Genetically Engineered Bacteria: Electrochemical Sensing Systems for Antimonite and Arsenite

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...

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

“…The regulatory protein of the operon, ArsR, has been shown to be a trans-acting repressor that senses environmental As(III) (32). The ArsR protein contains a very specific binding site toward As(III) and can discriminate effectively against phosphate, sulfate, cobalt, and cadmium (28). ArsR has a strikingly high affinity, as even 10 Ϫ15 M As(III) could induce the ars promoter (22).…”

mentioning

confidence: 99%

Enhanced Arsenic Accumulation in Engineered Bacterial Cells Expressing ArsR

Košťál

Yang²,

et al. 2004

187

The metalloregulatory protein ArsR, which offers high affinity and selectivity toward arsenite, was overexpressed in Escherichia coli in an attempt to increase the bioaccumulation of arsenic. Overproduction of ArsR resulted in elevated levels of arsenite bioaccumulation but also a severe reduction in cell growth. Incorporation of an elastin-like polypeptide as the fusion partner to ArsR (ELP153AR) improved cell growth by twofold without compromising the ability to accumulate arsenite. Resting cells overexpressing ELP153AR accumulated 5-and 60-fold-higher levels of arsenate and arsenite than control cells without ArsR overexpression. Conversely, no significant improvement in Cd 2؉ or Zn 2؉ accumulation was observed, validating the specificity of ArsR. The high affinity of ArsR allowed 100% removal of 50 ppb of arsenite from contaminated water with these engineered cells, providing a technology useful to comply with the newly approved U.S. Environmental Protection Agency limit of 10 ppb. These results open up the possibility of using cells overexpressing ArsR as an inexpensive, high-affinity ligand for arsenic removal from contaminated drinking and ground water.Arsenic (As) is an extremely toxic metalloid that adversely affects human health. Both highly toxic trivalent arsenite [As(III)] and less-toxic pentavalent arsenate [As(V)] have been associated with increased risk of skin, kidney, lung, and bladder cancer (12). The toxicity of arsenic is attributed to the substitution of As(V) for phosphate, affinity of As(III) for protein thiol groups, and protein-DNA and DNA-DNA crosslinking (20). Arsenic enters the water supply primarily from geochemical sources, such as the mining of arsenopyrite gold ores (16), which constitute about one-third of world gold reserves. Additional contaminations arise from anthropomorphic sources such as arsenical-containing herbicides, pesticides, and the widely used wood preservative chromated copper arsenic (17, 21). Untreated, highly toxic arsenic effluent has been disposed of in rivers and ended up in groundwater. An estimated 20 million Americans consume water containing arsenic at levels presenting a potentially fatal cancer risk (9). Worldwide, 57 million people have been exposed to arsenic through contaminated wells in Bangladesh (18). These incidents again serve as a reminder of the toxic consequences of arsenic mobilization and the needs for efficient removal of arsenic in aquatic systems (19).Because of the toxicity, the regulatory limit on arsenic in the United States is currently set at 10 ppb. There are a variety of methods currently available for removal of arsenic from contaminated water. Conventional technologies, such as coagulation, do not discriminate between arsenic and other elements and involve alteration of the water chemistry and addition of other chemicals (7). Current technologies, such as activated alumina sorption, polymeric anion exchange, and polymeric ligand exchange (6), are more effective for As(V) than As(III), and most commonly used methods require pri...

“…crtIBS exhibited Ͼ500-fold-lower sensitivity to arsenate. Despite the fact that we used Pars and arsR from E. coli, the sensitivity of crtIBS to arsenate was significantly lower than the sensitivities of E. coli-based luciferase biosensors (23,26). It has been reported that arsenate is reduced to arsenite by the reductase in E. coli, and arsenite induces light emission.…”

Section: Discussionmentioning

confidence: 91%

“…Such systems indicate the presence of a target molecule by expressing a reporter protein, such as green fluorescent protein, luciferase, or ␤-galactosidase. Several arsenite biosensors harboring a reporter gene fused with the operator-promoter region of the ars operon have been constructed (4,20,23,26,28,29). The ars operon of Escherichia coli contains two regulatory genes (arsR and arsD) and three structural genes (arsA, arsB, and arsC) and has been shown to confer resistance to arsenite in its host (5,22).…”

mentioning

confidence: 99%

Novel Carotenoid-Based Biosensor for Simple Visual Detection of Arsenite: Characterization and Preliminary Evaluation for Environmental Application

Yoshida

Inoue

Takahashi

et al. 2008

A novel whole-cell arsenite biosensor was developed using the photosynthetic bacterium Rhodopseudomonas palustris no. 7 and characterized. A sensor plasmid containing the operator-promoter region of the ars operon and arsR gene from Escherichia coli and the crtI gene from R. palustris no. 7 was introduced into a blue-green mutant with crtI deleted, R. palustris no. 711. The biosensor changed color in response to arsenite, and the change was obvious to the naked eye after 24 h without further manipulation. Real-time reverse transcription-PCR showed that the crtI mRNA was induced 3-fold at 3 h and 2.5-fold at 6 h after addition of 50 g/liter arsenite compared with the no-arsenite control, and consistent with this, the relative levels of lycopene and rhodopin also increased compared with the control. Colorimetric analysis of the bacteria showed that the hue angle had clearly shifted from greenyellow toward red in an arsenic dose-dependent manner at 24 h after arsenite addition. This obvious shift occurred irrespective of the culture conditions before arsenite was added, indicating that the color change of the biosensor is stable in water samples containing various concentrations of dissolved oxygen. Finally, assays using samples prepared in various types of mineral water indicated that this biosensor could be used to screen groundwater samples for the presence of arsenite in a variety of locations, even where electricity is not available.