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.     

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.     

Illuminating the detection chain of bacterial bioreporters

Engineering bacteria for measuring chemicals of environmental or toxicological concern (bioreporter bacteria) has grown slowly into a mature research area. Despite many potential advantages, current bioreporters do not perform well enough to comply with environmental detection standards. Basically, the reasons for this are the lack of engineering principles in the detection chain in the bioreporters. Here, we dissect critical steps in the detection chain and illustrate how bioreporter design could be improved by mutagenizing specificity and selectivity of the sensing and regulatory proteins, by newer expression strategies and application of different signalling networks. Furthermore, we describe how redesigning bioreporter assays with respect to pollutant transport into the cells and application of other detection devices can decrease detection limits and increase the speed of detection.     

Miniaturized bacterial biosensor system for arsenic detection holds great promise for making integrated measurement device

Combining bacterial bioreporters with microfluidics systems holds great promise for in-field detection of chemical or toxicity targets. Recently we showed how Escherichia coli cells engineered to produce a variant of green fluorescent protein after contact to arsenite and arsenate can be encapsulated in agarose beads and incorporated into a microfluidic chip to create a device for in-field detection of arsenic, a contaminant of well known toxicity and carcinogenicity in potable water both in industrialized and developing countries. Cell-beads stored in the microfluidics chip at -20°C retained inducibility up to one month and we were able to reproducibly discriminate concentrations of 10 and 50 μg arsenite per L (the drinking water standards for European countries and the United States, and for the developing countries, respectively) from the blank in less than 200 minutes. We discuss here the reasons for decreasing bioreporter signal development upon increased storage of cell beads but also show how this decrease can be reduced, leading to a faster detection and a longer lifetime of the device.     

Towards the development of a new generation of whole-cell bioreporters to sense iron bioavailability in oceanic systems—learning from the case of Synechococcus sp. PCC7002 iron bioreporter

Whole-cell bioreporters are genetically modified micro-organisms designed to sense bioavailable forms of nutrients or toxic compounds in aquatic systems. As they represent the most promising cost-efficient tools available for such purpose, engineering and use of bioreporters is rapidly growing in association with wide applicability. Bioreporters are urgently needed to determine phytoplankton iron (Fe) limitation, which has been reported in up to 30% of the ocean, with consequences affecting Earth's global carbon cycle and climate. This study presents a critical evaluation and optimization of the only Cyanobacteria bioreporter available to sense Fe limitation in marine systems (Synechococcus sp. PCC7002). The nonmonotonic biphasic dose–response curve between the bioreporters’ signal and Fe bioavailability impairs an appropriate data interpretation, highlighting the need for new carefully designed bioreporters. Here, limitations under low Fe concentrations were related to cellular energy stress, nonlinear expression of the targeted promoter and siderophore expression. Furthermore, we provide critical standard criteria for the development of new Fe bioreporters. Finally, based on gene expression data under a range of marine Fe concentrations, we propose novel sensor genes for the development of new Cyanobacteria Fe bioreporters for distinct marine regions.     

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.     

Silicon photomultiplier (SPM) detection of low-level bioluminescence for the development of deployable whole-cell biosensors: possibilities and limitations

Whole-cell bacterial bioreporters await miniaturized photon counting modules with high sensitivity and robust compatible hardware to fulfill their promise of versatile, on-site biosensor functionality. In this study, we explore the photon counting readout properties of the silicon photomultiplier (SPM) with a thermoelectric cooler and the possibilities of detecting low-level bioluminescent signals. Detection performance was evaluated through a simulated LED light source and the bioluminescence produced by the genetically engineered Pseudomonas fluorescens bacterial bioreporter 5RL. Compared with the conventional photomultiplier tube (PMT), the results revealed that the cooled SPM exhibits a wider linear response to inducible substrate concentrations (salicylate) ranging from 250 to 5000 ppb. Although cooling of the SPM lowered dark count rates and improved the minimum detectable signal, and the application of a digital filter enhanced the signal-to-noise ratio, the detection of very low light signals is still limited and remains a challenge in the design of compact photon counting systems.     

Development of Bioluminescent Bioreporters for In Vitro and In Vivo Tracking of Yersinia pestis

Yersinia pestis causes an acute infection known as the plague. Conventional techniques to enumerate Y. pestis can be labor intensive and do not lend themselves to high throughput assays. In contrast, bioluminescent bioreporters produce light that can be detected using plate readers or optical imaging platforms to monitor bacterial populations as a function of luminescence. Here, we describe the development of two Y. pestis chromosomal-based luxCDABE bioreporters, Lux_PtolC and Lux_PcysZK. These bioreporters use constitutive promoters to drive expression of luxCDABE that allow for sensitive detection of bacteria via bioluminescence in vitro. Importantly, both bioreporters demonstrate a direct correlation between bacterial numbers and bioluminescence, which allows for bioluminescence to be used to compare bacterial numbers. We demonstrate the use of these bioreporters to test antimicrobial inhibitors (Lux_PtolC) and monitor intracellular survival (Lux_PtolC and Lux_PcysZK) in vitro. Furthermore, we show that Y. pestis infection of the mouse model can be monitored using whole animal optical imaging in real time. Using optical imaging, we observed Y. pestis dissemination and differentiated between virulence phenotypes in live animals via bioluminescence. Finally, we demonstrate that whole animal optical imaging can identify unexpected colonization patterns in mutant-infected animals.     

Modulating the sensing properties of Escherichia coli-based bioreporters for cadmium and mercury

Applied Microbiology and Biotechnology - Despite the large number of bioreporters developed to date, the ability to detect heavy metal(loid)s with bioreporters has thus far been limited owing to...     

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.     

A simple solid phase assay for the detection of 2,4-D in soil

Contaminated soils are usually characterized using chemical analyses. However, these do not assess the bioavailability of pollutants, a factor which may be important in estimating the risks associated with contamination. Thus there is a need to support chemical analyses with information on biological effects to determine the potential risks a pollutant may pose in the soil. Although bacterial bioreporters have been used to detect the presence of contaminants in soils, in general these studies have been carried out in slurries or soil extracts rather than soil itself. The following study presents the development of a simple solid-phase bioassay for the direct detection of the herbicide 2,4-dichlorophenoxy acetic acid (2,4-D) in soil using Ralstonia eutropha JMP 134-32, a luxCDABE-based 2,4-D whole cell bioreporter. The bioreporter was spotted onto glass microfibre filter discs that allowed its retrieval and analysis after exposure to 2,4-D amended soils. These disc-fixed cells responded in a concentration dependent manner to 2,4-D in solution (0-25 mg/L) and in spiked soil (0-50 mg/kg). The influence of environmental factors on bioavailability was demonstrated in soil with a low moisture content which prevented 2,4-D-induced bioluminescence but which did not affect bioluminescence from already induced cells. This rapid and low cost bioassay provides a proof of concept demonstrating that retrievable disk-fixed cells can be induced in soil, thus providing a measure of solid-phase bioavailability. This method overcomes some of the limitations associated with the inoculation and monitoring of bioreporters directly in soil. Additionally, this simple system should be amenable to use with other bioreporters.     

OPTIMIZATION OF IRON‐DEPENDENT CYANOBACTERIAL (SYNECHOCOCCUS,CYANOPHYCEAE) BIOREPORTERS TO MEASURE IRON BIOAVAILABILITY1

Complex chemistry and biological uptake pathways render iron bioavailability particularly difficult to assess in natural waters. Bioreporters are genetically modified organisms that are useful tools to directly sense the bioavailable fractions of solutes. In this study, three cyanobacterial bioreporters derived from Synechococcus PCC 7942 were examined for the purpose of optimizing the response to bioavailable Fe. Each bioreporter uses a Fe‐regulated promoter (isiAB, irpA and mapA), modulated by distinct mechanisms under Fe deficiency, fused to a bacterial luciferase (luxAB). In order to provide a better understanding of the way natural conditions may affect the ability of the bioreporter to sense iron bioavailability, the effect of relevant environmental parameters on the response to iron was assessed. Optimal conditions (and limits of applicability) for the use of these bioreporters on the field were determined to be: a 12 h (12–24 h) exposure time, temperature of 15°C (15°C–22°C), photon flux density of 100 μmol photons·m^?2·s^?1 (37–200 lmol photons·m^?2·s^?1), initial biomass of 0.6–0.8 lg chlorophyll a (chl a)·L^?1 (0.3–1.5 lg chl a·L^?1) or approximately 10⁵ bioreporter cells·mL^?1, high phosphate (10 lM), and low micronutrients (absent). The measured luminescence was optimal with an exogenous addition of 60 lM aqueous decanal substrate allowing a 5 min reaction time in the dark before analysis. This study provides important considerations relating to the optimization in the use of bioreporters under field conditions that can be used for method development of other algal and cyanobacterial bioreporters in aquatic systems.     

Microbial reporters of metal bioavailability

When attempting to assess the extent and the implications of environmental pollution, it is often essential to quantify not only the total concentration of the studied contaminant but also its bioavailable fraction: higher bioavailability, often correlated with increased mobility, signifies enhanced risk but may also facilitate bioremediation. Genetically engineered microorganisms, tailored to respond by a quantifiable signal to the presence of the target chemical(s), may serve as powerful tools for bioavailability assessment. This review summarizes the current knowledge on such microbial bioreporters designed to assay metal bioavailability. Numerous bacterial metal‐sensor strains have been developed over the past 15 years, displaying very high detection sensitivities for a broad spectrum of environmentally significant metal targets. These constructs are based on the use of a relatively small number of gene promoters as the sensing elements, and an even smaller selection of molecular reporter systems; they comprise a potentially useful panel of tools for simple and cost‐effective determination of the bioavailability of heavy metals in the environment, and for the quantification of the non‐bioavailable fraction of the pollutant. In spite of their inherent advantages, however, these tools have not yet been put to actual use in the evaluation of metal bioavailability in a real environmental remediation scheme. For this to happen, acceptance by regulatory authorities is essential, as is a standardization of assay conditions.     

Separation and confirmation of anabolic steroids with quadrupole ion trap tandem mass spectrometry

Gas chromatography-mass spectrometry (GC-MS) is the method of choice for separation and detection of anabolic steroids in urine. Recently, there have been advances in the areas of gas chromatography columns, tandem mass spectrometry using ion traps, and large volume sample injection that have promise for lowering detection limits and extending the utility of GC-MS for steroid analysis. In this work, a Varian Saturn III GC-MS system has been used in its tandem mass spectrometry mode to detect low picogram levels of model steroids in standard solution and the urine matrix. Application of MS-MS-MS provided structurally informative spectra for 3′-hydroxystanozolol at concentrations of 1 ng/ml. In addition, four polysilphenylene-polydimethylsiloxane capillary columns were examined for background and selectivity. The columns had bleed several-fold lower than conventional polysiloxane columns. The columns also exhibited significant differences in selectivity for structurally similar steroids. Finally, a new temperature-programmed split-splitless injector was used to inject as much as 25 μl on column. The resulting limits of detection were 5 pg/ml for norandrosterone.     

Functionalization of whole‐cell bacterial reporters with magnetic nanoparticles

We developed a biocompatible and highly efficient approach for functionalization of bacterial cell wall with magnetic nanoparticles (MNPs). Three Acinetobacter baylyi ADP1 chromosomally based bioreporters, which were genetically engineered to express bioluminescence in response to salicylate, toluene/xylene and alkanes, were functionalized with 18 ± 3 nm iron oxide MNPs to acquire magnetic function. The efficiency of MNPs functionalization of Acinetobacter bioreporters was 99.96 ± 0.01%. The MNPs‐functionalized bioreporters (MFBs) can be remotely controlled and collected by an external magnetic field. The MFBs were all viable and functional as good as the native cells in terms of sensitivity, specificity and quantitative response. More importantly, we demonstrated that salicylate sensing MFBs can be applied to sediments and garden soils, and semi‐quantitatively detect salicylate in those samples by discriminably recovering MFBs with a permanent magnet. The magnetically functionalized cells are especially useful to complex environments in which the indigenous cells, particles and impurities may interfere with direct measurement of bioreporter cells and conventional filtration is not applicable to distinguish and harvest bioreporters. The approach described here provides a powerful tool to remotely control and selectively manipulate MNPs‐functionalized cells in water and soils. It would have a potential in the application of environmental microbiology, such as bioremediation enhancement and environment monitoring and assessment.     

Acinetobacter baylyi ADP1: transforming the choice of model organism

For more than 25 years, Acinetobacter baylyi ADP1 has been used in molecular biology studies that address a broad range of questions. Recently, the rapid accumulation of data from DNA sequencing, gene expression, protein structure, and other high-throughput methodology has increased the ability to tackle complex topics using sophisticated approaches to metabolic and genetic engineering. While the genetic malleability of ADP1 makes it an ideal organism for such investigations, A. baylyi ADP1 has yet to become a common choice for bacterial studies. This review describes examples of ADP1-based studies that exploit its highly efficient system for natural transformation and chromosomal incorporation of exogenous DNA. These studies focus on a wide array of problems, including gene duplication and amplification, horizontal gene transfer, bioreporters, and metabolic reconstruction. Interesting results in these diverse areas highlight the utility of using A. baylyi in laboratory and industrial settings.