Lux-biosensors for detection of SOS-response, heat shock, and oxidative stress

Kotova, V. Yu.; Манухов, И. В.; Завильгельский, Г Б

doi:10.1134/s0003683810080089

Cited by 63 publications

(40 citation statements)

References 17 publications

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“…To examine whether endogenous H 2 S protects bacteria from DNA damage caused by the Fenton reaction, we first examined its effect on the global response to DNA damage (SOS). We used a pColD′::lux reporter plasmid to directly monitor SOS activation in response to DNA damage (25). (40 μM).…”

Section: Resultsmentioning

confidence: 99%

“…The SOS response was examined using a pColD′::lux hybrid plasmid (38), a derivative of the pDEW201 vector containing luxCDABE from Photorhabdus luminescens under the control of the LexA-regulated Pcda promoter (25). Overnight cultures of strains containing the pColD′::lux plasmid were diluted to a concentration of 10 7 cells per 1 mL in fresh Luria-Bertani broth medium and grown under aeration at 30°C until the early exponential growth phase; 200-μL aliquots were transferred into special cuvettes, one of which served as a control (4 mL distilled water was added to the control cuvette), and 4 mL peroxide was introduced at various concentrations into the other cuvettes.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Mechanism of H ₂ S-mediated protection against oxidative stress in Escherichia coli

Mironov

Серегина

Nagornykh

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

172

147

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Endogenous hydrogen sulfide (H 2 S) renders bacteria highly resistant to oxidative stress, but its mechanism remains poorly understood. Here, we report that 3-mercaptopyruvate sulfurtransferase (3MST) is the major source of endogenous H 2 S in Escherichia coli. Cellular resistance to H 2 O 2 strongly depends on the activity of mstA, a gene that encodes 3MST. Deletion of the ferric uptake regulator (Fur) renders ΔmstA cells hypersensitive to H 2 O 2 . Conversely, induction of chromosomal mstA from a strong pLtetO-1 promoter (P tet -mstA) renders Δfur cells fully resistant to H 2 O 2 . Furthermore, the endogenous level of H 2 S is reduced in Δfur or ΔsodA ΔsodB cells but restored after the addition of an iron chelator dipyridyl. Using a highly sensitive reporter of the global response to DNA damage (SOS) and the TUNEL assay, we show that 3MST-derived H 2 S protects chromosomal DNA from oxidative damage. We also show that the induction of the CysB regulon in response to oxidative stress depends on 3MST, whereas the CysB-regulated L-cystine transporter, TcyP, plays the principle role in the 3MST-mediated generation of H 2 S. These findings led us to propose a model to explain the interplay between L-cysteine metabolism, H 2 S production, and oxidative stress, in which 3MST protects E. coli against oxidative stress via L-cysteine utilization and H 2 S-mediated sequestration of free iron necessary for the genotoxic Fenton reaction.hydrogen sulfide | oxidative stress | cysteine | sulfur metabolism | antibiotics

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Mechanism of H ₂ S-mediated protection against oxidative stress in Escherichia coli

Mironov

Серегина

Nagornykh

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

172

147

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“…To study the effects of AR on heat shock protein (molecular chaperone) synthesis, recombinant E. coli pibpA'::luxCDABE AmpR strain, in which a complete lux gene cassette of P. luminescence had been cloned under the ibpA gene promoter, was used [16]. Such gene arrangement allows for a relatively low back ground luminescence level, which increases multifold upon heat treatment of a bacterial cell as a result of synchronous induction of both the ibpA chaperone gene and the bioluminescence genes under its control.…”

Section: Methodsmentioning

confidence: 99%

“…Experiments of this part of the study were performed using a recombinant E. coli strain pibpA'::luxCDABE AmpR which responded to heat treatment by the synchronous induction of both the ibpA chaperone gene and the bioluminescence genes under its promoter. Therefore, measurement of the luminescence intensity of the test strain allowed for real time quantitative evaluation of the dynamics of heat shock protein synthesis, the rate of which was proportional to protein denaturation induced by heat treatment [16].…”

Section: Effect Of Alkylhydroxybenzenes On Luciferase Ther Mal Stabilmentioning

confidence: 99%

Effect of alkylresorcinols on thermal denaturation and refolding of bacterial luciferase and synthesis of heat shock proteins revealed in the luminescent molecular and cellular test systems

et al. 2014

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Molecular and cellular luminescent biotests were used to reveal the effects of five alkylresorcinol homologues (C 7 , C 9 , C 11 , C 12 , and C 18 AR) on the thermally induced denaturation and refolding of bac terial luciferases, as well as on the synthesis of heat shock proteins. The ARs activities were found to depend on their fine structure and concentration. The direct heat protective effect of short chain C 7 and C 9 AR on the chromatographically pure Photobactrium leiognathii luciferase/oxidoreductase was shown within a broad range of concentrations (10 -6 -10 -3 M). The long chain ARs homologues exhibited a similar heat protective effect at micromolar concentrations only, while their millimolar concentrations increased the sensitivity of the model proteins to thermal treatment. The recombinant strain Escherichia coli K12 MG1655, bearing con stitutively expressed Vibrio fischeri luxAB genes was used to investigate the ARs effect on the intracellular chaperone independent refolding of bacterial luciferase. The functional activity of heat inactivated enzyme was restored by micromolar concentrations of short chain ARs, while long chain homologues inhibited refolding over the wide concentration range. The recombinant luminescent E. coli strain bearing the induc ible ibpA'::luxCDABE genetic construction was used to determine the effect of ARs on the synthesis of heat shock proteins (HSP). The preincubation mode of bacterial cells with long chain alkylresorcinols led to the dose dependent stimulation of HSP synthesis (2.7 to 4 times), which confirmed that ARs function as "alar mones." Subsequent thermal treatment resulted in a 5 to 15 fold decrease of the following HSP induction compared to the control, while the number of viable cells opposite increased by 1.5 to 4 fold. Thus, pretreat ment of the bacterial cells with long chain ARs resulted in their preadaptation to subsequent thermally induced stress. Short chain ARs caused less pronounced HSP suppression, although this was still was accom panied by increased heat resistance of the AR pretreated bacterial cells.

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“…As with the hypoxia example [12], creating a synthetic promoter with multiple response elements increased the signal [30]. In E. coli the promoters for the catalase and SoxS transcription factor genes were used for a similar purpose [31] and in mouse embryonic stem cells, the Srxn1 promoter has been used to sense oxidative damage by toxins [32].…”

Section: Intrinsic Stressmentioning

confidence: 99%

Genetically-encoded biosensors for monitoring cellular stress in bioprocessing

Polizzi

Kontoravdi

2015

Current Opinion in Biotechnology

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With the current wealth of transcriptomic data, it is possible to design genetically-encoded biosensors for the detection of stress responses and apply these to high-throughput bioprocess development and monitoring of cellular health. Such biosensors can sense extrinsic factors such as nutrient or oxygen deprivation and shear stress, as well as intrinsic stress factors like oxidative damage and unfolded protein accumulation. Alongside, there have been developments in biosensing hardware and software applicable to the field of genetically-encoded biosensors in the near future. This review discusses the current state-of-the-art in biosensors for monitoring cultures during biological manufacturing and the future challenges for the field. Connecting the individual achievements into a coherent whole will enable the application of genetically-encoded biosensors in industry.

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Lux-biosensors for detection of SOS-response, heat shock, and oxidative stress

Cited by 63 publications

References 17 publications

Mechanism of H ₂ S-mediated protection against oxidative stress in Escherichia coli

Mechanism of H ₂ S-mediated protection against oxidative stress in Escherichia coli

Effect of alkylresorcinols on thermal denaturation and refolding of bacterial luciferase and synthesis of heat shock proteins revealed in the luminescent molecular and cellular test systems

Genetically-encoded biosensors for monitoring cellular stress in bioprocessing

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Lux-biosensors for detection of SOS-response, heat shock, and oxidative stress

Cited by 63 publications

References 17 publications

Mechanism of H 2 S-mediated protection against oxidative stress in Escherichia coli

Mechanism of H 2 S-mediated protection against oxidative stress in Escherichia coli

Effect of alkylresorcinols on thermal denaturation and refolding of bacterial luciferase and synthesis of heat shock proteins revealed in the luminescent molecular and cellular test systems

Genetically-encoded biosensors for monitoring cellular stress in bioprocessing

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Mechanism of H ₂ S-mediated protection against oxidative stress in Escherichia coli

Mechanism of H ₂ S-mediated protection against oxidative stress in Escherichia coli