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On the extracellular accumulation and isolation of D -ribulose in cultures of Agrobacterium tumefaciens
BIOCHEMICAL
Vol. 19, No. 5, 1965
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ONTHL RXT~CUlJLAR ACCUMJiATIONANDISOiLTIOlJ OF
T. Suzuki* and R.M. Hochster Microbiology Research Institute Canada Department of Agriculture Ottawa, Ontario, Canada
ReceivedApril16,
1965
During growth of Agrobacterium tumefaciens on i-glucose
as the
only source of carbon it was observed that free D_-ribulose accumulated in the mediumwith time. these findings
The purpose of this communication is to record
since they represent the first
extracellular
accumulation
of ribulose by a bacterium grown on glucose and to draw attention further
observation that there is a highly significant
difference
to a
quantitative
between certain tumorogenic and non-tumorogenic strains of
this organism with respect to the formation of free ribulose. Materials
and Methods
Agrobacterium tumefaciens strains Bo (tumoroganic), (tunorogenic)
and IIDw6
(non-tumorogenic) were isolates
obtained from the laboratory
and Tipson, 1936).
Q -ribulose-o-nitrophenylhydrasone
Biochemical Corporation and free e-ribulose
the latter
by hydrolysis
2.
Institute.
of monoacetone-D-xylulose (Levene P
California
1.
originally
of Dr. A.C. Braun, Rockefeller
D-xylulose was prepared by hydrolysis
IIBV~(k)
(Schmidt and Treiber,
was purchased from was obtained from
1933).
Contribution No. 586 National Research Council Post Doctorate Fellow in collaboration the Canada Department of Agriculture, 19634965.
637
with
Vol. 19, No. 5, 1965
BIOCHEMICAL
The synthetic
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
growth mediumof NcIntire,
Peterson and Riker
(19.42) was used throughout with the omission of manganese, ferric Growth from an initial
phosphate ions.
2l+ hour inoculum (1 ml added to
a total
volume of 30 ml of fresh medium) was carried
culture
at 26~
out in shake
and was determined turbidimetrically.
For descending paper chromatography, water-saturated (Chang and Knight, 1961) and n-propanol-ethyl
acetate-water
Durbin, Grebner and Feingold, 1963) were used. chromatograms with aniline
phthalate
were located and distinguished orcinol
and aniline
phenol (F'ukui, Hochster
Aldoses were detected on
(Partridge,
1949) while ketopentoses
by meansof the trichloroacetic
acid-
phthalate combination sprays (Rochster, 1955).
Urea phosphate spray (Wise, Dimler, Davis and Rist, distinguish
and
ribulose from 3-ketoglucose since it
The
1955) was used to
reacts only with the
latter. Pentose was estimated quantitatively
according to Mejbaum (1939)
using a 40 minute heating period (Albaum and Umbreit, 1947) and ribulose by the method of Dische and Dorenfreund (1951). by the ltglucostat'l
technique (Saifer
Glucose was estimated
and Gerstenfeld,
1958).
Results Isolation medium
of P-ribulose In preliminary
large filter the culture
(as o-nitrophenylhydrazone)
experiments Q-ribulose was first
paper sheets after fluid
from the culture
by precipitation
with ethanol.
of such chromatograms showing the relative
isolated
both gave identical
on
polysaccharide had been removed from The supernatent
solution was then concentrated to a syrup -in vacua.
as controls is given in Table I.
identified
A typical
positions of related
Authentic e-ribulose
result sugars
and the material
colors when sprayed with the combination
spray for ketopentoses (Hochster, 1955).
638
BIOCHEMICAL
Vol. 19, No. 5, 1965
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
TABLE1 Chromatography of Sugars Rf-Values Solvent 1* Solvent.2
Sugars &+lucose
0.40
0.19
3&eto-&+Glucose
0.24
r&xylose
0.47
0.27
g-Ribose
0.64
0.33
g-Xylulose
0.60
0.42
g-Ribulose
0.68
0.38
Material
0.68
0.37
isolated
*Solvent 1 - Water-saturated phenol; Solvent 2 - npropanol: Ethyl acetate:water, 7:1:2.
Subsequently, 300 ml of culture mediumremoved after
growth of
the non-tumorogenic organism IIl%V6, was concentrated -in vacua (at 35%) to 50 ml and 250 ml of 95% ethanol was added. Following removal of the precipitated
polysaccharide by centrifugation
was concentrated to 5 ml and a further
the supernatant solution
quantity
of ethanol (50 ml) added
in the samemanner to remove small amounts of polysaccharide remaining. The final syrup.
supernatant solution was concentrated in -- vacua to a thick The latter
was
dissolved
in 10 ml of 0.05 g sodium tetraborate
and this soiution was passed through a 2 x 23 cm Dowex (1 x 2) column in
the
borate form at a flow rate of 25 ml/l0 min.
washed with 100 ml 0.05 4 sodium tetraborate with 0.02 g tetraborate material
emergedafter
column and was collected
was begun.
following which elution
Cysteine-carbazole-positive
about 2-4 litres over an elution
not eluted under these conditions,
The column was
of effluent
had come through the
span of 1 liter.
Glucose was
but remained on the column until
639
much
Vol. 19, No. 5, 1965
later.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The combined sJfsteine-carbazole-positive
fractions
were treated
with Dowex 50 (H+) to remove sodium and then concentrated to dryness at Borate was then removed with methanol (Zill,
35%
1953) and the residue reduced to a small volume. tion with charcoal (Darco G-60 previously and concentration
After
several recrystallizations,
melting points of authentic material,
constants were obtained:
melting point, (authentic
work of Glatthaar
optical after
(1933).
orcinol
and Reichstein (1935). Free as described the ratio
of
free ribulose was 0.90
treatment (Mejbaum, 1939) while the ratio
for the isolated,
free ribulose was 0.89 (Seegmiller and Horecker, 1952). rotation
The [cz]?
sample: -45.5O) which is in
Spectrophotometrically,
densities at 5&O/670 rq~ of authentic
165-166%,
165-1660~.
ribulose was then prepared from the o-nitrophenylhydrazone by Schmidt and Treiber
was
from absolute alcohol
ribulose-o-nitrophenylhydrazone,
was -47"
agreement with the original
first
the following
16&167'X,mixed
of the above derivative
o-nitrophenylhydrazone
described (Hochster, 1955).
and then from ethyl acetate,
isolated
Following decoloriza-
washedwith concentrated HCl)
to a syrup the crystalline
prepared as previously
Khym and Chenisl,
The optical
of the free ribulose isolated as above was -l7.2O compared
with the literature
value of -16.3O (Glatthaar
and Reichstein,
1935).
On the basis of the above physical constants the product obtained from the culture mediumwas g-ribulose. In the microbiological
conversion to g-ribulose
by this organism
it was observed that only 2-glucose served as a precursor, fructose,
2&eto+gluconate,
E-arabitol
g-xylose,
while D-
D-ribose, D_-arabinose and =
gave completely negative results.
The rate of conversion of It
should be pointed out
0_-glucose to P-ribulose
is given in Table II.
that highly significant
conversion appears to be a characteristic
of the
non-tumorogenic strain used while the tumorogenic strains produced relatively
little
D_-ribulose under these experimental conditions.
640
This
A.tumefaciens ( 1mv6)
(IIFW7K)
A,tumefaciens
tB6)
&turnefaciens
Strains
* Klett Units *5 Glucose concentration lOl;* Ribulose concentration
Nontumorogenic
Tumorogenic
Biological Activity
x 10m3 x lo- BM
x x lo-3M 10'3M
x 10'3M*
10e3M**
60 54.0 5.40
20 68.0 0.70
method)
56 57.5 1.20
84 58.5 0.55
30 68.0 0.33 30 68.0 0.51
10
of
0
by Intact Cells tumefaciens
of medium (glucostat technique) of medium (cysteine-carbaxole
Turbidity Glucose; Ribulose;
Turbidity Glucose; Ribulose;
Glucose; Hibulose;
x
of Ribulose Agrobacterium
Turbidity*
Formation
TAELF, II
107 40.2 8.25
90 55.2 1.72
128 56.7 0.60
24
Time (Hours)
170 29.3 3.85
49.0 1.38
190 21.5 3.02
190 36.5 0.83
210 38.7 0.38
195. 49.0 0.45 160
60 48
Vol. 19, No. 5, 1965
interesting
BIOCHEMICAL
difference
glucose to g-ribulose
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
as well as the mechanismof conversion of Qis now under investigation.
suggest the existence of a new non-phosphorvlative to E-ribulose
in which 3-keto-&glucose
Preiiminary
data
pathway from I&glucose
(Fukui and Hochster, 1963; Fukui,
1965) is the intermediate.
References Albaum, H.G. and Umbreit, W.W., J. Biol. Chem. m, 369 (1947) Chang, C. and Knight, S.G., Biochim. Biophys. Acta. 46, 271 (1961) Dische, 2. and Borenfreund, R., J. Biol. Cnem. 1 2, 583 (1951) Fukui, S., Biochem. Biophys. Res. Comm.I& 186--it-1965) F&ui, S. and Hochster, R.M., J. Am. Chem. Sot. a, 1697 (1963) Fulmi, S.,Hochster, R.M., Durbin, R., Grebner, x.3. and Feingold, D.S., Bull. Res. Count. of Israel, w, 262 (1963) Glatthaar, C. and Reichstein, T., Helv. Chim. Acta. l-8, 80 (1935) Hochster, R.M., Can. J. Microbial. 1, 346 (1955) Levene, P.A. and Tipson, R.S., J. Viol. Chem. u, 731 (1936) Mejbaum, W., Z, Physiol. Chem. a, 117 (1939) KcIntire, F.C., Peterson, W.H. and Ricker, A.J., J. Dial. Chem.l&, 491 (1942) Partridge, S.M., Nature I&, 443 (1949) Saifer, A. and Gerstenfeld, S., J. Lab. Clin. Med. 2, L&8 (1958) J. Biol. Chem. a, 261 (1952) Seegmiller, J.S. and Horecker, R.L., Wise, C. A., Dimler, R.J., Davis, H.A. and Rist, C.Z., Anal. Chem. 2, 33 (1955) Zill, L.P., Xhym, J.X. and Chenial, G.M., J. Am. Chem. SOC. & 1339 (1953)
642
Vol. 19, No. 5, 1965
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ONTHL RXT~CUlJLAR ACCUMJiATIONANDISOiLTIOlJ OF
T. Suzuki* and R.M. Hochster Microbiology Research Institute Canada Department of Agriculture Ottawa, Ontario, Canada
ReceivedApril16,
1965
During growth of Agrobacterium tumefaciens on i-glucose
as the
only source of carbon it was observed that free D_-ribulose accumulated in the mediumwith time. these findings
The purpose of this communication is to record
since they represent the first
extracellular
accumulation
of ribulose by a bacterium grown on glucose and to draw attention further
observation that there is a highly significant
difference
to a
quantitative
between certain tumorogenic and non-tumorogenic strains of
this organism with respect to the formation of free ribulose. Materials
and Methods
Agrobacterium tumefaciens strains Bo (tumoroganic), (tunorogenic)
and IIDw6
(non-tumorogenic) were isolates
obtained from the laboratory
and Tipson, 1936).
Q -ribulose-o-nitrophenylhydrasone
Biochemical Corporation and free e-ribulose
the latter
by hydrolysis
2.
Institute.
of monoacetone-D-xylulose (Levene P
California
1.
originally
of Dr. A.C. Braun, Rockefeller
D-xylulose was prepared by hydrolysis
IIBV~(k)
(Schmidt and Treiber,
was purchased from was obtained from
1933).
Contribution No. 586 National Research Council Post Doctorate Fellow in collaboration the Canada Department of Agriculture, 19634965.
637
with
Vol. 19, No. 5, 1965
BIOCHEMICAL
The synthetic
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
growth mediumof NcIntire,
Peterson and Riker
(19.42) was used throughout with the omission of manganese, ferric Growth from an initial
phosphate ions.
2l+ hour inoculum (1 ml added to
a total
volume of 30 ml of fresh medium) was carried
culture
at 26~
out in shake
and was determined turbidimetrically.
For descending paper chromatography, water-saturated (Chang and Knight, 1961) and n-propanol-ethyl
acetate-water
Durbin, Grebner and Feingold, 1963) were used. chromatograms with aniline
phthalate
were located and distinguished orcinol
and aniline
phenol (F'ukui, Hochster
Aldoses were detected on
(Partridge,
1949) while ketopentoses
by meansof the trichloroacetic
acid-
phthalate combination sprays (Rochster, 1955).
Urea phosphate spray (Wise, Dimler, Davis and Rist, distinguish
and
ribulose from 3-ketoglucose since it
The
1955) was used to
reacts only with the
latter. Pentose was estimated quantitatively
according to Mejbaum (1939)
using a 40 minute heating period (Albaum and Umbreit, 1947) and ribulose by the method of Dische and Dorenfreund (1951). by the ltglucostat'l
technique (Saifer
Glucose was estimated
and Gerstenfeld,
1958).
Results Isolation medium
of P-ribulose In preliminary
large filter the culture
(as o-nitrophenylhydrazone)
experiments Q-ribulose was first
paper sheets after fluid
from the culture
by precipitation
with ethanol.
of such chromatograms showing the relative
isolated
both gave identical
on
polysaccharide had been removed from The supernatent
solution was then concentrated to a syrup -in vacua.
as controls is given in Table I.
identified
A typical
positions of related
Authentic e-ribulose
result sugars
and the material
colors when sprayed with the combination
spray for ketopentoses (Hochster, 1955).
638
BIOCHEMICAL
Vol. 19, No. 5, 1965
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
TABLE1 Chromatography of Sugars Rf-Values Solvent 1* Solvent.2
Sugars &+lucose
0.40
0.19
3&eto-&+Glucose
0.24
r&xylose
0.47
0.27
g-Ribose
0.64
0.33
g-Xylulose
0.60
0.42
g-Ribulose
0.68
0.38
Material
0.68
0.37
isolated
*Solvent 1 - Water-saturated phenol; Solvent 2 - npropanol: Ethyl acetate:water, 7:1:2.
Subsequently, 300 ml of culture mediumremoved after
growth of
the non-tumorogenic organism IIl%V6, was concentrated -in vacua (at 35%) to 50 ml and 250 ml of 95% ethanol was added. Following removal of the precipitated
polysaccharide by centrifugation
was concentrated to 5 ml and a further
the supernatant solution
quantity
of ethanol (50 ml) added
in the samemanner to remove small amounts of polysaccharide remaining. The final syrup.
supernatant solution was concentrated in -- vacua to a thick The latter
was
dissolved
in 10 ml of 0.05 g sodium tetraborate
and this soiution was passed through a 2 x 23 cm Dowex (1 x 2) column in
the
borate form at a flow rate of 25 ml/l0 min.
washed with 100 ml 0.05 4 sodium tetraborate with 0.02 g tetraborate material
emergedafter
column and was collected
was begun.
following which elution
Cysteine-carbazole-positive
about 2-4 litres over an elution
not eluted under these conditions,
The column was
of effluent
had come through the
span of 1 liter.
Glucose was
but remained on the column until
639
much
Vol. 19, No. 5, 1965
later.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The combined sJfsteine-carbazole-positive
fractions
were treated
with Dowex 50 (H+) to remove sodium and then concentrated to dryness at Borate was then removed with methanol (Zill,
35%
1953) and the residue reduced to a small volume. tion with charcoal (Darco G-60 previously and concentration
After
several recrystallizations,
melting points of authentic material,
constants were obtained:
melting point, (authentic
work of Glatthaar
optical after
(1933).
orcinol
and Reichstein (1935). Free as described the ratio
of
free ribulose was 0.90
treatment (Mejbaum, 1939) while the ratio
for the isolated,
free ribulose was 0.89 (Seegmiller and Horecker, 1952). rotation
The [cz]?
sample: -45.5O) which is in
Spectrophotometrically,
densities at 5&O/670 rq~ of authentic
165-166%,
165-1660~.
ribulose was then prepared from the o-nitrophenylhydrazone by Schmidt and Treiber
was
from absolute alcohol
ribulose-o-nitrophenylhydrazone,
was -47"
agreement with the original
first
the following
16&167'X,mixed
of the above derivative
o-nitrophenylhydrazone
described (Hochster, 1955).
and then from ethyl acetate,
isolated
Following decoloriza-
washedwith concentrated HCl)
to a syrup the crystalline
prepared as previously
Khym and Chenisl,
The optical
of the free ribulose isolated as above was -l7.2O compared
with the literature
value of -16.3O (Glatthaar
and Reichstein,
1935).
On the basis of the above physical constants the product obtained from the culture mediumwas g-ribulose. In the microbiological
conversion to g-ribulose
by this organism
it was observed that only 2-glucose served as a precursor, fructose,
2&eto+gluconate,
E-arabitol
g-xylose,
while D-
D-ribose, D_-arabinose and =
gave completely negative results.
The rate of conversion of It
should be pointed out
0_-glucose to P-ribulose
is given in Table II.
that highly significant
conversion appears to be a characteristic
of the
non-tumorogenic strain used while the tumorogenic strains produced relatively
little
D_-ribulose under these experimental conditions.
640
This
A.tumefaciens ( 1mv6)
(IIFW7K)
A,tumefaciens
tB6)
&turnefaciens
Strains
* Klett Units *5 Glucose concentration lOl;* Ribulose concentration
Nontumorogenic
Tumorogenic
Biological Activity
x 10m3 x lo- BM
x x lo-3M 10'3M
x 10'3M*
10e3M**
60 54.0 5.40
20 68.0 0.70
method)
56 57.5 1.20
84 58.5 0.55
30 68.0 0.33 30 68.0 0.51
10
of
0
by Intact Cells tumefaciens
of medium (glucostat technique) of medium (cysteine-carbaxole
Turbidity Glucose; Ribulose;
Turbidity Glucose; Ribulose;
Glucose; Hibulose;
x
of Ribulose Agrobacterium
Turbidity*
Formation
TAELF, II
107 40.2 8.25
90 55.2 1.72
128 56.7 0.60
24
Time (Hours)
170 29.3 3.85
49.0 1.38
190 21.5 3.02
190 36.5 0.83
210 38.7 0.38
195. 49.0 0.45 160
60 48
Vol. 19, No. 5, 1965
interesting
BIOCHEMICAL
difference
glucose to g-ribulose
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
as well as the mechanismof conversion of Qis now under investigation.
suggest the existence of a new non-phosphorvlative to E-ribulose
in which 3-keto-&glucose
Preiiminary
data
pathway from I&glucose
(Fukui and Hochster, 1963; Fukui,
1965) is the intermediate.
References Albaum, H.G. and Umbreit, W.W., J. Biol. Chem. m, 369 (1947) Chang, C. and Knight, S.G., Biochim. Biophys. Acta. 46, 271 (1961) Dische, 2. and Borenfreund, R., J. Biol. Cnem. 1 2, 583 (1951) Fukui, S., Biochem. Biophys. Res. Comm.I& 186--it-1965) F&ui, S. and Hochster, R.M., J. Am. Chem. Sot. a, 1697 (1963) Fulmi, S.,Hochster, R.M., Durbin, R., Grebner, x.3. and Feingold, D.S., Bull. Res. Count. of Israel, w, 262 (1963) Glatthaar, C. and Reichstein, T., Helv. Chim. Acta. l-8, 80 (1935) Hochster, R.M., Can. J. Microbial. 1, 346 (1955) Levene, P.A. and Tipson, R.S., J. Viol. Chem. u, 731 (1936) Mejbaum, W., Z, Physiol. Chem. a, 117 (1939) KcIntire, F.C., Peterson, W.H. and Ricker, A.J., J. Dial. Chem.l&, 491 (1942) Partridge, S.M., Nature I&, 443 (1949) Saifer, A. and Gerstenfeld, S., J. Lab. Clin. Med. 2, L&8 (1958) J. Biol. Chem. a, 261 (1952) Seegmiller, J.S. and Horecker, R.L., Wise, C. A., Dimler, R.J., Davis, H.A. and Rist, C.Z., Anal. Chem. 2, 33 (1955) Zill, L.P., Xhym, J.X. and Chenial, G.M., J. Am. Chem. SOC. & 1339 (1953)
642