Effects of environmental stresses on the activities of the uspA, grpE and rpoS promoters of Escherichia coli O157:H7 (original) (raw)
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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...
Green fluorescent protein as a noninvasive stress probe in resting Escherichia coli cells
Applied and environmental microbiology, 1999
We constructed and characterized three stress probe plasmids which utilize a green fluorescent protein as a noninvasive reporter in order to elucidate Escherichia coli cellular stress responses in quiescent or resting cells. Cellular stress levels were easily detected by fusing three heat shock stress protein promoter elements, those of the heat shock transcription factor sigma32, the protease subunit ClpB, and the chaperone DnaK, to the reporter gene gfpuv. When perturbed by a chemical or physical stress (such as a heat shock, nutrient [amino acid] limitation, or addition of IPTG [isopropyl-beta-D-thiogalactopyranoside], acetic acid, ethanol, phenol, antifoam, or salt [osmotic shock]), the E. coli cells produced GFPuv, which was easily detected within the cells as emitted green fluorescence. Temporal and amplitudinal mapping of the responses was performed, and the results revealed regions where quantitative delineation of cell stress was afforded.
Selection of Escherichia coli heat shock promoters toward their application as stress probes
J Biotechnol., 2014
The mechanism of heat shock response of Escherichia coli can be explored to program novel biological functions. In this study, the strongest heat shock promoters were identified by microarray experiments conducted at different temperatures (37 • C and 45 • C, 5 min). The promoters of the genes ibpA, dnaK and fxsA were selected and validated by RT-qPCR. These promoters were used to construct and characterize stress probes using green fluorescence protein (GFP). Cellular stress levels were evaluated in experiments conducted at different shock temperatures during several exposure times. It was concluded that the strength of the promoter is not the only relevant factor in the construction of an efficient stress probe. Furthermore, it was found to be crucial to test and optimize the ribosome binding site (RBS) in order to obtain translational efficiency that balances the transcription levels previously verified by microarrays and RT-qPCR. These heat shock promoters can be used to trigger in situ gene expression of newly constructed biosynthetic pathways.
Journal of Experimental Biology and Agricultural Sciences, 2022
Response to heat stress (HSR) is a key stress response for endurance in Escherichia coli mediated by transcriptional factor σ-32. Even though there has been extensive investigation on the contribution of proteins and chaperones in retaining protein stability in cells under stress conditions, limited information is available regarding the dynamic nature of mechanisms regulating the activity of the highly conserved heat shock proteins (Hsps). Several gene expression-based studies suggest the pivotal role of Hsp70 (DnaK) in the regulation of the expression of heat shock genes (Hsg). Direct interaction of Hsp70 with σ-32 may regulate this function in E. coli. Recent studies revealed that localization of σ-32 to the membrane interior by SRP-dependent pathway enables them to function appropriately in their role as regulators. The contributions of different cellular components including cell membrane remain unknown. Other cellular components or σ-32 interfere with polypeptides which could...
Proceedings of the National Academy of Sciences, 1997
A gene encoding the rice 16.9-kDa class I low-molecular-mass (LMM) heat-shock protein (HSP), Oshsp16.9, was introduced into Escherichia coli using the pGEX-2T expression vector to analyze the possible function of this LMM HSP under heat stress. It is known that E. coli does not normally produce class I LMM HSPs. We compared the survivability of E. coli XL1-Blue cells transformed with a recombinant plasmid containing a glutathione S -transferase (GST)–Oshsp16.9 fusion protein (pGST-FL cells) with the control E. coli cells transformed with the pGEX-2T vector (pGST cells) under heat-shock (HS) after isopropyl β- d -thiogalactopyranoside induction. The pGST-FL cells demonstrated thermotolerance at 47.5°C, a treatment that was lethal to the pGST cells. When the cell lysates from these two E. coli transformants were heated at 55°C, the amount of protein denatured in the pGST-FL cells was 50% less than that of the pGST cells. Similar results as pGST-FL cells were obtained in pGST-N78 cells...
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