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Nutritional conditions affecting induced enzyme synthesis in Streptomyces violaceus
L~7
FEMS Microbiology Lctlers 7 (1980) 257- 260 © Copyright Fcderaflon of European Microbiological Societies Published by F.lsevier/North-HoUand BiomedicalPress
NUTRITIONAL CONDITIONS AFFECTING INDUCED ENZYME SYNTHESIS IN STREPTOM YCES VIOLACEUS JESUS SANCHEZ * and CARLOS HARDISSON ** Departamento de Mlcrobiologfo, Univer~dadde Oviedo. Oviedo, Spa~n Received 21 December 1979 Accepted I~ January 1980
I. Introduction
2. Material and Methods
The synthesis of enzymes related to antibiotic production in Streptomyces is sensitive to repression by glucose and other sugars (see [1 ] for review). Catab. olite repre~ion has been suggested also to affect the synthesis of enzymes of primary metabolism in that genus, although, there are few investigations on this subject [2,3]. S. viotaceus produces a ~.galactosidaselike enzyme induced by galactose during the exponential phase of growth [4]; this enzyme hydrolyzes o.nitrophenyl~.O-galactoside and lactose, the latter with low affinity. Its physiological function is yet unclear. The mechanism of control of the galactoseinduced enzymatic synthesis by glucose seems tO be exerted through an inhibition of the galactos¢ uptake [5]. Other regulatory mechanisms, as catabolite repression or catabolite inactivation have not been so far demonstrated ia the former system. We describe in this work divene nutritional con. ditons which affect the basal activity level and the galactos¢induced level of ~.galactosidasein 5. violaeeus. Some of the nutritional situationsproduce inhibitory effects which can be explained by catabolite repression mechanisms.
2.1. Organism and culture conditions 5. violaceus MR 3196 was obtained from C.E.C.T. (Colecci6n Espafiola de Cultivos Tipo, Bilbao, Spain). The synthetic medium used for induction e:~pedments, the conditions of growth and the me~,sureof specific growth rate K,have been described previously
{4]. 2.2. Cell-free extracts and ~alactosgdase assay
Cell-free extracts were obtained from washed cells by sonic disruption [4]. ~43ahctosida~ activity was assayed in these extracts by the method of Lederberg [6] using o.nitrophenyl.~.D.galactodde (ONPG) as substrate, as described previously [4]. One unit of enzymatic activity (EU) represents the amount of enzyme which hydrolyses I 9tool of ONPG in i rain. Specific activity is expressed as units per mg of cellutar dry weight. 2. 3. Carbohydrate assay
The concentration ofgalac[ose in the medium was determined by the method of Somogy[ [7]. 2.4. Chemicalt
"Present address: Eidgen6stischeTechnische Hochschule, MikrobJologisches Institut, CH-8092 ZUrich, Switzerland. "* To whom reprint iequests ~hould be a~dretted.
Sugars were obtained from Sigma Chemical Co., St. Louis, USA. Trypfone was obtained from Difco Lab., Detroit, USA, and caseinhydrolysat e (addhydrolysed, vitamin.iRe) was from Merck, Darm.
258 stadt, F.R.G, All other reagents were of analytical grade, QI °
3. Results attd Diu:us,don
im
~00
o
[. 3.1, Effect o;'gmwih olt ghwose on lhc basal ~.galac. toxidese h,rel
~L
~,. °i
The maximum level of basal B.galactosidase on 1% glycerol as ~;ole catbou source (0.02), reached twice the value of the ntaxinmm specific activity obtained in I% glucose medium (0.O1), suggesting a catabo|ite represser effect by glucose or a catabolite inactivation meehauisnt. In both media, the maximum specific activity was achieved at the onset of the stationary phase of growth {.data not shown). Tile growth rate in glucose was somewaht higher than in glycerol (K: 0.15 h "t and K: 0.14 h "t respectlvely), The same effect of glucose on basal ettzytuatic activity with respect to glycerol and other less readily utilizable carbott sources was initially described for Ihe ,~galactosidas¢ [8,9] and galactokinase [10] systems of k: Coll. Tiffs effect has been commonly attributed to catabolile repressiotL
3,2. Induced en:ymatic syntl~esis ht poor growth conditions in a typical experiment of~l-galactotidase indue. tion by galactoze (e.g. see control curve in Fig. 2), an increase [n the enzymatic activity of the cellular extracts can be observed 2-21 h after the addition of galaclose; t he synthesis proceeds tmtil 3-7 It, decreasing from this point on. The substitution of the usual nitrogen source (asparagine and ammonium phosphate) by L.proline (2 $/1), cau~d slow growth (specific growth rate of about K: 0.08 h -z , as compared with K: 0.17 h -t in the normal medium), and retarded the beginning ofa-galactosidase increase in the extracts tmtil after 9 h (Fig, 1). Thereafter, the synthesis proceeds with a slope about tO times less than in the normal medium (see control curve of Fig. 2). Gatactose consutnption at 9 h was apple×. 12% in L-proline r0edium,compared with 22% in the normal medium. L.Proline "per se" did not significantly affect induced ~.galactosidase synthesis nor ~.gataclotida,~ activity (Table 1). The primary effect
I la
oa ot
; In
IO
JO
Tim. tNautsl Fi~;. |. Synthesis of~-galactosidase on 1% galactose reed|urn with L,protine (2 g/l) as tt~e nitrogen source. Symboh: ~. a-gMa¢tosiciase activity (total units); ~, specit'te activity (enzymatic unitdmg dry weight);o, growth (log dry we|ghO; ~, galaetose (~) in supematant.
of L.proline as nitrogen source lies probably in a slower rate of protein synthesis, which should be also responsible of the slow growth rate; also, a catabolite repre~or effect can be postulated to take place, as has been suggested in b.: colt/Lgalactosidase [ 11 ], Streptomyces a-mannosidase ~[2] and Streptomyccs /~-1,3 glucanase [3j in analogous limiting.growth conditions.
3.3. Effect o/some amino acid5 on gdactose induced [J~galactosidase synthesis and enzymatic activity Some amino acids, such as L.tryptophan or Lalanine showed a significam inhibitory effect on induction when added to the galactote complete medium at the onset of incubation (Table 1). The same occurred with casein hydrolysate and tryptone (discussed later on). L-~rin¢ also affects the induction, although with a weaker effect. None of the assayed compounds inhibited the enzymatic activity in ~e cellular extracts.
3.4. Effect of compounds which increase growth on eozyrnatf¢ synthesis When 0.5% cabin hydrolysate, 0.5% tryptone or 0.2% yeast extract were added to the 1% galactose
239 TABLE 1 Effect of some compoundsof induceda-galactosidasesynthesisa and enzymaticactivityb Added compound Control L-Praline L-Sertne L-Alanine L-TWptophan Casein
hydmlymte Tryptone
Concentrationlndaeed Enzymatic synthesis activity 30 mM 30 mM 30 mM 30 m M
100 91 83 39
!00 100 100 100
0.5%
5 4
I00 108
0.5%
3
tO8
O
I j
~ 2
L~
.! t ° Io0
@
& A
a The ceils wezeinoculatedon syntheticmediumwith 0.5% gaiactose (control), or galactos¢plus the compoundtested at the indicatedconcentration.After 5 h of incubation, specificactivitywas measuredand related (%),to the control (0.3 EU/mgdry weight). b Enzymaticactivity in the inducedcellswas measuredin absence (control),or presence of the mentionedcompoundsin the reaction mixture. Activityin the control (100%) was 3.5 EO/min.
synthetic medium at the onset of incubation, a transient repression of about 3 h (with casein itydrotysate and tryptone) and of I h (with yeast extract) was observed (Fig. 2). After this lag, the synthesis started with a slope simitar to that of the control. The initial specific growth rate rwas K: 0.24 h -t with yeast extract or tryptone, and K: 0.20 h "t with casein hydrolysate (this latter not shown in the figute). We have shown that a similar effect of transitory repression, in 5. violaceus, of~-galactosidase synthesis by glucose when this sugar was added to the galactose medium at the onset of theincubation, was mediated by an inhibition of the galactose uptake [5]. Thus, the possibility that the same phenomenon oc~ arred in the presence Of the former compounds was tested. When 3H4abeUed galactose (i mM final concentration) was added io the induced celts in q~thepresen¢,~ of 0.5% casein hydrolysate or 0.5% tryptone, no inhibition of sugar incorporation was observed (data not shown), thus discarding any effect on gahctose uptake. O-Galactosidas¢ activity in the cellular extract was also not inhibited by the previous compounds (Table 1), It could be suggested, as in E. coli in analogous nutritional Conditions [ 12,13], that the
J__: 0
2
4
~
B
DO
Time Ihours)
Fig. 2. Synthesisof p-galactosidasein I% ga|actosemedium plus gtowth-stimulatlngcompounds.Symbols:m,,~-galacto. sidase activityIn the control (total units);~, t~,m,0.galacto. sidase activityin presen~ of 0.2% yeast extract,0.5% casein hydrolysateor 0.5% tryptone; o, growth in control (logdry w,:ight);~, srowth in the presenceof uyptone or yeast extract.
inhibition of the induction takes place through a catabolite-mediated repression mechanism. Moreover, the inhibition of the induction shown by individual amino acids, as L-tryptophan and L.alanlne, but not by L-praline (Table 1), could also indicate that only some of them (perhaps readily metal, oils. able) present in the casein hydrolysate, caused the repression. The reduction of the particular amino acid concentration could re]ieve that, as has been postu. ]ated for a similar transitory casein hydrolysate effect observed on the enzymes of the lac [ 12) and gal operon [12,13] in E. coll. From the above results it can be postulated that a catabolite-like repressor effect takes place in tile ~-galactosidase system of& violaceus in nutritional conditions which produce the same effect in &: coll. However, more detailed investigations, such as the effect of cyclic nucleotides on repre~ion and the possible existence of catabolite inactivation mechanisms, are needed to clarify the former observations.
260 References I l I I)¢main, A.L., Kennel, Y,M, nnd Aharonowltz, Y. (I 979) in; Microbial Techlto|ogy: Current State, I:utut*: I'roslx'¢ls (Bull. A.T.. El!wood. D,C. and Ratledge, C., Fds.). 29th Syltlp. So¢. Gen. Mlcrobiol., pp. t63-I85, Cambridge Llnlversity Press, London. I2t tnamin¢, F., Lago. B.D. and Ih:main, A.L. (1969) In: l:crmenlationt Advances (Perlman, D., Ed.). pp, 19922 I. AtxllJen~iePress, New York. [3l Litiey. G. and Bull, A.T. (1974) J. Gen. MicrobtoL 83, 123-133. [4] S2nche.,, J. and Ilardi~son, C. (1979) Can. J. Microbiol. 25, 833-840.
151 S;in~-IlCz,J. and Ilardis~n, C. (1980) Arch. Mlcrobiol. ltq Press. 16] Lederberg, J. (1950) J. B-cteriol.60, 381-392. 17l Somogyi, M. (1945) J. BioL Chem. 160, 61-68, 181 Marasanik, B. (1961) Cold Spring llarb. Syrup, Ouant, Biol. 26, 249-256. [91 Okinaka, R,T. and Dobrogosz, WJ , (1967) J. Bactedol. 93, 1644-1650. |10] Buttin, G, (1963) J. MoL Biol. 7, I64-182. [l I I Mandelstam. J. (1961) Bloehem. J. 79,489-496. I121 Paigen, K. (1963) Biochim. Biophys. Acta 77, 318328. 113l Jordan, E;. Saedler, It., Lengeler, J. and Stafltnger, P, (1967) MoL Gen. Genet. 100, 203-209.
FEMS Microbiology Lctlers 7 (1980) 257- 260 © Copyright Fcderaflon of European Microbiological Societies Published by F.lsevier/North-HoUand BiomedicalPress
NUTRITIONAL CONDITIONS AFFECTING INDUCED ENZYME SYNTHESIS IN STREPTOM YCES VIOLACEUS JESUS SANCHEZ * and CARLOS HARDISSON ** Departamento de Mlcrobiologfo, Univer~dadde Oviedo. Oviedo, Spa~n Received 21 December 1979 Accepted I~ January 1980
I. Introduction
2. Material and Methods
The synthesis of enzymes related to antibiotic production in Streptomyces is sensitive to repression by glucose and other sugars (see [1 ] for review). Catab. olite repre~ion has been suggested also to affect the synthesis of enzymes of primary metabolism in that genus, although, there are few investigations on this subject [2,3]. S. viotaceus produces a ~.galactosidaselike enzyme induced by galactose during the exponential phase of growth [4]; this enzyme hydrolyzes o.nitrophenyl~.O-galactoside and lactose, the latter with low affinity. Its physiological function is yet unclear. The mechanism of control of the galactoseinduced enzymatic synthesis by glucose seems tO be exerted through an inhibition of the galactos¢ uptake [5]. Other regulatory mechanisms, as catabolite repression or catabolite inactivation have not been so far demonstrated ia the former system. We describe in this work divene nutritional con. ditons which affect the basal activity level and the galactos¢induced level of ~.galactosidasein 5. violaeeus. Some of the nutritional situationsproduce inhibitory effects which can be explained by catabolite repression mechanisms.
2.1. Organism and culture conditions 5. violaceus MR 3196 was obtained from C.E.C.T. (Colecci6n Espafiola de Cultivos Tipo, Bilbao, Spain). The synthetic medium used for induction e:~pedments, the conditions of growth and the me~,sureof specific growth rate K,have been described previously
{4]. 2.2. Cell-free extracts and ~alactosgdase assay
Cell-free extracts were obtained from washed cells by sonic disruption [4]. ~43ahctosida~ activity was assayed in these extracts by the method of Lederberg [6] using o.nitrophenyl.~.D.galactodde (ONPG) as substrate, as described previously [4]. One unit of enzymatic activity (EU) represents the amount of enzyme which hydrolyses I 9tool of ONPG in i rain. Specific activity is expressed as units per mg of cellutar dry weight. 2. 3. Carbohydrate assay
The concentration ofgalac[ose in the medium was determined by the method of Somogy[ [7]. 2.4. Chemicalt
"Present address: Eidgen6stischeTechnische Hochschule, MikrobJologisches Institut, CH-8092 ZUrich, Switzerland. "* To whom reprint iequests ~hould be a~dretted.
Sugars were obtained from Sigma Chemical Co., St. Louis, USA. Trypfone was obtained from Difco Lab., Detroit, USA, and caseinhydrolysat e (addhydrolysed, vitamin.iRe) was from Merck, Darm.
258 stadt, F.R.G, All other reagents were of analytical grade, QI °
3. Results attd Diu:us,don
im
~00
o
[. 3.1, Effect o;'gmwih olt ghwose on lhc basal ~.galac. toxidese h,rel
~L
~,. °i
The maximum level of basal B.galactosidase on 1% glycerol as ~;ole catbou source (0.02), reached twice the value of the ntaxinmm specific activity obtained in I% glucose medium (0.O1), suggesting a catabo|ite represser effect by glucose or a catabolite inactivation meehauisnt. In both media, the maximum specific activity was achieved at the onset of the stationary phase of growth {.data not shown). Tile growth rate in glucose was somewaht higher than in glycerol (K: 0.15 h "t and K: 0.14 h "t respectlvely), The same effect of glucose on basal ettzytuatic activity with respect to glycerol and other less readily utilizable carbott sources was initially described for Ihe ,~galactosidas¢ [8,9] and galactokinase [10] systems of k: Coll. Tiffs effect has been commonly attributed to catabolile repressiotL
3,2. Induced en:ymatic syntl~esis ht poor growth conditions in a typical experiment of~l-galactotidase indue. tion by galactoze (e.g. see control curve in Fig. 2), an increase [n the enzymatic activity of the cellular extracts can be observed 2-21 h after the addition of galaclose; t he synthesis proceeds tmtil 3-7 It, decreasing from this point on. The substitution of the usual nitrogen source (asparagine and ammonium phosphate) by L.proline (2 $/1), cau~d slow growth (specific growth rate of about K: 0.08 h -z , as compared with K: 0.17 h -t in the normal medium), and retarded the beginning ofa-galactosidase increase in the extracts tmtil after 9 h (Fig, 1). Thereafter, the synthesis proceeds with a slope about tO times less than in the normal medium (see control curve of Fig. 2). Gatactose consutnption at 9 h was apple×. 12% in L-proline r0edium,compared with 22% in the normal medium. L.Proline "per se" did not significantly affect induced ~.galactosidase synthesis nor ~.gataclotida,~ activity (Table 1). The primary effect
I la
oa ot
; In
IO
JO
Tim. tNautsl Fi~;. |. Synthesis of~-galactosidase on 1% galactose reed|urn with L,protine (2 g/l) as tt~e nitrogen source. Symboh: ~. a-gMa¢tosiciase activity (total units); ~, specit'te activity (enzymatic unitdmg dry weight);o, growth (log dry we|ghO; ~, galaetose (~) in supematant.
of L.proline as nitrogen source lies probably in a slower rate of protein synthesis, which should be also responsible of the slow growth rate; also, a catabolite repre~or effect can be postulated to take place, as has been suggested in b.: colt/Lgalactosidase [ 11 ], Streptomyces a-mannosidase ~[2] and Streptomyccs /~-1,3 glucanase [3j in analogous limiting.growth conditions.
3.3. Effect o/some amino acid5 on gdactose induced [J~galactosidase synthesis and enzymatic activity Some amino acids, such as L.tryptophan or Lalanine showed a significam inhibitory effect on induction when added to the galactote complete medium at the onset of incubation (Table 1). The same occurred with casein hydrolysate and tryptone (discussed later on). L-~rin¢ also affects the induction, although with a weaker effect. None of the assayed compounds inhibited the enzymatic activity in ~e cellular extracts.
3.4. Effect of compounds which increase growth on eozyrnatf¢ synthesis When 0.5% cabin hydrolysate, 0.5% tryptone or 0.2% yeast extract were added to the 1% galactose
239 TABLE 1 Effect of some compoundsof induceda-galactosidasesynthesisa and enzymaticactivityb Added compound Control L-Praline L-Sertne L-Alanine L-TWptophan Casein
hydmlymte Tryptone
Concentrationlndaeed Enzymatic synthesis activity 30 mM 30 mM 30 mM 30 m M
100 91 83 39
!00 100 100 100
0.5%
5 4
I00 108
0.5%
3
tO8
O
I j
~ 2
L~
.! t ° Io0
@
& A
a The ceils wezeinoculatedon syntheticmediumwith 0.5% gaiactose (control), or galactos¢plus the compoundtested at the indicatedconcentration.After 5 h of incubation, specificactivitywas measuredand related (%),to the control (0.3 EU/mgdry weight). b Enzymaticactivity in the inducedcellswas measuredin absence (control),or presence of the mentionedcompoundsin the reaction mixture. Activityin the control (100%) was 3.5 EO/min.
synthetic medium at the onset of incubation, a transient repression of about 3 h (with casein itydrotysate and tryptone) and of I h (with yeast extract) was observed (Fig. 2). After this lag, the synthesis started with a slope simitar to that of the control. The initial specific growth rate rwas K: 0.24 h -t with yeast extract or tryptone, and K: 0.20 h "t with casein hydrolysate (this latter not shown in the figute). We have shown that a similar effect of transitory repression, in 5. violaceus, of~-galactosidase synthesis by glucose when this sugar was added to the galactose medium at the onset of theincubation, was mediated by an inhibition of the galactose uptake [5]. Thus, the possibility that the same phenomenon oc~ arred in the presence Of the former compounds was tested. When 3H4abeUed galactose (i mM final concentration) was added io the induced celts in q~thepresen¢,~ of 0.5% casein hydrolysate or 0.5% tryptone, no inhibition of sugar incorporation was observed (data not shown), thus discarding any effect on gahctose uptake. O-Galactosidas¢ activity in the cellular extract was also not inhibited by the previous compounds (Table 1), It could be suggested, as in E. coli in analogous nutritional Conditions [ 12,13], that the
J__: 0
2
4
~
B
DO
Time Ihours)
Fig. 2. Synthesisof p-galactosidasein I% ga|actosemedium plus gtowth-stimulatlngcompounds.Symbols:m,,~-galacto. sidase activityIn the control (total units);~, t~,m,0.galacto. sidase activityin presen~ of 0.2% yeast extract,0.5% casein hydrolysateor 0.5% tryptone; o, growth in control (logdry w,:ight);~, srowth in the presenceof uyptone or yeast extract.
inhibition of the induction takes place through a catabolite-mediated repression mechanism. Moreover, the inhibition of the induction shown by individual amino acids, as L-tryptophan and L.alanlne, but not by L-praline (Table 1), could also indicate that only some of them (perhaps readily metal, oils. able) present in the casein hydrolysate, caused the repression. The reduction of the particular amino acid concentration could re]ieve that, as has been postu. ]ated for a similar transitory casein hydrolysate effect observed on the enzymes of the lac [ 12) and gal operon [12,13] in E. coll. From the above results it can be postulated that a catabolite-like repressor effect takes place in tile ~-galactosidase system of& violaceus in nutritional conditions which produce the same effect in &: coll. However, more detailed investigations, such as the effect of cyclic nucleotides on repre~ion and the possible existence of catabolite inactivation mechanisms, are needed to clarify the former observations.
260 References I l I I)¢main, A.L., Kennel, Y,M, nnd Aharonowltz, Y. (I 979) in; Microbial Techlto|ogy: Current State, I:utut*: I'roslx'¢ls (Bull. A.T.. El!wood. D,C. and Ratledge, C., Fds.). 29th Syltlp. So¢. Gen. Mlcrobiol., pp. t63-I85, Cambridge Llnlversity Press, London. I2t tnamin¢, F., Lago. B.D. and Ih:main, A.L. (1969) In: l:crmenlationt Advances (Perlman, D., Ed.). pp, 19922 I. AtxllJen~iePress, New York. [3l Litiey. G. and Bull, A.T. (1974) J. Gen. MicrobtoL 83, 123-133. [4] S2nche.,, J. and Ilardi~son, C. (1979) Can. J. Microbiol. 25, 833-840.
151 S;in~-IlCz,J. and Ilardis~n, C. (1980) Arch. Mlcrobiol. ltq Press. 16] Lederberg, J. (1950) J. B-cteriol.60, 381-392. 17l Somogyi, M. (1945) J. BioL Chem. 160, 61-68, 181 Marasanik, B. (1961) Cold Spring llarb. Syrup, Ouant, Biol. 26, 249-256. [91 Okinaka, R,T. and Dobrogosz, WJ , (1967) J. Bactedol. 93, 1644-1650. |10] Buttin, G, (1963) J. MoL Biol. 7, I64-182. [l I I Mandelstam. J. (1961) Bloehem. J. 79,489-496. I121 Paigen, K. (1963) Biochim. Biophys. Acta 77, 318328. 113l Jordan, E;. Saedler, It., Lengeler, J. and Stafltnger, P, (1967) MoL Gen. Genet. 100, 203-209.