Selection of Escherichia coli heat shock promoters toward their application as stress probes (original) (raw)
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Molecular Microbiology, 1998
The expression of heat shock genes in Escherichia coli is regulated by the antagonistic action of the transcriptional activator, the 32 subunit of RNA polymerase, and negative modulators. Modulators are the DnaK chaperone system, which inactivates and destabilizes 32 , and the FtsH protease, which is largely responsible for 32 degradation. A yet unproven hypothesis is that the degree of sequestration of the modulators through binding to misfolded proteins determines the level of heat shock gene transcription. This hypothesis was tested by altering the modulator concentration in cells expressing dnaK, dnaJ and ftsH from IPTG and arabinose-controlled promoters. Small increases in levels of DnaK and the DnaJ co-chaperone (< 1.5-fold of wild type) resulted in decreased level and activity of 32 at intermediate temperature and faster shut-off of the heat shock response. Small decreases in their levels caused inverse effects and, furthermore, reduced the refolding efficiency of heat-denatured protein and growth at heat shock temperatures. Fewer than 1500 molecules of a substrate of the DnaK system, structurally unstable firefly luciferase, resulted in elevated levels of heat shock proteins and a prolonged shutoff phase of the heat shock response. In contrast, a decrease in FtsH levels increased the 32 levels, but the accumulated 32 was inactive, indicating that sequestration of FtsH alone cannot induce the heat shock response efficiently. DnaK and DnaJ thus constitute the primary stress-sensing and transducing system of the E. coli heat shock response, which detects protein misfolding with high sensitivity.
Journal of food protection, 2003
Heat shock proteins play an important role in protecting bacterial cells against several stresses, including starvation. In this study, the promoters for two genes encoding heat shock proteins involved in many stress responses, UspA and GrpE, were fused with the green fluorescent protein (gfp) gene. Thus, the expression of the two genes could be quantified by measuring the fluorescence emitted by the cells under different environmental conditions. The heat resistance levels of starved and nonstarved cells during storage at 5, 10, and 37 degrees C were compared with the levels of expression of the uspA and grpE genes. D52-values (times required for decimal reductions in count at 52 degrees C) increased by 11.5, 14.6, and 18.5 min when cells were starved for 3 h at 37 degrees C, for 24 h at 10 degrees C, and for 2 days at 5 degrees C, respectively. In all cases, these increases were significant (P < 0.01), indicating that the stress imposed by starvation altered the ability of E. c...
Escherichia coli Heat Shock Protein DnaK: Production and Consequences in Terms of Monitoring Cooking
Applied and Environmental Microbiology, 2003
Through use of commercially available DnaK proteins and anti-DnaK monoclonal antibodies, a competitive enzyme-linked immunosorbent assay was developed to quantify this heat shock protein in Escherichia coli ATCC 25922 subjected to various heating regimens. For a given process lethality (F 70 10 of 1, 3, and 5 min), the intracellular concentration of DnaK in E. coli varied with the heating temperature (50 or 55°C). In fact, the highest DnaK concentrations were found after treatments at the lower temperature (50°C) applied for a longer time. Residual DnaK after heating was found to be necessary for cell recovery, and additional DnaK was produced during the recovery process. Overall, higher intracellular concentrations of DnaK tended to enhance cell resistance to a subsequent lethal stress. Indeed, E. coli cells that had undergone a sublethal heat shock (105 min at 55°C, F 70 10 ؍ 3 min) accompanied by a 12-h recovery (containing 76,786 ؎ 25,230 molecules/cell) resisted better than exponentially growing cells (38,500 ؎ 6,056 molecules/cell) when later heated to 60°C for 50 min (F 70 10 ؍ 5 min). Results reported here suggest that using stress protein to determine cell adaptation and survival, rather than cell counts alone, may lead to more efficient heat treatment.
The heat shock response of Escherichia coli
International Journal of Food Microbiology, 2000
A large variety of stress conditions including physicochemical factors induce the synthesis of more than 20 heat shock proteins (HSPs). In E. coli, the heat shock response to temperature upshift from 30 to 428C consists of the rapid induction of these HSPs, followed by an adaptation period where the rate of HSP synthesis decreases to reach a new steady-state level. Major HSPs are molecular chaperones, including DnaK, DnaJ and GrpE, and GroEL and GroES, and proteases. They constitute the two major chaperone systems of E. coli (15-20% of total protein at 468C). They are important for cell survival, since they play a role in preventing aggregation and refolding proteins.The E. coli heat shock response is positively 32 controlled at the transcriptional level by the product of the rpoH gene, the heat shock promoter-specific s subunit of RNA 32 polymerase. Because of its rapid turn-over, the cellular concentration of s is very low under steady-state conditions (10-30 copies / cell at 308C) and is limiting for heat shock gene transcription. The heat shock response is induced as a consequence 32 32 of a rapid increase in s levels and stimulation of s activity. The shut off of the response occurs as a consequence of 32 32 declining s levels and inhibition of s activity Stress-dependent changes in heat shock gene expression are mediated by . 32 32 the antagonistic action of s and negative modulators which act upon s . These modulators are the DnaK chaperone 32 32
International Journal of Food Microbiology, 2005
Heat shock proteins and RNA polymerase sigma factor play an important role in protecting cells against environmental stresses, including starvation, osmotic and oxidative stresses, and cold shock. In this study, the effect of environmental stresses on activity of the auto-fluorescent Escherichia coli O157:H7 generated by the fusion of gfp uv to E. coli uspA, grpE and rpoS promoters were examined. Osmotic shock caused about a 4-fold increase in green fluorescence of E. coli O157:H7 harboring uspADgfp uv or rpoSDgfp uv at 37 8C and room temperature whereas osmotic shock at 5 8C did not induce green fluorescence. When starved, E. coli O157:H7 possessing grpEDgfp uv was more sensitive for evaluating stress at low temperature while uspADgfp uv was better suited for detecting the stress response at higher temperature. The uspA, grpE and rpoS promoters were up-regulated to varying degrees by stresses commonly encountered during food processing.