Microbiol. Immunol. Vol. 36 (11), 1119-1128, 1992
Melibiose
Transport Chiyuki
System in Lactobacillus
TAMURA, * and
Osamu
plantarum
MATSUSHITA
Department of Microbiology,Okayama UniversityMedical School, Okayama, Okayama 700, Japan (Accepted for publication, August 28, 1992)
Abstract at
Lactobacillus
37
C,
8014
grown
In
30
C,
least
3
hr
not at
37
than
the
inducer
failed
to
synthesis,
when a
no
was
that
C,
was
previous
the
be
can
the
which 30 both
transport system induced
described transport
C, but 30
by
L. plantarum (13)
the
the
induced C
and
activity was
in below
active melibiose
37 was at
to
lactose C.
4)
both at
1)
30 30
C was
30 the
30 C
the
C, and stable
transport
melibiose
raffinose
strain
30
C,
the
37
strain not
37
C. for
111 9
C.
was at
but
6) at
least
not
8014,
of
L.
and
(EC by
on
The
5)
hr
at
37
C.
37
revealed
only active
melibiose
melibiose transport C.
2.3.1.22),
was
melibiose,
The
melibiose 3
the
consisted
melibiose
3) ƒ¿-Galactosidase
C.
at
plantarum
induced
grown
37
of
ATCC
temperature-sensitive
ATCC
was
One
L. plantarum
2) ƒ¿-Galactosidase
galactose,
but
system;
(10). in
at of
metabolism
C or
system studies
transport
sugars
sugars
wild-type
and
When at
the
mechanism
processes.
glucose
in
essentially
Inhibition
and
Melibiose
at either
induced
on
the
is
and ƒ¿-galacto-
melibiose
of these
grew
hydrolysis
sugar by
it
enzyme lactose
raffinose.
melibiose
with
.
of and
utilization
i.e.,
and
hydrolyzes
substrate
ferment
by
enzyme system
induced
lactose
analyzed
also
at
organism
The
(and the
for rather
The
melibiose.
of
melibiose
stable
cytoplasm
for ƒ¿-galactosidase.
weak
be
o-nitrophenyl-ƒ¿-D-galactopyranoside
Raffinose
the
the
transport
and
by to
mutant transport
induced
21
induction
melibiose
inducer
a
that
Tamura
utilization
facts
C.
with
(13)
paper,
melibiose
30
may
is temperature-sensitive, a
21,
good
demonstrated
described
the
NTG
lactose-
The
organism. on
that
A
melibiose
in
not
suggesting
found
the
grown in
melibiose
a
plantarum
have
when
that
NTG
at
was
in C
galactose
indicating
grown it
37
by
galactose,
galactose
Lactobacillus
at
was
process
temperature-sensitive
In
while
inhibition
authors
at
37
induction
but ATCC
isolated.
was
itself
C,
system.
normal
activity
activity the
slowly,
was
retained
transport
that
induced
by
strain
little,
revealed
at
at
induced
the
only
of
was
8014
but
30
temperature.
transport
ATCC
transport
is
lactose
at
either
melibiose
a
from
cx-galactosidase
even
were
by
melibiose
at
accumulated
melibiose
temperature-sensitive.
sidase
C
on
lactose
transport,
The
grew
or
derived
the C.
entrance form
8014)
37
suggesting
however,
ATCC
In
C,
or
lactose
21, 37
8014
galactose
transported 21,
in
at
ATCC
on 30
NTG
NTG
but
also
8014
be
deficient
activity.
not
at
partly
mutant, totally
grew
lactose
may
negative
at
it
on
melibiose
was
plantarum
although
These
transport system findings
112 0
C. TAMURA
strongly
suggest
that
galactosidase, may
is
not
grown at
on
transport to
C
the
by
in
NTG
21,
lactose
from
2A).
as
in
transport
in
8014,
and
detail,
it
In this
the
substrate
7,
is
this
using
including
(4,
strain
actually due
the
11,
obtain
a
isolated
some and
There-
to we
mutant,
lactose
14).
desirable
paper,
was
induced
to
9,
of ƒ¿-
enzymes
the
was
coli
not
these
when
probably
E.
system.
such
characteristics
inducer
specificities
investigated.
MATERIALS Bacterial
strains
plantarum
with
30
C
8014
1%
cells
the
experiments.
were
extract,
ISL
3 g of
tryptose,
0.12
g of
For
making
agar,
EMBL-ISL
0.04%
eosin of
absorbance
grew
slightly
in of
exceeded
ISL
20 the
mm
a
buffer,
stationary
not
an
at
37
Assay
a final
C.
EMBL-ISL
cells.
for
The agar
fermenting
whole
C in
ISL
Cells
concentration
was
C
was After
isolated,
of
washed 1 mg
30
serially
and
min,
the
1.5%
plantarum
by
utilizing
the
turbidity
carbohydrate.
8014
was
with
grown
to
glucose,
in
the
diluted
suspended cells
per
at
same
C
washed and
the for
NTG
72
volume
2 ml The
of assay
buffer, hr,
with
and a
the
incubated spread
white
colony
21.
of ƒ¿-galactosidase in
ml.
were glucose,
with 30
the
washed
(NTG).
cells with
1 %.
monitoring
when
suspended
designated
Activity and
wet
for
incubation
of ƒ¿-galactosidase. were
with
by
any
supplemented
6.8
of
L.
supplemented and
pH
lactose.
only
ATCC
6.0),
FeSO4.
to
carbohydrate +
for yeast
citrate, g of
adjusted
measured
supplementing
fermentation.
(pH
37
culture
1%
any as
of
N-methyl-N'-nitro-N-nitrosoguanidine
medium
plate.
lactose activity
at
with
g
triammonium
spectrophotometer.
expressed
used
5
supplemented
was
growth,
4 C and
concentration
and
growth
medium
of
at
was
was
incubated of
0.034
final
lactose supple-
and
peptone,
medium
blue
U-2000
buffer
ISL
10 min
a
in
phase
2 g of
at
Lactobacillus
medium
medium,
MgSO4•2H20,
medium
was
ISL
trypticase
The
without
200ƒÊg/m1
into
of
supplemented
lactose
incubation
inoculated
Hitachi
in
phosphate
containing
overnight on
sodium
37
g
g of
ISL
culture
deficient
fresh
were deficient
C in
x g for
methylene
Growth
at
2,500
Bacterial
a not
control
phase
buffer
After
medium
a
liter.
plates,
in
the
carbohydrate
growth.
nm
mutants
per
was
37
3 g of KH2PO4, 0.575
study
mid-exponential
10
80,
0.0065%
medium.
of of
mid-exponential with
650
the
that
Isolation
agar
bacterial
at
components
desired
yellow,
Measurement the
at
contained
2H20 a
with
this
which at
the
centrifugation
MnSO4. with
21,
times
3 g of K2HPO4,
supplemented
in
overnight
During
1 g of Tween
used
NTG
1,000
medium
acetate,
and
derivative
stated. by
Strains
precultured
diluted
harvested
1 g of sodium 7H20
its
were
otherwise
AND METHODS
conditions.
and
glucose,
unless
the
and
growth
Bacteria
mented at
and
ATCC
fermentation.
of
of
for
hand,
is
but
coding
activity
case
system
system
other
This
the
system,
genes
transport
Fig.
ATCC
transport
that the
melibiose
also
transport
the
transport
hence On
lactose
melibiose
melibiose
were
of
see
MATSUSHITA
melibiose
operon.
level
(13;
induced
deficient
the
of and
same
low
37
study
mutant,
of
a
or
system
mutant a
the
lactose, 30
induction
temperature-sensitive,
constitute
either
fore,
the
AND O.
an
was assay mixture
assayed
mixture contained
to
with obtain 1 mm
MELIBIOSE
TRANSPORT
o-nitrophenyl-a-D-galactopyranoside (pH
7.0).
2 ml
of
After 0.5
NML
liberated 420 per
per
mg
of
according
to
solution
of
buffer
phosphate
column at
(pH
(1.6
a
of
flow
cm
rate
of
authentic
20
collected,
diluted
wet to
per
give
At
a
final
(0.65 ƒÊm
pore
of
the
Tesque,
Inc.),
was
All
from
other
the
added
Sartorius
o-nitrophenol at liberated
and
the in
reduced an
invertase
Pont
chemicals
Fine
were
at
4 C . by
to was an
filter
was
scintillator in
a
from
Chemicals.
preliminary
melibiose
and
the
50
mm
containing was 1 .5
mg
cell
suspension
incubated MF-Millipore quickly
grade
and
were
at
30 C. filter
washed I
with (Nacalai counter
.
(TMG)
,
Co ., Sephadex
and
Amersham
were
experiments. with
Chemical
[3H]raffinose
Products
special
Sigma
7 .0)
positions
, Clear Sol scintillation
liquid
G-15 (pH
The
ISL medium suspension
added
inactivate
water
to
mixture through
measured
to
Sephadex
distilled
in this
of
The
purchased
Research of
a
for
M potassium
3 min
to
acetate
methyl-ƒÀ-D-thiogalactoside
were
NEN
0.5
determined
was
emulsifying
was
of
for
aqueous
incubated
, and used for the Cells were washed
lactose.
mm
M sodium
corresponding
pressure.
of
ml
with
[3H]raffinose
0.5
and
applied
were
, and the and filtered
taken
2 ml
0 .6
collected
[14C]lactose
100 ƒÊm
0 .2
bath
were
of
3 .2.1.26)
was
fructose
melibiose
from ml of
with
Fractions
or
Pharmacia Du
adding
spectrophotometer
: 0 .3
eluted
fractions
radioactivity
and from
obtained
a
of
prepared
(EC
NTG, ƒ¿-ONPG, ƒÀ-0NPG,
chloramphenicol
buffer by
o-nitrophenol
volume
melibiose
of
under
same
and
and
were
soaked
and
phosphate stopped through
of
(14)
(pH 7.0), and suspended Final concentration
aliquots
size)
buffer,
Chemicals.
G-15
of
buffer
al
mixture
column.
[3H]melibiose
100 ƒÊl
filtration Absorbance
a boiling-water
the
2 ml
same
concentration
intervals,
1 ml
and
uptake
ml.
in
ice,
non-radioactive
phosphate chloramphenicol.
cells
was
melibiose
of
by .
was
the
pre-equilibrated
the
with
Measurement potassium 50ƒÊg/ml
was
U-2000
invertase
heated on
ml/hr,
on
sodium
as ƒÊmoles
et
with
mixture
and
cm)
raffinose,
chromatography
mm
reaction
Hitachi
Tanaka
mixed
cooling
x 90
of
the
7.0)
After
a
expressed
100 ƒÊg/m1
Then
buffer
enzyme.
size)
[3H]melibiose
method was
C.
removed
in
was
20
the
pore
measured
containing
50
in
min,
were
melibiose.
modified
4.9)
at
cells
30
112 1
cells.
[3H]
[3H]raffinose
(pH min
a
C for
(0.45ƒÊm
activity
wet
of
30
the
was
specific
Preparation
the
and
membrane
The
min
90
at
from ƒ¿-ONPG
nm.
IN L. PLANTARUM
(ƒ¿-ONPG)
incubation
M Na2CO3,
Mini-sart
SYSTEM
[14C]lactose Japan
obtained
from
were
, respectively. commercial
sources.
RESULTS
Characterization
of
Mutant growth
on
sidase
under
ATCC
8014
only was
at
30
observed
NTG
Mutant 21
galactose, the grew C.
well
lactose respective on
Similar on
NTG as
lactose
21 as
the
and
parental
melibiose,
and
conditions
galactose
and
results even
30
C.
In
with ATCC
for
1).
at either
obtained
ATCC
also
(Table
lactose
were at
strain
8014 the
As 30
or
NTG
were
induction
reported 37
C 21
examined
for
of ƒ¿-galactopreviously
(13),
, but grew on melibiose except that no growth
8014, ƒ¿-galactosidase
was
induced
112 2
C. TAMURA
Table
Fig.
1. Cells
Lactose were
1.
at
MATSUSHITA
Growth and induction of a-galactosidase in L. plantarum ATCC 8014 and its mutant NTG 21
uptake grown
AND 0.
by 30
L. C
in
plantarum ISL
ATCC
medium
8014 supplemented
( •œ)
and with
its
mutant
lactose.
NTG Assayed
21 at
(•›). 30
C.
under all the growth conditions except that melibiose failed to induce the enzyme when the strain was grown at 37 C. In NTG 21, a-galactosidase was induced normally by galactose and melibiose, but practically not by lactose. Lactose uptake was measured with NTG 21 and ATCC 8014 grown on lactose at 30 C (Fig. 1). In contrast to ATCC 8014, NTG 21 scarcely accumulated lactose.
MELIBIOSE
TRANSPORT
SYSTEM
IN L. PLANTARUM
112 3
Melibiose transport activity was assayed with NTG 21 (Fig. 2B) and ATCC 8014 (Fig. 2A) grown on melibiose or lactose at 30 or 37 C. ATCC 8014 grown on melibiose at 30 C rapidly accumulated melibiose, but that grown at 37 C practically
A
B
Fig.
2.
Melibiose
uptake
Cells
were
grown
with
lactose
at
30
in
ISL
C
(•¡)
by
L.
plantarum
medium and
supplemented 37
C
(• ).
ATCC
8014 with
(A) melibiose
and
its at
30
mutant C
(•œ)
NTG and
21 37
C
(B). (•›);
112 4
C. TAMURA
Fig.
3. on
Fig.
Effect
of
melibiose.
4. Cells
temperature
Assayed
Stability were
supplemented
removed
as
at
hr, 30
and
a
C
50
mm
with
for
expressed
melibiose
( •œ)
and
transport
with
control,
assayed
C were
30
of melibiose
washed
medium
3
on
at
AND 0.
and
the
melibiose by
per
transport 37
C
system
remainder uptake. cent
in
NTG
phosphate
NTG
was Amounts
21
buffer
chloramphenicol.
of control.
mutant
21
grown
at
30
C
(•›).
of mutant
potassium
50 ƒÊg/m1
MATSUSHITA
A
incubated of
at
grown 7.0),
part
of
37
[3H]melibiose
at
(pH
C,
30
C
on
melibiose.
suspended this
sampled accumulated
in
suspension at
0,
ISL was
1, 2 in
4
and min
MELIBIOSE
TRANSPORT
SYSTEM
IN L. PLANTARUM
112 5
did not. Similar results were obtained with NTG 21. Low levels of melibiose uptake were observed with ATCC 8014 grown on lactose at 30 or 37 C. Such activities were not detected in NTG 21 grown on lactose, suggesting that these activities are due to the lactose transport system which is intact in ATCC 8014. All these data indicate that NTG 21 is deficient in lactose transport system which may cross-react with melibiose, but retains normal melibiose transport activity .
Effectsof Temperatureon Melibiose TransportSystem Melibiose uptake by NTG 21 grown on melibiose at 30 C was measured at 30 and 37 C (Fig. 3). The initial rates of uptake under the two conditions were very similar to each other, suggesting that the transport system has a broad temperature optimum ranging from 30 to 37 C. The transport system was quite stable at 37 C for at least 3 hr (Fig. 4). Inhibition of Melibiose Transport by Various Carbohydrates Inhibition NTG
21
of
grown
significantly
by
inhibition
was
of
raffinose,
II
of
E.
Inducer
(Table melibiose, the
same
strain of
was
melibiose
raffinose
30
7, 9,
were
11,
melibiose
and
NTG
In ATCC raffinose, ƒ¿-ONPG except
that
deficient
appeared
Table
to
2.
21
lactose
transport be
carbohydrates
2).
was
Melibiose
not
by
similar
and
was
that
Melibiose
tested
tested was
and
TMG.
observed
Transport in
raffinose With
with
.
the TMG
with
inhibited Some
exception permease
with
system
was inducer
did
for
to the
as
well
as
of ƒ¿-galactosidase
carbohydrates induced with
not
activity. similar
21
various was Results
and ƒÀ-ONPG
System
NTG
a-galactosidase and ƒÀ-0NPG.
transport
Inhibition mutant
to
system
lactose
a poor
lactose or
was uptake
14).
transport
8014,
in
various
(Table
practically
of ƒ¿-Galactosidase of
by C
with ƒ¿-ONPG, ƒÀ-0NPG
profiles (4,
8014 3).
but
observed
K-12
Specificity
ATCC
at
galactose,
inhibitor coli
transport
melibiose
also
Induction in
melibiose
on
by NTG
induce In
that transport
as
the
the
galactose, 21 were enzyme
NTG
21 , inducer of ƒ¿-galactosidase
lactose, essentially since
the
specificity except that
system.
of melibiose transport by various carbohydrates NTG 21 grown at 30 C on melibiose
inducers
in
112 6
C. TAMURA
Table 3.
AND 0.
MATSUSHITA
Inducer specificity of cc-galactosidase and melibiose transport system in parental (ATCC 8014) and mutant (NTG 21) strains
DISCUSSION
Melibiose metabolism has been studied in some detail with Escherichia coli K-12. The melibiose system of E. coli consists of its transport and hydrolysis (8). α-Galactosidase,
which
hydrolyzes
the
sugar
to glucose
and
galactose,
is induced
and active at either 30 or 37 C (12). In E. coli, melibiose is transported via two distinct systems, namely, thiomethylgalactoside (TMG) permease I (lactose permease), coded by lacY gene in the lactose operon (6) and TMG permease II (9). The former is induced by lactose and melibiose, and stable at 37 C, while the latter is induced by melibiose, and unstable at the temperature (4, 9, 11, 15). Therefore, wild-type E. coli K-12 can grow on melibiose at either 30 or 37 C since melibiose is taken up by TMG permease I, even when TMG permease II is inactivated at 37 C. On the other hand, mutants deficient in TMG permease I, cannot grow on melibiose at 37 C although they grow normally at 30 C (1, 2). Genes coding for α-galactosidase and TMG permease II are components of the same operon (5). In contrast to E. coli K-12, in spite of possessing both lactose and melibiose transport system, even a wild-type strain L. plantarum ATCC 8014 failed to grow on melibiose at 37 C. The same strain grew on lactose and galactose at either temperature (13; see also Table 1). When grown at 30 C on melibiose, a mutant NTG 21, deficient in lactose transport system, took up melibiose equally at either 30 or 37 C (Fig. 3), and the melibiose transport system was stable for at least 3 hr at 37 C (Fig. 4). These results are consistent with that observed in ATCC 8014 (13), and provide the evidence that L. plantarum differs from E. coli K-12 in that the
MELIBIOSE
TRANSPORT
SYSTEM
IN
L. PLANTARUM
112 7
induction of melibiose transport system is temperature-sensitive (Fig. 2) although the transport system itself is stable at 37 C (Fig.4). Lactose transport system of L. plantarum might not be induced by melibiose since, as mentioned above, even a wild-type strain ATCC 8014 failed to grow on melibiose at 37 C, while the transport system seems to be induced at either temperature and transport melibiose (Fig. 2A). In contrast to the melibiose transport system, a-galactosidase of L. plantarum was induced at 37 C when galactose or lactose served as the inducer (Table 1). Therefore, it is highly probable that the syntheses of these two proteins are subjected to separate genetic controls. In NTG 21, raffinose induced the melibiose transport system only a little, while it was a good inducer for a-galactosidase. This observation that the inducer specificities for these proteins are not exactly the same might favor the hypothesis. However, it was reported that cx-galactosidase synthesis in E. coli organisms is controlled not only by the melibiose operon in the genome DNA, but also by the raffinose operon in a plasmid (3). Therefore, more biochemical and genetic works will be needed to know the genetic control for the melibiose metabolism. Wewouldliketo thankProf.Y. Kanemasaforthe valuableadviceand encouragementthroughout this study. REFERENCES
1) Beckwith, J. 1964. Restoration of operon activity by suppressors. Biochim. Biophys. Acta 76: 162-164. 2) Beckwith, J. 1964. A deletion analysis of the lac operator region in Escherichia coli. J. Mol. Biol. 8: 427-430. 3) Burkardt, H. J., Mattes, R., Schmid, K., and Schmitt, R. 1978. Profiles of two conjugative plasmids mediating tetracycline resistance, raffinose catabolism and hydrogen sulfide production in Escherichiacoli. Mol. Gen. Genet. 166: 75-84. 4) Burstein, C., and Kepes, A. 1985. The melibiose permease system of Escherichia coli K12. Biochimie 67: 59-67. 5) Hanatani, M., Yazyu, H., Shiota-Niiya, S., Moriyama, Y., Kanazawa, H., Futai, M., and Tsuchiya, T. 1984. Physical and genetic characterization of the melibiose operon and identification of the gene products in Escherichia coli. J. Biol. Chem. 259: 1807-1812. 6) Jacob, F., and Monod, J. 1958. Genetic and physical determination of chromosomal segments in Escherichia coli. Symp. Soc. Exp. Biol. 12: 75-92. 7) Lopilato, J., Tsuchiya, T., and Wilson, T.H. 1978. Role of Na+ and Li+ in thiomethylgalactoside transport by the melibiose transport system of Escherichia coli. J. Bacteriol. 134: 147-156. 8) Pardee, A.B. 1957. An inducible mechanism for accumulation of melibiose in Escherichia coli. J. Bacteriol. 73: 376-385. 9) Prestidge, L.S., and Pardee, A.B. 1965. A second permease for methyl-thio-j9-n-galactoside in Escherichiacoli. Biochim. Biophys. Acta 100: 591-593. 10) Rogosa, M. 1974. Genus I. Lactobacillus, p. 576-593. In Buchanan, R. E., and Gibbons, N.E. (eds), Bergey's manual of determinative bacteriology, 8 ed, The Williams and Wilkins Company, Baltimore. 11) Rotman, B., Ganesan, A.K., and Guzman, R. 1968. Transport systems for galactose and galactosides in Escherichia coli. II. Substrate and inducer specificities. J. Mol. Biol. 36: 247-260. 12) Schmitt, R., and Rotman, B. 1966. oc-Galactosidase activity in cell-free extracts of Escherichia coli. Biochem. Biophys. Res. Commun. 22: 473-479.
112 8
C. TAMURA
AND O.
MATSUSHITA
13)
Tamura, C. 1991. Analysis on the mechanism of the temperature-sensitive melibiose metabolism in Lactobacillusplantarum. Okayama I. Z. 103: 101-110 (in Japanese with English summary). 14) Tanaka, K., Niiya, S., and Tsuchiya, T. 1980. Melibiose transport in Escherichia coli. J. Bacteriol. 141: 1030-1036. 15) Tsuchiya, T., Lopilato, J., and Wilson, T.H. 1978. Effect of lithium ion on melibiose transport in Escherichia coli.J. Membrane Biol. 42: 45-59. (Received for publication, March 9, 1992; in revised form, August 24, 1992)
Melibiose
Transport Chiyuki
System in Lactobacillus
TAMURA, * and
Osamu
plantarum
MATSUSHITA
Department of Microbiology,Okayama UniversityMedical School, Okayama, Okayama 700, Japan (Accepted for publication, August 28, 1992)
Abstract at
Lactobacillus
37
C,
8014
grown
In
30
C,
least
3
hr
not at
37
than
the
inducer
failed
to
synthesis,
when a
no
was
that
C,
was
previous
the
be
can
the
which 30 both
transport system induced
described transport
C, but 30
by
L. plantarum (13)
the
the
induced C
and
activity was
in below
active melibiose
37 was at
to
lactose C.
4)
both at
1)
30 30
C was
30 the
30 C
the
C, and stable
transport
melibiose
raffinose
strain
30
C,
the
37
strain not
37
C. for
111 9
C.
was at
but
6) at
least
not
8014,
of
L.
and
(EC by
on
The
5)
hr
at
37
C.
37
revealed
only active
melibiose
melibiose transport C.
2.3.1.22),
was
melibiose,
The
melibiose 3
the
consisted
melibiose
3) ƒ¿-Galactosidase
C.
at
plantarum
induced
grown
37
of
ATCC
temperature-sensitive
ATCC
was
One
L. plantarum
2) ƒ¿-Galactosidase
galactose,
but
system;
(10). in
at of
metabolism
C or
system studies
transport
sugars
sugars
wild-type
and
When at
the
mechanism
processes.
glucose
in
essentially
Inhibition
and
Melibiose
at either
induced
on
the
is
and ƒ¿-galacto-
melibiose
of these
grew
hydrolysis
sugar by
it
enzyme lactose
raffinose.
melibiose
with
.
of and
utilization
i.e.,
and
hydrolyzes
substrate
ferment
by
enzyme system
induced
lactose
analyzed
also
at
organism
The
(and the
for rather
The
melibiose.
of
melibiose
stable
cytoplasm
for ƒ¿-galactosidase.
weak
be
o-nitrophenyl-ƒ¿-D-galactopyranoside
Raffinose
the
the
transport
and
by to
mutant transport
induced
21
induction
melibiose
inducer
a
that
Tamura
utilization
facts
C.
with
(13)
paper,
melibiose
30
may
is temperature-sensitive, a
21,
good
demonstrated
described
the
NTG
lactose-
The
organism. on
that
A
melibiose
in
not
suggesting
found
the
grown in
melibiose
a
plantarum
have
when
that
NTG
at
was
in C
galactose
indicating
grown it
37
by
galactose,
galactose
Lactobacillus
at
was
process
temperature-sensitive
In
while
inhibition
authors
at
37
induction
but ATCC
isolated.
was
itself
C,
system.
normal
activity
activity the
slowly,
was
retained
transport
that
induced
by
strain
little,
revealed
at
at
induced
the
only
of
was
8014
but
30
temperature.
transport
ATCC
transport
is
lactose
at
either
melibiose
a
from
cx-galactosidase
even
were
by
melibiose
at
accumulated
melibiose
temperature-sensitive.
sidase
C
on
lactose
transport,
The
grew
or
derived
the C.
entrance form
8014)
37
suggesting
however,
ATCC
In
C,
or
lactose
21, 37
8014
galactose
transported 21,
in
at
ATCC
on 30
NTG
NTG
but
also
8014
be
deficient
activity.
not
at
partly
mutant, totally
grew
lactose
may
negative
at
it
on
melibiose
was
plantarum
although
These
transport system findings
112 0
C. TAMURA
strongly
suggest
that
galactosidase, may
is
not
grown at
on
transport to
C
the
by
in
NTG
21,
lactose
from
2A).
as
in
transport
in
8014,
and
detail,
it
In this
the
substrate
7,
is
this
using
including
(4,
strain
actually due
the
11,
obtain
a
isolated
some and
There-
to we
mutant,
lactose
14).
desirable
paper,
was
induced
to
9,
of ƒ¿-
enzymes
the
was
coli
not
these
when
probably
E.
system.
such
characteristics
inducer
specificities
investigated.
MATERIALS Bacterial
strains
plantarum
with
30
C
8014
1%
cells
the
experiments.
were
extract,
ISL
3 g of
tryptose,
0.12
g of
For
making
agar,
EMBL-ISL
0.04%
eosin of
absorbance
grew
slightly
in of
exceeded
ISL
20 the
mm
a
buffer,
stationary
not
an
at
37
Assay
a final
C.
EMBL-ISL
cells.
for
The agar
fermenting
whole
C in
ISL
Cells
concentration
was
C
was After
isolated,
of
washed 1 mg
30
serially
and
min,
the
1.5%
plantarum
by
utilizing
the
turbidity
carbohydrate.
8014
was
with
grown
to
glucose,
in
the
diluted
suspended cells
per
at
same
C
washed and
the for
NTG
72
volume
2 ml The
of assay
buffer, hr,
with
and a
the
incubated spread
white
colony
21.
of ƒ¿-galactosidase in
ml.
were glucose,
with 30
the
washed
(NTG).
cells with
1 %.
monitoring
when
suspended
designated
Activity and
wet
for
incubation
of ƒ¿-galactosidase. were
with
by
any
supplemented
6.8
of
L.
supplemented and
pH
lactose.
only
ATCC
6.0),
FeSO4.
to
carbohydrate +
for yeast
citrate, g of
adjusted
measured
supplementing
fermentation.
(pH
37
culture
1%
any as
of
N-methyl-N'-nitro-N-nitrosoguanidine
medium
plate.
lactose activity
at
with
g
triammonium
spectrophotometer.
expressed
used
5
supplemented
was
growth,
4 C and
concentration
and
growth
medium
of
at
was
was
incubated of
0.034
final
lactose supple-
and
peptone,
medium
blue
U-2000
buffer
ISL
10 min
a
in
phase
2 g of
at
Lactobacillus
medium
medium,
MgSO4•2H20,
medium
was
ISL
trypticase
The
without
200ƒÊg/m1
into
of
supplemented
lactose
incubation
inoculated
Hitachi
in
phosphate
containing
overnight on
sodium
37
g
g of
ISL
culture
deficient
fresh
were deficient
C in
x g for
methylene
Growth
at
2,500
Bacterial
a not
control
phase
buffer
After
medium
a
liter.
plates,
in
the
carbohydrate
growth.
nm
mutants
per
was
37
3 g of KH2PO4, 0.575
study
mid-exponential
10
80,
0.0065%
medium.
of of
mid-exponential with
650
the
that
Isolation
agar
bacterial
at
components
desired
yellow,
Measurement the
at
contained
2H20 a
with
this
which at
the
centrifugation
MnSO4. with
21,
times
3 g of K2HPO4,
supplemented
in
overnight
During
1 g of Tween
used
NTG
1,000
medium
acetate,
and
derivative
stated. by
Strains
precultured
diluted
harvested
1 g of sodium 7H20
its
were
otherwise
AND METHODS
conditions.
and
glucose,
unless
the
and
growth
Bacteria
mented at
and
ATCC
fermentation.
of
of
for
hand,
is
but
coding
activity
case
system
system
other
This
the
system,
genes
transport
Fig.
ATCC
transport
that the
melibiose
also
transport
the
transport
hence On
lactose
melibiose
melibiose
were
of
see
MATSUSHITA
melibiose
operon.
level
(13;
induced
deficient
the
of and
same
low
37
study
mutant,
of
a
or
system
mutant a
the
lactose, 30
induction
temperature-sensitive,
constitute
either
fore,
the
AND O.
an
was assay mixture
assayed
mixture contained
to
with obtain 1 mm
MELIBIOSE
TRANSPORT
o-nitrophenyl-a-D-galactopyranoside (pH
7.0).
2 ml
of
After 0.5
NML
liberated 420 per
per
mg
of
according
to
solution
of
buffer
phosphate
column at
(pH
(1.6
a
of
flow
cm
rate
of
authentic
20
collected,
diluted
wet to
per
give
At
a
final
(0.65 ƒÊm
pore
of
the
Tesque,
Inc.),
was
All
from
other
the
added
Sartorius
o-nitrophenol at liberated
and
the in
reduced an
invertase
Pont
chemicals
Fine
were
at
4 C . by
to was an
filter
was
scintillator in
a
from
Chemicals.
preliminary
melibiose
and
the
50
mm
containing was 1 .5
mg
cell
suspension
incubated MF-Millipore quickly
grade
and
were
at
30 C. filter
washed I
with (Nacalai counter
.
(TMG)
,
Co ., Sephadex
and
Amersham
were
experiments. with
Chemical
[3H]raffinose
Products
special
Sigma
7 .0)
positions
, Clear Sol scintillation
liquid
G-15 (pH
The
ISL medium suspension
added
inactivate
water
to
mixture through
measured
to
Sephadex
distilled
in this
of
The
purchased
Research of
a
for
M potassium
3 min
to
acetate
methyl-ƒÀ-D-thiogalactoside
were
NEN
0.5
determined
was
emulsifying
was
of
for
aqueous
incubated
, and used for the Cells were washed
lactose.
mm
M sodium
corresponding
pressure.
of
ml
with
[3H]raffinose
0.5
and
applied
were
, and the and filtered
taken
2 ml
0 .6
collected
[14C]lactose
100 ƒÊm
0 .2
bath
were
of
3 .2.1.26)
was
fructose
melibiose
from ml of
with
Fractions
or
Pharmacia Du
adding
spectrophotometer
: 0 .3
eluted
fractions
radioactivity
and from
obtained
a
of
prepared
(EC
NTG, ƒ¿-ONPG, ƒÀ-0NPG,
chloramphenicol
buffer by
o-nitrophenol
volume
melibiose
of
under
same
and
and
were
soaked
and
phosphate stopped through
of
(14)
(pH 7.0), and suspended Final concentration
aliquots
size)
buffer,
Chemicals.
G-15
of
buffer
al
mixture
column.
[3H]melibiose
100 ƒÊl
filtration Absorbance
a boiling-water
the
2 ml
same
concentration
intervals,
1 ml
and
uptake
ml.
in
ice,
non-radioactive
phosphate chloramphenicol.
cells
was
melibiose
of
by .
was
the
pre-equilibrated
the
with
Measurement potassium 50ƒÊg/ml
was
U-2000
invertase
heated on
ml/hr,
on
sodium
as ƒÊmoles
et
with
mixture
and
cm)
raffinose,
chromatography
mm
reaction
Hitachi
Tanaka
mixed
cooling
x 90
of
the
7.0)
After
a
expressed
100 ƒÊg/m1
Then
buffer
enzyme.
size)
[3H]melibiose
method was
C.
removed
in
was
20
the
pore
measured
containing
50
in
min,
were
melibiose.
modified
4.9)
at
cells
30
112 1
cells.
[3H]
[3H]raffinose
(pH min
a
C for
(0.45ƒÊm
activity
wet
of
30
the
was
specific
Preparation
the
and
membrane
The
min
90
at
from ƒ¿-ONPG
nm.
IN L. PLANTARUM
(ƒ¿-ONPG)
incubation
M Na2CO3,
Mini-sart
SYSTEM
[14C]lactose Japan
obtained
from
were
, respectively. commercial
sources.
RESULTS
Characterization
of
Mutant growth
on
sidase
under
ATCC
8014
only was
at
30
observed
NTG
Mutant 21
galactose, the grew C.
well
lactose respective on
Similar on
NTG as
lactose
21 as
the
and
parental
melibiose,
and
conditions
galactose
and
results even
30
C.
In
with ATCC
for
1).
at either
obtained
ATCC
also
(Table
lactose
were at
strain
8014 the
As 30
or
NTG
were
induction
reported 37
C 21
examined
for
of ƒ¿-galactopreviously
(13),
, but grew on melibiose except that no growth
8014, ƒ¿-galactosidase
was
induced
112 2
C. TAMURA
Table
Fig.
1. Cells
Lactose were
1.
at
MATSUSHITA
Growth and induction of a-galactosidase in L. plantarum ATCC 8014 and its mutant NTG 21
uptake grown
AND 0.
by 30
L. C
in
plantarum ISL
ATCC
medium
8014 supplemented
( •œ)
and with
its
mutant
lactose.
NTG Assayed
21 at
(•›). 30
C.
under all the growth conditions except that melibiose failed to induce the enzyme when the strain was grown at 37 C. In NTG 21, a-galactosidase was induced normally by galactose and melibiose, but practically not by lactose. Lactose uptake was measured with NTG 21 and ATCC 8014 grown on lactose at 30 C (Fig. 1). In contrast to ATCC 8014, NTG 21 scarcely accumulated lactose.
MELIBIOSE
TRANSPORT
SYSTEM
IN L. PLANTARUM
112 3
Melibiose transport activity was assayed with NTG 21 (Fig. 2B) and ATCC 8014 (Fig. 2A) grown on melibiose or lactose at 30 or 37 C. ATCC 8014 grown on melibiose at 30 C rapidly accumulated melibiose, but that grown at 37 C practically
A
B
Fig.
2.
Melibiose
uptake
Cells
were
grown
with
lactose
at
30
in
ISL
C
(•¡)
by
L.
plantarum
medium and
supplemented 37
C
(• ).
ATCC
8014 with
(A) melibiose
and
its at
30
mutant C
(•œ)
NTG and
21 37
C
(B). (•›);
112 4
C. TAMURA
Fig.
3. on
Fig.
Effect
of
melibiose.
4. Cells
temperature
Assayed
Stability were
supplemented
removed
as
at
hr, 30
and
a
C
50
mm
with
for
expressed
melibiose
( •œ)
and
transport
with
control,
assayed
C were
30
of melibiose
washed
medium
3
on
at
AND 0.
and
the
melibiose by
per
transport 37
C
system
remainder uptake. cent
in
NTG
phosphate
NTG
was Amounts
21
buffer
chloramphenicol.
of control.
mutant
21
grown
at
30
C
(•›).
of mutant
potassium
50 ƒÊg/m1
MATSUSHITA
A
incubated of
at
grown 7.0),
part
of
37
[3H]melibiose
at
(pH
C,
30
C
on
melibiose.
suspended this
sampled accumulated
in
suspension at
0,
ISL was
1, 2 in
4
and min
MELIBIOSE
TRANSPORT
SYSTEM
IN L. PLANTARUM
112 5
did not. Similar results were obtained with NTG 21. Low levels of melibiose uptake were observed with ATCC 8014 grown on lactose at 30 or 37 C. Such activities were not detected in NTG 21 grown on lactose, suggesting that these activities are due to the lactose transport system which is intact in ATCC 8014. All these data indicate that NTG 21 is deficient in lactose transport system which may cross-react with melibiose, but retains normal melibiose transport activity .
Effectsof Temperatureon Melibiose TransportSystem Melibiose uptake by NTG 21 grown on melibiose at 30 C was measured at 30 and 37 C (Fig. 3). The initial rates of uptake under the two conditions were very similar to each other, suggesting that the transport system has a broad temperature optimum ranging from 30 to 37 C. The transport system was quite stable at 37 C for at least 3 hr (Fig. 4). Inhibition of Melibiose Transport by Various Carbohydrates Inhibition NTG
21
of
grown
significantly
by
inhibition
was
of
raffinose,
II
of
E.
Inducer
(Table melibiose, the
same
strain of
was
melibiose
raffinose
30
7, 9,
were
11,
melibiose
and
NTG
In ATCC raffinose, ƒ¿-ONPG except
that
deficient
appeared
Table
to
2.
21
lactose
transport be
carbohydrates
2).
was
Melibiose
not
by
similar
and
was
that
Melibiose
tested
tested was
and
TMG.
observed
Transport in
raffinose With
with
.
the TMG
with
inhibited Some
exception permease
with
system
was inducer
did
for
to the
as
well
as
of ƒ¿-galactosidase
carbohydrates induced with
not
activity. similar
21
various was Results
and ƒÀ-ONPG
System
NTG
a-galactosidase and ƒÀ-0NPG.
transport
Inhibition mutant
to
system
lactose
a poor
lactose or
was uptake
14).
transport
8014,
in
various
(Table
practically
of ƒ¿-Galactosidase of
by C
with ƒ¿-ONPG, ƒÀ-0NPG
profiles (4,
8014 3).
but
observed
K-12
Specificity
ATCC
at
galactose,
inhibitor coli
transport
melibiose
also
Induction in
melibiose
on
by NTG
induce In
that transport
as
the
the
galactose, 21 were enzyme
NTG
21 , inducer of ƒ¿-galactosidase
lactose, essentially since
the
specificity except that
system.
of melibiose transport by various carbohydrates NTG 21 grown at 30 C on melibiose
inducers
in
112 6
C. TAMURA
Table 3.
AND 0.
MATSUSHITA
Inducer specificity of cc-galactosidase and melibiose transport system in parental (ATCC 8014) and mutant (NTG 21) strains
DISCUSSION
Melibiose metabolism has been studied in some detail with Escherichia coli K-12. The melibiose system of E. coli consists of its transport and hydrolysis (8). α-Galactosidase,
which
hydrolyzes
the
sugar
to glucose
and
galactose,
is induced
and active at either 30 or 37 C (12). In E. coli, melibiose is transported via two distinct systems, namely, thiomethylgalactoside (TMG) permease I (lactose permease), coded by lacY gene in the lactose operon (6) and TMG permease II (9). The former is induced by lactose and melibiose, and stable at 37 C, while the latter is induced by melibiose, and unstable at the temperature (4, 9, 11, 15). Therefore, wild-type E. coli K-12 can grow on melibiose at either 30 or 37 C since melibiose is taken up by TMG permease I, even when TMG permease II is inactivated at 37 C. On the other hand, mutants deficient in TMG permease I, cannot grow on melibiose at 37 C although they grow normally at 30 C (1, 2). Genes coding for α-galactosidase and TMG permease II are components of the same operon (5). In contrast to E. coli K-12, in spite of possessing both lactose and melibiose transport system, even a wild-type strain L. plantarum ATCC 8014 failed to grow on melibiose at 37 C. The same strain grew on lactose and galactose at either temperature (13; see also Table 1). When grown at 30 C on melibiose, a mutant NTG 21, deficient in lactose transport system, took up melibiose equally at either 30 or 37 C (Fig. 3), and the melibiose transport system was stable for at least 3 hr at 37 C (Fig. 4). These results are consistent with that observed in ATCC 8014 (13), and provide the evidence that L. plantarum differs from E. coli K-12 in that the
MELIBIOSE
TRANSPORT
SYSTEM
IN
L. PLANTARUM
112 7
induction of melibiose transport system is temperature-sensitive (Fig. 2) although the transport system itself is stable at 37 C (Fig.4). Lactose transport system of L. plantarum might not be induced by melibiose since, as mentioned above, even a wild-type strain ATCC 8014 failed to grow on melibiose at 37 C, while the transport system seems to be induced at either temperature and transport melibiose (Fig. 2A). In contrast to the melibiose transport system, a-galactosidase of L. plantarum was induced at 37 C when galactose or lactose served as the inducer (Table 1). Therefore, it is highly probable that the syntheses of these two proteins are subjected to separate genetic controls. In NTG 21, raffinose induced the melibiose transport system only a little, while it was a good inducer for a-galactosidase. This observation that the inducer specificities for these proteins are not exactly the same might favor the hypothesis. However, it was reported that cx-galactosidase synthesis in E. coli organisms is controlled not only by the melibiose operon in the genome DNA, but also by the raffinose operon in a plasmid (3). Therefore, more biochemical and genetic works will be needed to know the genetic control for the melibiose metabolism. Wewouldliketo thankProf.Y. Kanemasaforthe valuableadviceand encouragementthroughout this study. REFERENCES
1) Beckwith, J. 1964. Restoration of operon activity by suppressors. Biochim. Biophys. Acta 76: 162-164. 2) Beckwith, J. 1964. A deletion analysis of the lac operator region in Escherichia coli. J. Mol. Biol. 8: 427-430. 3) Burkardt, H. J., Mattes, R., Schmid, K., and Schmitt, R. 1978. Profiles of two conjugative plasmids mediating tetracycline resistance, raffinose catabolism and hydrogen sulfide production in Escherichiacoli. Mol. Gen. Genet. 166: 75-84. 4) Burstein, C., and Kepes, A. 1985. The melibiose permease system of Escherichia coli K12. Biochimie 67: 59-67. 5) Hanatani, M., Yazyu, H., Shiota-Niiya, S., Moriyama, Y., Kanazawa, H., Futai, M., and Tsuchiya, T. 1984. Physical and genetic characterization of the melibiose operon and identification of the gene products in Escherichia coli. J. Biol. Chem. 259: 1807-1812. 6) Jacob, F., and Monod, J. 1958. Genetic and physical determination of chromosomal segments in Escherichia coli. Symp. Soc. Exp. Biol. 12: 75-92. 7) Lopilato, J., Tsuchiya, T., and Wilson, T.H. 1978. Role of Na+ and Li+ in thiomethylgalactoside transport by the melibiose transport system of Escherichia coli. J. Bacteriol. 134: 147-156. 8) Pardee, A.B. 1957. An inducible mechanism for accumulation of melibiose in Escherichia coli. J. Bacteriol. 73: 376-385. 9) Prestidge, L.S., and Pardee, A.B. 1965. A second permease for methyl-thio-j9-n-galactoside in Escherichiacoli. Biochim. Biophys. Acta 100: 591-593. 10) Rogosa, M. 1974. Genus I. Lactobacillus, p. 576-593. In Buchanan, R. E., and Gibbons, N.E. (eds), Bergey's manual of determinative bacteriology, 8 ed, The Williams and Wilkins Company, Baltimore. 11) Rotman, B., Ganesan, A.K., and Guzman, R. 1968. Transport systems for galactose and galactosides in Escherichia coli. II. Substrate and inducer specificities. J. Mol. Biol. 36: 247-260. 12) Schmitt, R., and Rotman, B. 1966. oc-Galactosidase activity in cell-free extracts of Escherichia coli. Biochem. Biophys. Res. Commun. 22: 473-479.
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C. TAMURA
AND O.
MATSUSHITA
13)
Tamura, C. 1991. Analysis on the mechanism of the temperature-sensitive melibiose metabolism in Lactobacillusplantarum. Okayama I. Z. 103: 101-110 (in Japanese with English summary). 14) Tanaka, K., Niiya, S., and Tsuchiya, T. 1980. Melibiose transport in Escherichia coli. J. Bacteriol. 141: 1030-1036. 15) Tsuchiya, T., Lopilato, J., and Wilson, T.H. 1978. Effect of lithium ion on melibiose transport in Escherichia coli.J. Membrane Biol. 42: 45-59. (Received for publication, March 9, 1992; in revised form, August 24, 1992)
