkler and deHaan,
1950). It seemed possible that the action of this
inhibitor was to prevent the formation of HMC. In
order to duplicate the resultsof Rutten et al.,it was
229
found necessary to grow B in the sulfanilamidemetabolite media and to infect in this media. It is
important to note that the metabolites provided do
not restore growth rate to more than 30 per cent
of that in the absence of sulfanilamide. Further, if
cells are grown in this fashion, and infected in
glucose-mineral medium without sulfanilamide, T4r
for instance, may multiply, albeit with a longer lag
in the inception of net DNA synthesis and a lower
rate for this function. When these cells are infected in the complete medium containing sulfanilamide and metabolites, T4r does not multiply, and
the DNA content of the cells falls to about 60 per
cent of their original value. The addition of HMC,
HMU, or HMC deoxyriboside at 3 T base per ce
still does not permit virus multiplication, as can be
seen in Figure 5.
THE ASSIMILATION OF PYRIMIDINE DERIVATIVES
When cytosine deoxyriboside or cytosine is incubated with strain B during growth or during infection with T2, a marked shift and reduction in the
spectra of these substances may be observed. The
spectra were determined in two per cent perchloric
acid extracts of the cultures. The shifts may be
seen after correction for the spectral changes in
control cultures prepared in the absence of added
pyrimidine in the medium. The amounts of supplement added to the medium are limited by the optical densities of the acid extracts from unsupplemented cultures, and by the amounts of substance
whose assimilation can be detected under the physi-
70t
6o
20.
,o.
o
_:
9 NO
,sMf:
L:
INHIBITOR(SM)
o ox , os,o
"--..
2b 4:o 6b 8'0 16o
MINUTES
FIGURE5. The lack of effect of hydroxymethylated derivatives on T4r-infected cultures of B, in media containing
SM (sulfanilamide and metabolites--see text).
Downloaded from symposium.cshlp.org on May 8, 2016 - Published by Cold Spring Harbor Laboratory Press
230
SEYMOUR S. COHEN
ological conditions of our experiments. Typical
transformations of these compounds by infected
cells are presented in Figure 6. It appears that the
compounds are both deaminated and assimilated in
these systems. Under comparable conditions, uracil is assimilated, but significant uptake of HMC,
HMU, 5-methyl cytosine, and thymine cannot be
detected. Although the latter is surprising, it appears that the amounts incorporated into DNA
under the conditions used, are at about the limits
of detection by this procedure. In any case, neither
HMC nor 5-methyl cytosine are deaminated by infected or growing B.
The deoxyriboside of HMC does appear to be
assimilated and deaminated with the accumulation
of a compound with the absorption of the HMU or
4
HMC
.3
.2
.1
i
Z
hi
t3
:3-
HMC
-0
DEOXYRIBOSIDE
.2
CYTOSINE
9
Oo,
0
30'
x 60'
o
.3
230 2 .0 250 260 2- 0
.2
FIGURE 7. The fate of HMC and the deoxyriboside of
HMC in cultures of B infected by T2r+. B = 3 X 108
per cc; T r + = 1.5 X 109 per cc.
.1"
:t-
metrically as described by Wang, Sable, and Lampen (1950). A typical result is presented in Figure
8. It was found that HMC and 5-methyl cytosine
DEOXYCYTIDI NE
z
bJ
O
.2
.1' ~
290 3oo
m~
o~
_
.9.
~
81
/
\OEAM,NAO
CYTOSINE
230 240 2.% 260 2?0 28o L:xjo 300
m~.
F[CURE 6. The deamination and assimilation of cytosine
and deoxycytidine by B infected by T2r+. B = 3 X 10s
per cc; T r + _-- 1.5 X 109 per cc.
/J
its deoxyriboside. These curves are presented in
Figur~ 7.
I
>.-
I.-
"o. _.d(~YTOS I N E
STUDIES ON THE ENZYMATIC DEAMINATION OF H M C
AND THE HMC DEOXYRIBOSIDE
Cytoxine deaminase has been reported in yeast,
deoxycytidine deaminase in E. coli strain W. Since
We- grows better on uracil than cytosine, it appeared likely that the utilization of cytosine was
effected throuzh a deamination to uracil. We- was
grown in media containing cytosine and cell-free
extracts were obtained by the alumina procedure
from organisms harvested during exponential
growth. The extracts proved to have both deaminases, of which the cytosine deaminase was quite
labile. In tests of the latter enzvme 5 g moles of
base in 0.8 cc of .05 M tris pH 7.0 were incubated
with 0.2 cc of extract for 90 minutes at 37 ~ and
the reaction product was examined spectrophoto-
W
r~
9
I
\
FIGURE 8. Speetrophotometrie analysis of the deamination of cytosine by deaminase of extracts of We-.
Downloaded from symposium.cshlp.org on May 8, 2016 - Published by Cold Spring Harbor Laboratory Press
METABOLISM OF VIRUS INFECTED BACTERIA
TABLE 5. COMPOUNDSPERMITTINGGROWTHOF We"
AND 15,~Compound
Strain Wr
Uracil ..................................................
Dihydrouracil ....................................
Orotic acid ......................................
Uracil deoxyriboside ..................
5-hydroxymethyl uracil (HMU) ....
HMU deoxyriboside .........................
Cytosine ............................................
Cytosine deoxyriboside ...................
HMC ................................................
HMC deoxyriboside ......................
Thymine .........................................
Thymidine .......................................
5-methyl cytosine ..............................
Strain 15~-
+
--+
--+
+
------
-4"4-
were inert to the cytosine deaminase of E. coli. It
had been reported (Kream and Chargaff) that the
cytosine deaminase of baker's yeast will deaminate
both cytosine and 5-methyl cytosine. This enzyme
was, therefore, prepared in cell-free extract and
tested as described above. In Figure 9, it can be
seen that cytosine had been converted to uracil, 5methyl cytosine to thymine, whereas the spectrum
of HMC did not reveal appreciable deamination.
The deoxycytidine deaminase of E. colt is normally a very active enzyme in this organism and is
about two and a half times more active on this
substrate than on cytidine. The enzyme has a broad
91
230
240
250
260
270
280
290
300
m~t
FIGURE 9. Spectrophotometrie analysis of the products of
incubation of cytosine, 5-methyl cytosine, and HMC with
the cytosine deaminase of baker's yeast.
231
pH range for optimal activity. It is convenient to
follow the course of a deamination by following
the decrease in density at 282 m/z in tris buffer at
pH 7.0. However, the addition of a cell free extract
of E. coli, rich in nucleic acid and protein, to limited amounts of the deoxyribosides of cytidine and
HMC obscures such a measurement. Furthermore,
the extract increases its density in the absence of
substrate, perhaps as a function of nuclease activity on the nucleic acids of the extract. Also it has
been shown (Wang, Sable and Lampen, 1950) that
the deamination product may be cleaved to the
free pyrimidine. It is, therefore, desirable to purify
the nucleoside deaminase.
The following procedure permitted the isolation
of an active stable preparation of deoxycytidine
deaminase low in absorption at 282 mg and virtually free of a hydrolytic activity under the conditions of our measurement. A wet pellet of cells
harvested during exponential growth was ground in
the cold with alumina (Cohen, 1951a) and extracted with a 10 fold volume of 0.05 M tris buffer
at pH 7. The extract was sedimented at 5000 RPM
for 30 minutes and 40,000 RPM for two hours. To
the supernatant fluid was added one tenth volume
of 0.25 M MnC12, and after 30 minutes Mn nu.
cleates were removed by centrifugation. The
supernatant fluid was made 89 saturated with
(NH4)2SO4 at pH 7.6 and the precipitate was discarded. The precipitate formed between 50 and 75
per cent saturation, (NH4)zS04 at pH 7.6 was
separated by centrifugation, and was dissolved in
one seventh the original volume. It possessed one
tenth of the original nucleic acid content and contained about 85 per cent of the activity of the extract. The enzyme preparation could be stored in
the frozen state for months without apparent loss of
activity.
Complete deamination of 0.1 micromole of deoxycytidine per cc 0.05 M tris pH 7.0 may be
effected by 0.01 cc of the deaminase in ten minutes. After three hours, the reaction product still
behaves as deoxyuridine, since the position of the
maximum at pH 7 and 12 are essentially identical.
The deamination of the deoxyriboside of HMC by
the same amount of enzyme proceeded at a rate
only two to four per cent of the rate on deoxycytidine. This was measured spectrophotometrically,
and corrected for differences in density changes per
mole at 282 mg, assuming ~ma~ X 10 -4 - - 0.9 at
pH 7.0 for both substances. The coefficients determined from the spectra were then ~2s2 X 10 -4 - 0.60 for deoxycytidine, and c2s2 X 10 -4 = 0.72
for the deoxyriboside of HMC. It was found that
the deamination was complete in four hours, and
the reaction products behaved as the deoxyribo.
side of HMU, as presented in Figure 10.
If these materials are deaminated at five- to tenfold concentrations with proportionately larger
Downloaded from symposium.cshlp.org on May 8, 2016 - Published by Cold Spring Harbor Laboratory Press
232
SEYMOUR S. COHEN
DF'AMINATION PP,ODUCT
\~.,.
o,/ /
"~.'~DEOXYRIBOSIDE
42
>. 3
Z2
u.l
23,0 240 250 260 2~/0 28,0 290 360
m.u,
FIGURE 10. Spectrophotometrlc analysis of the deamination product of the deoxyriboside of HMC, effected by a
purified deoxycytidine deaminase of E. coll.
amounts of enzyme for three-hour intervals at 37 ~
they may then be readily separated by paper chromatography with butanol-HzO. The Rt values of
the nucleosides in this solvent are: deoxycytidine
--0.23, deoxyuridine--0.36, HMC deoxyriboside
--0.14, and HMU deoxyriboside --0.24. Starting
materials and end products showed only a single
spot in these experiments. Thus the enzymatic
technique permits the preparation of deoxyuridine
and the deoxyriboside of HMU, products whose
formation was indicated during experiments on the
assimilation of the aminated pyrimidine deoxyribosides.
POSSIBLE SIGNIFICANCEOF THE METABOLIC DATA
Although the isotope experiments suggest the
conversion of host cytosine to virus HMC, the
metabolic data presented suggest that
a. the conversion is effected as the deoxyribosides, whereas HMC and HMU are inert, and
b. two routes are theoretically available for the
conversion, either
CH20
1. deoxycytidine
) deoxyriboside of
HMC, or
-NH3
CH~O
2. deoxycytidine
> deoxyuridine
>
+NH3
deoxyriboside of HMU ~
deoxyriboside of HMC.
It is not considered likely that isotope experiments alone will clarify this situation, and the study
of the mechanism of hydroxymethylation in cellfree extracts would appear to be required. Although
the activity of the deoxycytidine deaminase of B in
intact cells suggests that deoxyuridine is readily
formed from deoxycytidine, and may, therefore, be
an active intermediary in the formation of HMC,
it is not at all clear that deoxyuridine is the actual
substrate for the hydroxyrnethylation. If a competition for amination of the uracil and HMU
nucleosides (route 2) were established in a system
containing deoxycytidine deaminase, it can he seen
that, other factors being equal, the deoxyriboside
of HMC would tend to accumulate, owing to the
resistance of this compound to the deaminase. Indeed, if this compound were then organized into
the enzyme resistant nucleotides characteristic of
phage DNA, it is evident that all the known relations of HMC facilitate the winning out of phage
products, without requiring any additional inhibitory action on the cytosine metabolism of the host.
In addition, it will be recalled that the deoxyribosides of hydroxymethyl derivatives do not fill
uracil or cytosine requirements in We-. The hydroxyrnethylation would seem, therefore, to be
essentially irreversible and this would magnify the
selection for hydroxymethyl compounds in the
postulated competitive relations. However, since
the irreversibility of the hydroxymethylation appears so complete, how are we to explain the winning out of cytosine and uracil derivatives in normal metabolism?
It is possible that the deoxyriboside of HMU is
not a normal metabolic intermediate, even though
on paper it has the additional advantage of also
providing a possible precursor for thymidine as
well. In this connection, it should be noted that
the task of removing the oxygen from the hydroxymethyl group at this type of carbon atom has no
known biochemical analogies. However, a deoxygenation must occur somewhere, although it is
known that thyrnidine may be generated by transglycosidation from thymine and deoxyuridine. The
conversion of hydroxymethyl to methvl may occur
at some other metabolic system and an intact methyl
group may be placed on uracil or deoxyuridine.
The problem of the formation of thymidine is thus
seen to be tied up with the problem of a possible
physiological role for the deoxyriboside of HMU.
If the formation of a hydroxyrnethylated pyrimidine deoxyriboside is to be explained as an abnormal product, and the relative inertness of HMC and
HMU deoxyriboside as contrasted to deoxycytidine
and deoxyuridine is taken to mean that these hy.
droxymethylated compounds do not serve as inter.
rnediary rnetabolites, two additional possibilities
may he mentioned:
a. The enzymes responsible for the hydroxymethylation of pyrimidine nucleosides are evolutionary vestiges in E. coli. It may be supposed
that these enzymes are normally not active in the
life of the bacterium, but are activated by infection.
b. The virus supplies the enzyme or other com.
portent essential for these particular hydroxymethylations.
Downloaded from symposium.cshlp.org on May 8, 2016 - Published by Cold Spring Harbor Laboratory Press
METABOLISM OF VIRUS INFECTED BACTERIA
ON THE GENERATION OF " N E w " FUNCTIONS
BY VIRUS INFECTION
Having confronted the possibility that a virus
may confer a new metabolic activity upon the
infected cell, we then proceeded, quite by chance,
to find such an activity in an apparently unrelated
system. Dr. Zinder will dicuss "infective heredity"
or "transduction" as it was known in the ancient
literature (Zinder and Lederberg, 1952). The production of toxin by diphtheria bacillus as a function of infection has recently been reported (Groman, 1953). The systems used by Zinder and Groman are lysogenic, permitting genetic analysis of
the infected progeny. This is not possible in the
phenomenon I will briefly report.
The thymine or thymidine requirements of strain
15T- were described earlier. Strain 15x- will support
the multiplication of T2, but not T4 or T6. When
grown on broth, it absorbs T2 much better than
when grown on glucose, amino acids, and thymine.
This is not an adsorption cofactor effect; the organism appears to have nutritional requirements
for the formation of receptors. When 15~- is grown
in broth, suspended in a glucose-amino acid mixture in which it is multiply infected, and subsequently, transferred to media containing thymine
or free of thymine, it was found that the infected
ceils produced almost equivalent amounts of DNA
whether thymine were present in the medium or
not (Figure 11). T2r+ virus was isolated from
lysates of these cells infected in the absence of
thymine, and it was found that the isolated DNA of
this virus preparation was comparable to other
preparations of T2r+-DNA, possessing thymine and
HMC. In experiments comparable to those reported
by Hershey on base s?nthesis by infected cells, it
I00.
o
U
u3
80
rr 60"
W
n
o
Z 40200
NO THYMINE
IO~/ML THYMINE
o
2'o 4:o
sb
MINUTES
16o 6o
FI6UR~ 11. The synthesis of DNA by the thymine requiring strain of E. coli 15T-, when infected by T2r+ in the
presence or absence of thymine. 15~ = 3.2 X 108 per ce;
average multiplicity of infection = 8.3 T2r+ per bacterium.
233
was also found that HMC and thymine were synthesized immediately following infection.
Of course, it is not evident whether virus infection stimulated relatively inactive systems to normal activity (Hypothesis a.), or introduced the
ability to make thymine (Hypothesis b.), but it
seems that the possible validity of one of the
hypotheses of this type must be taken more seriously as a result of this finding. This system bears
some similarity to the stimulation of thymine synthesis in infection of strain B by T2. However, the
apparently absolute initial requirement for thymine
by 15a~ places a different aspect on the phenomenon.
SUMMARY
1. The site of inhibition of synthesis of host
nucleic acid in bacteria infected by T-even phages
is being studied. Although the effect is manifested
in pathways of carbohydrate metabolism leading to
ribose formation, the enzymes of this pathway in
infected cells are not directly inhibited by virus.
2. Base analyses for the r and r+ strains of the
T-even phages are reported, and are seen to be indistinguishabIe. The existence and identification of
a new pyrimidine, 5-hydroxymethyl cytosine
(HMC) in these phages are described. The absence
of cytosine in this material and of HMC in the
DNA of the host and other virus DNA is noted.
The possible correlation of the presence of HMC
and the inhibition of synthesis of host nucleic acid
is discussed.
3. The origin of HMC has been studied by isotope experiments. The t-carbon of serine is an
active precursor of the -CH2OH group.
4. An enzyme-resistant nucleotide fraction was
found to be particularly rich in HMC.
5. The isolation of HMC deoxyriboside is described.
6. The hydroxymethylated pyrimidines or their
derivatives were unable to fill nutritional requirements of certain pyrimidineless strains of E. coli,
or to permit synthesis of virus DNA in infected W,
and in infected B, inhibited by sulfanilamide.
7. The deamination and utilization of certain
pyrimidines and their derivatives by growing or
virus infected bacteria are described.
8. The cytosine deaminases of E. coli and yeast
were unable to deaminate HMC.
9. The enzymatic deamination of the deoxyriboside of HMC by the deoxycytidine deaminase of E.
coli is described. The preparation and some properties of the deoxyriboside of HMU are presented.
10. The possible significance of these metabolic
data in terms of the route of formation of HMC
deoxyriboside is discussed, as are some hypotheses
on the mechanism of control of pyrimidine metabolism.
11. The stimulation of thymine synthesis by T2
infection of the thymine-requiring strain 15r- is
described.
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234
S E Y M O U R S. C O H E N
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DISCUSSION
MANSON: I have done experiments similar to those
that Dr. Cohen has reported here today. I tested
the ability of glucose-grown E. coli B cells to use
intermediates in the oxidative pathway of glucose
metabolism for their ability to act as the sole carbon source during multiplication of T4r.
Cells were raised in a glucose-salts medium.
When the cell concentration had reached 2 X 10 s
p e r ml, they were centrifuged a n d washed in cold
saline. They were then resuspended in media in
wbich the sole c a r b o n source was glucose, gluco-
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METABOLISM OF VIRUS INFECTED BACTERIA
hate, ribose, phosphogluconate, or ribose-5-phosphate. One-step growth curves were carried out at
37 ~ with T4r. Burst sizes of gIucose-grown cells
with glucose were about 75, with phosphogluconate
about 50, and with ribose-5-phosphate about 30.
No measurable bursts were obtained with either
gluconate or ribose.
With cells raised on gluconate, the burst size with
glucose was about 80, with gluconate and phosphogluconate about 50, and with ribose-5-phosphate 25.
There was no burst with ribose.
This indicated that the intermediates of the oxidative pathway can be used for the synthesis of
phage materials. Also, the cells had no difficulty in
using phosphorylated intermediates as carbon
sources, since in the experiments carried out with
235
glucose grown-cells they could not use the dephosphorylated product.
GoTs: We have another example of a biochemically deficient mutant which is capable of phage
synthesis in an environment which cannot support
the growth of the non-infected bacteria. This is a
purine requiring mutant of E. coli B. In a saltsglucose medium containing amino acids, growth of
the bacteria will not occur unless a purine is added.
Yet, this medium will allow the production of phage
when the cells are infected with T1 and T5 coliphages, in a manner indistinguishable from the
wild type. T2, T3 and T4 will not be produced
unless a purine is added. T6 and T7 have not as
yet been examined.
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STUDIES ON CONTROLLING MECHANISMS IN THE
METABOLISM OF VIRUS-INFECTED BACTERIA
Seymour S. Cohen
Cold Spring Harb Symp Quant Biol 1953 18: 221-235
Access the most recent version at doi:10.1101/SQB.1953.018.01.033
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