Sensitive fluorescent microplate bioassay using recombinant Escherichia coli with multiple promoter–reporter units in tandem for detection of arsenic

Tani, Chiaki; Inoue, Kazuhiro; Tani, Yuri; Harun-ur-Rashid, Md.; Azuma, Norihiro; Ueda, Shunsaku; Yoshida, Kazuyuki; Maeda, Isamu

doi:10.1016/j.jbiosc.2009.05.014

Cited by 37 publications

(26 citation statements)

References 28 publications

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“…S5 in the supplemental material). Another undesirable property of biosensors is their elevated background signal (43,51,(53)(54)(55). The biosensors described here are characterized by very low background signals, and ␤-galactosidase activity of ϳ10 Miller units was observed in the absence of a ligand, translating into very favorable signal-tonoise ratios for arsenic measurements.…”

Section: Acidithiobacillus Ferrooxidansmentioning

confidence: 99%

Paralogous Regulators ArsR1 and ArsR2 of Pseudomonas putida KT2440 as a Basis for Arsenic Biosensor Development

Fernández

Morel

Ramos

et al. 2016

Appl Environ Microbiol

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The remarkable metal resistance of many microorganisms is related to the presence of multiple metal resistance operons. Pseudomonas putida KT2440 can be considered a model for these microorganisms since its arsenic resistance is due to the action of proteins encoded by the two paralogous arsenic resistance operons ARS1 and ARS2. Both operons contain the genes encoding the transcriptional regulators ArsR1 and ArsR2 that control operon expression. We show here that purified ArsR1 and ArsR2 bind the trivalent salt of arsenic (arsenite) with similar affinities (~30 M), whereas no binding is observed for the pentavalent salt (arsenate). Furthermore, trivalent salts of bismuth and antimony showed binding to both paralogues. The positions of cysteines, found to bind arsenic in other homologues, indicate that ArsR1 and ArsR2 employ different modes of arsenite recognition. Both paralogues are dimeric and possess significant thermal stability. Both proteins were used to construct whole-cell, lacZ-based biosensors. Whereas responses to bismuth were negligible, significant responses were observed for arsenite, arsenate, and antimony. Biosensors based on the P. putida arsB1 arsB2 arsenic efflux pump double mutant were significantly more sensitive than biosensors based on the wild-type strain. This sensitivity enhancement by pump mutation may be a convenient strategy for the construction of other biosensors. A frequent limitation found for other arsenic biosensors was their elevated background signal and interference by inorganic phosphate. The constructed biosensors show no interference by inorganic phosphate, are characterized by a very low background signal, and were found to be suitable to analyze environmental samples. IMPORTANCE Arsenic is at the top of the priority list of hazardous compounds issued by the U.S. Agency for Toxic Substances and Disease. The reason for the stunning arsenic resistance of many microorganisms is the existence of paralogous arsenic resistance operons.Pseudomonas putida KT2440 is a model organism for such bacteria, and their duplicated ars operons and in particular their ArsR transcription regulators have been studied in depth by in vivo approaches. Here we present an analysis of both purified ArsR paralogues by different biophysical techniques, and data obtained provide valuable insight into their structure and function. Particularly insightful was the comparison of ArsR effector profiles determined by in vitro and in vivo experimentation. We also report the use of both paralogues to construct robust and highly sensitive arsenic biosensors. Our finding that the deletion of both arsenic efflux pumps significantly increases biosensor sensitivity is of general relevance in the biosensor field.A rsenic is omnipresent in nature and found in many environmental biotopes such as seawater, freshwater, soils, and rocks. It is either released through various natural processes such as weathering or hydrothermal emissions or generated by a number of anthropogenic activities such as the use of arse...

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Section: Acidithiobacillus Ferrooxidansmentioning

confidence: 99%

Paralogous Regulators ArsR1 and ArsR2 of Pseudomonas putida KT2440 as a Basis for Arsenic Biosensor Development

Fernández

Morel

Ramos

et al. 2016

Appl Environ Microbiol

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“…9 An alternative strategy to achieve greater protein expression could be to utilize tandem promoters to maximize transcript levels. 23 However, the Figure 3. Construction of the plasmid, pGFLI.…”

Section: Discussionmentioning

confidence: 99%

Genetic engineering ofFrancisella tularensisLVS for use as a novel live vaccine platform againstPseudomonas aeruginosainfections

Robinson

Kobe

Schmitt³

et al. 2015

Bioengineered

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“…Expression host Cinnagen washed 2 times with 500 µL of phosphate-buffered saline (PBS) and resuspended in an appropriate amount of PBS (between 10 and 50 µL, depending on the amount of cells) (Stocker et al, 2003;Tani et al, 2009). …”

Section: E Coli Bl21 (De3) F-ompt Hsdsb(rb-mb-) Gal Dcm (De3)mentioning

confidence: 99%

“…E. coli BL21 (DE3) with plasmid pUC19 was used as a negative control. Cells were viewed using an epifluorescent microscope (Zeiss, Germany) equipped with a mercury lamp (100 W), an excitation filter (385-425 nm), a dichroic mirror (450 nm long pass), and an emission filter (500-540 nm) (Tani et al, 2009). …”

Section: E Coli Bl21 (De3) F-ompt Hsdsb(rb-mb-) Gal Dcm (De3)mentioning

confidence: 99%

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Designing a bacterial biosensor for detection of mercury in water solutions

Roointan¹,

Shabab²,

Karimi³

et al. 2015

Turk J Biol

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Due to increasing advances in physical and chemical techniques for the assessment of pollutants in the environment, there is an immediate demand for a bioassay that can report both the presence of an analyte and its biological effects. In accordance with this need, there has been a fast growth in whole-cell biosensor technology. In this study we aimed to design a whole-cell bacterial biosensor to detect mercury in liquid solutions. The Pseudomonas pBS228 merR gene and its related promoter/operator was synthesized by Bioneer. The green fluorescence protein (GFP) gene was used as a reporter. GFP was cloned downstream of the merR gene. The construct, including the merR promoter, gene, and GFP, was cloned in a pUC19 vector and transferred into E. coli BL21 (DE3) bacteria. Transformed bacteria were used as whole-cell biosensors for detecting mercury. Mercury detection was monitored by means of microscopy and fluorometry techniques. Transformed BL21 (DE3) biosensors responded mainly to Hg(II), with the lowest detectable concentration being 10 -8 M during a 3-h exposure induction period. Our results demonstrated that the noninfectious bacterial biosensors developed in the present study could be beneficial and enforceable in detection of mercury in contaminated water samples at concentrations as low as 10 -8 M.

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Sensitive fluorescent microplate bioassay using recombinant Escherichia coli with multiple promoter–reporter units in tandem for detection of arsenic

Cited by 37 publications

References 28 publications

Paralogous Regulators ArsR1 and ArsR2 of Pseudomonas putida KT2440 as a Basis for Arsenic Biosensor Development

Paralogous Regulators ArsR1 and ArsR2 of Pseudomonas putida KT2440 as a Basis for Arsenic Biosensor Development

Genetic engineering ofFrancisella tularensisLVS for use as a novel live vaccine platform againstPseudomonas aeruginosainfections

Designing a bacterial biosensor for detection of mercury in water solutions

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