Synergistic induction of the heat shock response in Escherichia coli by simultaneous treatment with chemical inducers

Abstract

Escherichia coli strains carrying transcriptional fusions of four 32-controlled E. coli heat shock promoters to luxCDABE or lacZ reporter genes were stressed by chemicals added singly or in pairs. Much more than additive induction resulted from combinations of cadmium chloride, copper sulfate, ethanol, formamide, 4-nitrophenol, and pentachlorophenol.Exposure of cells to sudden increases in temperature or other environmental stresses triggers increased expression of a set of proteins. This response, found in all cells, is known as the heat shock response. The spectrum of inducing agents varies from organism to organism; however, for any particular organism, the heat shock response can be induced by diverse stresses (9). For Escherichia coli, these include viral infection, antibiotics, methylating and alkylating agents, hydrogen peroxide (12), and various pollutant molecules (1, 17). The alternative sigma factor 32 coordinately controls induction of about 20 heat shock genes in E. coli (5,20).The Vibrio fischeri luxCDABE genes have been used as a reporter of transcription initiated from the promoter regions of the E. coli heat shock genes dnaK, grpE (17), and lon (this study). E. coli strains containing these fusions respond to the presence of a variety of chemicals by increasing bioluminescence and are useful to detect sublethal doses of environmental insults (16-18). It was of interest to determine how these biosensor strains would respond to combinations of chemicals, because environmental samples may contain mixtures of toxicants.Plasmids, strains, and bioluminescence measurement. E. coli MC4100 lysogenic for pF13-(PrpoD hs -lacZ) (19), WM1202 containing pRY002 (dnaKp::lux), and TV1061 containing pGrpELux5 (17) have been described. E. coli DPD1006 containing the lonЈ::lux fusion plasmid, pLonLux2, in host RFM443 (10) was constructed as previously described (17), by using these primers for PCR amplification: 5Ј-ACTTAAGGATCCAAGC GATGGCGCGTAAAA-3Ј and 5Ј-AGCAGCGAATTCATC GCCGCTTCCAGACAA-3Ј. These primers include, respectively, nucleotides Ϫ308 to Ϫ290 and ϩ203 to ϩ184 relative to the start point of lon transcription (2). The DNA sequences of the promotor regions of pLonLux2 and pRY002 were determined in one direction by a method previously used to verify the sequence of the grpE promoter region of pGrpELux5 (18). The sequences determined for pLonLux2 and pRY002 were identical to the promoter region sequences of lon (2) and dnaK (3), respectively. Bioluminescence was quantitated in a microtiter plate luminometer with log-phase cells in LB medium (11), as previously described (17).Synergistic increases in bioluminescence from a lon::lux gene fusion. Two chemical inducers of the heat shock response, pentachlorophenol and ethanol, were added singly and in combination to E. coli DPD1006. Figure 1A shows the bioluminescence at 26ЊC as a function of time. The combination of inducers as well as each individual inducer resulted in transient increases in light output, as previously observed with other strains containing hea...

A genomic approach to gene fusion technology

“…Bioluminescence and turbidity, recorded with a KlettSummerson colorimeter (24), of actively growing cultures were used for calculation of light production per 10 9 cells. For such experiments, transformants of P90 and 397C were grown at 30°C in LB medium containing 100 g͞ml ampicillin.…”

Roles of Effectors in XylS-Dependent Transcription Activation: Intramolecular Domain Derepression and DNA Binding

“…Interestingly, this behavior is similar to the activation mechanism of single-domain proteins in the AraC family, such as MarA and SoxS, where induction of the pathway in response to specific signals increases intracellular levels of the regulator and triggers activation of the target promoter (22,23). XylS shares with MarA and Rob the feature that they all respond to signal molecules that induce the stress response; in fact, both 3MB (the effector of XylS) and toluene (which triggers the cascade circuit leading to XylS overproduction) can induce the heat-shock response (8,39,75). Furthermore, XylS activation of the Pm promoter was functional with E 32 RNA polymerase ( Fig.…”

Biosensor-based comparison of the ecotoxicological contamination of the wastewaters of Southern Russia and Southern Germany

“…The biosensor with the PkatG promoter fixes production of hydroperoxides in a cell and with the PsoxS promotorsuperoxide anion and NO (Vollmer et al 1997;Belkin et al 2003;Lee and Gu 2003;Zavilgelsky et al 2007;Lushchak 2011). Biosensor strain with PibpA promotor responds to the substances damaging proteins (Van Dyk et al 1994, 1995. Biosensor strain with PfabA promotor responds to the substances damaging membranes (Choi and Gu 1999).…”

“…Usage of lux-biosensor batteries provides an opportunity to evaluate the presence of different toxic substances in ecosystems simultaneously: DNA-tropic compounds (Ptitsyn et al 1997;Vollmer et al 1997;Biran et al 2010), heavy metals (Lyngberg et al 1999;Hakkila et al 2004;Ivask et al 2009), polychlorinated biphenyls (Layton et al 1998), substances causing oxidative stress (Lee andGu 2003, Zavilgelsky et al 2007), damaging proteins (Van Dyk et al 1994, 1995 and membranes (Choi and Gu 1999), etc. Besides, there is an opportunity to make a preliminary conclusion about the mechanism of their action.…”