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Proline uptake in Streptomyces clavuligerus
FEMS Mierobiolog~ Letters 69 (1990) 27-30 Published by Elsevier
27
FEMSLE 039"/6
Proline uptake in Streptomyces clavuligerus Victoria Bascarhn, Carlos H a r d i s s o n and Alfredo F. Braha * Laboratorio de Microbiologi.. Dep~r~c~mentode B~ologia FunclonaL Facuhad de Medlcina, Universidad de O~,iedo, Oviedo. Spain
Received 23 l:k:cember t989 Accepted ~ January 1990
Key words: Proline uptake; Slrepwmyces clavuligert~
1. S U M M A R Y
gtreptomyces clavuligerus was able to accumulate proline intrac¢llularly throughout a wide range of external proline concentrations. Kinetic analysis of proline uptake indicated that this phenomenon is mediated by two saturable systems. One of them was a high-affinity system (Km = 11 FM), with low uptake capacity and specific for proline. The second system had lower affinity for proline (K m = 10.2 raM), higher uptake capacity, and was inhibited by several amino acids. Proline was not required as an inducer of the system~, which were neither repressed nor inhibited by ammonium.
2. I N T R O D U C T I O N The utilization of nitrogen sources by many microorganisms is often controlled by regulatory circuits that respond to nitrogen availability. Amino acid permeases, as a part of the enzymatic machinery that enables microorganisms to utilize amino acids as a nutrient, are sometimes regulated
Correspondem~eto: A.F. Braha, Laboralono de Microbiologia, Departaraenlo de Biologla Funcional, Facuhad de Medicina. Unviersidad de Oviedo, 331106Oviedo, Spain.
by these mechanisms [1,2], Previous studies on amino acid permeases in Streptomyces have revealed the presence of multiple transport systems with different specificity and properties [3-5]. Proline transport, in particular, has been studies in S. antibioticus, and it was found to be mediated by two systems. One of them was specific for proline and constitutive, whereas the other was nonspecific and induced by $lutamate [61. The possible influence of nitrogen control circuits on perincase formation was not reported in these studies. S. claeuligerus can utilize proline as the sole sources of nitrogen or as the sole source of carbon and nitrogen. Catabolism of this amino acid seems to be initiated by an inducible proline dehydrogenase that is not controlled by the nitrogen regulatory circuit detected in this bacterium [7,8]. In the present study we have tried to characterize the mechanisms of proline uptake in S. claouligerus as well as their possible control in relation to the availability of nitrogen.
3. MATERIALS A N D METHODS
3.1. Microorganism and culture conditions S. claouligerus N R R L 3585 (ATCC 27064) was grown at 20 o C in minimal medium (MF medium), as described previously [7]. The nature and con-
037g-1097/~1/$03.50 ~ 1990 Federutloa of European Microbiological Soci~:tie~
28 centration of the nitrogen sources are given in the text. Procedures for obtention of spores, growth determinations and strain maintemlnce can be found elsewhere [7,9].
3.2. Uptake of prolme Cells growth to exponential phase (about I mg dry wt/ml) were harvested by filtration, washed with 50 mM potassium phosphate buffer pH 7.0, co,;ahaing 1% (w/v) glycerol and 100 Fg/ml chloramphenicol, and resuspended in the same buffer at a cell density of 1.6 mg dry wt/mL Portions of 0.5 ml of this suspension were distributed among test tubes and incubated at 30°C in a reciprocal shaking water bath. After 5 rain, uptake was initiated by the addition of L-[4-~H]proline (New England Nuclear). The specific activities of labeled proline were 5 #Ci/#mol (185 kBq//~mol) for final assay concentrations up to 0.2 raM, 2 p.Ci/#mol (74 kBq//zmol) for final assay concentrations of 0.5 to 2 raM, and 0.5 # C i / # m d (18.5 kBq//~mol) for final assay concentrations higher than 2 raM. Uptake was terminated by adding to each tube 5 ml of 50 mM potassium phosphate buffer pH 7.0, containing 1 $ (w/v) glycerol and 10 mM proline, followed by immediate filtration through a glass fiber filler (Wbatman GF/C). The filters were washed three times with 5 ml of the latter buffer and, after drying, radioactivity was estimated in a toluenebased scintillation fluid [10]. For the cell concentration used, the uptake was linear with time for at least 2 rain. The values given were obtained from duplicate assays performed during 90 s. Corrections for nonspecific adsorption of labeled proline to the cells and to the filters were made by substra,aing radioactivity counts in samples taken immediately after the addition of L-[4-3H]proline.
3.3. Protein assay A fraction of 1 ml was taken from the cell suspensions prepared to initiate uptake. After centrlfugation (12000 × g, 5 rain), the pellet was resuspended in 1 M NaOH and heated to 100*C for 15 win. The solubilized protein was determined by the method of Lowry et ai. [11),
60
~. 45 K .E 30
15 I ":--0~ I V/Is]
IP~
• .... I
~
I
too
20o
300
[ (.tool/olin
X nlg proteln)(mM"~]
Fig. ], Eadi¢-Hofstee plol of prolin¢ aptake in S. clavufigerus.
4. RESULTS Proline uptake by ceils grown in minimal medium with 30 mM NH4CI as the nitrogen source, was determined with labeled proline supplied at concentrations ranging from 6.25 ~M to 20 raM. Kinetic analysis of the results using a Lineweaver-Burk plot or a Eadie.Hofstee plot (Fig. 1) suggested the presence of two saturable systems, At low proline concentrations, uptake seemed to be mediated by a high-affinity system with an apparent K,, of 11 ,aM, while at high proline concentrations a low-affinity component, with an apparent g m of 10.2 mM was deduced from the plot. The calculated Vn,~ values for these systems were 9 nmol/min-mg protein and 122 nmol/min, mg protein, respectively. Using cells previously grown with ammonium, and assuming an internal volume for Streptomyces mycelium of 1.56 #1 per mg of dry weight (L.M. Quir6s and J.A. Salas, unpublished results), we calculated the intracellular concentration of proline accumulated during 90 s in cells exposed to different external concentrations of this amino acid. The higher intracellular content found throughout the range of concentrations used indicates that proline uptake takes place against a concentration gradient (Table 1). Furthermore, the effect of the metabolic inhibitors sodium cyanide and 2A-dinitrophenol was tested in experiments designed to assay uptake mediated predominantly by one component. Thus, according tc. the kinetic
29 Table 1
Table 3
lntraeellulat accumulation of labeled proline in the presence of different external concentrations
Effect of the nitrogen source used for growth on proline uptake Nilrog,en source J
ProUne uptake h (nmol/min, mg protein)
Proline uptake ¢ (nmol/minmg protein)
Proline NH4CI Proline + NHaCI Proline ~- Glutamate Proline + Glulamale + N HaCI
2.t 4.0 3.6 2.9
8.7 50,0 32.2 ND
3.7
ND
Proline (raM)
Extracellular
Intracetlular
6.25 X 10 _a 12.50x 10 -~ 25.00× 10 J 50.00 × 10- ~ 0.1 0.2 1.0 2.0 S.O I0.0 20,0
0.6 1.3 1.9 2.3 2.5 2.5 3.4 4.4 12.5 18.2 23.4
a The concemration of each nitrogen source was 30 mM. b Measured with 0.l mM t-[3H]proline. = Measured with 5 mM L-I~H]proline. ND. not determined.
data, with a proline concentration of 0A mM, 87% of the proline uptake should be mediated by the high-affinity system, whereas with 5 mM proline, the low-affinity component would be responsible for 82~0 of the total uptake. In both cases the inhibitors interfered significantly, which indicates that it is an energy-dependent process (Table 2). The results of competition experiments in which proline uptake was determined in the presence of an excess of different amino acids or ammonium Table 2 Effect of metabolic inhibilors and nitrogen sources on proline uptake Addition a Sodium cyanide 2,4,Dinitrophenol NH4CI L-Alanin¢ t-Arginine t-Aspara~ne t-Aspartate L-Glutamate t-Lysine t-Threonine
Praline uptake b (~ inhibition}
( ~ inhibition)
40 51 l 89 0 2 7 4 0 7
4! 34 0 84 21 0 27 21 16 22
is also shown in Table 2. With 0.1 mM proline, the high.affinity system was only affected by alanine, but with 5 mM proline, several amino acids, besides alanine, had an interfering effect. Kinetic analysis of proline uptake in cells previously grown with 30 mM proline, 30 mM NH4C1, or a mixture of these compounds, showed the presence of the two components with different affinity. Km for either component remained constunt, independently of the nitrogen source used for growth, but the uptake capacity of the cells was maximal in cells grown with ammonium (Table 3). Addition of glutamate to the culture medium did not cause an enhanced uptake rate with 0.1 mM proline.
Proline uptake c
a Additions were made simultaneously witk the labeled proline. Sodium eyanJ.de was used at 2 mM and 2,4-dinitrophend at 0.5 m M . Nitro;~en sources we~ added at a concentration tenfold higher than the labeled protine. t~ Measured with 0.t mM t-13HIproline. Mee,~ured with 5 mM L-[3H|proline_
5. DISCUSSION This study shows that proline uptake by S. clavuligerus is mediated by two distinguishable carriers that probably fulfil different physiological roles. One of them is a high-affinity, low-capacity system specific for proline, which may enable the cells to scavenge low amounts of proline present in the medium. The second system has lower affinity and specificity for proline, but its high capacity may allow growth of S. clavuligerus with proline provided at higher concentrations as the only source of carbon and nitrogen or as the sole source of nitrogen. In addition, proline transport by these or any other, as yet unidentified system,
31}
may have a role in osmoregulation, as found in other bacteria I12,13] including Streptomyces [14]. The existence of more than one proline carrier with distinct characteristics has been described in other microorganisms [6,15,16]. Our results are partially similar to those found in S. antibioticus, where a carrier with high affinity and specificity for pro]ine and competitively inhibited by alanine was described [6]. However, the second system detected in S. antibioticus had a low K,~ for proline, a high transport capacity and was induced by glutamate, a result not found in S. daouligerus. Proline, apart from being the substrate for the uptake system studied in this work, is a permissive nitrogen source for the expression of enzymatic activities under nitrogen control [7]. Our results indicate that both components of proline uptake do not require proline as an inducer, and their formation is not interfered in conditions of nitrogen excess, i.e. in the presence of ammonium. This behaviour is in agreement with the previous find. ing that ammonium does not prevent proline consumption or proline dehydrogenase induction [7] and confirms that proline utilization is not submitred to nitrogen control in this strain.
REFERENCES Ill Magasanik, B. (1982) Ann, Rev, Genet. 16, 135-168. [2[ Courchesne, W.E. and Magasanik, B. (]983) Mol, Cell. Biol,3. 672-683,
[3] Gross, W. and Ring. K. (1971) Bi0chim. Biophys. Acta 233, 652-665, [4] Gross. W. and Burkhardl, K.L {19731 Biochlm. Iliophy,~ Acta 298, 4.37 4-q.5, [5] Ftltseh, J. and Gross, W. (1983) Z. Naturforsch. 38¢. 617-620. [6] Stratford May, W. and Formica, LV. 1"!978) J. Bacleriol. 134, 546-5~4. [71 Bascar~n, V., Hardisson, C~ and Brafia, A.F. (t959) J. Gen. Microbial 135, 2465-2474. [81 Bascarfin. V.. Hardi~son, C, and Bra~a, A.F. (1989) J. Gen. Microbial, 135, 2475-2482. [9] Braiia, A.F.. Paiva, N. and Dcmaln. A.L. (1986) J. Gcn. Microbial, 132, 1305-1317. [14)] Salas, J.A, and Hardisson. C. {1981) J, Gen. Microbial. 125.25-31. [11] Lowry, O.H., Rosebrough, NJ., Fair, A.L. and Randall, R.J. (1951) J. Blot.Chem. 193, 265-275~ I12] Csonka, L.N. 0982) J. Bacterial. 151, 1433-1443. [13] Gowrishanka¢, J. (1986) J. Bacleriol. 166, 331-333. [14] Killham. K. and Firestone, M.K. [1984} Appl. Environ. Microbial. 48, 239-241. [15] Lasko, P.F, and Brandlss, M.C. (1981) J. Bacterial. 148. 241-247. [16] Malay, S.R, [1987) in Escherichia coli and Salmonella lyphimurium (Neidhardt, F.C.. ed.), pp. 1513-1519. American Society for Microbiology, Washington DC.
27
FEMSLE 039"/6
Proline uptake in Streptomyces clavuligerus Victoria Bascarhn, Carlos H a r d i s s o n and Alfredo F. Braha * Laboratorio de Microbiologi.. Dep~r~c~mentode B~ologia FunclonaL Facuhad de Medlcina, Universidad de O~,iedo, Oviedo. Spain
Received 23 l:k:cember t989 Accepted ~ January 1990
Key words: Proline uptake; Slrepwmyces clavuligert~
1. S U M M A R Y
gtreptomyces clavuligerus was able to accumulate proline intrac¢llularly throughout a wide range of external proline concentrations. Kinetic analysis of proline uptake indicated that this phenomenon is mediated by two saturable systems. One of them was a high-affinity system (Km = 11 FM), with low uptake capacity and specific for proline. The second system had lower affinity for proline (K m = 10.2 raM), higher uptake capacity, and was inhibited by several amino acids. Proline was not required as an inducer of the system~, which were neither repressed nor inhibited by ammonium.
2. I N T R O D U C T I O N The utilization of nitrogen sources by many microorganisms is often controlled by regulatory circuits that respond to nitrogen availability. Amino acid permeases, as a part of the enzymatic machinery that enables microorganisms to utilize amino acids as a nutrient, are sometimes regulated
Correspondem~eto: A.F. Braha, Laboralono de Microbiologia, Departaraenlo de Biologla Funcional, Facuhad de Medicina. Unviersidad de Oviedo, 331106Oviedo, Spain.
by these mechanisms [1,2], Previous studies on amino acid permeases in Streptomyces have revealed the presence of multiple transport systems with different specificity and properties [3-5]. Proline transport, in particular, has been studies in S. antibioticus, and it was found to be mediated by two systems. One of them was specific for proline and constitutive, whereas the other was nonspecific and induced by $lutamate [61. The possible influence of nitrogen control circuits on perincase formation was not reported in these studies. S. claeuligerus can utilize proline as the sole sources of nitrogen or as the sole source of carbon and nitrogen. Catabolism of this amino acid seems to be initiated by an inducible proline dehydrogenase that is not controlled by the nitrogen regulatory circuit detected in this bacterium [7,8]. In the present study we have tried to characterize the mechanisms of proline uptake in S. claouligerus as well as their possible control in relation to the availability of nitrogen.
3. MATERIALS A N D METHODS
3.1. Microorganism and culture conditions S. claouligerus N R R L 3585 (ATCC 27064) was grown at 20 o C in minimal medium (MF medium), as described previously [7]. The nature and con-
037g-1097/~1/$03.50 ~ 1990 Federutloa of European Microbiological Soci~:tie~
28 centration of the nitrogen sources are given in the text. Procedures for obtention of spores, growth determinations and strain maintemlnce can be found elsewhere [7,9].
3.2. Uptake of prolme Cells growth to exponential phase (about I mg dry wt/ml) were harvested by filtration, washed with 50 mM potassium phosphate buffer pH 7.0, co,;ahaing 1% (w/v) glycerol and 100 Fg/ml chloramphenicol, and resuspended in the same buffer at a cell density of 1.6 mg dry wt/mL Portions of 0.5 ml of this suspension were distributed among test tubes and incubated at 30°C in a reciprocal shaking water bath. After 5 rain, uptake was initiated by the addition of L-[4-~H]proline (New England Nuclear). The specific activities of labeled proline were 5 #Ci/#mol (185 kBq//~mol) for final assay concentrations up to 0.2 raM, 2 p.Ci/#mol (74 kBq//zmol) for final assay concentrations of 0.5 to 2 raM, and 0.5 # C i / # m d (18.5 kBq//~mol) for final assay concentrations higher than 2 raM. Uptake was terminated by adding to each tube 5 ml of 50 mM potassium phosphate buffer pH 7.0, containing 1 $ (w/v) glycerol and 10 mM proline, followed by immediate filtration through a glass fiber filler (Wbatman GF/C). The filters were washed three times with 5 ml of the latter buffer and, after drying, radioactivity was estimated in a toluenebased scintillation fluid [10]. For the cell concentration used, the uptake was linear with time for at least 2 rain. The values given were obtained from duplicate assays performed during 90 s. Corrections for nonspecific adsorption of labeled proline to the cells and to the filters were made by substra,aing radioactivity counts in samples taken immediately after the addition of L-[4-3H]proline.
3.3. Protein assay A fraction of 1 ml was taken from the cell suspensions prepared to initiate uptake. After centrlfugation (12000 × g, 5 rain), the pellet was resuspended in 1 M NaOH and heated to 100*C for 15 win. The solubilized protein was determined by the method of Lowry et ai. [11),
60
~. 45 K .E 30
15 I ":--0~ I V/Is]
IP~
• .... I
~
I
too
20o
300
[ (.tool/olin
X nlg proteln)(mM"~]
Fig. ], Eadi¢-Hofstee plol of prolin¢ aptake in S. clavufigerus.
4. RESULTS Proline uptake by ceils grown in minimal medium with 30 mM NH4CI as the nitrogen source, was determined with labeled proline supplied at concentrations ranging from 6.25 ~M to 20 raM. Kinetic analysis of the results using a Lineweaver-Burk plot or a Eadie.Hofstee plot (Fig. 1) suggested the presence of two saturable systems, At low proline concentrations, uptake seemed to be mediated by a high-affinity system with an apparent K,, of 11 ,aM, while at high proline concentrations a low-affinity component, with an apparent g m of 10.2 mM was deduced from the plot. The calculated Vn,~ values for these systems were 9 nmol/min-mg protein and 122 nmol/min, mg protein, respectively. Using cells previously grown with ammonium, and assuming an internal volume for Streptomyces mycelium of 1.56 #1 per mg of dry weight (L.M. Quir6s and J.A. Salas, unpublished results), we calculated the intracellular concentration of proline accumulated during 90 s in cells exposed to different external concentrations of this amino acid. The higher intracellular content found throughout the range of concentrations used indicates that proline uptake takes place against a concentration gradient (Table 1). Furthermore, the effect of the metabolic inhibitors sodium cyanide and 2A-dinitrophenol was tested in experiments designed to assay uptake mediated predominantly by one component. Thus, according tc. the kinetic
29 Table 1
Table 3
lntraeellulat accumulation of labeled proline in the presence of different external concentrations
Effect of the nitrogen source used for growth on proline uptake Nilrog,en source J
ProUne uptake h (nmol/min, mg protein)
Proline uptake ¢ (nmol/minmg protein)
Proline NH4CI Proline + NHaCI Proline ~- Glutamate Proline + Glulamale + N HaCI
2.t 4.0 3.6 2.9
8.7 50,0 32.2 ND
3.7
ND
Proline (raM)
Extracellular
Intracetlular
6.25 X 10 _a 12.50x 10 -~ 25.00× 10 J 50.00 × 10- ~ 0.1 0.2 1.0 2.0 S.O I0.0 20,0
0.6 1.3 1.9 2.3 2.5 2.5 3.4 4.4 12.5 18.2 23.4
a The concemration of each nitrogen source was 30 mM. b Measured with 0.l mM t-[3H]proline. = Measured with 5 mM L-I~H]proline. ND. not determined.
data, with a proline concentration of 0A mM, 87% of the proline uptake should be mediated by the high-affinity system, whereas with 5 mM proline, the low-affinity component would be responsible for 82~0 of the total uptake. In both cases the inhibitors interfered significantly, which indicates that it is an energy-dependent process (Table 2). The results of competition experiments in which proline uptake was determined in the presence of an excess of different amino acids or ammonium Table 2 Effect of metabolic inhibilors and nitrogen sources on proline uptake Addition a Sodium cyanide 2,4,Dinitrophenol NH4CI L-Alanin¢ t-Arginine t-Aspara~ne t-Aspartate L-Glutamate t-Lysine t-Threonine
Praline uptake b (~ inhibition}
( ~ inhibition)
40 51 l 89 0 2 7 4 0 7
4! 34 0 84 21 0 27 21 16 22
is also shown in Table 2. With 0.1 mM proline, the high.affinity system was only affected by alanine, but with 5 mM proline, several amino acids, besides alanine, had an interfering effect. Kinetic analysis of proline uptake in cells previously grown with 30 mM proline, 30 mM NH4C1, or a mixture of these compounds, showed the presence of the two components with different affinity. Km for either component remained constunt, independently of the nitrogen source used for growth, but the uptake capacity of the cells was maximal in cells grown with ammonium (Table 3). Addition of glutamate to the culture medium did not cause an enhanced uptake rate with 0.1 mM proline.
Proline uptake c
a Additions were made simultaneously witk the labeled proline. Sodium eyanJ.de was used at 2 mM and 2,4-dinitrophend at 0.5 m M . Nitro;~en sources we~ added at a concentration tenfold higher than the labeled protine. t~ Measured with 0.t mM t-13HIproline. Mee,~ured with 5 mM L-[3H|proline_
5. DISCUSSION This study shows that proline uptake by S. clavuligerus is mediated by two distinguishable carriers that probably fulfil different physiological roles. One of them is a high-affinity, low-capacity system specific for proline, which may enable the cells to scavenge low amounts of proline present in the medium. The second system has lower affinity and specificity for proline, but its high capacity may allow growth of S. clavuligerus with proline provided at higher concentrations as the only source of carbon and nitrogen or as the sole source of nitrogen. In addition, proline transport by these or any other, as yet unidentified system,
31}
may have a role in osmoregulation, as found in other bacteria I12,13] including Streptomyces [14]. The existence of more than one proline carrier with distinct characteristics has been described in other microorganisms [6,15,16]. Our results are partially similar to those found in S. antibioticus, where a carrier with high affinity and specificity for pro]ine and competitively inhibited by alanine was described [6]. However, the second system detected in S. antibioticus had a low K,~ for proline, a high transport capacity and was induced by glutamate, a result not found in S. daouligerus. Proline, apart from being the substrate for the uptake system studied in this work, is a permissive nitrogen source for the expression of enzymatic activities under nitrogen control [7]. Our results indicate that both components of proline uptake do not require proline as an inducer, and their formation is not interfered in conditions of nitrogen excess, i.e. in the presence of ammonium. This behaviour is in agreement with the previous find. ing that ammonium does not prevent proline consumption or proline dehydrogenase induction [7] and confirms that proline utilization is not submitred to nitrogen control in this strain.
REFERENCES Ill Magasanik, B. (1982) Ann, Rev, Genet. 16, 135-168. [2[ Courchesne, W.E. and Magasanik, B. (]983) Mol, Cell. Biol,3. 672-683,
[3] Gross, W. and Ring. K. (1971) Bi0chim. Biophys. Acta 233, 652-665, [4] Gross. W. and Burkhardl, K.L {19731 Biochlm. Iliophy,~ Acta 298, 4.37 4-q.5, [5] Ftltseh, J. and Gross, W. (1983) Z. Naturforsch. 38¢. 617-620. [6] Stratford May, W. and Formica, LV. 1"!978) J. Bacleriol. 134, 546-5~4. [71 Bascar~n, V., Hardisson, C~ and Brafia, A.F. (t959) J. Gen. Microbial 135, 2465-2474. [81 Bascarfin. V.. Hardi~son, C, and Bra~a, A.F. (1989) J. Gen. Microbial, 135, 2475-2482. [9] Braiia, A.F.. Paiva, N. and Dcmaln. A.L. (1986) J. Gcn. Microbial, 132, 1305-1317. [14)] Salas, J.A, and Hardisson. C. {1981) J, Gen. Microbial. 125.25-31. [11] Lowry, O.H., Rosebrough, NJ., Fair, A.L. and Randall, R.J. (1951) J. Blot.Chem. 193, 265-275~ I12] Csonka, L.N. 0982) J. Bacterial. 151, 1433-1443. [13] Gowrishanka¢, J. (1986) J. Bacleriol. 166, 331-333. [14] Killham. K. and Firestone, M.K. [1984} Appl. Environ. Microbial. 48, 239-241. [15] Lasko, P.F, and Brandlss, M.C. (1981) J. Bacterial. 148. 241-247. [16] Malay, S.R, [1987) in Escherichia coli and Salmonella lyphimurium (Neidhardt, F.C.. ed.), pp. 1513-1519. American Society for Microbiology, Washington DC.