- Email: [email protected]
Anionic polymers of Bacillus subtilis cell wall modulate the folding rate of secreted proteins
FEMS Microbiology Letters 179 (1999) 43^47
Anionic polymers of Bacillus subtilis cell wall modulate the folding rate of secreted proteins R. Chambert *, M.F. Petit-Glatron Institut Jacques Monod, C.N.R.S., Universite¨s Paris 6 et Paris 7, Laboratoire Ge¨ne¨tique et Membranes, Tour 43, 2 place Jussieu, F-75251 Paris Cedex 05, France Received 9 June 1999; accepted 28 July 1999
Abstract In order to characterize the dynamics of the interaction between the emergent membrane translocated exoprotein and the components of Bacillus subtilis cell wall, we examined the kinetics of the in vitro refolding of levansucrase and K-amylase, at pH 7 and 37³C, in the presence of polyphosphates (polyP) of various chain lengths (2 9 n 9 65). These soluble anionic polymers are considered here to mimic the role of teichoic acids. Even in the absence of calcium, levansucrase rapidly refolded in the presence of polyP of n v 16. In contrast, polyP modulate indirectly the rate of K-amylase refolding via their affinity for calcium. These differential effects might explain that the rate of the cell wall translocation of K-amylase secretion was found to be half that of levansucrase. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Protein folding; Alpha-amylase; Levansucrase; Teichoic acid ; Cell wall translocation; Bacillus subtilis
1. Introduction It is now widely accepted that proteins are translocated under a non-native state [1^5]. In B. subtilis, proteins emerging on the trans side of the membrane encounter the cell wall, a highly cross-linked semiporous macromolecule composed of peptidoglycan and teichoic acids. The subsequent translocation of the extracellular proteins through the cell wall requires a rapid and e¤cient folding into their native conformation in order to prevent the proteolytic action of cell wall associated proteases [6,7] and to
* Corresponding author. Tel.: +33 (1) 44 27 47 19; Fax: +33 (1) 44 27 59 94; E-mail: [email protected]
minimize interactions with cell wall components. It was proposed that PrsA [8], a lipoprotein linked to the outer surface of the cytoplasmic membrane, assists the folding. Other possible cofactors of folding are metal ions such as calcium or iron which are concentrated at the membrane wall interface [9,10] and mediate the in vitro refolding of several proteins [11^13]. In the present work, we explore the hypothesis that structural components of cell wall could per se facilitate the folding of proteins en route to be secreted. We focused our attention on the possible role of teichoic acids. These phosphorylated polymers, which account for almost 50% of the cell wall dryweight [14], are linear polyanionic molecules that usually contain between 10 and 50 glycerophosphate
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 3 8 9 - 4
FEMSLE 8975 9-9-99
44
R. Chambert, M.F. Petit-Glatron / FEMS Microbiology Letters 179 (1999) 43^47
Fig. 1. Unfolding-refolding transition of levansucrase and K-amylase mediated by polyP45 in the absence of calcium. The transition was monitored by £uorescence measurements (A) or by protease sensitivity (B). 4 Wl levansucrase or K-amylase stock solution was mixed with 16 Wl of 6 M Gdn-HCl in bu¡er A (0.1 M sodium phosphate pH 7, containing 1 mM EDTA). After 10 min incubation at 37³C, 10 Wl denaturing mixture was diluted into 2 ml bu¡er A in the absence (a) or in the presence (b) of 1 mM polyP45 . A: Traces of changes in £uorescence were recorded at 343 nm (excitation wavelength was 283 nm). The dead-time for mixing was approximately 8 s. B: Protease sensitivity changes were estimated from 100 Wl aliquots withdrawn at 30 s and 720 s after initiation of refolding and mixed with 1 Wl of subtilisin 1 mg ml31 . Time 0 of refolding was obtained by mixing 1 Wl denaturing mixture with 200 Wl refolding bu¡er containing 10 Wg ml31 subtilisin. The samples were submitted to SDS-PAGE analysis. The gel was stained with Coomassie brilliant blue.
units, displaying a¤nity for metal ions such as Ca2 and possessing a KA of 4.5U104 M31 [15]. This compound is not commercially available and is di¤cult to obtain in an homogeneous and pure unmodi¢ed state from cell wall extracts [16]. Allowing for that, we chose inorganic short chain length polyphosphates (polyP) as models to mimic the properties of teichoic acids and their e¡ects on the refolding of K-amylase and levansucrase, two B. subtilis exocellular proteins of which the in vitro reversible un-
folding-folding transition at pH 7 and 37³C are well characterized [11,12].
2. Materials and methods 2.1. Materials PolyP (sodium salts) of various chain lengths were purchased from Sigma.
FEMSLE 8975 9-9-99
R. Chambert, M.F. Petit-Glatron / FEMS Microbiology Letters 179 (1999) 43^47
45
2.2. Puri¢cation and assays of proteins Levansucrase (stock solution 30 mg ml31 ) was puri¢ed from the culture supernatant of induced B. subtilis QB112 (degU32(Hy), sacA321) according to the published procedure [17]. K-Amylase (stock solution 30 mg ml31 ) was prepared from the culture supernatant of induced B. subtilis GM96101 (degU32(Hy), sacA321, vsacB, sacRamyE) [18], according to the published procedure [19]. 2.3. Fluorescence measurements Changes in intrinsic £uorescence and £uorescence spectra were recorded with a F2000 Hitachi thermoregulated spectro£uorimeter.
3. Results 3.1. Unfolding-refolding transition of levansucrase and K-amylase in the presence of polyP and in the absence of calcium
Fig. 2. Rate constants of unfolding-refolding transition of levansucrase and K-amylase in the presence of polyP of various chain lengths and a ¢xed concentration of calcium. Levansucrase and K-amylase were unfolded in 5 M Gdn-HCl as described in Fig. 1. The refolding at 37³C was initiated by diluting the denaturing mixture 200 times in 0.1 M. Sodium phosphate pH 7, 0.5 mM CaCl2 , 1 mM polyP of chain length indicated. The apparent ¢rst order rate constants of refolding were determined from the regression adjustment, by the least square method, of changes in the intensity of £uorescence at 343 nm as a function of time [11,12]. Levansucrase (a); K-amylase (b).
We showed previously [12,20] from in vitro studies that the unfolding-refolding transition of the two proteins was mediated by calcium at pH 7 and 37³C. In the absence of this metal, the proteins remained in an unfolded state after dilution of denaturing agents. We studied the e¡ect of the presence of 1 mM polyP of various chain lengths under such conditions. Refolding was monitored by measuring changes in the intrinsic £uorescence and the disappearance of proteolytic sensitivity of the refolded state. We observed that levansucrase rapidly and fully refolded in the presence of polyP of n v 16. In contrast, these polymers did not promote K-amylase refolding. The e¡ect of polyP45 (Fig. 1) illustrated this study. 3.2. Unfolding-refolding transition of levansucrase and K-amylase in the presence of both polyP and calcium The e¡ects of 1 mM polyP of various chain lengths on the unfolding-refolding transition of each protein, occurring in the presence of 0.5 mM
Fig. 3. Kinetics of unfolding-refolding transition of levansucrase and K-amylase with respect to polyP65 concentration and a ¢xed concentration of calcium. Levansucrase and K-amylase were unfolded in 5 M Gdn-HCl as described before. The refolding was initiated by diluting the denaturing mixture 200 times in 0.1 M sodium phosphate, pH 7, 0.5 mM CaCl2 containing 0^100 WM polyP65 . The rate constants of refolding were determined from changes in the £uorescence intensity at 343 nm as a function of time. Levansucrase (a); K-amylase (b).
FEMSLE 8975 9-9-99
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R. Chambert, M.F. Petit-Glatron / FEMS Microbiology Letters 179 (1999) 43^47
4. Discussion
Fig. 4. E¡ect of the addition of a large quantity of calcium on the inhibition of K-amylase refolding by polyP65 . K-Amylase was unfolded in 5 M Gdn-HCl as described above. 5 Wl of the denaturing mixture was diluted in 1 ml 0.2 M sodium acetate pH 7, 0.5 mM CaCl2 , containing 1 mM polyP65 preincubated at 37³C. The changes in intensity of £uorescence with respect to time were recorded. After 2 min, 25 Wl 0.1 M CaCl2 (25 mM ¢nal concentration) was added (arrow) and changes in the intensity of £uorescence of the protein recorded.
Ca2 , at pH 7 and 37³C, are shown in Fig. 2. The rate constants of levansucrase refolding increased with the chain length of polyP, whereas an opposite result was obtained for K-amylase. PolyP of n s 5 totally inhibited the refolding of this latter protein. The variation in the folding rate constant of each protein versus the concentration of polyP65 in the folding mixture was studied (Fig. 3). We noted that both proteins refolded at an equivalent rate in the absence of polyP65 . A gradual increase of polyP65 concentration decreased the folding rate constant of K-amylase, whereas it had the opposite e¡ect on the folding rate of levansucrase. 3.3. How does polyP inhibit K-amylase refolding? Two hypotheses can account for the inhibition of K-amylase refolding: either polyP stabilize the unfolded form of the protein through electrostatic interactions or polyP, via their high a¤nity to calcium (KA = 6.3U106 M31 for polyP60 [21]), decrease the amount of free calcium needed to promote protein refolding. To test the second hypothesis, we studied whether inhibition of refolding by polyP could be abolished by the addition of a large excess of calcium. This hypothesis seems to be correct, as shown in Fig. 4.
PolyP of n s 16 act as cofactors of levansucrase folding even in the absence of calcium, K-amylase, in contrast, needs free calcium to promote its folding. If polyP mimic in vitro, at least partially, the role played by teichoic acids in the cell wall environment, one would expect that levansucrase emerging from the translocase complex fold more rapidly than K-amylase. This could explain the di¡erence in the release kinetics of the two proteins, t1=2 of 1 min and 2 min, for levansucrase and K-amylase, respectively. Furthermore, we show that a high a¤nity of calcium for polyP inhibits K-amylase folding mediated by this metal ion. This might be an indication that modulation of teichoic acid anionic charge via alanylation [22] controls calcium a¤nity in such a way that free calcium remains available for an e¤cient folding of exoproteins that have properties in common with Kamylase. On the other hand, we cannot rule out the possibility that the polyP themselves contribute to the e¤ciency of levansucrase secretion. Although no counterpart of E. coli polyP kinase was found in B. subtilis [23], it is reasonable to suggest that di¡erent enzymic pathways might promote the synthesis of short chain length polyP in this bacterium [24].
Acknowledgements This work was supported in part by the European Commission (Biotechnology program, Contract BIO4-CT96-0097) and was carried out within the framework of the European Bacillus Secretion Group. We thank A. Krop¢nger for revision of the English text.
References [1] Randall, L.L. and Hardy, S.L. (1986) Correlation of competence for export with lack of tertiary structure of the mature species : a study in vivo of maltose-binding protein in E. coli. Cell 46, 921^928. [2] Eilers, M. and Schatz, G. (1988) Protein unfolding and the energetics of protein translocation across biological membranes. Cell 52, 481^483.
FEMSLE 8975 9-9-99
R. Chambert, M.F. Petit-Glatron / FEMS Microbiology Letters 179 (1999) 43^47 [3] Baker, J. and Craig, E.A. (1994) Heat-shock proteins as molecular chaperones. Eur. J. Biochem. 219, 11^23. [4] Kumamoto, C.A. (1991) Molecular chaperones and protein translocation across the Escherichia coli inner membrane. Mol. Microbiol. 5, 19^22. [5] Stuart, R.A., Cyr, D.M., Craig, E.A. and Neupert, W. (1994) Mitochondrial molecular chaperones: their role in protein translocation. Trends Biochem. Sci. 19, 87^92. [6] Stephenson, K. and Harwood, C.R. (1998) In£uence of a cell wall-associated protease on production of K-amylase by Bacillus subtilis. Appl. Environ. Microbiol. 64, 2875^2881. [7] Babe, L.M. and Schmidt, B. (1998) Puri¢cation and biochemical analysis of WprA, a 52-kDa serine protease secreted by Bacillus subtilis as an active complex with its 23-kDa propeptide. Biochim. Biophys. Acta 1386, 211^219. [8] Kontinen, V.P., Saris, P. and Sarvas, M. (1991) A gene (prsA) of Bacillus subtilis involved in a novel late stage of protein export. Mol. Microbiol. 5, 1273^1283. [9] Hughes, A.H., Hancock, I.C. and Baddiley, J. (1973) The function of teichoic acids in cation control in bacterial membranes. Biochem. J. 132, 83^93. [10] Beveridge, T.J. and Murray, R.G.E. (1976) Uptake and retention of metals by cell walls of Bacillus subtilis. J. Bacteriol. 127, 1502^1518. [11] Chambert, R. and Petit-Glatron, M.F. (1990) Reversible thermal unfolding of Bacillus subtilis levansucrase is modulated by Fe3 and Ca2 . FEBS Lett. 275, 61^64. [12] Haddaoui, E.A., Leloup, L., Petit-Glatron, M.F. and Chambert, R. (1997) Characterization of a stable intermediate trapped during reversible refolding of Bacillus subtilis K-amylase. Eur. J. Biochem. 249, 505^509. [13] Leloup, L., Le Saux, J., Petit-Glatron, M.F. and Chambert, R. (1999) Kinetics of the secretion of Bacillus subtilis levanase overproduced during the exponential phase of growth. Microbiology 145, 613^619. [14] Archibald, A.R., Hancock, I.C. and Harwood, C.R. (1993) Cell wall structure, synthesis and turnover. In: Bacillus subtilis and Other Gram positive Bacteria (Sonenshein, A.L., Hoch,
[15]
[16] [17] [18]
[19]
[20]
[21]
[22]
[23]
[24]
47
J.A. and Losick, R., Eds.), pp. 381^410. Biochemistry, Physiology and Molecular Genetics, American Society for Microbiology. Washington, DC. Doyle, R. (1989) How cell walls of Gram-positive bacteria interact with metal ions. In: Metal Ions and Bacteria (Beveridge, T.J. and Doyle, R.J., Eds.), pp. 275^293. John Wiley and Sons, New York. Archibald, A.R. (1974) The structure, biosynthesis and function of teichoic acid. Adv. Microb. Physiol. 11, 53^95. Dedonder, R. (1966) Levansucrase from Bacillus subtilis. Methods Enzymol. 8, 500^505. Leloup, L., Haddaoui, E., Chambert, R. and Petit-Glatron, M.F. (1997) Characterization of the rate limiting step of the secretion of Bacillus subtilis (K-amylase overproduced during the exponential phase of growth. Microbiology 143, 3295^ 3303. Haddaoui, E.A., Petit-Glatron, M.F. and Chambert, R. (1995) Characterization of a new cell-bound K-amylase in Bacillus subtilis 168 Marburg that is only immunologically related to the exocellular K-amylase. J. Bacteriol. 177, 5148^ 5150. Chambert, R., Haddaoui, E.A. and Petit-Glatron, M.F. (1995) Bacillus subtilis levansucrase: the e¤ciency of the second stage of secretion is modulated by external e¡ectors assisting folding. Microbiology 141, 997^1005. Sillen, L.G. and Martell, A. (1964) Stability Constants of Metal Ion Complexes, Special Publication No. 17. The Chemical Society, London. Shabarova, Z.A., Hughes, N.A. and Baddiley, J. (1962) The in£uence of adjacent phosphate and hydroxyl groups on amino acid esters. Biochem. J. 83, 216^219. Tzeng, C.M. and Kornberg, A. (1998) Polyphosphate kinase is highly conserved in many bacterial pathogens. Mol. Microbiol. 29, 381^382. Kornberg, A. (1995) Inorganic polyphosphate: toward making a forgotten polymer unforgettable. J. Bacteriol. 177, 491^ 496.
FEMSLE 8975 9-9-99
Anionic polymers of Bacillus subtilis cell wall modulate the folding rate of secreted proteins R. Chambert *, M.F. Petit-Glatron Institut Jacques Monod, C.N.R.S., Universite¨s Paris 6 et Paris 7, Laboratoire Ge¨ne¨tique et Membranes, Tour 43, 2 place Jussieu, F-75251 Paris Cedex 05, France Received 9 June 1999; accepted 28 July 1999
Abstract In order to characterize the dynamics of the interaction between the emergent membrane translocated exoprotein and the components of Bacillus subtilis cell wall, we examined the kinetics of the in vitro refolding of levansucrase and K-amylase, at pH 7 and 37³C, in the presence of polyphosphates (polyP) of various chain lengths (2 9 n 9 65). These soluble anionic polymers are considered here to mimic the role of teichoic acids. Even in the absence of calcium, levansucrase rapidly refolded in the presence of polyP of n v 16. In contrast, polyP modulate indirectly the rate of K-amylase refolding via their affinity for calcium. These differential effects might explain that the rate of the cell wall translocation of K-amylase secretion was found to be half that of levansucrase. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Protein folding; Alpha-amylase; Levansucrase; Teichoic acid ; Cell wall translocation; Bacillus subtilis
1. Introduction It is now widely accepted that proteins are translocated under a non-native state [1^5]. In B. subtilis, proteins emerging on the trans side of the membrane encounter the cell wall, a highly cross-linked semiporous macromolecule composed of peptidoglycan and teichoic acids. The subsequent translocation of the extracellular proteins through the cell wall requires a rapid and e¤cient folding into their native conformation in order to prevent the proteolytic action of cell wall associated proteases [6,7] and to
* Corresponding author. Tel.: +33 (1) 44 27 47 19; Fax: +33 (1) 44 27 59 94; E-mail: [email protected]
minimize interactions with cell wall components. It was proposed that PrsA [8], a lipoprotein linked to the outer surface of the cytoplasmic membrane, assists the folding. Other possible cofactors of folding are metal ions such as calcium or iron which are concentrated at the membrane wall interface [9,10] and mediate the in vitro refolding of several proteins [11^13]. In the present work, we explore the hypothesis that structural components of cell wall could per se facilitate the folding of proteins en route to be secreted. We focused our attention on the possible role of teichoic acids. These phosphorylated polymers, which account for almost 50% of the cell wall dryweight [14], are linear polyanionic molecules that usually contain between 10 and 50 glycerophosphate
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 3 8 9 - 4
FEMSLE 8975 9-9-99
44
R. Chambert, M.F. Petit-Glatron / FEMS Microbiology Letters 179 (1999) 43^47
Fig. 1. Unfolding-refolding transition of levansucrase and K-amylase mediated by polyP45 in the absence of calcium. The transition was monitored by £uorescence measurements (A) or by protease sensitivity (B). 4 Wl levansucrase or K-amylase stock solution was mixed with 16 Wl of 6 M Gdn-HCl in bu¡er A (0.1 M sodium phosphate pH 7, containing 1 mM EDTA). After 10 min incubation at 37³C, 10 Wl denaturing mixture was diluted into 2 ml bu¡er A in the absence (a) or in the presence (b) of 1 mM polyP45 . A: Traces of changes in £uorescence were recorded at 343 nm (excitation wavelength was 283 nm). The dead-time for mixing was approximately 8 s. B: Protease sensitivity changes were estimated from 100 Wl aliquots withdrawn at 30 s and 720 s after initiation of refolding and mixed with 1 Wl of subtilisin 1 mg ml31 . Time 0 of refolding was obtained by mixing 1 Wl denaturing mixture with 200 Wl refolding bu¡er containing 10 Wg ml31 subtilisin. The samples were submitted to SDS-PAGE analysis. The gel was stained with Coomassie brilliant blue.
units, displaying a¤nity for metal ions such as Ca2 and possessing a KA of 4.5U104 M31 [15]. This compound is not commercially available and is di¤cult to obtain in an homogeneous and pure unmodi¢ed state from cell wall extracts [16]. Allowing for that, we chose inorganic short chain length polyphosphates (polyP) as models to mimic the properties of teichoic acids and their e¡ects on the refolding of K-amylase and levansucrase, two B. subtilis exocellular proteins of which the in vitro reversible un-
folding-folding transition at pH 7 and 37³C are well characterized [11,12].
2. Materials and methods 2.1. Materials PolyP (sodium salts) of various chain lengths were purchased from Sigma.
FEMSLE 8975 9-9-99
R. Chambert, M.F. Petit-Glatron / FEMS Microbiology Letters 179 (1999) 43^47
45
2.2. Puri¢cation and assays of proteins Levansucrase (stock solution 30 mg ml31 ) was puri¢ed from the culture supernatant of induced B. subtilis QB112 (degU32(Hy), sacA321) according to the published procedure [17]. K-Amylase (stock solution 30 mg ml31 ) was prepared from the culture supernatant of induced B. subtilis GM96101 (degU32(Hy), sacA321, vsacB, sacRamyE) [18], according to the published procedure [19]. 2.3. Fluorescence measurements Changes in intrinsic £uorescence and £uorescence spectra were recorded with a F2000 Hitachi thermoregulated spectro£uorimeter.
3. Results 3.1. Unfolding-refolding transition of levansucrase and K-amylase in the presence of polyP and in the absence of calcium
Fig. 2. Rate constants of unfolding-refolding transition of levansucrase and K-amylase in the presence of polyP of various chain lengths and a ¢xed concentration of calcium. Levansucrase and K-amylase were unfolded in 5 M Gdn-HCl as described in Fig. 1. The refolding at 37³C was initiated by diluting the denaturing mixture 200 times in 0.1 M. Sodium phosphate pH 7, 0.5 mM CaCl2 , 1 mM polyP of chain length indicated. The apparent ¢rst order rate constants of refolding were determined from the regression adjustment, by the least square method, of changes in the intensity of £uorescence at 343 nm as a function of time [11,12]. Levansucrase (a); K-amylase (b).
We showed previously [12,20] from in vitro studies that the unfolding-refolding transition of the two proteins was mediated by calcium at pH 7 and 37³C. In the absence of this metal, the proteins remained in an unfolded state after dilution of denaturing agents. We studied the e¡ect of the presence of 1 mM polyP of various chain lengths under such conditions. Refolding was monitored by measuring changes in the intrinsic £uorescence and the disappearance of proteolytic sensitivity of the refolded state. We observed that levansucrase rapidly and fully refolded in the presence of polyP of n v 16. In contrast, these polymers did not promote K-amylase refolding. The e¡ect of polyP45 (Fig. 1) illustrated this study. 3.2. Unfolding-refolding transition of levansucrase and K-amylase in the presence of both polyP and calcium The e¡ects of 1 mM polyP of various chain lengths on the unfolding-refolding transition of each protein, occurring in the presence of 0.5 mM
Fig. 3. Kinetics of unfolding-refolding transition of levansucrase and K-amylase with respect to polyP65 concentration and a ¢xed concentration of calcium. Levansucrase and K-amylase were unfolded in 5 M Gdn-HCl as described before. The refolding was initiated by diluting the denaturing mixture 200 times in 0.1 M sodium phosphate, pH 7, 0.5 mM CaCl2 containing 0^100 WM polyP65 . The rate constants of refolding were determined from changes in the £uorescence intensity at 343 nm as a function of time. Levansucrase (a); K-amylase (b).
FEMSLE 8975 9-9-99
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R. Chambert, M.F. Petit-Glatron / FEMS Microbiology Letters 179 (1999) 43^47
4. Discussion
Fig. 4. E¡ect of the addition of a large quantity of calcium on the inhibition of K-amylase refolding by polyP65 . K-Amylase was unfolded in 5 M Gdn-HCl as described above. 5 Wl of the denaturing mixture was diluted in 1 ml 0.2 M sodium acetate pH 7, 0.5 mM CaCl2 , containing 1 mM polyP65 preincubated at 37³C. The changes in intensity of £uorescence with respect to time were recorded. After 2 min, 25 Wl 0.1 M CaCl2 (25 mM ¢nal concentration) was added (arrow) and changes in the intensity of £uorescence of the protein recorded.
Ca2 , at pH 7 and 37³C, are shown in Fig. 2. The rate constants of levansucrase refolding increased with the chain length of polyP, whereas an opposite result was obtained for K-amylase. PolyP of n s 5 totally inhibited the refolding of this latter protein. The variation in the folding rate constant of each protein versus the concentration of polyP65 in the folding mixture was studied (Fig. 3). We noted that both proteins refolded at an equivalent rate in the absence of polyP65 . A gradual increase of polyP65 concentration decreased the folding rate constant of K-amylase, whereas it had the opposite e¡ect on the folding rate of levansucrase. 3.3. How does polyP inhibit K-amylase refolding? Two hypotheses can account for the inhibition of K-amylase refolding: either polyP stabilize the unfolded form of the protein through electrostatic interactions or polyP, via their high a¤nity to calcium (KA = 6.3U106 M31 for polyP60 [21]), decrease the amount of free calcium needed to promote protein refolding. To test the second hypothesis, we studied whether inhibition of refolding by polyP could be abolished by the addition of a large excess of calcium. This hypothesis seems to be correct, as shown in Fig. 4.
PolyP of n s 16 act as cofactors of levansucrase folding even in the absence of calcium, K-amylase, in contrast, needs free calcium to promote its folding. If polyP mimic in vitro, at least partially, the role played by teichoic acids in the cell wall environment, one would expect that levansucrase emerging from the translocase complex fold more rapidly than K-amylase. This could explain the di¡erence in the release kinetics of the two proteins, t1=2 of 1 min and 2 min, for levansucrase and K-amylase, respectively. Furthermore, we show that a high a¤nity of calcium for polyP inhibits K-amylase folding mediated by this metal ion. This might be an indication that modulation of teichoic acid anionic charge via alanylation [22] controls calcium a¤nity in such a way that free calcium remains available for an e¤cient folding of exoproteins that have properties in common with Kamylase. On the other hand, we cannot rule out the possibility that the polyP themselves contribute to the e¤ciency of levansucrase secretion. Although no counterpart of E. coli polyP kinase was found in B. subtilis [23], it is reasonable to suggest that di¡erent enzymic pathways might promote the synthesis of short chain length polyP in this bacterium [24].
Acknowledgements This work was supported in part by the European Commission (Biotechnology program, Contract BIO4-CT96-0097) and was carried out within the framework of the European Bacillus Secretion Group. We thank A. Krop¢nger for revision of the English text.
References [1] Randall, L.L. and Hardy, S.L. (1986) Correlation of competence for export with lack of tertiary structure of the mature species : a study in vivo of maltose-binding protein in E. coli. Cell 46, 921^928. [2] Eilers, M. and Schatz, G. (1988) Protein unfolding and the energetics of protein translocation across biological membranes. Cell 52, 481^483.
FEMSLE 8975 9-9-99
R. Chambert, M.F. Petit-Glatron / FEMS Microbiology Letters 179 (1999) 43^47 [3] Baker, J. and Craig, E.A. (1994) Heat-shock proteins as molecular chaperones. Eur. J. Biochem. 219, 11^23. [4] Kumamoto, C.A. (1991) Molecular chaperones and protein translocation across the Escherichia coli inner membrane. Mol. Microbiol. 5, 19^22. [5] Stuart, R.A., Cyr, D.M., Craig, E.A. and Neupert, W. (1994) Mitochondrial molecular chaperones: their role in protein translocation. Trends Biochem. Sci. 19, 87^92. [6] Stephenson, K. and Harwood, C.R. (1998) In£uence of a cell wall-associated protease on production of K-amylase by Bacillus subtilis. Appl. Environ. Microbiol. 64, 2875^2881. [7] Babe, L.M. and Schmidt, B. (1998) Puri¢cation and biochemical analysis of WprA, a 52-kDa serine protease secreted by Bacillus subtilis as an active complex with its 23-kDa propeptide. Biochim. Biophys. Acta 1386, 211^219. [8] Kontinen, V.P., Saris, P. and Sarvas, M. (1991) A gene (prsA) of Bacillus subtilis involved in a novel late stage of protein export. Mol. Microbiol. 5, 1273^1283. [9] Hughes, A.H., Hancock, I.C. and Baddiley, J. (1973) The function of teichoic acids in cation control in bacterial membranes. Biochem. J. 132, 83^93. [10] Beveridge, T.J. and Murray, R.G.E. (1976) Uptake and retention of metals by cell walls of Bacillus subtilis. J. Bacteriol. 127, 1502^1518. [11] Chambert, R. and Petit-Glatron, M.F. (1990) Reversible thermal unfolding of Bacillus subtilis levansucrase is modulated by Fe3 and Ca2 . FEBS Lett. 275, 61^64. [12] Haddaoui, E.A., Leloup, L., Petit-Glatron, M.F. and Chambert, R. (1997) Characterization of a stable intermediate trapped during reversible refolding of Bacillus subtilis K-amylase. Eur. J. Biochem. 249, 505^509. [13] Leloup, L., Le Saux, J., Petit-Glatron, M.F. and Chambert, R. (1999) Kinetics of the secretion of Bacillus subtilis levanase overproduced during the exponential phase of growth. Microbiology 145, 613^619. [14] Archibald, A.R., Hancock, I.C. and Harwood, C.R. (1993) Cell wall structure, synthesis and turnover. In: Bacillus subtilis and Other Gram positive Bacteria (Sonenshein, A.L., Hoch,
[15]
[16] [17] [18]
[19]
[20]
[21]
[22]
[23]
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