Jul 28, 1994 - min showed thermotolerance at 74°C, Sulfolobus shibatae grown at 70°C and heat shocked at 88°C for 60 min showed thermotolerance at ...
JOURNAL OF BACrERIOLOGY, Oct. 1994, p. 6148-6152
Vol. 176, No. 19
0021-9193/94/$04.00+0 Copyright C 1994, American Society for Microbiology
Acquired Thermotolerance and Heat Shock Proteins in Thermophiles from the Three Phylogenetic Domains JONATHAN D. TRENT,`*
ME1TE GABRIELSEN,2 BO JENSEN,2 JAN NEUHARD,2
AND J0RGEN OLSEN2 Center for Mechanistic Biology and Biotechnology, Argonne National Laboratory, Argonne, Illinois 60439, 1 and Institute of Molecular Biology, University of Copenhagen, DK-3708 Copenhagen, Denmark2
Received 31 March 1994/Accepted 28 July 1994
Thermophilic organisms from each of the three phylogenetic domains (Bacteria, Archaea, and Eucarya) acquired thermotolerance after heat shock. Bacillus caldolyticus grown at 60°C and heat shocked at 69°C for 10 min showed thermotolerance at 74°C, Sulfolobus shibatae grown at 70°C and heat shocked at 88°C for 60 min showed thermotolerance at 95°C, and Thermomyces lanuginosus grown at 50T and heat shocked at 55°C for 60 min showed thermotolerance at 58°C. Determinations of protein synthesis during heat shock revealed differences in the dominant heat shock proteins for each species. For B. caldolytieus, a 70-kDa protein dominated while for S. shibatae, a 55-kDa protein dominated and for T. lanuginosus, 31- to 33-kDa proteins dominated. Reagents that disrupted normal protein synthesis during heat shock prevented the enhanced thermotolerance.
Acquired thermotolerance refers to the enhanced survival of organisms at lethal temperatures after a brief exposure to near-lethal temperatures (2, 13). This response correlates with the synthesis of a small number of proteins known as heatshock proteins (HSPs), which has led to the hypothesis that thermotolerance depends on one or more of these specific proteins (10, 12, 19). There have been many investigations to test this hypothesis and to clarify the role of specific HSPs in thermotolerance (1, 8, 15). Two generalizations that may be made from the result of this research are that (i) HSPs do indeed play an important role in acquired thermotolerance and (ii) different species use different strategies (different combinations of HSPs and/or other macromolecules) in acquiring thermotolerance. Extending our knowledge of thermotolerance and the associated HSPs to other species may therefore provide additional insights into similarities and differences in these strategies. Most previously published research in this area has focused on mesophilic organisms (20, 21, 27), with only a few reports about thermophiles (7, 25, 28). To expand our knowledge of thermophiles and to compare thermophiles from different domains, we have investigated thermotolerance, HSP synthesis, and the link between them in the thermophilic bacterium Bacillus caldolyticus, the hyperthermophilic archaeon Sulfolobus shibatae, and the thermophilic eukaryote Thernomyces lanuginosus. Acquired thermotolerance. Our standard procedure for monitoring thermotolerance was similar to procedures described elsewhere. Basically, one of two equal samples of an actively growing culture was heat shocked, while the other was maintained at the initial culture temperature. At the end of the heat shock period, both samples were shifted to a lethal temperature and survival was monitored as a function of time. The conditions for each of the three test organisms were as follows. Samples of B. caldolyticus (DSM strain 405) grown at 60'C with vigorous shaking in liquid medium (Bc medium, consisting of minimal medium 162 [3] supplemented with 0.5%
glucose, 0.2% Casamino Acids, and 10 mM NH4Cl) were heat shocked at 690C for 10 min and challenged at the lethal temperature of 740C. Viable cells were enumerated on Bc medium supplemented with 0.25% yeast extract, 0.25% tryptone, and 3% agar with overnight incubation at 600C. Samples of Sulfolobus shibatae (DSM strain 5389) (6) grown at 70'C with gentle agitation in liquid medium (Ss medium, consisting of Brock's salts and 0.2% dextrin [25]) were heat shocked at 880C for 60 min and challenged at 950C. Viable cells were enumerated by endpoint dilutions in Ss medium (25). Finally, samples of conidia of T. lanuginosus (4) incubated for 4 h at 50°C with agitation in a modified YPS medium (T1 medium) (4), which sprouted over 90% of them, were heat shocked at 55°C for 60 min and challenged at 58°C. Viable conidia were enumerated by counting mycelial colonies that grew from dilutions on T1 medium solidified with 2% agar after 48 h at
450C. Under our experimental conditions, all three thermophilic species acquired thermotolerance, although their response times and the magnitudes of their tolerances varied (Fig. 1). The difference in survival of cells with and without heat shock was sufficiently large that despite variations between experiments, there was no doubt that some molecular adaptation had occurred. To determine whether this adaptation was associated with changes in protein synthesis like those observed in mesophilic species, we monitored changes in protein synthesis during the heat shock treatments of these thermophiles. Heat shock protein synthesis and gel electrophoresis. Protein synthesis at normal and heat shock temperatures was determined by pulse-labeling cells with L-[35S]methionine. Five-milliliter samples of B. caldolyticus in log-phase growth (1 X 10' to 2 x 107 cells/ml) were pulse-labeled for 5 min with 10 ,Ci of L-[35S]methionine in Bc medium modified by replacing the Casamino Acids with a methionine-free amino acid mixture (Methionine Assay Medium, Difco) supplemented with 4 mg of methionine per liter. One-milliliter samples of S. shibatae in late log-phase growth (5 X 108 cells/ml) were labeled with 10 ,uCi of L-[35S]methionine for 15 min in their standard medium. Five-milliliter samples of sprouted conidia of T lanuginosus washed in salt solution were labeled with 100 ,uCi
* Corresponding author. Mailing address: CMB/202, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, IL 60439. Phone: (708) 252-3917. Fax: (708) 252-3387.
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