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Secretion of proteins by Bacillus subtilis 168 grown in the presence of membrane active agents (alcohols)
Journal of’ Chromatography, Elsevier Science Publishers CHROM.
Institute
in The Netherlands
20 244
SECRETION PRESENCE
SVETLA
440 (1988) 379-383 B.V.. Amsterdam - Printed
OF PROTEINS OF MEMBRANE
BY BACILLUS SUBTZLZS 168 GROWN ACTIVE AGENTS (ALCOHOLS)
IN THE
BAYKOUSHEVA
qf Microbiology.
Bulgarian
Academy of Sciences,I I13 Sofia (Bulgaria)
SUMMARY
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis was used to study the composition of proteins secreted by the Gram-positive microorganism Bacillus suhtilis strain 168 when the latter was grown in the presence of primary alcohols (methanol, ethanol, 1-propanol and I-butanol). These membrane-active agents had different effects on the pattern of proteins exported by B. subtilis. The secretion of some proteins was inhibited by the alkanols while that of others was stimulated, depending on the type of drug used. All alcohols were found to decrease the activity of extracellular enzymes such as a-amylase and serine protease without affecting significantly the activity of these enzymes when tested in vitro. The observed effects might be due to the ability of these agents to perturb the structure of biological membranes, thus interfering with the process of protein translocation through the lipid bilayers.
INTRODUCTION
The Gram-positive microorganism Bacillus subtilis secretes a wide variety of proteins including the enzymes serine protease (subtilisin) and a-amylase, widely used in the fermentation industry 1-3 In the past few years genetic and biochemical studies with bacteria have contributed significantly to our understanding of the mechanisms of protein secretion and localization 1J,4. Nevertheless, some aspects of the problem remain controversial, for example, the question of whether proton motive force5 and/or ATP6 are necessary for a protein to be translocated through the membrane and the role of the latter in this process. The phospholipid composition of bacterial membranes also seems to be essential in this respect’. Bacteria offer excellent opportunities for studying this problem as they allow manipulation of the physical and chemical properties of the membranes simply by growing the cells in the presence of membrane-active agents*. Solvents, detergents and specific inhibitors of lipid synthesis are able to modify the lipid composition and the lipid to protein ratio of bacterial membranes. When the lipid composition of these structures was changed by an inhibitor of lipid synthesis, the antibiotic cerulenin, the export of proteins was inhibited9+12, the rate of autolysis of cells was greatly reduced13 and sporulation was affectedl4. 0021-9673/88/$03.50
0
1988 Elsevier Science Publishers
B.V.
SECRETION TABLE
OF PROTEINS
BY BACILLUS
381
SUBTZLZS 168
I
EFFECT
OF ALCOHOLS
Alcohol added
ON THE SECRETION
Concen[ration
Methanol Ethanol I-Propanol I -Butanol Control
OF PROTEINS
BY B. SUBTZLZS 168
Extracellular cc-amylase
(M)
Total protein (mg/rnl)
U/ml
Spec$c activity (U/mg protein)
U/ml
Specific activity (Ujmg prolein)
1.0 0.3 0.1 0.04 0
5.63 4.58 4.73 5.18 4.14
158.4 255.2 211 158.4 281.6
28 56 45 31 68
4.00 3.12 2.90 2.60 5.39
0.53 0.80 0.60 0.50 1.52
Extracellular serine protease
not affect the latter were chosen for performing the subsequent experiments (Table I). These concentrations decreased with increasing chain length. The amount of total protein secreted in the presence of alcohols was slightly higher than that of the control (culture without alcohol). Fig. 1 shows the separation of secreted proteins by SDSPAGE. The number of protein bands observed in the control (Fig. 1, lane a) was 35 whereas in the presence of alcohols it was lower (Fig. 1, lanes b-e). In some instances the proportion of a protein was lower whereas for other proteins it was higher compared with the control. It was shown that all alcohols decreased the activities of extracellular serine protease and a-amylase to different extents, which was confirmed by SDS-PAGE (Fig. 1). The bands corresponding to serine protease and a-amylase with molecular
-7
BS-
C-
-AMY
_.L -
GD-
MP
Q
b
c
d
e
Fig. 1. SDS-PAGE of proteins secreted by B. subtilis 168 (a) in the absence or in the presence of (b) methanol, (c) ethanol, (d) 1-propanol or (e) I-butanol. BS, bovine serum albumin (MW 67 000); OV, ovalbumin (MW 45 000); GD, Glyceraldehyde-3-phosphate dehydrogenase (MW 36 000); SP, serine protease (MW 29 000); AMY, cc-amylase (MW 59 500); MP. marker proteins. Concentration of acrylamide, 10%.
382
S. BAYKOUSHEVA
weights of about 29 000 and 59 500, respectively, were less pronounced when alcohols were present in the growth medium. In order to confirm that this decrease in extracellular activities was not due to a direct effect of alcohols on the enzymes, we tested this effect in vitro under the same conditions (i.e., concentrations of alcohols, duration of contact with the enzymes, temperature and shaking) as those used to study protein secretion (data not shown). The influence of these agents was not significant as they reduced the enzyme activities by about 10% in all instances. DISCUSSION
We have shown previously that some membrane-active agents such as the nonionic detergents Triton X-100 and octyl glucoside decreased the activities of extracellular serine protease and a-amylase of B. subtilis 16gz8. Some other results that we obtained demonstrated that the modification of the membranes of this microorganism (by cerulenin, for example) lowered the activity of a membrane-bound enzyme, i.e., ATPase, which was proved by both biochemical and ultracytochemical methodsz9. The use of membrane-active agents to modify the properties of bacterial membranes allows us to study the role of the membrane in the translocation of proteins through this hydrophobic barrier. The results presented here show that alcohols might be used as such agents at concentrations that do not affect growth but affect significantly different biological functions of the cells. The production of exoproteins might be dependent on the formation of a proper functional link between the ribosomes and the membrane and hence is expected to be vulnerable to subtle alterations in membrane organizationlo. As alcohols are able to interact directly with the biological membranes Is they might affect (prevent) the formation of such a link and thus inhibit the secretion of proteins. This is a reasonable explanation of the results presented here but the first possibility outlined above (i.e., that protein secretion was affected by changes in the lipid composition of bacterial membranes) should not be neglected. REFERENCES 1 F. G. Priest, Bacterial. Rev., 41 (1977) 711. 2 F. G. Priest, in J. Chaloupka and V. Krumphazl (Editors), Extracellular Enzymes of Microorganisms, Plenum Press, New York, 1987, p. 23. 3 V. G. Debabov, in D. Dubnau (Editor), Molecular Biology of the Bacilli, Vol. I, Bacillus subtilis, Academic Press, New York, 1982, p. 331. 4 M. Sarvas, Curr. Topics Microbial. Immunol., 125 (1986) 103. 5 L. L. Randall, Methods Enzymol., 125 (1986) 129. 6 P. C. Tai, Curr. Topics Microbial. Immunol., 125 (1986) 43. 7 M. A. Nesmeyanova, FEBS Left., 142 (1982) 189. 8 J. E. Cronan and E. P. Gelmann, Bacterial. Rev., 39 (1975) 232. 9 J. C. Paton, B. K. May and W. H. Elliott, J. Gen. Microbial., 118 (1980) 179. IO Y. Fishman, S. Rottem and N. Citri, J. Bacterial., 141 (1980) 1435. 11 M. P. Caulfield, R. C. W. Berkeley, E. A. Pepper and J. Melling, J. Bacterial., 138 (1979) 345. 12 M.-F. Petit-Glatron and R. Chambert, Eur. J. Biochem., 119 (1981) 603. 13 H. J. Rogers and G. Wright, J. Gen. Microbial., 132 (1986) 1221.
SECRETION 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
OF PROTEINS
BY BAClLLUS
SUBTILIS
168
383
N. Cherepova, S. Baykousheva and K. Ilieva, Microbios Lett., 28 (1985) 127. M. K. Jain, D. G. Toussaint and E. H. Cordes, J. Membr. Biol., 14 (1973) 1. L. 0. Ingram and T. M. Buttle. Adv. Microb. Physiol., 25 (1984) 253. B. Berger, C. E. Carty and L. 0. Ingram, J. Bucterinl., 142 (1980) 1040. D. Rigomier, J.-P. Bohin and B. Lubochinsky, J. Gen. Microbial., 121 (1980) 139. J.-P. Bohin, D. Rigomier and P. Schaeffer, J. Bucteriol.. 127 (1976) 934. S. Lory, P. C. Tai and B. D. Davis, J. Bacterial., 156 (1983) 695. E. T. Palva, T. R. Hirst, S. J. S. Hardy., J. Holmgren and L. L. Randall, J. Bacterial., 146 (1981) 325. B. K. May and W. H. Elliott, Biockim. BiophJ,s. Actu. 166 (1968) 532. J. Millet, J. Appl. Bucteriol., 33 (1970) 207. 0. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, J. Biol. Cbem., 193 (1951) 265. U. K. Laemmli, Mature (Lotdon), 227 (1970) 680. P. H. O’Farrell, J. Biol. Chem., 250 (1975) 4007. J. Heukeshoven and R. Dernick, Electrophoresis, 6 (1985) 103. S. Baykousheva and K. Ilieva, Acta Microbial. Bulg., 21 (1987) 10. S. Baykousheva, N. Cherepova and K. Ilieva, in J. Chaloupka and V. Krumphanzl. (Editors), Extracdlulur Enzymes of Microorgunisms, Plenum Press, New York, 1987, p. 39.
Institute
in The Netherlands
20 244
SECRETION PRESENCE
SVETLA
440 (1988) 379-383 B.V.. Amsterdam - Printed
OF PROTEINS OF MEMBRANE
BY BACILLUS SUBTZLZS 168 GROWN ACTIVE AGENTS (ALCOHOLS)
IN THE
BAYKOUSHEVA
qf Microbiology.
Bulgarian
Academy of Sciences,I I13 Sofia (Bulgaria)
SUMMARY
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis was used to study the composition of proteins secreted by the Gram-positive microorganism Bacillus suhtilis strain 168 when the latter was grown in the presence of primary alcohols (methanol, ethanol, 1-propanol and I-butanol). These membrane-active agents had different effects on the pattern of proteins exported by B. subtilis. The secretion of some proteins was inhibited by the alkanols while that of others was stimulated, depending on the type of drug used. All alcohols were found to decrease the activity of extracellular enzymes such as a-amylase and serine protease without affecting significantly the activity of these enzymes when tested in vitro. The observed effects might be due to the ability of these agents to perturb the structure of biological membranes, thus interfering with the process of protein translocation through the lipid bilayers.
INTRODUCTION
The Gram-positive microorganism Bacillus subtilis secretes a wide variety of proteins including the enzymes serine protease (subtilisin) and a-amylase, widely used in the fermentation industry 1-3 In the past few years genetic and biochemical studies with bacteria have contributed significantly to our understanding of the mechanisms of protein secretion and localization 1J,4. Nevertheless, some aspects of the problem remain controversial, for example, the question of whether proton motive force5 and/or ATP6 are necessary for a protein to be translocated through the membrane and the role of the latter in this process. The phospholipid composition of bacterial membranes also seems to be essential in this respect’. Bacteria offer excellent opportunities for studying this problem as they allow manipulation of the physical and chemical properties of the membranes simply by growing the cells in the presence of membrane-active agents*. Solvents, detergents and specific inhibitors of lipid synthesis are able to modify the lipid composition and the lipid to protein ratio of bacterial membranes. When the lipid composition of these structures was changed by an inhibitor of lipid synthesis, the antibiotic cerulenin, the export of proteins was inhibited9+12, the rate of autolysis of cells was greatly reduced13 and sporulation was affectedl4. 0021-9673/88/$03.50
0
1988 Elsevier Science Publishers
B.V.
SECRETION TABLE
OF PROTEINS
BY BACILLUS
381
SUBTZLZS 168
I
EFFECT
OF ALCOHOLS
Alcohol added
ON THE SECRETION
Concen[ration
Methanol Ethanol I-Propanol I -Butanol Control
OF PROTEINS
BY B. SUBTZLZS 168
Extracellular cc-amylase
(M)
Total protein (mg/rnl)
U/ml
Spec$c activity (U/mg protein)
U/ml
Specific activity (Ujmg prolein)
1.0 0.3 0.1 0.04 0
5.63 4.58 4.73 5.18 4.14
158.4 255.2 211 158.4 281.6
28 56 45 31 68
4.00 3.12 2.90 2.60 5.39
0.53 0.80 0.60 0.50 1.52
Extracellular serine protease
not affect the latter were chosen for performing the subsequent experiments (Table I). These concentrations decreased with increasing chain length. The amount of total protein secreted in the presence of alcohols was slightly higher than that of the control (culture without alcohol). Fig. 1 shows the separation of secreted proteins by SDSPAGE. The number of protein bands observed in the control (Fig. 1, lane a) was 35 whereas in the presence of alcohols it was lower (Fig. 1, lanes b-e). In some instances the proportion of a protein was lower whereas for other proteins it was higher compared with the control. It was shown that all alcohols decreased the activities of extracellular serine protease and a-amylase to different extents, which was confirmed by SDS-PAGE (Fig. 1). The bands corresponding to serine protease and a-amylase with molecular
-7
BS-
C-
-AMY
_.L -
GD-
MP
Q
b
c
d
e
Fig. 1. SDS-PAGE of proteins secreted by B. subtilis 168 (a) in the absence or in the presence of (b) methanol, (c) ethanol, (d) 1-propanol or (e) I-butanol. BS, bovine serum albumin (MW 67 000); OV, ovalbumin (MW 45 000); GD, Glyceraldehyde-3-phosphate dehydrogenase (MW 36 000); SP, serine protease (MW 29 000); AMY, cc-amylase (MW 59 500); MP. marker proteins. Concentration of acrylamide, 10%.
382
S. BAYKOUSHEVA
weights of about 29 000 and 59 500, respectively, were less pronounced when alcohols were present in the growth medium. In order to confirm that this decrease in extracellular activities was not due to a direct effect of alcohols on the enzymes, we tested this effect in vitro under the same conditions (i.e., concentrations of alcohols, duration of contact with the enzymes, temperature and shaking) as those used to study protein secretion (data not shown). The influence of these agents was not significant as they reduced the enzyme activities by about 10% in all instances. DISCUSSION
We have shown previously that some membrane-active agents such as the nonionic detergents Triton X-100 and octyl glucoside decreased the activities of extracellular serine protease and a-amylase of B. subtilis 16gz8. Some other results that we obtained demonstrated that the modification of the membranes of this microorganism (by cerulenin, for example) lowered the activity of a membrane-bound enzyme, i.e., ATPase, which was proved by both biochemical and ultracytochemical methodsz9. The use of membrane-active agents to modify the properties of bacterial membranes allows us to study the role of the membrane in the translocation of proteins through this hydrophobic barrier. The results presented here show that alcohols might be used as such agents at concentrations that do not affect growth but affect significantly different biological functions of the cells. The production of exoproteins might be dependent on the formation of a proper functional link between the ribosomes and the membrane and hence is expected to be vulnerable to subtle alterations in membrane organizationlo. As alcohols are able to interact directly with the biological membranes Is they might affect (prevent) the formation of such a link and thus inhibit the secretion of proteins. This is a reasonable explanation of the results presented here but the first possibility outlined above (i.e., that protein secretion was affected by changes in the lipid composition of bacterial membranes) should not be neglected. REFERENCES 1 F. G. Priest, Bacterial. Rev., 41 (1977) 711. 2 F. G. Priest, in J. Chaloupka and V. Krumphazl (Editors), Extracellular Enzymes of Microorganisms, Plenum Press, New York, 1987, p. 23. 3 V. G. Debabov, in D. Dubnau (Editor), Molecular Biology of the Bacilli, Vol. I, Bacillus subtilis, Academic Press, New York, 1982, p. 331. 4 M. Sarvas, Curr. Topics Microbial. Immunol., 125 (1986) 103. 5 L. L. Randall, Methods Enzymol., 125 (1986) 129. 6 P. C. Tai, Curr. Topics Microbial. Immunol., 125 (1986) 43. 7 M. A. Nesmeyanova, FEBS Left., 142 (1982) 189. 8 J. E. Cronan and E. P. Gelmann, Bacterial. Rev., 39 (1975) 232. 9 J. C. Paton, B. K. May and W. H. Elliott, J. Gen. Microbial., 118 (1980) 179. IO Y. Fishman, S. Rottem and N. Citri, J. Bacterial., 141 (1980) 1435. 11 M. P. Caulfield, R. C. W. Berkeley, E. A. Pepper and J. Melling, J. Bacterial., 138 (1979) 345. 12 M.-F. Petit-Glatron and R. Chambert, Eur. J. Biochem., 119 (1981) 603. 13 H. J. Rogers and G. Wright, J. Gen. Microbial., 132 (1986) 1221.
SECRETION 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
OF PROTEINS
BY BAClLLUS
SUBTILIS
168
383
N. Cherepova, S. Baykousheva and K. Ilieva, Microbios Lett., 28 (1985) 127. M. K. Jain, D. G. Toussaint and E. H. Cordes, J. Membr. Biol., 14 (1973) 1. L. 0. Ingram and T. M. Buttle. Adv. Microb. Physiol., 25 (1984) 253. B. Berger, C. E. Carty and L. 0. Ingram, J. Bucterinl., 142 (1980) 1040. D. Rigomier, J.-P. Bohin and B. Lubochinsky, J. Gen. Microbial., 121 (1980) 139. J.-P. Bohin, D. Rigomier and P. Schaeffer, J. Bucteriol.. 127 (1976) 934. S. Lory, P. C. Tai and B. D. Davis, J. Bacterial., 156 (1983) 695. E. T. Palva, T. R. Hirst, S. J. S. Hardy., J. Holmgren and L. L. Randall, J. Bacterial., 146 (1981) 325. B. K. May and W. H. Elliott, Biockim. BiophJ,s. Actu. 166 (1968) 532. J. Millet, J. Appl. Bucteriol., 33 (1970) 207. 0. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, J. Biol. Cbem., 193 (1951) 265. U. K. Laemmli, Mature (Lotdon), 227 (1970) 680. P. H. O’Farrell, J. Biol. Chem., 250 (1975) 4007. J. Heukeshoven and R. Dernick, Electrophoresis, 6 (1985) 103. S. Baykousheva and K. Ilieva, Acta Microbial. Bulg., 21 (1987) 10. S. Baykousheva, N. Cherepova and K. Ilieva, in J. Chaloupka and V. Krumphanzl. (Editors), Extracdlulur Enzymes of Microorgunisms, Plenum Press, New York, 1987, p. 39.