Download Maltose uptake and its regulation in Bacillus subtilis

FEMS MicrobiologyLetters 97 (1992) 191-196
© 1992 Federation of European MicrobiologicalSocieties 0378-11197/92/$tl5.110
Published by Elsevier
FEMSLE 05088
Maltose uptake and its regulation in Bacillus subtilis
M a r t i n T a n g n e y , C a l l u m J. B u c h a n a n ~, F e r g u s G, Priest a n d W i i f r i d J. M i t c h e l l
Department of Biological &'iemes. th'riot.Watt Unirer~it3. Ricrarttm. Edinburgh. UK
Received 26 June 19'42
Accepted 15 July 1092
Key words: Maltose uptake; Bacillus .°: btilis: Phosphotransferase system
1. S U M M A R Y
Extracts prepared from cultures of Bacillus
subtilis, grown on maltose as the sole carbon
source, lacked maltose phosphotransferase system activity. T h e r e was, however, evidence for a
maltose phosphorylase activity, and such extracts
also possessed both glucokinase and glucose
phosphotransferase system activities. Maltose was
accumulated by whole cells of B. subtilis by an
energy-dependent mechanism. This uptake was
sensitive to the effects of uncouplers, suggesting a
role for the proton-motive force in maltose transport. Accumulation of maltose was inhibited in
the presence of glucose, and there was no accumulation of maltose by a strain carrying the ptsl6
null-mutation. A strain carrying the temperaturesensitive p t s l l mutation accumulated maltose
normally at 37°C but, in contrast to the wild-type,
was devoid of maltose transport activity at 47°C.
Correspondence to: .M. Tangney, Department of Biological
Sciences. Heriot-Watt University, Riccarton, Edinburgh Ell14
4AS, UK.
i Present address: Gastrointestinal Laboratories, Department
of Medicine, University of Edinb~rgh Western General
Hospital, Crewe Road South, Edinburgh EH4 2XU. UK.
The results indicate a role for the phosphotransferase system in the regulation of maltose transport activity in this organism.
2. I N T R O D U C T I O N
The active transport of carbohydrates in bacteria occurs via a number of distinct mechanisms,
which can be energised by one of several forms of
potential energy stored in the cell. Proton symport systems utilise the trattsmembrane proton
gradient, which is stored across the cell membrane, as the driving force [I]. These transport
systems are therefore particularly sensitive to the
effects of uncouplers, such as tetrachlorosalicylap, i',!de (TCS) and dinitrophenol (DNP), which
can collapse this gradient. O t h e r systems utilise
the energy stored in high-energy phosphate bonds
such as are found in A T P and phosphoenolpyruvate (PEP). An important and widespread example is the PEP-dependent phosphotransferase
system (PTS). The PTS is typically composed of
two cytoplasmic components, Enzyme ! and HPr,
as well as a sugar-specific protein, Enzyme !!, in
the cytoplasmic membrane. Enzyme I transfers
the phosphate from PEP to HPr which, in turn,
192
phosphorylates the membrane protein that ultimately phosphorylates the substrate concomitant
with its translocation into the cell [2]. The substrate therefore enters the cytoplasm as a phosphorylated carbohydrate. In some cases an additional polypeptide chain, termed Enzyme Ill or
ll-A [3], mediates phospho-transfer from HPr to
Enzyme II. In enteric bacteria the Enzyme lIl
specific for glucose (Ill Gjc) has been shown to
play a central role in the inhibition of transport
of non-PTS substrates such as glycerol and maltose [4].
Bacillus subtilis has been shown to transport
glucose, sucrose, fructose, mannose and mannitol
via a PTS mechanism [5], and there is also evidence for PTS-mediated regulation of the transport of glycerol, which is not a PTS substrate [6].
Tbele are, however, no reports concerning the
transport of maltose. Here we present evidence
that maltose is not a PTS substrate in B. subtilis
but that its transport, which may be proton-motive force (PMF)-dependent, appears to be regulated by the PTS. We also show evidence for the
existence of a maltose phosphorylase activity in
this organism.
3. MATERIALS AND METHODS
3.1. Organism and culture conditions
Bacillus subtilis Marburg was obtained from K.
Bott, University of North Carolina. The related
strains, PG554 carrying the ptsl6 mutation [5,7],
and PG587 carrying the temperature-sensitive
ptsll mutation [7], were both obtained from G.
Rapoport, Institute Pasteur, France. Cultures
were routinely grown in SS minimal medium broth
[8], supplemented with 1% (w/v) peptone. Carbon sources were added separately at a concentration of 0.5% (w/v).
3.2. Preparation of cell-free extracts
Extracts were prepared essentially by the
method of Mitchell and Booth [9]. Once cultures
had reached late log phase (OD~ 0 of approx. 0.9)
cells were harvested and washed twice in PIPES
buffer (100 mM, pH 6.6) before resuspending at a
ratio of 4 ml per g (wet weight). Cells were
ruptured by passage through a French pressure
cell at 20000 Ib per square inch. Cell debris and
any remaining intact cells were removed by eentrifugation at 10000×g for 30 rain, and the
supernatant recovered and stored at -20°C until
used. The entire procedure was carried out at
0-4°C.
3.3. Measurement of sugar uptake by whole cells
Cells used in uptake assays were prepared
from cultures grown in 100 ml of medium in a 500
mi flask (containing a coiled spring) shaken in an
orbital incubator at 37°C. Ceils were harvested at
mid log-phase by centrifugation at 6000 × g for
15 min at 4°C. The pellet was resuspended and
washed twice in 100 mM potassium phosphate
buffer at pH 6.6. The pellet was finally resuspended to the required cell density using the
relationship, mg dry weight/ml = OD680 x 0.33
[10], such as to give 1 mg/ml in the assay. Cells
were allowed to equilibrate at 37°C for 3 rain and
then the appropriate ~4C-labelled sugar (9.5 mM,
1.05 Ci/mol) was added to give a final concentration of 0.21 mM. At the indicated times, 0.15 ml
samples were removed (from a total assay volume
of 1 ml), filtered through glass fibre discs (Whatman G F / F ) and washed twice with 5 ml of the
assay buffer. The filters were dried under a heat
lamp, and finally counted in 4 ml of scintillation
cocktail O (BDH Ltd.).
3.4. Sugar phosphorylation assays in cell-free extracts
Sugar phosphorylation assays were carried out
by the method of Gachelin [11]. Routinely 0.4-ml
aliquots of extract were diluted into the assay
mix, which contained 100 mM PIPES buffer and
5 mM MgCI 2. Where appropriate, the assay mix
also contained either 1 mM ATP, 1 mM PEP or
100 mM inorganic phosphate, and the total assay
volume in all cases was 1 ml. The mix was equilibrated at 37°C for 3 min prior to assay. Radiolabelled sugar (9.5 mM; 1.05 Ci/mol) was added to
0.21 mM, and 0.15-ml samples ~vere removed at
stated intervals for estimation of sugar phosphate. Samples were added to 2 ml 1% (w/v)
barium bromide in 80% (v/v) ethanol. The resulting phosphate precipitates which formed were
grown on maltose as the sole carbon source, for
ATP- and P E P - d e p e n d e n t phosphorylation of
glucose and maltose. In the absence of a high-energy phosphate donor, there was no detectable
phosphorylation of either sugar. In the case of
glucose, both ATP- and PEP-stimulated phosphorylation, demonstrating the presence of both
kinase and PTS activity, respectively, for this substrate (Fig. IA). In contrast, maltose phosphorylation was not stimulated by either A T P or P E P
(Fig. 1B). As we have previously d e m o n s t r a t e d
the existence of a maltose phosphorylase activity
in the closely related organism B. licheniformis
[12], we also assayed for maltose phosphorolysis
in the presence of inorganic phosphate. In contrast to the results obtained with the high-energy
phosphate donors, there was stimulation of sugar
phosphate formation from maltose by inorganic
phosphate (Fig. 1B). T h e most plausible interpretation of these data is that a maltose phosphoryl-
removed by filtration on glass fibre discs (Whatm a n G F / F ) and washed with 5 ml of 80% ethanol.
T h e filters were dried u n d e r a heat lamp, and
finally counted in 4 ml of scintillation cocktail O.
3.5. Chemicals
T h e laC-labelled sugars were obtained from
A m e r s h a m ; P E P and D N P from Sigma; and A T P
from Boehringer. TCS was a gift from I.R. Booth,
University of A b e r d e e n , UK. All o t h e r chemicals
were of the highest available purity.
4. R E S U L T S
4.1. P T S activity in cell extracts •
T h e presence of PTS activity can be readily
revealed as P E P - d e p e n d e n t sugar phosphorylation in cell-free extracts. We assayed cell-free
extracts, which were p r e p a r e d from cultures
A
15
/
12
9
v
6
0
i
0
i
2
i
i
4
t
i
6
i
)
8
0
2
4
6
8
Time (min)
Fig. 1. PTS activity in cell-free extracts of B. subtilis. Extracts of B. subtilis were prepared in 100 mM PIPES buffer from cultures
which had been grown on maltose as the sole carbon source. Extracts were assayed for phospborylation of glucose (A) or maltose
(B) as described in MATERIALSANDMETHODSand results are presented as nmol of sugar phosphate produced per ml of assay mix.
The assays contained the followingadditions; no addition (e), 1 mM ATP ( • ), 1 mM PEP (11), or 100 mM PO~ (El).
194
ase enzyme also exists in B. subtilis which is
capable of catalysing the phosphorolysis of maltose using inorganic phosphate. What is evident is
that cultures grown on maltose, although possessing a functional glucose PTS, are devoid of maltose PTS activity.
4.2. Effects o f uncouplers on maltose uptake by
whole cells
The finding of a phosphotransferase system for
glucose, but not maltose, demonstrated that maltose must be transported in B. subtilis by an
alternative mechanism. In other organisms, maltose transport has been variously reported to be
via an ATP-dependent system, as in E. coil [13],
or driven by the PMF, as in B. licheniformis [12].
A characteristic of energy-dependent transport
systems which utilise the PMF is their sensitivity
to uncoupling agents which abolish the proton
gradient and hence PMF-dependent uptake. To
determine if maltose transport in B. subtilis was
dependent on the PMF, the accumulation of
[14C]maltose by resting cells was followed in the
"~
1:I /
/I.A
4
~t /
o
~
4
6
s
T i m e (rain)
Fig. 2. Effects of uncouplers on maltose uptake by whole cells
of B. subtilis. Whole-cellsuspensions of B. subtilis which had
been grown on maltose as the sole carbon source were prepared and assayed for maltose uptake as described in MATERIALSANDMETHODS.Maltose accumulationwas followedin the
absence of uncouplers (•). or in the presence of either 5
g.g/ml TCS (11), or l mM DNP (o).
presence and absence of a number of uncouplers.
The sugar was accumulated by the cells at a
significant rate in the absence of an exogenous
energy source, but this uptake was severely inhibited by the presence of both DNP and TCS (Fig.
2). In contrast, glucose uptake by these cells was
only partially inhibited by the uncouplers, consistent with a PTS mode of transport for this substrate (data not shown). This clearly demonstrates
that maltose is transported by an energy dependent mechanism in this organism and strongly
implicate~ a role for the PMF in this process.
4.3. Effects of PTS activity on maltose uptake
Transport of maltose and other sugars in E.
colt, which occurs via several non-PTS transport
mechanisms, has been shown to be subject to
inhibition by the glucose PTS [4], while a similiar
phenomenon has been reported for non-PTS mediated glycerol uptake in B. subtilis [6]. Given the
presence of glucose PTS activity in cultures of B.
subtilis grown on maltose as the carbon source, it
was of interest to determine if the non-PTS transport of maltose in this organism was also subject
to such PTS-mediated regulation. That glucose
could in fact inhibit maltose transport was
demonstrated directly using resting cells (Fig. 3A).
Evidence for the involvement of the PTS in this
inhibition was obtained using a ptsl mutant,
PG554. This strain was found to be devoid of
maltose transport activity (Fig. 3B), despite the
fact that we have established that maltose transport is not via a PTS mechanism in B. subtilis.
These results were further supported using a
temperature-sensitive ptsI mutant, PG587. While
both the wild-type and PG587 accumulated maltose to a comparable degree when assayed at
37°C, only the wild-type accumulated the sugar at
47°C (Table 1). The most probable conclusion
from these experiments is that maltose transport,
although non-PTS, is regulated by the PTS in B.
subtilis.
5. DISCUSSION
Maltose transport in Gram-positive organisms
has been reported to occur both via a PTS mech-
Table I
from studies of the effects of uncouplers on maltose uptake suggests that maltose transport may
be energised by the PMF, and that it could occur
via a proton symport mechanism, although we
have no direct proof for the operation of such a
system. Nevertheless, if such a system were operative, then the free maltose accumulated within
the cell by this process would have to be hydrolysed somehow internally, in this respect the detection of maltose phosphorolysis activity in the
presence of inorganic phosphate may be significant, although the cellular location of this enzyme
activity is not known.
In Staphylococcus aureus, maltose transport
occurs via a non-PTS mechanism, although pts
mutations result in inhibition of maltose uptake
and metabolism [17,18]. Likewise, glycerol uptake
in B. subtilis, which too is via a non-PTS mechanism, is modulated by the PTS [6]. Here we
present evidence for a similar phenomenon for
"
:"0piake of maltose by resting cel;s of strains of B. subtili.~
Organism
Rate of maltose
uptake at 37°C
(pmol/min/mg)
Rate of mal: ,se
uptake at 47°C
(pmol/min/mg)
B. subtilis Marburg
B. subtilis PG587
750
730
775
Not detected
anism, such as in a number of streptococci [14,15],
and non-PTS systems, such as in Bacillus licheniformis and B. popillae [12,16]. in this work we
have studied the transport of maltose in B. subtilis and have found no evidence for the operation
of a maltose PTS. The organism does, however,
synthesise a functional glucose PTS when grown
on maltose as the sole carbon source, indicating
the presence of both HPr and Enzyme 1 in such
cells, and suggesting that B. subtilis does not
possess an Enzyme 11 for maltose. The evidence
A
10
/8
6
~-~,~
4
0
0
2
4
6
8
0
2
4
6
8
Time (min)
Fig. 3. Maltose accumulation by whole cells of B. subtilis. Whole-cell suspensions of wild-type and pts mutant strains of B. subtilis
which had been grown on maltose as the sole carbon source were prepared and assayed for maltose uptake as described in
MATERIALSaNt) METHODS. In (A) accumulation by the wild-type was followed in the presence re) and absence ( • ) of I mM glucose.
In (B) accumulation of maltose by the wild-type ( • ) was compared with the Enzyme I-defective strain PG554 (Ill).
196
the transport of maltose in B. subtilis. Tight ptsl
mutations in enteric bacteria have been shown to
prevent the transport of non-PTS carbohydrates,
such as maltose [4]. We have observed the same
effect on maltose transport in B. subtilis PG554,
which carries the ptsl6 mutation. Furthermore, a
strain carrying a temperature-sensitive Enzyme I
was found to be unaffected for maltose transport
at the permissive t e m p e r a t u r e of 37°C, but devoid
of maltose transport activity at the thermo-inactivating t e m p e r a t u r e of 47°C, a t e m p e r a t u r e at
which maltose transport in the wild-type was little
affected. These results are consistent with a regulatory role for the PTS in the transport of maltose
in B. subtilis. Reizer et ai. have examined the
PTS-dependent regulation of glycerol uptake in
this organism, and have [4,6] shown it to be quite
similar phenotypically to that observed in enteric
bacteria, where Enzyme III °It plays a critical
role. However, it has since b e e n shown that, in
contrast to the situation with E. coli, ptsG mutations, which eliminate the equivalent Enzyme
III ° c functiou in B. subtilis, fail to restore glycerol transport in ptsI mutants [19]. Therefore,
despite the phenotypic similarities between the
regulation of non-PTS activity in these two organisms, the mechanism by which it is achieved,
while clearly involving the PTS in each case, must
be different. It will be of interest to determine
precisely how this PTS-mediated regulation is
achieved in B. subtilis and w h e t h e r maltose and
glycerol uptake are subject to the same type of
control in this organism.
ACKNOWLEDGEMENTS
This work was supported by a grant to M.T.
from the Commission of the E u r o p e a n Communities u n d e r the B R I D G E programme.
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