Internal arsenite bioassay calibration using multiple bioreporter cell lines

Bioassays with bioreporter bacteria are usually calibrated with analyte solutions of known concentrations that are analysed along with the samples of interest. This is done as bioreporter output (the intensity of light, fluorescence or colour) does not only depend on the target concentration, but also on the incubation time and physiological activity of the cells in the assay. Comparing the bioreporter output with standardized colour tables in the field seems rather difficult and error‐prone. A new approach to control assay variations and improve application ease could be an internal calibration based on the use of multiple bioreporter cell lines with drastically different reporter protein outputs at a given analyte concentration. To test this concept, different Escherichia coli‐based bioreporter strains expressing either cytochrome c peroxidase (CCP, or CCP mutants) or β‐galactosidase upon induction with arsenite were constructed. The reporter strains differed either in the catalytic activity of the reporter protein (for CCP) or in the rates of reporter protein synthesis (for β‐galactosidase), which, indeed, resulted in output signals with different intensities at the same arsenite concentration. Hence, it was possible to use combinations of these cell lines to define arsenite concentration ranges at which none, one or more cell lines gave qualitative (yes/no) visible signals that were relatively independent of incubation time or bioreporter activity. The discriminated concentration ranges would fit very well with the current permissive (e.g. World Health Organization) levels of arsenite in drinking water (10 µg l^?1).     

Characterization and validation of a bioluminescent bioreporter for the direct detection of Escherichia coli

A two-component bacteriophage-based bioluminescent reporter system was developed for the detection of Escherichia coli in environmental samples. The bioreporter system consists of a luxI integrated lambda bacteriophage and a lux-based bioluminescent reporter cell that responds to the infection event through acyl-homoserine lactone (AHL) mediated quorum sensing and bioluminescent signal stimulation. This work addresses the ability of the bioreporter system to detect and quantify the target pathogen in response to two analytical challenges: (1) detection of target cells in the presence of lactonase-producing non-target organisms that could interrupt AHL signal transduction, and (2) detection of sub-lethally injured or physiologically stressed target cells. The bioreporter system was able to autonomously respond to lambda phage infection events with a target host E. coli at 1x10(8) cfu/mL against a background of lactonase-producing Arthrobacter globiformis at cell densities ranging from 1 to 1x10(8) cfu/mL. E. coli target cells stressed by carbon-limitation for 2 weeks (i.e., starvation) or exposure to iodine for 1 week at 2 and 20 ppm (i.e., disinfection) yield a reduced, but detectable, biosensor response. Conversely, short-term iodine exposure produces a significant increase in bioreporter response within the first 24 h. The signal response and limit of detection for the two-component bioreporter system were affected by the physiology and environment of the target, but the bioreporter maintained target specificity demonstrating its potential application for remote sensing of pathogens.     

Escherichia coli as a bioreporter in ecotoxicology

Ecotoxicological assessment relies to a large extent on the information gathered with surrogate species and the extrapolation of test results across species and different levels of biological organisation. Bacteria have long been used as a bioreporter for genotoxic testing and general toxicity. Today, it is clear that bacteria have the potential for screening of other toxicological endpoints. Escherichia coli has been studied for years; in-depth knowledge of its biochemistry and genetics makes it the most proficient prokaryote for the development of new toxicological assays. Several assays have been designed with E. coli as a bioreporter, and the recent trend to develop novel, better advanced reporters makes bioreporter development one of the most dynamic in ecotoxicology. Based on in-depth knowledge of E. coli, new assays are being developed or existing ones redesigned, thanks to the availability of new reporter genes and new or improved substrates. The technological evolution towards easier and more sensitive detection of different gene products is another important aspect. Often, this requires the redesign of the bacterium to make it compatible with the novel measuring tests. Recent advances in surface chemistry and nanoelectronics open the perspective for advanced reporter based on novel measuring platforms and with an online potential. In this article, we will discuss the use of E. coli-based bioreporters in ecotoxicological applications as well as some innovative sensors awaited for the future.     

New family of biosensors for monitoring BTX in aquatic and edaphic environments

Benzene, toluene, ethylbenzene and xylenes (BTEX) contamination is a serious threat to public health and the environment, and therefore, there is an urgent need to detect its presence in nature. The use of whole‐cell reporters is an efficient, easy‐to‐use and low‐cost approach to detect and follow contaminants outside specialized laboratories; this is especially important in oil spills that are frequent in marine environments. The aim of this study is the construction of a bioreporter system and its comparison and validation for the specific detection of monocyclic aromatic hydrocarbons in different host bacteria and environmental samples. Our bioreporter system is based on the two component regulatory system TodS–TodT of P. putida DOT‐T1E, and the P_todX promoter fused to the GFP protein as the reporter protein. For the construction of different biosensors, this bioreporter was transferred into three different bacterial strains isolated from three different environments, and their performance was measured. Validation of the biosensors on water samples spiked with petrol, diesel and crude oil on contaminated waters from oil spills and on contaminated soils demonstrated that they can be used in mapping and monitoring some BTEX compounds (specifically benzene, toluene and two xylene isomers). Validation of biosensors is an important issue for the integration of these devices into pollution‐control programmes.     

Microbial sensor cell arrays

Motivated by the advantages endowed by high-throughput analysis, researchers have succeeded in incorporating multiple reporter cells into a single platform; the technology now allows the simultaneous scrutiny of a large collection of sensor strains. We review current aspects in cell array technology with emphasis on microbial sensor arrays. We consider various techniques for patterning live cells on solid surfaces, describe different array-based applications and devices, and highlight recent efforts for live cell storage. We review mathematical approaches for deciphering the data emanating from bioreporter collections, and discuss the future of single cell arrays. Innovative technologies for cell patterning, preservation and interpretation are continuously being developed; when they all mature, cell arrays may become an efficient analytical tool, in a scope resembling that of DNA microarray biochips.     

PHYSIOLOGICAL CHARACTERIZATION OF A SYNECHOCOCCUS SP. (CYANOPHYCEAE) STRAIN PCC 7942 IRON‐DEPENDENT BIOREPORTER FOR FRESHWATER ENVIRONMENTS1

The complex chemical speciation of Fe in aquatic systems and the uncertainties associated with biological assimilation of Fe species make it difficult to assess the bioavailability of Fe to phytoplankton in relation to total dissolved Fe concentrations in natural waters. We developed a cyanobacterial Fe‐responsive bioreporter constructed in Synechococcus sp. strain PCC 7942 by fusing the Fe‐responsive isiAB promoter to Vibrio harveyi luxAB reporter genes. A comprehensive physiological characterization of the bioreporter has been made in defined Fraquil medium at free ferric ion concentrations ranging from pFe 21.6 to pFe 19.5. Whereas growth and physiological parameters are largely constrained over this range of Fe bioavailability, the bioreporter elicits a luminescent signal that varies in response to Fe deficiency. A dose‐response characterization of bioreporter luminescence made over this range of Fe^{3 +} bioavailability demonstrates a sigmoidal response with a dynamic linear range extending between pFe 21.1 and pFe 20.6. The applicability of using this Fe bioreporter to assess Fe availability in the natural environment has been tested using water samples from Lake Huron (Laurentian Great Lakes). Parallel assessment of dissolved Fe and bioreporter response from these samples reinforces the idea that measures of dissolved Fe should not be considered alone when assessing Fe availability to phytoplankton communities.     

利用蓝藻生物报告体检测水体中可利用铁的研究

鱼腥藻PCC 7120 中的alr2581 基因编码的蛋白质在缺铁胁迫时显著上调。将该基因的启动子Palr2581和费氏弧菌的luxAB 基因融合, 通过同源单交换, 整合到鱼腥藻PCC 7120 的基因组上, 构建了可以感知环境中铁的生物报告体Palr2581-luxAB。该藻株在不含铁的BG11 中培养时, 启动子Palr2581 的转录活性增强, LuxAB酶活显著升高。通过测定Palr2581-luxAB 藻株在不同铁浓度Fraquil 培养基中的LuxAB 酶活, 得到了铁浓度pFe(-lgFe3+)与 LuxAB 酶活的剂量反应曲线。结果显示, 12h 时, LuxAB 酶活随培养基中Fe3+浓度增加呈S形递减关系, 其中在pFe=20.7—21.2 范围内有很好的线性关系。根据这一特性, 我们利用Palr2581-luxAB 作为铁的生物报告体, 测定了武汉市东湖水体中可利用的铁浓度为10-20.56 mol/L。研究显示, 通过这一方法可以较方便地监测各种淡水中可利用的铁浓度。       

Visualisation of gradients in arsenic concentrations around individual roots of Zea mays L. using agar-immobilized bioreporter bacteria

The classical concept of arsenic transfer into plants through arsenate uptake via phosphate transporters, reduction to arsenite, complexation and compartmentation within vacuoles is challenged by recent identification of bidirectional transporters for arsenite and their potential role in plant As status regulation. Soil-based studies with chemical analysis of soil solution require root mat formation amplifying root effects on their surroundings and additionally denying investigations along individual roots differing in age and function. We tried to overcome these shortcomings by using bioreporter bacteria to visualise the spatial distribution of inorganic arsenic along roots and to characterize inorganic arsenic gradients in the rhizosphere concurrent with root age and branching. Therefore we developed an agar-based carrier element ensuring intimate contact between bioreporters and root-soil system and enabling fast and easy reporter output analysis. We show that inorganic arsenic distribution is related to root development with the highest bioreporter signal induction around lateral roots, which are known to show the highest expression of transporters responsible for bidirectional arsenite flux. Since there is so far no evidence for an arsenate efflux mechanism this is a strong indicator that we observed rather arsenite than arsenate efflux. No signal was detected along the distal region of young adventitious roots, i.e. the region of extension growth and root hair formation. The novel bioreporter assay may thus complement conventional measurements by providing information on the spatial distribution of inorganic arsenic on mm to cm-scale.     

Transforming cyanobacteria into bioreporters of biological relevance

Microbial bioreporters play an important role in environmental monitoring and ecotoxicology. Microorganisms that are genetically modified with reporter genes can be used in various formats to determine the bioavailability of chemicals and their effect on living organisms. Cyanobacteria are abundant in the photosynthetic biosphere and have considerable potential with regards to broadening bioreporter applications. Two recent studies described novel cyanobacterial reporters for the detection of environmental toxicants and iron availability.     

Linking bacteriophage infection to quorum sensing signalling and bioluminescent bioreporter monitoring for direct detection of bacterial agents

AIM: To incorporate into the lambda phage genome, a luxI-based acyl-homoserine lactone (AHL) synthase genetic construct and exploit the autoamplified power of quorum sensing to translate a phage infection event into a chemical signature detectable by a lux-based bioluminescent bioreporter, with focus towards facile detection of microbial pathogens. METHODS AND RESULTS: The luxI gene from Vibrio fischeri was inserted into the lambda phage genome to construct a model phage-based biosensor system for the general detection of Escherichia coli. The AHL signalling molecules synthesized upon phage infection are detected by an AHL-specific bioluminescent bioreporter based on the luxCDABE gene cassette of V. fischeri. The assay generates target-specific visible light signals with no requisite addition of extraneous substrate. This binary reporter system was able to autonomously respond to lambda phage infection events at target E. coli concentrations ranging from 1 x 10(8) to 1 CFU ml(-1) within 1.5-10.3 h, respectively, in pure culture. When assayed against artificially contaminated lettuce leaf washings, detection within an E. coli inoculum range from 1 x 10(8) to 130 CFU ml(-1) was achieved within 2.6-22.4 h, respectively. CONCLUSIONS: The initial feasibility of binary phage-based reporter assays indicates that quorum sensing can be used to translate a phage infection event into an autoamplified chemical signature. SIGNIFICANCE AND IMPACT OF STUDY: With further modification, binary phage-based reporter assays may be capable of rapidly and cost effectively detecting pathogenic agents at very low population densities.     

Whole-cell living biosensors—are they ready for environmental application?

Since the development of the first whole-cell living biosensor or bioreporter about 15 years ago, construction and testing of new genetically modified microorganisms for environmental sensing and reporting has proceeded at an ever increasing rate. One and a half decades appear as a reasonable time span for a new technology to reach the maturity needed for application and commercial success. It seems, however, that the research into cellular biosensors is still mostly in a proof-of-principle or demonstration phase and not close to extensive or commercial use outside of academia. In this review, we consider the motivations for bioreporter developments and discuss the suitability of extant bioreporters for the proposed applications to stimulate complementary research and to help researchers to develop realistic objectives. This includes the identification of some popular misconceptions about the qualities and shortcomings of bioreporters.     

A green fluorescent protein-based whole-cell bioreporter for the detection of phenylacetic acid

Phenylacetic acid (PAA) is produced by many bacteria as an antifungal agent and also appears to be an environmentally toxic chemical. The object of this study was to detect PAA using Pseudomonas putida harboring a reporter plasmid that has a PAA-inducible promoter fused to a green fluorescent protein (GFP) gene. Pseudomonas putida KT2440 was used to construct a green fluorescent protein-based reporter fusion using the paaA promoter region to detect the presence of PAA. The reporter strain exhibited a high level of gfp expression in minimal medium containing PAA; however, the level of GFP expression diminished when glucose was added to the medium, whereas other carbon sources, such as succinate and pyruvate, showed no catabolic repression. Interestingly, overexpression of a paaF gene encoding PAACoA ligase minimized catabolic repression. The reporter strain could also successfully detect PAA produced by other PAA-producing bacteria. This GFP-based bioreporter provides a useful tool for detecting bacteria producing PAA.     

Molecular characterization of autoinduction of bioluminescence in the Microtox indicator strain Vibrio fischeri ATCC 49387

Repeated attempts to clone the luxI from Vibrio fischeri ATCC 49387 failed to produce a clone carrying a functional LuxI. Sequence data from the clones revealed the presence of a polymorphism when compared with previously published luxI sequences, prompting further characterization of bioluminescence regulation in V. fischeri ATCC 49387. Further investigation of V. fischeri ATCC 49387 revealed that its LuxI protein lacks detectable LuxI activity due to the presence of a glutamine residue at position 125 in the deduced amino acid sequence. Specific bioluminescence in V. fischeri ATCC 49387 increases with increasing cell density, indicative of a typical autoinduction response. However, conditioned medium from this strain does not induce bioluminescence in an ATCC 49387 luxR-plux-based acyl homoserine lactone reporter strain, but does induce bioluminescence in ATCC 49387. It has been previously shown that a V. fischeri MJ-1 luxI mutant exhibits autoinduction of bioluminescence through N-octanoyl-L-homoserine lactone, the product of the AinS autoinducer synthase. However, a bioreporter based on luxR-plux from V. fischeri ATCC 49387 responded poorly to conditioned medium from V. fischeri ATCC 49387 and also responded poorly to authentic N-octanoyl-DL-homoserine lactone. A similar MJ-1-based bioreporter showed significant induction under the same conditions. A putative ainS gene cloned from ATCC 49387, unlike luxI from ATCC 49387, expresses V. fischeri autoinducer synthase activity in Escherichia coli. This study suggests that a regulatory mechanism independent of LuxR and LuxI but possibly involving AinS is responsible for the control of autoinduction of bioluminescence in V. fischeri ATCC 49387.     

Highly Sensitive,Highly Specific Whole-Cell Bioreporters for the Detection of Chromate in Environmental Samples

Microbial bioreporters offer excellent potentialities for the detection of the bioavailable portion of pollutants in contaminated environments, which currently cannot be easily measured. This paper describes the construction and evaluation of two microbial bioreporters designed to detect the bioavailable chromate in contaminated water samples. The developed bioreporters are based on the expression of gfp under the control of the chr promoter and the chrB regulator gene of TnOtChr determinant from Ochrobactrum tritici 5bvl1. pCHRGFP1 Escherichia coli reporter proved to be specific and sensitive, with minimum detectable concentration of 100 nM chromate and did not react with other heavy metals or chemical compounds analysed. In order to have a bioreporter able to be used under different environmental toxics, O. tritici type strain was also engineered to fluoresce in the presence of micromolar levels of chromate and showed to be as specific as the first reporter. Their applicability on environmental samples (spiked Portuguese river water) was also demonstrated using either freshly grown or cryo-preserved cells, a treatment which constitutes an operational advantage. These reporter strains can provide on-demand usability in the field and in a near future may become a powerful tool in identification of chromate-contaminated sites.     

Bioreporters: gfp versus lux revisited and single-cell response

Genetically engineered organisms expressing spectroscopically active reporter molecules in response to chemical effectors display great potential as living transducers in sensing applications. Green fluorescent protein (gfp gene) bioreporters have distinct advantages over luminescent couterparts (lux gene), including applicability at the single-cell level, but are typically less sensitive. Here we describe a gfp-bearing bioreporter that is sensitive to naphthalene (a poorly water soluble pollutant behaving like a large class of hydrophobic compounds), is suitable for use in chemical assays and bioavailability studies, and has detection limits comparable to lux-bearing bioreporters for higher efficiency detection strategies. Simultaneously, we find that the exploitation of population response data from single-cell analysis is not an algorithmic conduit to enhanced signal detection and hence lower effector detection limits, as normally assumed. The assay reported functions to equal effect with or without biocide.     

Whole‐cell bacterial bioreporter for actively searching and sensing of alkanes and oil spills

Acinetobacter baylyi ADP1 was found to tolerate seawater and have a special ability of adhering to an oil–water interface of 10–80 µm emulsified mineral and crude oil droplets. These properties make ADP1 an ideal bacterial chassis for constructing bioreporters that are able to actively search and sense oil spill in water and soils. Acinetobacter baylyi bioreporter ADPWH_alk was developed and applied to the detection of alkanes and alkenes in water, seawater and soils. Bioreporter ADPWH_alk was able to detect a broad range of alkanes and alkenes with carbon chain length from C7 to C36. So far, ADPWH_alk is the only bioreporter that is able to detect alkane with carbon chain length greater than C18. This bioreporter responded to the alkanes in about 30 min and it was independent to the cell growth phase because of two point mutations in alkM promoter recognized by alkane regulatory protein ALKR. ADPWH_alk was applied to detect mineral oil, Brent, Chestnut and Sirri crude oils in water and seawater in the range 0.1–100 mg l^?1, showing that the bioreporter oil detection was semi‐quantitative. This study demonstrates that ADPWH_alk is a rapid, sensitive and semi‐quantitative bioreporter that can be useful for environmental monitoring and assessment of oil spills in seawater and soils.     

Improving the sensitivity of bacterial bioreporters for heavy metals

Whole-cell bacterial bioreporters represent a convenient testing method for quantifying the bioavailability of contaminants in environmental samples. Despite the fact that several bioreporters have been constructed for measuring heavy metals, their application to environmental samples has remained minimal. The major drawbacks of the available bioreporters include a lack of sensitivity and specificity. Here, we report an improvement in the limit of detection of bacterial bioreporters by interfering with the natural metal homeostasis system of the host bacterium. The limit of detection of a Pseudomonas putida KT2440-based Zn/Cd/Pb-biosensor was improved by a factor of up to 45 by disrupting four main efflux transporters for Zn/Cd/Pb and thereby causing the metals to accumulate in the cell. The specificity of the bioreporter could be modified by changing the sensor element. A Zn-specific bioreporter was achieved by using the promoter of the cadA1 gene from P. putida as a sensor element. The constructed transporter-deficient P. putida reporter strain detected Zn(2+) concentrations about 50 times lower than that possible with other available Zn-bioreporters. The achieved detection limits were significantly below the permitted limit values for Zn and Pb in water and in soil, allowing for reliable detection of heavy metals in the environment.     

A high sensitivity iron-dependent bioreporter used to measure iron bioavailability in freshwaters

A Nostoc sp. PCC 7120 iron bioreporter containing iron-regulated schizokinen transporter gene alr0397 promoter fused to the luxAB genes was examined to optimize its response to bioavailable iron. Dose-response relationships between luciferase activity and free ferric ion (Fe(3+) ) concentrations pFe (-lg [Fe(3+) ]) were generated by measuring luciferase activities of the bioreporter in trace metal-buffered Fraquil medium with various incubation times. The results were best demonstrated by sigmoidal curves (pFe 18.8-21.7, Fe(3+) =?10(-18.8) -10(-21.7) M) with the linear range extending from pFe 19.6-21.5 (Fe(3+) =?10(-19.6) -10(-21.5) M) after a 12-h incubation time. Optimal conditions for the use of this bioreporter to sense the iron bioavailability were determined to be: a 12-h exposure time, initial cell density of OD(730?nm) =?0.06, high nitrate (100?μM), high phosphate (10?μM), moderate Co(2+) (0.1-22.5?nM), Zn(2+) (0.16-12?nM), Cu(2+) (0.04-50?nM), and wide range of Mn(2+) concentration (0.92-2300?nM). The applicability of using this iron bioreporter to assess iron availability in the natural environment has been tested using water samples from eutrophic Taihu, Donghu, and Chaohu lakes. It is indicated that the bioreporter is a useful tool to assess bioavailable iron in various water quality samples, especially in eutrophic lakes with high bioavailable iron.     

Bioreporters and biosensors for arsenic detection. Biotechnological solutions for a world-wide pollution problem

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Highlights► A wide variety of bioreporter and biosensor assays for arsenic detection exists. ► Assay detection limits are mostly in the range of 10–50 μg As per L and below. ► New focus is on reporter integration into microdevices for more optimal detection. ► A number of case studies show realistic field applicability of bioreporter assays.