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
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.
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-
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
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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
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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
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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.
REFERENCES [I] Booth, I.R. (1988) In: Bacterial energy Iransduction (C. Anthony, Ed.), pp. 377-428. Academic Press, New York, NY. [2] Meadow, N.D., Fox, D.K. and Roseman, S. (1990) Annu. Rev, Biochem. 59, 497-542. [3] Saier, M.H. Jr. and Reizer, J. (1992) J. Bacteriol. 174, 1433-1438. [4l Saier, M.H. Jr. (1989) Microbiol. Rev. 53, 109-120. 15] Gay, P.. Cordier, P., Marquet, M. and Delobbe. A. (1973) Mol. Gen. Genet. 121,355-368. [6] Reizer, J., Novotny, M.J., Stuiver, !. and Saier M.H. Jr. (1984) J. Bacteriol. 159, 243-250. [7l Niaudet, B., Gay, P. and Dedonder, R. (1975) Mol. Gen. Genet. 136, 337-349. [8] Spizizen, J. and Anagnostopoulos, C. (1961) J. Baeteriol. 81,741-746. [9] Mitchell, W.J. and Booth I.R. (1984) J. Gen. Microbiol. 130, 2193-2200. [10] Harris, P. and Ko:'nberg, H.L. (1972) Proc. R. Soc. Lond. B. 182, 159-170. [11] Gachelin, G. (1969) Biochem. Biophys. Res. Commun. 34, 382-387. [12] Tangney, M., Smith, P., Priest, F.G. and Mitchell, W.J. (1992) J. Gen. Microbiol. 138, in press. [13] Hengge, R. and Boos, W. (1983) Biochim. Biophys. Acta 737, 443-478. [14] Martin, S.A. and Russel, J.B. (1987) Appl. Environ. Mierohiol. 53, 2388-2393. [15] Wursch, P. and Koellreuter, B. (1985) Caries Res. 19, 439-449. [16] Taylor, D.C. and Costilow, R.N. (1977) Appl. Environ. Microbiol. 34, 102-104. [17] Button, D.K., Egan, J.B., Hengstenberg, W. and Morse, M.L. (1973) Biochem. Biophys. Res. Commun. 52, 850855. [18] Reizer, J., Sutrina, S.L., Saier M.H. Jr., Stewart. G.C., Peterkofs~, A. and Reddy, P. (1989) EMBO J. 8, 21112120. [19] Gonzy-Treboul, G., de Waard, J.H., Zagorec, M. and Postma, P.W. (1991) Mol. Microbiol. 5, 1241-!249.
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
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.
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-
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
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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
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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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