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Amino acid-dependent control of the transport of α-methyl glucoside in E. coli
Vol. 34, No. 1, 1969
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
AMINO ACID-DEPENDENT CONTROL OF THE TRANSPORT OF a-METHYL GLUCOSIDE IN E. COLI
Yoshihiro
Sokawa
and Yoshito
Department of Chemistry, University of Tokyo,
Received
December
control
Stent
and the
the
presence
In this of a-methyl system
paper
the
the
and Cohen,
relaxed
amino
chloramphenicol(CM) starvation
1962),
fail
in stringent
and culture
is
analog
14
to accumulate continue
amino
acid
C-c(-MG during
to uptake
14
C-a-MG
of RNA and lipid
the transport
of c+MG under
on the
Consequently, to RNA synthesis,
by RC gene.
a non-metabolizable under
acid
1968).
dependent
limited
that
to amino
unpublished).
observation
also
and Prestidge,
Nakao and Kaziro,
recent
is
our
the
demonstrated
subject
be regulated
complement for
Pardee
was shown to be also
might
that
the transport
of glucose control
permeation
in E. coli.
amino acid in the
starvation
absence
synthesis, conditions
of
addition of amino
strains. MATERIALS
The strains
(Sokawa,
As in the case restores
responsible 1955;
fraction
by RC gene is not
to report
strains
acids.
lipid
reactions
(a-MG),
is
of a full
we have recently
Nakao and Kaziro,
control
we wish
strains
into
of E. coli
(Sokawa,
Rglucoside
The stringent
required
that
However,
of carbohydrate
biosynthetic
(Kepes
whereas
strains
chromosome
Ryan and Rockenbach,
C-acetate
acids
was suggested
and many other
Science, Tokyo.
on the presence
"RC!" on its
1961).
synthesis
of amino
is dependent
(Borek,
14
of
in stringent
Moreover,
acid
locus
and Brenner,
incorporation
control
it
in E. coli
of RNA synthesis
1956; the
acids
of Medical Minatoku,
2, 1968
RNA synthesis of amino
Institute Takanawa,
Kaziro
AND METHODS
medium used
99
in the present
study
were
described
of
Vol. 34, No. 1, 1969
previously
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(Sokawa
et al.,
1968).
leucine
and threonine
as well
relaxed
one requiring
methionine.
The cells harvested
were grown
as thiamine
cells
were
for
strain
and washed resuspended
twice
in the
of E. coli
growth.
with
with
W677 requires
The strain
in M9 medium supplemented
by centrifugation
The washed
The stringent
58-161
necessary
is
a
requirements,
M9 medium without
glucose.
above medium to a density
of 3-5 x lo8
cells/ml.
cell
The uptake
of l4 C-a-MG
suspension
were
was measured
added
1 wnole
glucoside
(glucose-UL-14C)
Florida),
and 50 ug each of the
specified.
(2.4
The final
shaking
volume
HA filters
required
The filters
and the
radioactivities
a toluene-based
were
counting
were
were
0.025
times,
twice
with
dried
in liquid
of a-methyl
After
at various
washed
above D-
works,
Orlando,
and 25 ug of CM when
to 1.0 ml.
immediately
counted
wnole
ml of the
chemical
amino acids
were taken cells
To 0.85
(Mallinchrodt
was adjusted
and the
room temperature.
of D-glucose,
UC/ umole)
at 37O, 0.2 ml aliquots
Millipore
as follows:
incubation
with
filtered
through
2 ml of M9 medium at
under
an infrared
scintillation
lamp
spectrometer
using
mixture. RESULTS
Figure CM on the RC
str
uptake
of
14
effect
C-a-MG
) and 5%161(Met-,
ct-MG into
the
by removal hand,
cells
of the
a relaxed
absence with
1 shows the
of methionine
B1 - ' RCStr) It required
(Fig.
strains
amino acids
The same stimulatory
that
effect
leucine
Thr-,
the
RC locus:
Arg-,
CM is able
l-A,
His-,
B1-,
to release
stringent
100
the transport completely
of
abolished
On the
amount
other
of a-MG in the
same results CP 78(Leu-,
were Thr-,
obtained Arg-,
His-,
RCrel). in the
(Aronson
and Spiegelman,
synthesis
of
Bl-,
RNA synthesis
strains
of CM on lipid
Thr-,
and threonine.
considerable
except
end of addition
W677(Leu-,
in Fig.
Essentially
1-B).
in E. coli
auxotrophs
acid
W677 was almost
accumulated
isogenic
and CP 79(Leu-,
was reported
strain
amino acids,
58-161
of amino
As indicated
by a stringent required
removal
by two E. coli
RCrel).
strain
two E. coli
of the
was observed
absence
in amino
of 1961). acid-
BIOCHEMICAL
Vol. 34, No. 1, 1969
AND. BIOPHYSICAL RESEARCH COMMUNICATIONS
2.5 x-7 I 2" 2.0 & 2 P 1.5 A 2 j 1.0 3"
0.5
0
20 10 Tlme(min)
0
0
30
10
20
30
Tlme(mln)
Fig. 1. Effect of removal of amino acid and addition of chloramphenicol (CM) on o-methyl ILglucoside(oc-MG) uptake by the stringent and relaxed strains of E. coli. (A) and (a): W677 (stringent strain), requiring leucine and threonine; (B) and (b): 58-161 (relaxed strain), requiring methionine. For details see text. 3 in the presence of amino acids 0 0 -A
*
A-A
starved on the required by the
culture uptake amino addition
in the
of CM in the presence of CM in the
strains
the uptake
relaxed
strain
with
58-161
(Fig.
of methionine
at about
half
result
further
in the
in Figs.
of amino
1968).
et al., l-a
same level
l-b),
the
of the
acids
uptake
(Fig.
rate.
101
The effect
l-a).
and the
of CM
absence
of
W677 was elevated to the
control
In the
case of
of CLMG continued
maximum rate,
of the
strain
as when CM was added
amino
acids
In the
and l-b.
of a-MG by stringent
required
increase
of amino acids
absence
(Sokawa
illustrated
of CM to the
supplemented
acids
addition
of a-MG is
culture
of amino
addition
of stringent
acids
absence
addition
in the
absence
of CM did
not
Vol. 34, No. 1, 1969
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DISCUSSION In a previous is
starved
report,
of amino
1968).
acids,
The addition
level
glucose stimulatory These
effect
control
is
is
not
The fact regulation
on the
also
substrates
is
a sole
carbon of glucose
reactions
in the
of a-MG,
the
into
cellular
that
the
recent
necessarily
report
system
The possibility, during
starvation
failure the
pool other
however, of amino
of the transport
biosynthetic
is
acids
serves
we have
than
of glucose
of
of the
cause
and leads
entry
Since
that
the
are also
source
impairment
only
entry
that
as well
the
than
to the
general
trans-
biosynthetic
of RNA precursors acid
suggests
RC gene control. the decrease
rather
as
the transport
of amino under
in the
of the
of all
measured
are under
of extra-
acids.
inhibition
to be examined
is the
same
synthesis
in E. coli the
by deprivation
glucose
remains
that
the
so far
(1968)
blocked
of
the
stringency
as an energy
in the
by Nierlich
that
of amino
synthesis,
result
Although
nucleotide
transpcrt
glucose
macromolecular
acids.
synthesis. the
reactions
absence
study,
amino
same
of RNA.
inhibited
cells.
required
that
indicate
biosynthetic
in the
to the
CM displayed
observation
by assuming
for
would
all,
et al.,
of OC-MG and therefore
can be explained
present
source
not
(Sokawa
of c+MG as on RNA and lipid
same control,
of E. coli
synthesis
with
Here again
to the synthesis if
of lipid
transport
control.
strain
reduced
supplemented
an unpublished the
most,
medium used in the
port
under
activity
the
transport
restricted that
the
that
when a stringent is extremely
culture
acid
with
by RC gene,
cellular
to the
amino
together
carbohydrate
synthesis
demonstrate
under
results,
lipid
added
findings
is also
was shown that
of CM elevated
as when CM is
The present
it
the
result impairment
in ATP of the of all
reactions. SUMMARY
The transport
of a-MG was examined
E. coli.
The stringent
starvation
whereas
strain
the relaxed
failed strain
in the
stringent
to accumulate continued
and relaxed c+MG during
to uptake
amino
o(-MG in the
strains acid absence
of
Vol. 34, No. 1, 1969
of the
required
transport
of a-W
strain.
These
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
amino under results
acid.
Furthermore,
conditions indicate
the
of amino that
the
addition
acid
transport
starvation
of CM restored in the
of o-MG is under
the
stringent the
rule
of RC gene in E. coli. REFERENCES Aronson, A.I., and Spiegelman, S., Biuchim. Biophys. Acta, 2, 70 (1961). Borek, E ., Ryan, A., and Rockenbach, J., J. Bacterial., 69, 460 (1955). Keoes. ed. I.C. Gunsalus and R.Y. Stanier, ^ , A.*, and Cohen, G.N., in The Bacteria, Vo1.4, p.179-222 (1962), New York and London, Academic Press. Nierlich, D.P., Proc. Natl. Acad. Sci. U.S., 60, 1345 (1968). Pardee, A.B., and Prestidge, L-S., J. Bacterial., 7l, 677 (1956). Sokawa, Y., Nakao, E., and Kaziro, Y., Biochem. Biophys. Res. Commun., 33, 108 (1968). Stent, G.S., and Brenner, S., Proc. Natl. Acad. Sci. U.S., 47, 2005 (1961).
103
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
AMINO ACID-DEPENDENT CONTROL OF THE TRANSPORT OF a-METHYL GLUCOSIDE IN E. COLI
Yoshihiro
Sokawa
and Yoshito
Department of Chemistry, University of Tokyo,
Received
December
control
Stent
and the
the
presence
In this of a-methyl system
paper
the
the
and Cohen,
relaxed
amino
chloramphenicol(CM) starvation
1962),
fail
in stringent
and culture
is
analog
14
to accumulate continue
amino
acid
C-c(-MG during
to uptake
14
C-a-MG
of RNA and lipid
the transport
of c+MG under
on the
Consequently, to RNA synthesis,
by RC gene.
a non-metabolizable under
acid
1968).
dependent
limited
that
to amino
unpublished).
observation
also
and Prestidge,
Nakao and Kaziro,
recent
is
our
the
demonstrated
subject
be regulated
complement for
Pardee
was shown to be also
might
that
the transport
of glucose control
permeation
in E. coli.
amino acid in the
starvation
absence
synthesis, conditions
of
addition of amino
strains. MATERIALS
The strains
(Sokawa,
As in the case restores
responsible 1955;
fraction
by RC gene is not
to report
strains
acids.
lipid
reactions
(a-MG),
is
of a full
we have recently
Nakao and Kaziro,
control
we wish
strains
into
of E. coli
(Sokawa,
Rglucoside
The stringent
required
that
However,
of carbohydrate
biosynthetic
(Kepes
whereas
strains
chromosome
Ryan and Rockenbach,
C-acetate
acids
was suggested
and many other
Science, Tokyo.
on the presence
"RC!" on its
1961).
synthesis
of amino
is dependent
(Borek,
14
of
in stringent
Moreover,
acid
locus
and Brenner,
incorporation
control
it
in E. coli
of RNA synthesis
1956; the
acids
of Medical Minatoku,
2, 1968
RNA synthesis of amino
Institute Takanawa,
Kaziro
AND METHODS
medium used
99
in the present
study
were
described
of
Vol. 34, No. 1, 1969
previously
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(Sokawa
et al.,
1968).
leucine
and threonine
as well
relaxed
one requiring
methionine.
The cells harvested
were grown
as thiamine
cells
were
for
strain
and washed resuspended
twice
in the
of E. coli
growth.
with
with
W677 requires
The strain
in M9 medium supplemented
by centrifugation
The washed
The stringent
58-161
necessary
is
a
requirements,
M9 medium without
glucose.
above medium to a density
of 3-5 x lo8
cells/ml.
cell
The uptake
of l4 C-a-MG
suspension
were
was measured
added
1 wnole
glucoside
(glucose-UL-14C)
Florida),
and 50 ug each of the
specified.
(2.4
The final
shaking
volume
HA filters
required
The filters
and the
radioactivities
a toluene-based
were
counting
were
were
0.025
times,
twice
with
dried
in liquid
of a-methyl
After
at various
washed
above D-
works,
Orlando,
and 25 ug of CM when
to 1.0 ml.
immediately
counted
wnole
ml of the
chemical
amino acids
were taken cells
To 0.85
(Mallinchrodt
was adjusted
and the
room temperature.
of D-glucose,
UC/ umole)
at 37O, 0.2 ml aliquots
Millipore
as follows:
incubation
with
filtered
through
2 ml of M9 medium at
under
an infrared
scintillation
lamp
spectrometer
using
mixture. RESULTS
Figure CM on the RC
str
uptake
of
14
effect
C-a-MG
) and 5%161(Met-,
ct-MG into
the
by removal hand,
cells
of the
a relaxed
absence with
1 shows the
of methionine
B1 - ' RCStr) It required
(Fig.
strains
amino acids
The same stimulatory
that
effect
leucine
Thr-,
the
RC locus:
Arg-,
CM is able
l-A,
His-,
B1-,
to release
stringent
100
the transport completely
of
abolished
On the
amount
other
of a-MG in the
same results CP 78(Leu-,
were Thr-,
obtained Arg-,
His-,
RCrel). in the
(Aronson
and Spiegelman,
synthesis
of
Bl-,
RNA synthesis
strains
of CM on lipid
Thr-,
and threonine.
considerable
except
end of addition
W677(Leu-,
in Fig.
Essentially
1-B).
in E. coli
auxotrophs
acid
W677 was almost
accumulated
isogenic
and CP 79(Leu-,
was reported
strain
amino acids,
58-161
of amino
As indicated
by a stringent required
removal
by two E. coli
RCrel).
strain
two E. coli
of the
was observed
absence
in amino
of 1961). acid-
BIOCHEMICAL
Vol. 34, No. 1, 1969
AND. BIOPHYSICAL RESEARCH COMMUNICATIONS
2.5 x-7 I 2" 2.0 & 2 P 1.5 A 2 j 1.0 3"
0.5
0
20 10 Tlme(min)
0
0
30
10
20
30
Tlme(mln)
Fig. 1. Effect of removal of amino acid and addition of chloramphenicol (CM) on o-methyl ILglucoside(oc-MG) uptake by the stringent and relaxed strains of E. coli. (A) and (a): W677 (stringent strain), requiring leucine and threonine; (B) and (b): 58-161 (relaxed strain), requiring methionine. For details see text. 3 in the presence of amino acids 0 0 -A
*
A-A
starved on the required by the
culture uptake amino addition
in the
of CM in the presence of CM in the
strains
the uptake
relaxed
strain
with
58-161
(Fig.
of methionine
at about
half
result
further
in the
in Figs.
of amino
1968).
et al., l-a
same level
l-b),
the
of the
acids
uptake
(Fig.
rate.
101
The effect
l-a).
and the
of CM
absence
of
W677 was elevated to the
control
In the
case of
of CLMG continued
maximum rate,
of the
strain
as when CM was added
amino
acids
In the
and l-b.
of a-MG by stringent
required
increase
of amino acids
absence
(Sokawa
illustrated
of CM to the
supplemented
acids
addition
of a-MG is
culture
of amino
addition
of stringent
acids
absence
addition
in the
absence
of CM did
not
Vol. 34, No. 1, 1969
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DISCUSSION In a previous is
starved
report,
of amino
1968).
acids,
The addition
level
glucose stimulatory These
effect
control
is
is
not
The fact regulation
on the
also
substrates
is
a sole
carbon of glucose
reactions
in the
of a-MG,
the
into
cellular
that
the
recent
necessarily
report
system
The possibility, during
starvation
failure the
pool other
however, of amino
of the transport
biosynthetic
is
acids
serves
we have
than
of glucose
of
of the
cause
and leads
entry
Since
that
the
are also
source
impairment
only
entry
that
as well
the
than
to the
general
trans-
biosynthetic
of RNA precursors acid
suggests
RC gene control. the decrease
rather
as
the transport
of amino under
in the
of the
of all
measured
are under
of extra-
acids.
inhibition
to be examined
is the
same
synthesis
in E. coli the
by deprivation
glucose
remains
that
the
so far
(1968)
blocked
of
the
stringency
as an energy
in the
by Nierlich
that
of amino
synthesis,
result
Although
nucleotide
transpcrt
glucose
macromolecular
acids.
synthesis. the
reactions
absence
study,
amino
same
of RNA.
inhibited
cells.
required
that
indicate
biosynthetic
in the
to the
CM displayed
observation
by assuming
for
would
all,
et al.,
of OC-MG and therefore
can be explained
present
source
not
(Sokawa
of c+MG as on RNA and lipid
same control,
of E. coli
synthesis
with
Here again
to the synthesis if
of lipid
transport
control.
strain
reduced
supplemented
an unpublished the
most,
medium used in the
port
under
activity
the
transport
restricted that
the
that
when a stringent is extremely
culture
acid
with
by RC gene,
cellular
to the
amino
together
carbohydrate
synthesis
demonstrate
under
results,
lipid
added
findings
is also
was shown that
of CM elevated
as when CM is
The present
it
the
result impairment
in ATP of the of all
reactions. SUMMARY
The transport
of a-MG was examined
E. coli.
The stringent
starvation
whereas
strain
the relaxed
failed strain
in the
stringent
to accumulate continued
and relaxed c+MG during
to uptake
amino
o(-MG in the
strains acid absence
of
Vol. 34, No. 1, 1969
of the
required
transport
of a-W
strain.
These
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
amino under results
acid.
Furthermore,
conditions indicate
the
of amino that
the
addition
acid
transport
starvation
of CM restored in the
of o-MG is under
the
stringent the
rule
of RC gene in E. coli. REFERENCES Aronson, A.I., and Spiegelman, S., Biuchim. Biophys. Acta, 2, 70 (1961). Borek, E ., Ryan, A., and Rockenbach, J., J. Bacterial., 69, 460 (1955). Keoes. ed. I.C. Gunsalus and R.Y. Stanier, ^ , A.*, and Cohen, G.N., in The Bacteria, Vo1.4, p.179-222 (1962), New York and London, Academic Press. Nierlich, D.P., Proc. Natl. Acad. Sci. U.S., 60, 1345 (1968). Pardee, A.B., and Prestidge, L-S., J. Bacterial., 7l, 677 (1956). Sokawa, Y., Nakao, E., and Kaziro, Y., Biochem. Biophys. Res. Commun., 33, 108 (1968). Stent, G.S., and Brenner, S., Proc. Natl. Acad. Sci. U.S., 47, 2005 (1961).
103