Biochimie génétique, Institut Jacques Monod, Université Paris 7, 2 place Jussieu, 75251 Paris Cedex 05, France1
Laboratoire des Biomembranes, ERS571/CNRS, Université Paris Sud, bat. 430, 91405 Orsay, France2
Author for correspondence: Gilbert Richarme. Tel: +33 1 44 27 50 98. Fax: +33 1 44 27 35 80. e-mail: richarme{at}ccr.jussieu.fr
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: glycine betaine, choline, osmoprotectant, thermoprotectant, heat-shock protein DnaK
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Thermal aggregation of citrate synthase.
The native enzyme (80 µM) was diluted 100-fold in 40 mM HEPES, 50 mM KCl, 10 mM (NH4)2SO4, 2 mM potassium acetate, pH 8·0 at 43 °C in the absence or in the presence of glycine betaine or choline. Citrate synthase aggregation was monitored by measuring the A650 as described by Richarme & Caldas (1997 ).
Thermal inactivation of citrate synthase and ß-galactosidase.
Citrate synthase was diluted to a final concentration of 80 nM at 49 °C in the absence or presence of glycine betaine or choline. Citrate synthase activity was determined as described by Richarme & Caldas (1997 ). ß-Galactosidase was diluted to a final concentration of 1·5 µM at 56 °C in the absence or in the presence of glycine betaine. Its activity was determined by measuring the A420 of samples containing ß-galactosidase and 2·8 mM ONPG in sodium phosphate buffer pH 7·3 at 25 °C.
Refolding of citrate synthase.
Denaturation and renaturation reactions were carried out at 20 °C. Renaturation was initiated by pouring the renaturation solvent onto the unfolded protein, under vortex agitation, in Eppendorf polyethylene tubes. Citrate synthase was denatured at a concentration of 10 µM in 8 M urea, 50 mM Tris/HCl, 2 mM EDTA, 20 mM dithiothreitol, pH 8·0 for 50 min. Renaturation was initiated by a 100-fold dilution in 40 mM HEPES, 50 mM KCl, 10 mM (NH4)2SO4, 2 mM potassium acetate, pH 8·0. The enzymic activity of citrate synthase was measured as described by Richarme & Caldas (1997 ). DnaK was prepared as described by Richarme & Caldas (1997
).
Transport measurements.
For glycine betaine (May et al., 1986 ) and choline uptake (Lanfald & Strom, 1986
), cells were grown in minimal medium M63 (Miller, 1972
), supplemented with 0·4% glycerol or 0·4% glucose, respectively, as carbon source, and with the required amino acids added at 50 µg ml-1. Bacteria were harvested in the exponential phase of growth, washed once with the culture medium and diluted in the same medium supplemented with 300 mM NaCl to an OD600 of 0·2. The cell suspension was equilibrated at 22 °C for 10 min, unless otherwise indicated. Transport was performed aerobically (200 r.p.m.), and was initiated by mixing 2 ml cells with [14C]glycine betaine [synthesized as described by Ikuta et al. (1977
), and used at 10 mCi mmol-1 (370 MBq mmol-1) at the concentrations indicated in the text] or [3H]choline [obtained from Amersham, and used at 50 mCi mmol-1 (1850 MBq mmol-1), at the concentrations indicated in the text]. Samples (200 µl) were removed, filtered through Millipore filters, washed with 3x1 ml transport medium and their radioactivity measured. To measure the effect of temperature shift-up on the rate of glycine betaine or choline uptake, exponential-phase cultures of E. coli were transferred from 30 to 42 °C, and transport activities were measured at 22 °C at several times before and after the temperature shift-up. Glycine betaine uptake was measured at 2 µM glycine betaine, at which there is a major contribution of ProU, and at 200 µM glycine betaine, at which both ProU and ProP are effective. Choline uptake was measured at 10 µM and 200 µM choline.
Colony-forming ability of the dnaK mutant.
The colony-forming ability of the dnaK deletion strain GW4813 was studied by plating 100 µl of a dilution of exponentially growing cells in M63 medium supplemented with 0·4% glucose, the required amino acids at 50 µg ml-1 and 1 mM glycine betaine or choline, as indicated, containing 200 bacteria (assuming 6x108 bacteria ml-1 at OD600=1: Stock et al., 1977 ), onto minimal medium agar plates (M63 medium supplemented with 0·4% glucose, the required amino acids at 50 µg ml-1, and 1 mM glycine betaine or choline, as indicated).
Materials.
Citrate synthase (from porcine heart) was obtained from Sigma. All other chemicals were from Sigma and were reagent grade.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Suppression of the thermosensitive phenotype of a dnaK deletion mutant by glycine betaine and choline
The dnaK deletion mutant GW4813 is deficient for growth and viability at 42 °C (Paek & Walker, 1987 ), probably as a consequence of protein aggregation. Samples (100 µl) of a dilution of the dnaK mutant were plated on M63-glucose agar plates, in the absence or presence of 1 mM glycine betaine or choline, at 30 and 42 °C. The colony-forming abilities of the dnaK mutant at 30 and 42 °C were compared. The colony-forming ability of the mutant at 42 °C rose from 0% (of that at 30 °C) in the absence of additives, to 11% and 29% (mean value of three experiments) in the presence of 1 mM glycine betaine and choline respectively (Fig. 4
) (glycine betaine and choline had no effect on the colony-forming ability of the parental strain; not shown). Since choline is quantitatively metabolized into glycine betaine in vivo (Lanfald & Strom, 1986
), we conclude that intracellular glycine betaine can partially compensate for the deficiency of the dnaK mutant at 42 °C, probably by reducing its known protein aggregation defect at elevated temperatures.
|
We measured the dependence of the colony-forming ability of the dnaK deletion mutant on the external concentrations of glycine betaine and choline (in the experimental conditions of Fig. 4). Half-maximal stimulation occurred at 2 µM glycine betaine (not shown) and 6 µM choline (Fig. 5
).These concentrations are similar to the Km of the ProU and BetT transport systems for glycine betaine and choline, respectively (May et al., 1986
; Lamark et al., 1991
; Boch et al., 1994
).
|
Implications
Our results suggest that glycine betaine and its precursor choline are involved in bacterial thermoprotection. In vitro, glycine betaine and choline protect citrate synthase against thermodenaturation and stimulate its renaturation, with an efficiency similar to that of protein chaperones, although at much higher concentrations. However, the glycine betaine concentrations which protect citrate synthase against thermodenaturation in vitro (around 50 mM) are similar to those found in bacteria grown in usual minimal media (this study). Although the protection of proteins by osmolytes in vitro often requires high concentrations of the latter (Yancey et al., 1982 ; Arakawa & Timasheff, 1985
), the protective effect of trimethylamine against the cold lability of phosphofructokinase shows a concentration threshold of around 50 mM (Hand & Somero, 1982
), similar to that observed in our work. The amount of glycine betaine accumulated in our study (48 nmol per mg cell protein after 30 min) is not significantly different from that obtained by others in similar conditions (35 nmol per mg cell protein after 30 min: Perroud & Le Rudelier, 1985
). The amounts of glycine betaine accumulated after an osmotic upshock and during the stationary phase are severalfold higher (Conska & Epstein, 1996
; Hengge-Aronis, 1996
; Perroud & Le Rudelier, 1985
), and the thermoprotectant effect of glycine betaine should thus be even more effective in these physiological conditions. Although glycine betaine and choline are not synthesized endogenously, and have to be taken up from the external medium, they are often present in the natural environment of enterobacteria (Conska & Epstein, 1996
). Our results show that glycine betaine and choline partially restore the viability and growth deficiency of a dnaK deletion mutant at 42 °C. Suppressors of dnaK null mutants that grow at 42 °C have been isolated (Paek & Walker, 1987
), suggesting that cells do not have an absolute requirement for the DnaK function for growth at 42 °C. The present study shows that one of the so-called chemical chaperones (Talzelt et al., 1996
), glycine betaine, can partially assume the function of the dnaK gene product, and emphasizes the role played by small molecules such as polyols (Hengge-Aronis et al., 1991
; Singer & Lindquist, 1998
; Yancey et al., 1982
), trimethylamines (Talzelt et al., 1996
; Yancey et al., 1982
) and amino acids (Yancey et al., 1982
) in the protection of cells against stress.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Boch, J., Kempf, B. & Bremer, E. (1994). Osmoregulation in Bacillus subtilis: synthesis of the osmoprotectant glycine betaine from exogenously provided choline. J Bacteriol 176, 5364-5371.[Abstract]
Buchner, J., Schmidt, M., Fuchs, M., Jaenicke, R., Rudolph, R., Schmid, F. & Kiefhaber, T. (1991). GroE facilitates refolding of citrate synthase by suppressing aggregation. Biochemistry 30, 1586-1591.[Medline]
Chambers, S. & Cunin, C. M. (1985). The osmoprotective effect of betaine and human urine against low pH and high concentrations of electrolytes, sugars, and urea. J Infect Dis 152, 1308-1316.[Medline]
Conska, L. N. & Epstein, W. (1996). Osmoregulation. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, pp. 12101223. Edited by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.
Georgopoulos, C., Liberek, K., Zylicz, M. & Ang, D. (1994). Properties of the heat shock proteins of Escherichia coli and the autoregulation of the heat shock response. In The Biology of the Heat Shock Proteins and Molecular Chaperones, pp. 209-250. Edited by R. I. Morimoto, A. Tissieres & C. Georgopoulos. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Gowrishankar, J. (1986). proP-mediated proline transport also plays a role in Escherichia coli osmoregulation. J Bacteriol 166, 331-333.[Medline]
Hand, S. C. & Somero, G. N. (1982). Urea and methylamine effects on rabbit muscle phosphofructokinase. J Biol Chem 257, 734-741.
Hendrick, J. P. & Hartl, F. U. (1993). Molecular chaperone functions of heat shock proteins. Annu Rev Biochem 62, 349-384.[Medline]
Hengge-Aronis, R. (1996). Regulation of gene expression during entry into stationary phase. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, pp. 14971512. Edited by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.
Hengge-Aronis, R., Klein, W., Lange, R., Rimmele, M. & Boos, W. (1991). Trehalose synthesis genes are controlled by the putative sigma factor encoded by rpoS and are involved in stationary phase thermotolerance in Escherichia coli. J Bacteriol 173, 7918-7924.[Medline]
Ikuta, S., Matuura, K., Imamura, S., Misaki, H. & Horiuti, Y. (1977). Oxidative pathway of choline to betaine in the soluble fraction prepared from Arthrobacter globiformis. J Biochem 82, 157-163.[Abstract]
Lamark, T., Kaasen, I., Eshoo, M. W., Falkenberg, P., McDougall, J. & Strom, A. R (1991). DNA sequence and analysis of the bet genes encoding the osmoregulatory choline-glycine betaine pathway of Escherichia coli. Mol Microbiol 5, 1049-1064.[Medline]
Lanfald, B. & Strom, A. R. (1986). Choline-glycine pathway confers a high level of osmotic tolerance in Escherichia coli. J Bacteriol 165, 849-855.[Medline]
May, G., Faatz, E., Villarejo, M. & Bremer, E. (1986). Binding protein dependent transport of glycine betaine and its osmotic regulation in Escherichia coli K12. Mol Gen Genet 205, 225-233.[Medline]
Miller, J. H. (1972). Experiments in Molecular Genetics, p. 431. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Milner, J. L. S., Grothe, S. & Wood, J. M. (1988). Proline porter II is activated by a hyperosmotic shift in both whole cells and membrane vesicles of Escherichia coli K-12. J Biol Chem 263, 14900-14905.
Moses, V. & Sharp, P. B. (1970). Intermediates between metabolic intermediates and ß-galactosidase of Escherichia coli. Biochem J 118, 491-495.[Medline]
Paek, K.-H. & Walker, G. C. (1987). Escherichia coli dnaK null mutants are inviable at high temperature. J Bacteriol 169, 283-290.[Medline]
Perroud, B. & Le Rudelier, D. (1985). Glycine betaine transport in Escherichia coli: osmotic modulation. J Bacteriol 161, 393-401.[Medline]
Pollard, A. & Wyn Jones, R. G. (1979). Metabolic engineering of glycine betaine synthesis. Planta 144, 291-298.
Richarme, G. & Caldas, T. (1997). Chaperone properties of the bacterial periplasmic substrate-binding proteins. J Biol Chem 272, 15607-15612.
Singer, M. A. & Lindquist, S. (1998). Multiple effects of trehalose on protein folding in vitro and in vivo. Mol Cell 1, 639-648.[Medline]
Stirling, D. A., Hulton, C. S. J., Wadell, L., Park, S. F., Stuart, G. S. A. B., Booth, I. R. & Higgins, C. F. (1989). Molecular characterization of the proU loci of Salmonella typhimurium and Escherichia coli encoding osmoregulated glycine betaine transport systems. Mol Microbiol 3, 1025-1038.[Medline]
Stock, J. B., Rauch, B. & Roseman, S. (1977). Periplasmic space in Salmonella typhimurium and Escherichia coli. J Biol Chem 252, 7850-7861.[Abstract]
Strom, A. R., Falkenberg, P. & Landfald, B. (1986). Genetics of osmoregulation in Escherichia coli uptake and biosynthesis of organic osmolytes. FEMS Microbiol Rev 39, 79-86.
Talzelt, J., Prusiner, S. B. & Welch, W. J. (1996). Chemical chaperones interfere with the formation of scrapie protein. EMBO J 15, 6363-6373.[Abstract]
Yancey, P. H. & Somero, G. N. (1979). Counteraction of urea destabilization of protein structure by methylamine osmoregulatory compounds of elasmobranch fishes. Biochem J 183, 317-323.[Medline]
Yancey, P. H., Clark, M. E., Hand, S. T., Bowlus, P. D. & Somero, G. N. (1982). Living with water stress: evolution of osmolyte systems. Science 217, 1214-1222.[Medline]
Received 21 December 1998;
revised 28 April 1999;
accepted 21 May 1999.