Derivation of pb(II)-sensing Escherichia coli cell-based biosensors from arsenic responsive genetic systems

Heavy metal-responsive operons were used for the generation of Escherichia coli cell-based biosensors. The selectivity and specificity of the biosensors were determined based on the interaction between heavy metals and regulatory proteins; thereby, the modulating target selectivity of biosensors could be achieved by changing target sensing properties of regulatory proteins. The results of this study demonstrated that Pb(II)-sensing biosensors could be generated from an arsenic-responsive genetic system, which was originally used for arsenic-sensing biosensors. The amino acids around to As(III)-binding sites of ArsR were mutated and cysteine residues were relocated to modulate the metal selectivity. In addition, genes encoding metal ion-translocating P-type ATPases, such as copA and zntA, were deleted to enhance the specificity by increasing the intercellular levels of divalent metal ions. Based on the results, channel protein deleted E. coli cells harboring a pair of recombinant genes, engineered ArsR and arsAp::egfp, showed enhanced responses upon Pb exposure and could be used to quantify the amount of Pb(II) in artificially contaminated water and plants grown in media containing Pb(II). Although we focused on generating Pb(II)-specific biosensors in this study, the proposed strategy has a great potential for the generation of diverse heavy metal-sensing biosensors and risk assessment of heavy metals in environmental samples as well as in plants.

A new CQDs/f-MWCNTs/GO nanocomposite electrode for arsenic (10−12 M) quantification in bore-well water and industrial effluents

Strategic combination of CQDs/f-MWCNTs/GO/GCE for pico-molar arsenic sensing.

Arsenic Directly Binds to and Activates the Yeast AP-1-Like Transcription Factor Yap8

The AP-1-like transcription factor Yap8 is critical for arsenic tolerance in the yeastSaccharomyces cerevisiae. However, the mechanism by which Yap8 senses the presence of arsenic and activates transcription of detoxification genes is unknown. Here we demonstrate that Yap8 directly binds to trivalent arsenite [As(III)]in vitroandin vivoand that approximately one As(III) molecule is bound per molecule of Yap8. As(III) is coordinated by three sulfur atoms in purified Yap8, and our genetic and biochemical data identify the cysteine residues that form the binding site as Cys132, Cys137, and Cys274. As(III) binding by Yap8 does not require an additional yeast protein, and Yap8 is regulated neither at the level of localization nor at the level of DNA binding. Instead, our data are consistent with a model in which a DNA-bound form of Yap8 acts directly as an As(III) sensor. Binding of As(III) to Yap8 triggers a conformational change that in turn brings about a transcriptional response. Thus, As(III) binding to Yap8 acts as a molecular switch that converts inactive Yap8 into an active transcriptional regulator. This is the first report to demonstrate how a eukaryotic protein couples arsenic sensing to transcriptional activation.

Identification of an Arsenic Resistance and Arsenic-Sensing System in Campylobacter jejuni

ABSTRACT Arsenic is commonly present in the natural environment and is also used as a feed additive for animal production. Poultry is a major reservoir for Campylobacter jejuni, a major food-borne human pathogen causing gastroenteritis. It has been shown that Campylobacter isolates from poultry are highly resistant to arsenic compounds, but the molecular mechanisms responsible for the resistance have not been determined, and it is unclear if the acquired arsenic resistance affects the susceptibility of Campylobacter spp. to other antimicrobials. In this study, we identified a four-gene operon that contributes to arsenic resistance in Campylobacter. This operon encodes a putative membrane permease (ArsP), a transcriptional repressor (ArsR), an arsenate reductase (ArsC), and an efflux protein (Acr3). PCR analysis of various clinical C. jejuni isolates indicated a significant association of this operon with elevated resistance to arsenite and arsenate. Gene-specific mutagenesis confirmed the role of the ars operon in conferring arsenic resistance. It was further shown that this operon is subject to regulation by ArsR, which directly binds to the ars promoter and inhibits the transcription of the operon. Arsenite inhibits the binding of ArsR to the ars promoter DNA and induces the expression of the ars genes. Mutation of the ars genes did not affect the susceptibility of C. jejuni to commonly used antibiotics. These results identify the ars operon as an important mechanism for arsenic resistance and sensing in Campylobacter.