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Since the initial introduction of whole-cell bioreporters (WCBs) nearly 30 years ago, their high sensitivity, selectivity, and suitability for on-site detection have rendered them highly promising for environmental monitoring, medical diagnosis, food safety, biomanufacturing, and other fields. Especially in the environmental field, the technology provides a fast and efficient way to assess the bioavailability of pollutants in the environment. Despite these advantages, the technology has not been commercialized. This lack of commercialization is confusing, given the broad application prospects of WCBs. Over the years, numerous research papers have focused primarily on enhancing the sensitivity and selectivity of WCBs, with little attention paid to their wider commercial applications. So far, there is no a critical review has been published yet on this topic. Therefore, in this article we critically reviewed the research progress of WCBs over the past three decades, assessing the performance and limitations of current systems to understand the barriers to commercial deployment. By identifying these obstacles, this article provided researchers and industry stakeholders with deeper insights into the challenges hindering market entry and inspire further research toward overcoming these barriers, thereby facilitating the commercialization of WCBs as a promising technology for environmental monitoring., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Legume nodulation requires the detection of flavonoids in the rhizosphere by rhizobia to activate their production of Nod factor countersignals. Here we investigated the flavonoids involved in nodulation of Medicago truncatula. We biochemically characterized five flavonoid-O-methyltransferases (OMTs) and a lux-based nod gene reporter was used to investigate the response of Sinorhizobium medicae NodD1 to various flavonoids. We found that chalcone-OMT 1 (ChOMT1) and ChOMT3, but not OMT2, 4, and 5, were able to produce 4,4'-dihydroxy-2'-methoxychalcone (DHMC). The bioreporter responded most strongly to DHMC, while isoflavones important for nodulation of soybean (Glycine max) showed no activity. Mutant analysis revealed that loss of ChOMT1 strongly reduced DHMC levels. Furthermore, chomt1 and omt2 showed strongly reduced bioreporter luminescence in their rhizospheres. In addition, loss of both ChOMT1 and ChOMT3 reduced nodulation, and this phenotype was strengthened by the further loss of OMT2. We conclude that: the loss of ChOMT1 greatly reduces root DHMC levels; ChOMT1 or OMT2 are important for nod gene activation in the rhizosphere; and ChOMT1/3 and OMT2 promote nodulation. Our findings suggest a degree of exclusivity in the flavonoids used for nodulation in M. truncatula compared to soybean, supporting a role for flavonoids in rhizobial host range., (© 2024 The Authors. New Phytologist © 2024 New Phytologist Foundation.)

Here, we demonstrate the beneficial effect of surfactant-producing pseudomonads on Pantoea eucalypti 299R. We conducted a series of experiments in environments of increasing complexity. P. eucalypti 299R (Pe299R), and Pseudomonas sp. FF1 (Pff1) or Pe299R and surfactant-production deficient Pseudomonas sp. FF1::ΔviscB (Pff1ΔviscB) were co-inoculated in broth, on swarming agar plates, and on plants. In broth, there were no differences in the growth dynamics of Pe299R when growing in the presence of Pff1 or Pff1ΔviscB. By contrast, on swarming agar plates, Pe299R was able to co-swarm with Pff1 which led to a significant increase in Pe299R biomass compared to Pe299R growing with Pff1ΔviscB or in monoculture. Finally in planta, and using the single-cell bioreporter for reproductive success (CUSPER), we found a temporally distinct beneficial effect of Pff1 on co-inoculated Pe299R subpopulations that did not occur in the presence of Pff1ΔviscB. We tested three additional surfactant-producing pseudomonads and their respective surfactant knockout mutants on PE299R on swarming agar showing similar results. This led us to propose a model for the positive effect of surfactant production during leaf colonization. Our results indicate that co-motility might be common during leaf colonization and adds yet another facet to the already manyfold roles of surfactants., (© 2024. The Author(s).)

Fluorescent proteins have revolutionized science since their discovery in 1962. They have enabled imaging experiments to decipher the function of proteins, cells, and organisms, as well as gene regulation. Green fluorescent protein and all its derivatives are now standard tools in cell biology, immunology, molecular biology, and microbiology laboratories around the world. A common feature of these proteins is their dioxygen (O 2 )-dependent maturation allowing fluorescence, which precludes their use in anoxic contexts. In this work, we report the development and in cellulo characterization of genetic circuits encoding the O 2 -independent KOFP-7 protein, a flavin-binding fluorescent protein. We have optimized the genetic circuit for high bacterial fluorescence at population and single-cell level, implemented this circuit in various plasmids differing in host range, and quantified their fluorescence under both aerobic and anaerobic conditions. Finally, we showed that KOFP-7-based constructions can be used to produce fluorescing cells of Vibrio diazotrophicus , a facultative anaerobe, demonstrating the usefulness of the genetic circuits for various anaerobic bacteria. These genetic circuits can thus be modified at will, both to solve basic and applied research questions, opening a highway to shed light on the obscure anaerobic world.IMPORTANCEFluorescent proteins are used for decades, and have allowed major discoveries in biology in a wide variety of fields, and are used in environmental as well as clinical contexts. Green fluorescent protein (GFP) and all its derivatives share a common feature: they rely on the presence of dioxygen (O 2 ) for protein maturation and fluorescence. This dependency precludes their use in anoxic environments. Here, we constructed a series of genetic circuits allowing production of KOFP-7, an O 2 -independant flavin-binding fluorescent protein. We demonstrated that Escherichia coli cells producing KOFP-7 are fluorescent, both at the population and single-cell levels. Importantly, we showed that, unlike cells producing GFP, cells producing KOFP-7 are fluorescent in anoxia. Finally, we demonstrated that Vibrio diazotrophicus NS1, a facultative anaerobe, is fluorescent in the absence of O 2 when KOFP-7 is produced. Altogether, the development of new genetic circuits allowing O 2 -independent fluorescence will open new perspective to study anaerobic processes., Competing Interests: The authors declare no conflict of interest.

In this study, the disrupting effects of glyphosate (GLY), aminomethylphosphonic acid (AMPA), and three glyphosate-based herbicides (GBHs) on vitellogenesis in a non-concentration-dependent manner are reported for the first time in 120 h of acute exposure of zebrafish at environmentally relevant concentrations. GBHs are commonly used worldwide in weed control management. Due to their extensive application, they frequently occur in aquatic ecosystems and may affect various organisms. The active substance GLY and its major by-product, AMPA, are the most thoroughly studied chemicals; however, the adverse effects of the complex formulas of GBHs with diverse and unknown content of co-formulants are still not sufficiently researched. This study focused on the embryotoxicity, sublethal malformations, and estrogenic potency of GLY, AMPA, and four commonly used GBHs on zebrafish embryos using a wild type and an estrogen-sensitive, transgenic zebrafish line (Tg(vtg1:mCherry)). After 120 h of exposition, AMPA did not cause acute toxicity, while the LC 50 of GLY was 160 mg/L. The GBHs were more toxic with LC 50 values ranging from 31 to 111 GLY active equivalent (a.e.) mg/L. Exposure to 0.35-2.8 mg/L GBHs led to sublethal abnormalities: typical symptoms were structural deformation of the lower jaw and anomalies in the olfactory region. Deformity rates were 10-30% in the treated groups. In vivo, fluorescently expressed vtg1 mCherry protein in embryonic liver was detected by a non-invasive microscopic method indicating estrogenic action through vitellogenin production by GLY, AMPA, and GBHs. To confirm the in vivo findings, RT-qPCR method was performed to determine the levels of the estrogenicity-related vtg1 mRNA. After 120 h of exposure to GLY, AMPA, and three GBHs at a concentration of 0.35 mg/L, the expression of vtg1 gene was significantly up-regulated. Our results highlight the risk that short-term GLY and GBH exposure can cause developmental malformations and disrupt the hormonal balance in zebrafish embryos., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The water eutrophication, resulting from the discharge of industrial and agricultural wastewater, leads to ecological degradation. However, to date, how to assess and manage the risks of water pollution, especially nitrogen pollution, remains a particularly noteworthy issue. Nitrate, the most important nitrogen compound, has become a bottleneck restricting total nitrogen management. The development of bioreporters monitoring nitrate pollution contributes to the estimation of water quality, especially the availability of nutrients. In this study, we obtained 9 bioreporters from 40 cyanobacterial derivatives which were constructed based on different hosts, copy numbers, and sensing elements and evaluated the performance of bioreporters. The results showed that single-celled Synechocystis was more sensitive to nitrate than filamentous Anabaena, that the reporter gene luxABCDE responded faster than sfgfp in most bioreporters, and that relatively medium-copy plasmid improved the performance of sensing elements. Nine bioreporters performed well in bioavailable nitrate detection, of which AD-AS-X and AR-NI-X, activated by nitrate repletion, had the shortest response time (2 h) and the widest response range (20-800 μM), respectively. Moreover, SR-GLN-SG, activated by nitrate deficiency, exhibited the best linear response (R 2  = 0.998). After parameter optimization, exponential growth phase bioreporters, culture temperature of 30 °C, sample volume of 200 μL were determined as optimal monitoring conditions. We found that common water contaminants (copper, cadmium, and phosphorus) had no impact on the performance of bioreporters, indicating the stability of bioreporters. Six out of 9 bioreporters, especially the SR-NB-X, were highly effective in detecting the bioavailable nitrate in wastewater sample. This study provides valuable references for developing more cyanobacterial bioreporters and their practical application in nitrate detection., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

The aim of this research is to define optimal conditions to improve the stability of gold and silver nanoparticles' anti-zearalenone antibody conjugates for their utilisation in lateral flow immunochromatographic assay (LFIA). The Turkevich-Frens method was used to synthesise gold nanoparticles (AuNPs), which were between 10 and 110 nm in diameter. Silver nanoparticles (AgNPs) with a size distribution of 2.5 to 100 nm were synthesised using sodium borohydride as a reducing agent. The onset of AuNP and AgNP aggregation occurred at 150 mM and 80 mM NaCl concentrations, respectively. Stable Au and Ag nanoparticle-antibody conjugates were achieved at 1.2 mM of K 2 CO 3 concentration, which corresponds to the pH value of ≈7. Lastly, the highest degree of conjugation between Au and Ag nanoparticles and anti-zearalenone antibodies was at 4 and 6 µg/mL of antibody concentrations. The optimisation of the conjugation conditions can contribute to better stability of nanoparticles and their antibody conjugate and can improve the reproducibility of results of bioreporter molecules in biosensing lateral flow devices.

For three years, a large amount of manufactured pollutants such as plastics, antibiotics and disinfectants has been released into the environment due to COVID-19. The accumulation of these pollutants in the environment has exacerbated the damage to the soil system. However, since the epidemic outbreak, the focus of researchers and public attention has consistently been on human health. It is noteworthy that studies conducted in conjunction with soil pollution and COVID-19 represent only 4 % of all COVID-19 studies. In order to enhance researchers' and the public awareness of the seriousness on the COVID-19 derived soil pollution, we propose the viewpoint that "pandemic COVID-19 ends but soil pollution increases" and recommend a whole-cell biosensor based new method to assess the environmental risk of COVID-19 derived pollutants. This approach is expected to provide a new way for environmental risk assessment of soils affected by contaminants produced from the pandemic., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Oil spill incidents occur frequently and threaten ecosystems and human health. Solid-phase microextraction allows direct alkane extraction from environmental matrices to improve the limit of detection but is unable to measure alkanes on site. A biological-phase microextraction and biosensing (BPME-BS) device was developed by immobilising an alkane chemotactic Acinetobacter bioreporter ADPWH_alk in agarose gel to achieve online alkane quantification with the aid of a photomultiplier. The BPME-BS device had a high enrichment factor (average 7.07) and a satisfactory limit of detection (0.075 mg/L) for alkanes. The quantification range was 0.1-100 mg/L, comparable to a gas chromatography flame ionisation detector and better than a bioreporter without immobilisation. ADPWH_alk cells in the BPME-BS device maintained good sensitivity under a wide range of environmental conditions, including pH (4.0-9.0), temperature (20-40 °C), and salinity (0.0-3.0%), and its response remained stable within 30 days at 4 °C. In a 7-day continual measurement, the BPME-BS device successfully visualised the dynamic concentration of alkanes, and a 7-day field test successfully captured an oil spill event, helping in source apportionment and on-scene law enforcement. Our work proved that the BPME-BS device is a powerful tool for online alkane measurement, showing substantial potential for fast detection and rapid response to oil spills on site and in situ., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Introduction. Klebsiella pneumoniae is a major threat to public health worldwide. It is the causative agent for multiple disease presentations including urinary tract infection, septicemia, liver abscess, wound infection and respiratory tract infection. K. pneumoniae causes community- and hospital-acquired pneumonia, which is a devastating disease associated with high mortality rates. Hypothesis. There is a growing concern about the emergence of multidrug-resistant K. pneumoniae strains complicating the treatment with the current available therapeutics; therefore, there is an urgent need for the development of new antimicrobial agents. Aim. K. pneumoniae causes an acute respiratory disease in mice and in the current work we investigated the capability to perform non-invasive monitoring of bioluminescent Klebsiella to monitor therapeutic efficacy. Methodology. We engineered a bioluminescence reporter strain of K. pneumoniae to monitor the impact of antibiotics in a murine respiratory disease model. Results. We demonstrate that bioluminescence correlates with bacterial numbers in host tissues allowing for a non-invasive enumeration of bacterial replication in vivo . Light production is directly linked to bacterial viability, and this novel bioluminescent K. pneumoniae strain enabled monitoring of the efficacy of meropenem therapy in arresting bacterial proliferation in the lung. Conclusion. The use of non-invasive bioluminescent imaging improves preclinical animal model testing to detect study outcome earlier and with higher sensitivity.

The contamination of lakes by industrial emissions is an issue of international concern. Traditional paleolimnology examines sedimentary micro-fossils to infer the biological response to natural and anthropogenic stressors over time. Here, we calculate a theoretical biological effect for historic sediment sections using Probable Effect Concentration Quotient (PEC-Q) and arsenic specific quotient methods and develop novel time-constrained sediment toxicity test methods using a cultured Daphnia sp. combined with a whole cell microbial biosensor to assess the toxicity of past industrial contamination with modern testing methods. These methods were developed using sediments collected from Pocket Lake (Northwest Territories, Canada), a lake known to have exhibited a significant ecological shift following input from nearby gold smelter emissions during the mid 20th century. We then applied these methods to near-, mid-, and far-field sites to assess the response of Daphnia sp. to varying contaminant load. Daphnia sp. mortality exposed to dated sediments indicated a strong concordance with the timing of mining activities, and a strong concordance with PEC-Q and arsenic specific toxicity quotients. In contrast, a decrease in Daphnia mortality was observed during pre-, and post-mining periods when the contaminant burden was lower. Initial assessments of bioavailability using a microbial biosensor indicated that arsenic in porewater is 72-96% bioavailable, and limited evidence that oxidative stress may contribute to the Daphnia sp. toxic response. These results indicate that lake sediment archives can be used to infer missing biomonitoring data in sites of legacy anthropogenic influence, which will be useful for those seeking to conduct cost-effective and efficient preliminary environmental risk assessments., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Creating unique microenvironments, hyphal surfaces and their surroundings allow for spatially distinct microbial interactions and functions at the microscale. Using a microfluidic system and pH-sensitive whole-cell bioreporters (Synechocystis sp. PCC6803) attached to hyphae, we spatially resolved the pH along surfaces of growing hyphae of the basidiomycete Coprinopsis cinerea. Time-lapse microscopy analysis of ratiometric fluorescence signals of >2400 individual bioreporters revealed an overall pH drop from 6.3 ± 0.4 (n = 2441) to 5.0 ± 0.3 (n = 2497) within 7 h after pH bioreporter loading to hyphal surfaces. The pH along hyphal surfaces varied significantly (p < 0.05), with pH at hyphal tips being on average ~0.8 pH units lower than at more mature hyphal parts near the entrance of the microfluidic observation chamber. Our data represent the first dynamic in vitro analysis of surface pH along growing hyphae at the micrometre scale. Such knowledge may improve our understanding of spatial, pH-dependent hyphal processes, such as the degradation of organic matter or mineral weathering.

The bioavailability of pollutants is a key factor affecting environmental risk. Whole-cell bioreporters are a demonstratedly effective tool for the investigation of pollutant bioavailability in water and soil/sediment. Unlike aqueous samples, transmittance of bioreporter optical signal is reduced in direct-contact assays with soil/sediment, which affects the accuracy of bioreporter-detected pollutant bioavailability. No studies have measured the magnitude and variability of soil/sediment effects on signal in direct-contact assays or how associated uncertainties influence results. In this study, we investigate the optical effects of soil/sediment particles in suspensions on bioreporter signal transmittance and quantify how variable these optical effects are from sample-to-sample. We find that neglecting bioreporter signal diminution by soil/sediment, as many studies do, can lead to order-of-magnitude errors in results, underestimating risk. Correction based on methods in ad hoc use (e.g. comparison to signal from non-inducible reporter or use of reference soil/sediment) are also problematic for some types of experiment, and could lead to errors in excess of 30%. Our findings have a sound basis in theory, and we provide recommendations concerning the most suitable type of approach to use for different experimental settings. Generally, if best accuracy is not needed to quantify bioavailability, for samples that have been ground, sieved, and are of reasonably uniform color, it may be possible to use a single or average correction factor, particularly for experiments performed at a single slurry concentration. For investigations studying bioavailability under varying solid-phase:water ratios (e.g., sorption/desorption), detailed compensation measurements are needed for independent variables, including each specific soil/sediment sample, slurry concentration, and in some cases bioreporter signal intensity. Our measurements and calculations indicate that best results are obtained when working in the region of ballistic photon transmittance. Findings herein will be useful in areas that require information on bioavailability, such as ecotoxicology and environmental risk assessment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

A possible solution for the standoff detection of buried landmines is based on the use of microbial bioreporters, genetically engineered to emit a remotely detectable optical signal in response to trace amounts of explosives' signature chemicals, mostly 2,4-dinitrotoluene (DNT). Previously developed DNT sensor strains were based on the fusion of a DNT-inducible gene promoter to a reporting element, either a fluorescent protein gene or a bacterial bioluminescence gene cassette. In the present study, a different approach was used: the DNT-inducible promoter activates, in Escherichia coli , the quorum-sensing luxI and luxR genes of Aliivibrio fischeri . N-Acyl homoserine lactone (AHL), synthesized by LuxI, combines with LuxR and activates the bioluminescence reporter genes. The resulting bioreporter displayed a dose-dependent luminescent signal in the presence of DNT. Performance of the sensor strain was further enhanced by manipulation of the sensing element (combining the E. coli DNT-inducible azoR and yqjF gene promoters), by replacing the luminescence gene cassette of Photorhabdus luminescens luxCDABE with A. fischeri luxCDABEG , and by introducing two mutations, eutE and ygdD , into the host strain. DNT detection sensitivity of the final bioreporter was over 340-fold higher than the original construct., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors. Engineering in Life Sciences published by Wiley‐VCH GmbH.)

Detection of buried landmines is a dangerous and complicated task that consumes large financial resources and poses significant risks to the personnel involved. A potential alternative to conventional detection methodologies is the use of microbial bioreporters, capable of emitting an optical signal upon exposure to explosives, thus revealing to a remote detector the location of buried explosive devices. We have previously reported the design, construction, and optimization of an Escherichia coli -based bioreporter for the detection of 2,4,6-trinitrotoluene (TNT) and its accompanying impurity 2,4-dinitrotoluene (DNT). Here we describe the further enhancement of this bioreporter by the directed evolution of YhaJ, the transcriptional activator of the yqjF gene promoter, the sensing element of the bioreporter's molecular circuit. This process resulted in a 37-fold reduction of the detection threshold, as well as significant enhancements to signal intensity and response time, rendering this sensor strain more suitable for detecting the minute concentrations of DNT in the soil above buried landmines. The capability of this enhanced bioreporter to detect DNT buried in sand is demonstrated., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Elad, Shemer, Simanowitz, Kabessa, Mizrachi, Gold, Shpigel, Agranat and Belkin.)

Characterizing waste ecotoxicity is laborious because of both the undefined nature of environmental samples and the diversity of contaminants that can be present. With regard to these limitations, traditional approaches do not provide information about the nature of the pollution encountered. To improve such assessments, a fluorescent library of 1870 transcriptomic reporters from Escherichia coli K12 MG1655 was used to report the ecotoxic status of environmental samples. The reliability of the approach was evaluated with 6 metallic pollutants (As, Cu, Cd, Hg, Pb, Zn) used alone and in mixture in pure and complex matrices. A total of 18 synthetic samples were used to characterize the specificity of the resulting metallic contamination fingerprints. Metallic contamination impacted 4.5 to 10.2% of the whole transcriptomic fingerprint of E. coli. The analysis revealed that a subset of 175 transcriptomic reporters is sufficient to characterize metallic contamination, regardless of the nature of the sample. A statistical model distinguished patterns due to metallic contamination and provided information about the level of toxicity with 93 to 98% confidence. The use of the transcriptomic assessment was validated for 17 complex matrices with various toxicities and metal contaminants, such as activated sludge, wastewater effluent, soil, wood and river water. The presence of metals and their associated toxicity, which seems linked to their bioavailabilities, were thereby determined. This method constitutes a possible tool to screen unknown complex samples for their metallic status and identify those for which a deeper characterization must be achieved by the use of traditional biosensors and analytical methods., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Arsenic detoxification systems can be found in a wide range of organisms, from bacteria to humans. In a previous study, we discovered an arsenic-responsive transcriptional regulator in the thermophilic bacterium Thermus thermophilus HB27 ( Tt SmtB). Here, we characterize the arsenic resistance system of T. thermophilus in more detail. We employed Tt SmtB-based pulldown assays with protein extracts from cultures treated with arsenate and arsenite to obtain an S -adenosyl-l-methionine (SAM)-dependent arsenite methyltransferase ( Tt ArsM). In vivo and in vitro analyses were performed to shed light on this new component of the arsenic resistance network and its peculiar catalytic mechanism. Heterologous expression of TtarsM in Escherichia coli resulted in arsenite detoxification at mesophilic temperatures. Although Tt ArsM does not contain a canonical arsenite binding site, the purified protein does catalyze SAM-dependent arsenite methylation with formation of monomethylarsenites (MMAs) and dimethylarsenites (DMAs). In addition, in vitro analyses confirmed the unique interaction between Tt ArsM and Tt SmtB. Next, a highly efficient ThermoCas9-based genome-editing tool was developed to delete the Tt ArsM-encoding gene on the T. thermophilus genome and to confirm its involvement in the arsenite detoxification system. Finally, the TtarsX efflux pump gene in the T. thermophilus Δ TtarsM genome was substituted by a gene encoding a stabilized yellow fluorescent protein (sYFP) to create a sensitive genome-based bioreporter system for the detection of arsenic ions. IMPORTANCE We here describe the discovery of an unknown protein by using a proteomics approach with a transcriptional regulator as bait. Remarkably, we successfully obtained a novel type of enzyme through the interaction with a transcriptional regulator controlling the expression of this enzyme. Employing this strategy, we isolated Tt ArsM, the first thermophilic prokaryotic arsenite methyltransferase, as a new enzyme of the arsenic resistance mechanism in T. thermophilus HB27. The atypical arsenite binding site of Tt ArsM categorizes the enzyme as the first member of a new arsenite methyltransferase type, exclusively present in the Thermus genus. The enzyme methylates arsenite-producing MMAs and DMAs. Furthermore, we developed an hyperthermophilic Cas9-based genome-editing tool, active up to 65°C. The tool allowed us to perform highly efficient, marker-free modifications (either gene deletion or insertion) in the T. thermophilus genome. With these modifications, we confirmed the critical role of Tt ArsM in the arsenite detoxification system and developed a sensitive whole-cell bioreporter for arsenic ions. We anticipate that the developed tool can be easily adapted for editing the genomes of other thermophilic bacteria, significantly boosting fundamental and metabolic engineering in hyperthermophilic microorganisms.

Quorum sensing (QS) helps the Xanthomonas group of phytopathogens to infect several crop plants. The vascular phytopathogen Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot disease on Brassicaceae leaves, where a typical v-shaped lesion spans both vascular and mesophyll regions with progressive leaf chlorosis. Recently, the role of QS has been elucidated during Xcc early infection stages. However, a detailed insight into the possible role of QS-regulated bacterial invasion in host chlorophagy during late infection stages remains elusive. In this study, using QS-responsive whole-cell bioreporters of Xcc, we present a detailed chronology of QS-facilitated Xcc colonization in the mesophyll region of cabbage (Brassica oleracea) leaves. We report that QS-enabled localization of Xcc to parenchymal chloroplasts triggers leaf chlorosis and promotion of systemic infection. Our results indicate that the QS response in the Xanthomonas group of vascular phytopathogens maximizes their population fitness across host tissues to trigger stage-specific host chlorophagy and establish a systemic infection., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Biofilms disperse in response to specific environmental cues, such as reduced oxygen concentration, changes in nutrient concentration and exposure to nitric oxide. Interestingly, biofilms do not completely disperse under these conditions, which is generally attributed to physiological heterogeneity of the biofilm. However, our results suggest that genetic heterogeneity also plays an important role in the non-dispersing population of P. aeruginosa in biofilms after nutrient starvation., Results: In this study, 12.2% of the biofilm failed to disperse after 4 d of continuous starvation-induced dispersal. Cells were recovered from the dispersal phase as well as the remaining biofilm. For 96 h starved biofilms, rugose small colony variants (RSCV) were found to be present in the biofilm, but were not observed in the dispersal effluent. In contrast, wild type and small colony variants (SCV) were found in high numbers in the dispersal phase. Genome sequencing of these variants showed that most had single nucleotide mutations in genes associated with biofilm formation, e.g. in wspF, pilT, fha1 and aguR. Complementation of those mutations restored starvation-induced dispersal from the biofilms. Because c-di-GMP is linked to biofilm formation and dispersal, we introduced a c-di-GMP reporter into the wild-type P. aeruginosa and monitored green fluorescent protein (GFP) expression before and after starvation-induced dispersal. Post dispersal, the microcolonies were smaller and significantly brighter in GFP intensity, suggesting the relative concentration of c-di-GMP per cell within the microcolonies was also increased. Furthermore, only the RSCV showed increased c-di-GMP, while wild type and SCV were no different from the parental strain., Conclusions: This suggests that while starvation can induce dispersal from the biofilm, it also results in strong selection for mutants that overproduce c-di-GMP and that fail to disperse in response to the dispersal cue, starvation., (© 2021. The Author(s).)

Due to the public health concerns of endocrine-disrupting chemicals, there is an increasing demand to develop improved high-throughput detection assays for enhanced exposure control and risk assessment. A substrate-free, autobioluminescent HEK293 ARE/Gal4-Lux assay was developed to screen compounds for their ability to induce androgen receptor (AR)-mediated transcriptional activation. The assay was validated against a group of 40 recommended chemicals and achieved an overall 87.5% accuracy in qualitatively classifying positive and negative AR agonists. The HEK293 ARE/Gal4-Lux assay was demonstrated as a suitable tool for Tier 1 AR agonist screening. By eliminating exogenous substrate, this assay provided a significant advantage over traditional reporter assays by enabling higher-throughput screening with reduced testing costs while maintaining detection accuracy.

The ongoing COVID-19 pandemic has highlighted the immediate need for the development of antiviral therapeutics targeting different stages of the SARS-CoV-2 life cycle. We developed a bioluminescence-based bioreporter to interrogate the interaction between the SARS-CoV-2 viral spike (S) protein and its host entry receptor, angiotensin-converting enzyme 2 (ACE2). The bioreporter assay is based on a nanoluciferase complementation reporter, composed of two subunits, large BiT and small BiT, fused to the S receptor-binding domain (RBD) of the SARS-CoV-2 S protein and ACE2 ectodomain, respectively. Using this bioreporter, we uncovered critical host and viral determinants of the interaction, including a role for glycosylation of asparagine residues within the RBD in mediating successful viral entry. We also demonstrate the importance of N-linked glycosylation to the RBD's antigenicity and immunogenicity. Our study demonstrates the versatility of our bioreporter in mapping key residues mediating viral entry as well as screening inhibitors of the ACE2-RBD interaction. Our findings point toward targeting RBD glycosylation for therapeutic and vaccine strategies against SARS-CoV-2., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021. Published by Elsevier Inc.)

A series of stable polysaccharide derivatives that spontaneously self-assemble into nanocarriers was synthesized by applying a reductive amination on chitosan. The prepared nanocarriers were comprehensively studied and found to allow encapsulation of molecular cargo in both aqueous and lipidic media and deliver this cargo across biological barriers. The nanocarriers have demonstrated effective transdermal delivery of diclofenac (Voltaren), a nonsteroidal anti-inflammatory drug, by increasing its skin permeation up to 100 vs the tested control. The modified polysaccharides were studied with a panel of three types of bioreporter bacteria sensitive to genotoxic and cytotoxic stresses. These studies showed the general safety of the prepared nanocarriers and provided insights concerning their activity in collaboration with the aliphatic side chain length. The described nanocarriers could be applied as tunable biocompatible vehicles for the delivery of medicines, cosmetic agents, and in other applications.

Bioluminescent yeast assays BLYES and BLYAS are whole-cell bioassays that utilize genetically modified Saccharomyces cerevisiae bioreporters to detect estrogenic and androgenic activities, respectively. The bioreporter strains chromosomally express human estrogen receptor alpha (BLYES) or androgen receptor (BLYAS) and contain a reporter plasmid expressing the complete bacterial luciferase gene cassette (luxCDABE) under the control of an estrogen- or androgen-responsive promoter. Exposure to endocrine-disrupting compounds activates the receptor which subsequently turns on the expression of the reporter genes, resulting in dose-dependent bioluminescence (i.e., light) emission. These yeast whole-cell bioassays provide rapid, cost-effective, and high-throughput detection of endocrine-disrupting activities in environmental samples. This protocol will provide a detailed description of the standard assay procedures as well as a framework for data analysis.

The detection of oxidative stress caused by emerging pollutants in aquatic systems is essential to carry out toxicological analysis since they can bring us information about the mechanisms of toxic action of the pollutants, which might be useful to address this contamination. To achieve this goal, two self-bioluminescent strains that respond to oxidative stress based on the filamentous cyanobacterium Nostoc sp. PCC7120, which has a high ecological relevance in aquatic continental systems, have been constructed. Nostoc sp. PCC7120 pBG2172 harbours the promoter region of the 2-cys-prx gene (P2-cys-prx), encoding a cytoplasmic peroxiredoxin, fused to luxCDABE genes of the bacterium Photorhabdus luminescens. Nostoc sp. PCC7120 pBG2173 harbours the promoter region of the KatA gene (PkatA), a cytoplasmic catalase, also fused to luxCDABE genes. Both strains have been characterized by exposing them to H 2 O 2 : Nostoc sp. PCC7120 pBG2172 responded while Nostoc sp. PCC7120 pBG2173 did not respond to this pollutant. In order to know their specificity, they were exposed to methyl viologen (MV), an herbicide that produces superoxide anion (O 2 - ) and a bioluminescence response was observed in both strains. Besides, the utility of these strains for the detection of H 2 O 2 and MV in natural water samples, both pristine and wastewater samples has been tested by spiking experiments. Finally, the possible application of these strains for the detection of the emerging pollutant triclosan has also been tested showing to be suitable bioreporters to study oxidative stress in aquatic environments., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Aim: Escherichia coli is the conventional choice as the host strain for whole-cell bioreporter construction due to its well-understood genetics and well-established cloning protocols. However, for real-world environmental biosensing applications, it is often beneficial to use a bacterial strain derived directly from the environment under study to better ensure chemical target specificity and optimal response time. The aim of this study was to develop a whole-cell bioreporter for detection of bioavailable mercury by replacing E. coli with a wild-type bacterial host derived from a soil environment., Materials and Results: In this study, an Enterobacter cloacae strain isolated from soil derived from a municipal and electronic waste dumping site was engineered to serve as a bioluminescent bioreporter for mercury toxicity by linking its merR-like gene and promoter sequence to a reorganized luxABCDE gene cassette from Photorhabdus luminescens. This bioreporter, designated as E. cloacae DWH4 lux , detected mercury (HgCl 2 ) at a minimum concentration of 0·2 µg l -1 with a linear response profile being maintained between a range of 0·4-1600 µg l -1 (R 2  = 0·9604) with a peak bioluminescent response occurring within 1 h after exposure. No significant synergistic or antagonistic influences were observed on the bioluminescent response by other contaminating metal elements. Enterobacter cloacae DWH4 lux was also demonstrated to detect mercury effectively in artificially contaminated water sample with linear correlation (R 2  = 0·9623)., Conclusions: The results indicated that E. cloacae DWH4 lux could detect mercury in quantities below the US Environmental Protection Agency's permitted limit values (2 µg l -1 ). Hence, it is concluded that E. cloacae DWH4 lux has the potential to serve as an effective whole-cell bioreporter for the environmental monitoring of mercury contamination., Significance and Impact of the Study: This study provides new insight into the recruitment of mercury-tolerant bacterial hosts derived from environmental samples over the conventional lab-based E. coli host for the construction of mercury bioreporters. With improved response time and selectivity, the environmentally sourced bacteria can serve as an alternative host choice to improve biosensing technology in the near future., (© 2019 The Society for Applied Microbiology.)

Today, two types of lamp systems dominate the UV disinfection industry: low-pressure (LP) UV lamps and medium-pressure (MP) polychromatic lamps. Both lamp types have their advantages and disadvantages in microorganism inactivation, with LP lamps being cheaper, having longer life, and working at lower temperature, hence reducing fouling, and MP lamps showing better inactivation per germicidal dose for certain microorganisms. Bacterium-based biosensors were used to compare LP and MP irradiation. These biosensors were Escherichia coli bacteria carrying the lux operon genes under the control of different stress-responding promoters, where activation of the specific promoter is manifested as bioluminescence. MP irradiation, considerably more than LP irradiation, resulted in activation of the superoxide dismutase expression, indicating the formation of superoxide radicals inside the cells. Accordingly, pre-exposure (immunization) of the bacteria to an activator that produces superoxide radicals resulted in lower inactivation and increased resistance to MP irradiation, but not to LP irradiation. This study shows that the difference in germicidal efficiency may result from the production of intracellular superoxide radicals by MP irradiation, at wavelengths other than 254 nm, as emitted by LP lamps., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

This review summarizes the development of whole-cell biosensors with a special focus on device development and cell immobilization. Integration of biosensor functions in a device will pave the way for field applications in remote areas and resource-limited settings. Firstly, an introduction to the field of whole-cell biosensors is provided, followed by examples of genetic engineering of cells in order to fulfill sensor functions. A framework of requirements to enable future field applications of biosensors is elaborated. A special focus is on different cell immobilization techniques ranging from polymers, to microfluidic devices, immobilization on paper and combinations of these methods. Looking at globally successfully implemented point of care devices such as a home pregnancy test or a blood glucose meter, we conclude the review with thoughts on long-term stability, portability, ease of use and user safety design guidelines for whole-cell biosensor devices.

A synthetic bacterial luciferase-based autobioluminescent bioreporter, HEK293ERE/Gal4-Lux, was developed in a human embryonic kidney (HEK293) cell line for the surveillance of chemicals displaying endocrine disrupting activity. Unlike alternative luminescent reporters, this bioreporter generates bioluminescence autonomously without requiring an external light-activating chemical substrate or cellular destruction. The bioreporter's performance was validated against a library of 76 agonistic and antagonistic estrogenic endocrine disruptor chemicals and demonstrated reproducible half maximal effective concentration (EC50) values meeting the U.S. Environmental Protection Agency (EPA) guidelines for Tier 1 endocrine disrupting chemical screening assays. For model compounds, such as the estrogen receptor (ER) agonist 17β-estradiol, HEK293ERE/Gal4-Lux demonstrated an EC50 value (7.9 × 10-12 M) comparable to that of the current EPA-approved HeLa-9903 firefly luciferase-based estrogen receptor transcription assay (4.6 × 10-12 M). Screening against an expanded array of common ER agonists likewise produced similar relative effect potencies as compared with existing assays. The self-initiated autobioluminescent signal of the bioreporter permitted facile monitoring of the effects of endocrine disrupting chemicals, which decreased the cost and hands-on time required to perform these assays. These characteristics make the HEK293ERE/Gal4-Lux bioreporter potentially suitable as a high-throughput human cell-based assay for screening estrogenic activity., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Biological systems with intrinsic luminescent properties serve as powerful and noninvasive bioreporters for real-time and label-free monitoring of cell physiology. This study employs the bioluminescent marine bacterium Vibrio fischeri to investigate the effects of separated microwave electric (E) and magnetic (H) fields. Using a cylindrical TM010 mode aluminum resonant cavity, designed to spatially separate E and H fields of a pulsed microwave (2.45 GHz) input, we sampled at 100-ms intervals the 490-nm emission of bioluminescence from suspensions of the V. fischeri. E-field exposure (at 4.24 and 13.4 kV/m) results in rapid and sensitive responses to 100-ms pulses. H-field excitation elicits no measurable responses, even at 100-fold higher power input levels (equivalent to 183 A/m). The observed effects on bacterial light output partially correlate with measured E-field-induced temperature increases. In conclusion, the endogenous bioluminescence of V. fischeri provides a sensitive and noninvasive method to assess the biological effects of microwave fields.

Vibrio campbellii BAA-1116 is renowned for its bioluminescence properties, and genetic tools are available to genetically track this strain. However, many other ecologically important V. harveyi strains exist, for which only few genetic tools are available. In this study, a rapid electroporation protocol was developed to transform replicative plasmids in various environmental V. harveyi and Pseudoalteromonas strains. Moreover, a mini-Tn7 delivery system was modified to site-specifically integrate mini-transposons in the genome of V. harveyi ORM4. As a proof-of-principle, replicative plasmids carrying bioreporters were introduced by electroporation in V. harveyi ORM4 cells, and gene expression was followed at the single cell level. We could demonstrate that a flagellar gene is subjected to bimodal gene expression in V. harveyi ORM4, being highly expressed in 10% of the population in stationary phase. This study extends the possibilities to study environmental Vibrio strains and uncovers the occurrence of phenotypic heterogeneity in flagellar expression in Vibrio., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Based on the sole information of structural genes of the 2-nitrobenzoate (2NBA) utilizing catabolic gene cluster ( onbX1X2FCAR1EHJIGDBX3 ), 2NBA-sensing bioreporters were constructed by incorporating egfp into the onb gene cluster of Cupriavidus sp. strain ST-14. Incorporation of reporter gene in proximal to the hypothesized promoter region in conjunction with the disruption of the gene encoding inducer-metabolizing enzyme was turned out to be advantageous in reporter gene expression at low inducer concentration. The bioreporter strain was capable of expressing EGFP from the very 1st hour of induction and could detect 2NBA at (sub) nanomolar level exhibiting a strict specificity toward 2NBA, displaying no response to EGFP expression from its meta - and para -isomers as well as from a number of structurally related compounds. The present study is a successful demonstration of the development of a 2NBA-sensing bioreporter with respect to ease of construction, inducer specificity, and sensitivity, without prior knowledge of the associated inducer-responsive promoter-regulator elements. The present approach can be used as a model for the development of bioreporters for other environmental pollutants.

An autonomously bioluminescent Saccharomyces cerevisiae BLYAhS bioreporter was developed in this study for the simple and rapid detection of dioxin-like compounds (DLCs) and aryl hydrocarbon receptor (AhR) agonists. This recombinant yeast reporter was based on a synthetic bacterial luciferase reporter gene cassette (lux) that can produce the luciferase as well as the enzymes capable of self-synthesizing the requisite substrates for bioluminescent production from endogenous cellular metabolites. As a result, bioluminescent signal production is generated continuously and autonomously without cell lysis or exogenous reagent addition. By linking the expression of the autobioluminescent lux reporter cassette to AhR activation via the use of a dioxin-responsive promoter, the S. cerevisiae BLYAhS bioreporter emitted a bioluminescent signal in response to DLC exposure in a dose-responsive manner. The model dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), could be detected within 4 h with a half maximal effective concentration (EC 50 ) of ~ 8.1 nM and a lower detection limit of 500 pM. The autobioluminescent response of BLYAhS to other AhR agonists, including 2,3,7,8-tetrachlorodibenzofuran (TCDF), polychlorinated bisphenyl congener 126 (PCB-126) and 169 (PCB-169), 1,2,3,6,7,8-hexachlorodibenzo-p-dioxin (HxCDD), 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (HpCDD), benzo[a]pyrene (BaP), and β-naphthoflavone (bNF), were also characterized in this study. The non-destructive and reagent-free nature of the BLYAhS reporter assay facilitated near-continuous, automated signal acquisition without additional hands-on effort and cost, providing a simple and cost-effective method for rapid DLC detection.

The bright bioluminescence catalyzed by Photinus pyralis firefly luciferase (Fluc) enables a vast array of life science research such as bio imaging in live animals and sensitive in vitro diagnostics. The effectiveness of such applications is improved using engineered enzymes that to date have been constructed using amino acid substitutions. We describe ΔFlucs: consecutive single amino acid deletion mutants within six loop structures of the bright and thermostable ×11 Fluc. Deletion mutations are a promising avenue to explore new sequence and functional space and isolate novel mutant phenotypes. However, this method is often overlooked and to date there have been no surveys of the effects of consecutive single amino acid deletions in Fluc. We constructed a large semi-rational ΔFluc library and isolated significantly brighter enzymes after finding ×11 Fluc activity was largely tolerant to deletions. Targeting an "omega-loop" motif (T352-G360) significantly enhanced activity, altered kinetics, reduced Km for D-luciferin , altered emission colors, and altered substrate specificity for redshifted analog DL-infraluciferin. Experimental and in silico analyses suggested remodeling of the Ω-loop impacts on active site hydrophobicity to increase light yields. This work demonstrates the further potential of deletion mutations, which can generate useful Fluc mutants and broaden the palette of the biomedical and biotechnological bioluminescence enzyme toolbox., (© 2017 Wiley Periodicals, Inc.)

Aims: Evaluate the use of polyethyleneimines (PEIs) as membrane permeabilizers to improve the responses and sensitivity of a bacterial bioreporter strain to viologens., Methods and Results: The responses from E. coli str. EBS, i.e., E. coli BW25113 carrying plasmid pSDS, when exposed to five different viologens were characterized, as were the toxicities of seven different PEIS, including two linear and five branched species. Based on these results, benzyl viologen led to the greatest responses, and 0·8-kDa branched PEI (BPEI) was the least toxic of the PEIs tested and, therefore, both were selected for the subsequent tests. The bioluminescence and relative responses from E. coli str. EBS exposed to various concentrations of 0·8 kDa BPEI identified 400 mg l -1 as the optimal concentration. Using this concentration, tests were performed with all five of the viologens., Conclusions: The responses from E. coli str. EBS to the viologens were improved, with the maximum relative bioluminescence values increasing between 5·6 and 16·5-fold. The minimum detectable levels for four of the viologens were likewise improved 2- to 4-fold., Significance and Impact of Study: Improving bacterial membrane permeability in a controlled manner using BPEIs can improve biosensing of toxic compounds, as well as be used in biofuel and bioenergy applications where membrane permeability to a solute is important., (© 2017 The Society for Applied Microbiology.)

This study examined the applicability of a freeze-dried bioluminescent bioreporter, Pseudomonas putida mt-2 KG1206 (called KG1206), to the biomonitoring of groundwater samples. Samples were collected from the monitoring wells of gas station tanks or old pipeline leakage sites in Korea. In general, the freeze-dried strain in the presence of pure inducer chemicals showed low bioluminescence activity and a different activity order compared with that of the subcultured strain. The effects of KNO 3 as a bioluminescence stimulant were observed on the pure inducers and groundwater samples. The stimulation rates varied according to the type of inducers and samples, ranging from 2.2 to 20.5 times (for pure inducers) and from 1.1 to 11 times (for groundwater samples) the total bioluminescence of the control. No considerable correlations were observed between the bioluminescence intensity of the freeze-dried strain and the inducer concentrations in the samples (R 2  < 0.1344). However, samples without a high methyl tertiary butyl ether (MTBE) level and those from the gas station leakage site showed reasonable correlations with the bioluminescence activity with R 2 values of 0.3551 and 0.4131, respectively. These results highlight the potential of using freeze-dried bioluminescent bacteria as a rapid, simple, and portable tool for the preliminary biomonitoring of specific pollutants at contaminated sites.

Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a dynamic intracellular signaling molecule that plays a central role in the biofilm life cycle. Current methodologies for the quantification of c-di-GMP are typically based on chemical extraction, representing end point measurements. Chemical methodologies also fail to take into consideration the physiological heterogeneity of the biofilm and thus represent an average c-di-GMP concentration across the entire biofilm. To address these problems, a ratiometric, image-based quantification method has been developed based on expression of the green fluorescence protein (GFP) under the control of the c-di-GMP-responsive cdrA promoter (Rybtke, M. T., Borlee, B. R., Murakami, K., Irie, Y., Hentzer, M., Nielsen, T. E., Givskov, M., Parsek, M. R., and Tolker-Nielsen, T. (2012) Appl. Environ. Microbiol. 78, 5060-5069). The methodology uses the cyan fluorescent protein (CFP) as a biomass indicator and the GFP as a c-di-GMP reporter. Thus, the CFP/GFP ratio gives the effective c-di-GMP per biomass. A binary mask was applied to alleviate background fluorescence, and fluorescence was calibrated against known c-di-GMP concentrations. Using flow cells for biofilm formation, c-di-GMP showed a non-uniform distribution across the biofilm, with concentrated hot spots of c-di-GMP. Additionally, c-di-GMP was found to be localized at the outer boundary of mature colonies in contrast to a uniform distribution in early stage, small colonies. These data demonstrate the application of a method for the in situ, real time quantification of c-di-GMP and show that the amount of this biofilm-regulating second messenger was dynamic with time and colony size, reflecting the extent of biofilm heterogeneity in real time., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

We report a transgenic zebrafish (Danio rerio) designed to respond to heavy metals using a metal-responsive promoter linked to a fluorescent reporter gene (DsRed2). The metallothionein MT-Ia1 promoter containing metal-responsive elements was derived from the Asian green mussel, Perna viridis. The promoter is known to be induced by a broad spectrum of heavy metals. The promoter-reporter cassette cloned into the Tol2 transposon vector was microinjected into zebrafish embryos that were then reared to maturity. A transgene integration rate of 28 % was observed. The confirmed transgenics were mated with wild-type counterparts, and pools of F1 embryos were exposed to sub-lethal doses of Cd(2+), Cu(2+), Hg(2+), Pb(2+) and Zn(2+). The red fluorescence response of zebrafish embryos was observed 8 h post- exposure to these sub-lethal doses of heavy metals using a fluorescence microscope. Reporter expression estimated by real-time PCR revealed eightfold, sixfold and twofold increase on exposure to highest concentrations of Hg(2+), Cd(2+) and Cu(2+), while Pb(2+) and Zn(2+) had no effect. This biosensor could be a first-level screening method for confirming aquatic heavy metal bio-toxicity to eukaryotes.

The β-proteobacterium Aromatoleum aromaticum degrades the aromatic ketone acetophenone, a key intermediate of anaerobic ethylbenzene metabolism, either aerobically or anaerobically via a complex ATP-dependent acetophenone carboxylase and a benzoylacetate-CoA ligase. The genes coding for these enzymes (apcABCDE and bal) are organized in an apparent operon and are expressed in the presence of the substrate acetophenone. To study the conditions under which this operon is expressed in more detail, we constructed a reporter strain by inserting a gene fusion of apcA, the first gene of the apc-bal operon, with the gene for the fluorescent protein mCherry into the chromosome of A. aromaticum. The fusion protein indeed accumulated consistently with the expression pattern of the acetophenone-metabolic enzymes under various growth conditions. After evaluating and quantifying the data by fluorescence microscopy, fluorescence-based flow cytometry and immunoblot analysis, mCherry production was found to be proportional to the applied acetophenone concentrations. The reporter strain allowed quantification of acetophenone within a concentration range of 50 μM (detection limit) to 250 μM after 12 and 24 h. Moreover, production of the Apc-mCherry fusion protein in the reporter strain was highly specific and responded to acetophenone and both enantiomers of 1-phenylethanol, which are easily converted to acetophenone. Other analogous substrates showed either a significantly weaker response or none at all. Therefore, the reporter strain provides a basis for the development of a specific bioreporter system for acetophenone with an application potential reaching from environmental monitoring to petroleum prospecting.

Toxicants in water sources are of concern. We developed a tool that is affordable and easy-to-use for monitoring toxicity in water. It is a biosensor composed of disposable bioreporter pads (calcium alginate matrix with immobilized bacteria) and a non-disposable CMOS photodetector. Various parameters to enhance the sensor's signal have been tested, including the effect of alginate and bacterium concentrations. The effect of various toxicants, as well as, environmental samples were tested by evaluating their effect on bacterial luminescence. This is the first step in the creation of a sensitive and simple operative tool that may be used in different environments., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The aim of this study was to evaluate the biological toxicity of cellulose nanocrystals (CNCs) using the constitutively bioluminescent luxCDABE-based bioreporter Escherichia coli 652T7. The effects of CNCs on E. c oli 652T7 biotoxicity were investigated at different CNC concentrations, reaction times, and IC50 values. CNC toxicity was also compared with and without ultrasonic dispersion to establish dispersibility effects. The results demonstrated that CNCs were not significantly toxic at concentrations at or below 250 mg/L. At concentrations higher than 300 mg/L, toxicity increased linearly as CNC concentrations increased up to 2000 mg/L. IC50 calculations demonstrated an increase in cytotoxicity as CNC exposure times increased, and elevated dispersibility of the CNCs were shown to increase cytotoxicity effects. These results suggest that CNCs can impact microbial populations if elevated concentration thresholds are met.

A luxCDABE-based genetically engineered bacterial bioreporter (Escherichia coli ARL1) was used to detect bioavailable ionic mercury (Hg(II)) and investigate the effects of humic acids and ethylenediaminetetraacetic acid (EDTA) on the bioavailability of mercury in E. c oli. Results showed that the E. c oli ARL1 bioreporter was sensitive to mercury, with a detection limit of Hg(II) of 0.5 µg/L and a linear dose/response relationship up to 2000 µg Hg(II)/L. Humic acids and EDTA decreased the Hg(II)-induced bioluminescent response of strain ARL1, suggesting that the two organic ligands reduced the bioavailability of Hg(II) via complexation with Hg(II). Compared with traditional chemical methods, the use of E. c oli ARL1 is a cost-effective, rapid, and reliable approach for measuring aqueous mercury at very low concentrations and thus has potential for applications in field in situ monitoring.

Main Conclusion: The activation and level of expression of an endogenous, stress-responsive biosensor (bioreporter) can be visualized in real-time and non-destructively using highly accessible equipment (fluorometer). Biosensor output can be linked to computer-controlled systems to enable feedback-based control of a greenhouse environment. Today's agriculture requires an ability to precisely and rapidly assess the physiological stress status of plants in order to optimize crop yield. Here we describe the implementation and utility of a detection system based on a simple fluorometer design for real-time, continuous, and non-destructive monitoring of a genetically engineered biosensor plant. We report the responses to heat stress of Arabidopsis thaliana plants expressing a Yellow Fluorescent Protein bioreporter under the control of the DREB2A temperature-sensing promoter. Use of this bioreporter provides the ability to identify transient and steady-state behavior of gene activation in response to stress, and serves as an interface for novel experimental protocols. Models identified through such experiments inform the development of computer-based feedback control systems for the greenhouse environment, based on in situ monitoring of mature plants. More broadly, the work here provides a basis for informing biologists and engineers about the kinetics of bioreporter constructs, and also about ways in which other fluorescent protein constructs could be integrated into automated control systems.

Bacteria based ecotoxicology assessment of manufactured nanoparticles is largely restricted to Escherichia coli bioreporters in laboratory media. Here, toxicity effects of model OECD nanoparticles (Ag NM-300K, ZnO NM-110 and TiO2 NM-104) were assessed using the switch-off luminescent Pseudomonas putida BS566::luxCDABE bioreporter in Luria Bertani (LB) medium and artificial wastewater (AW). IC50 values ∼4 mg L(-1), 100 mg L(-1) and >200 mg L(-1) at 1 h were observed in LB for Ag NM-300K, ZnO NM-110 and TiO2 NM-104, respectively. Similar results were obtained in AW for Ag NM-300K (IC50∼5 mg L(-1)) and TiO2 NM-104 (IC50>200 mg L(-1)) whereas ZnO NM-110 was significantly higher (IC50>200 mg L(-1)). Lower ZnO NM-110 toxicity in AW compared to LB was associated with differences in agglomeration status and dissolution rate. This work demonstrates the importance of nanoecotoxicological studies in environmentally relevant matrices., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Tetracyclines are a large class of antimicrobials used most extensively in livestock feeding operations. A large portion of tetracyclines administered to livestock is excreted in manure and urine which is collected in waste lagoons. Subsequent land application of these wastes introduces tetracyclines into the soil environment, where they could exert selective pressure for the development of antibiotic resistance genes in bacteria. Tetracyclines form metal-complexes in natural waters, which could reduce their bioavailability for bacterial uptake. We hypothesized that many naturally-occurring organic acids could effectively compete with tetracyclines as ligands for metal cations, hence altering the bioavailability of tetracyclines to bacteria in a manner that could enhance the selective pressure. In this study, we investigated the influence of acetic acid, succinic acid, malonic acid, oxalic acid and citric acid on tetracycline uptake from water by Escherichia coli bioreporter construct containing a tetracycline resistance gene which induces the emission of green fluorescence when activated. The presence of the added organic acid ligands altered tetracycline speciation in a manner that enhanced tetracycline uptake by E. coli. Increased bacterial uptake of tetracycline and concomitant enhanced antibiotic resistance response were quantified, and shown to be positively related to the degree of organic acid ligand complexation of metal cations in the order of citric acid > oxalic acid > malonic acid > succinic acid > acetic acid. The magnitude of the bioresponse increased with increasing aqueous organic acid concentration. Apparent positive relation between intracellular tetracycline concentration and zwitterionic tetracycline species in aqueous solution indicates that (net) neutral tetracycline is the species which most readily enters E. coli cells. Understanding how naturally-occurring organic acid ligands affect tetracycline speciation in solution, and how speciation influences tetracycline uptake by bacteria, allows more accurate assessment of the selective pressure from trace levels of antibiotics in the environment on microbial communities for preserving and developing antibiotic resistance., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Copper (Cu) is known to accumulate in agricultural soils receiving urban waste products as fertilizers. We here report the use of the leucine incorporation technique to determine pollution-induced community tolerance (Leu-PICT) to Cu in a long-term agricultural field trial. A significantly increased bacterial community tolerance to Cu was observed for soils amended with organic waste fertilizers and was positively correlated with total soil Cu. However, metal speciation and whole-cell bacterial biosensor analysis demonstrated that the observed PICT responses could be explained entirely by Cu speciation and bioavailability artifacts during Leu-PICT detection. Hence, the agricultural application of urban wastes (sewage sludge or composted municipal waste) simulating more than 100 years of use did not result in sufficient accumulation of Cu to select for Cu resistance. Our findings also have implications for previously published PICT field studies and demonstrate that stringent PICT detection criteria are needed for field identification of specific toxicants., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Expression of bacterial luciferase enzyme (lux) in eukaryotic cells would provide a new bioreporter system for in vivo imaging and diagnostics technology. In spite of this, until now only a few efforts have been made to express bacterial luciferase enzyme in eukaryotic cells. We attempted to synthesize an expression construct of luxA and luxB genes from Vibrio fischeri. The luxA and luxB genes were cloned into the MCS of pTZ57R via the 5' kpnI, BamHI and BamHI, EcoRI restriction sites to generate pTZ57R/luxA and pTZ57R/luxB respectively, then newly synthesized constructs were cleaved with the same enzymes and respectively cloned into the pcDNA3.1(+) (Hyg) and pcDNA3.1(+) (Neo) expression vectors to create pcDNA3.1(+) (Hyg)/luxA and pcDNA3.1(+) (neo)/luxB. Recombinant constructs were cotransfected to the NIH3T3 cell line. Gene expression was confirmed by reverse transcription-polymerase chain reaction, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting; in addition, bioluminescence characteristics of transfected NIH3T3 cell lines were evaluated by decanal supplement. In conclusion, in the current research, separate vector systems were constructed, which are composed of bacterial luciferase genes (luxA and luxB) that accordingly have not already been reported. These results hold promise toward the potential development of an autonomous light-generating lux reporter system in eukaryotic cells., (Copyright © 2013 John Wiley & Sons, Ltd.)

The effects of C60 on mercury bioavailability and sorption were investigated at different C60 dosages, reaction times, and pH ranges using the merR::luxCDABE bioluminescent bioreporter Escherichia coli ARL1. The results demonstrated that the bioavailability of mercury (Hg(2+)) decreased with increasing C60 dosage. Approximately 30% of aqueous mercury became biologically unavailable 2h after interaction with C60 at a mass ratio of C60 to mercury as low as 0.01. However, this reduction in bioavailability plateaued at a mass ratio of C60 to mercury of 10 with a further increase in C60 concentrations resulting in only a 20% additional decrease in bioavailability. If this reduction in bioluminescence output is attributable to mercury sorption on C60, then each one log-order increase in C60 concentration resulted in a 0.86 log-order decrease in the mercury partitioning coefficient (Kd). This relationship implies the presence of high mercury-affinitive sites on C60. The length of reaction time was found to play a more important role than C60 dosage in reducing Hg(2+) bioavailability, suggesting an overall slow kinetics of the C60-Hg interactions. In addition, lowering the pH from 7.2 to 5.8 decreased mercury bioavailability due likely to the increase in mercury's association with C60. These results suggest that C60 may be useful in capturing soluble mercury and thus reducing mercury biotoxicity., (Published by Elsevier Ltd.)

Bioluminescent imaging is an emerging biomedical surveillance strategy that uses external cameras to detect in vivo light generated in small animal models of human physiology or in vitro light generated in tissue culture or tissue scaffold mimics of human anatomy. The most widely utilized of reporters is the firefly luciferase ( luc ) gene; however, it generates light only upon addition of a chemical substrate, thus only generating intermittent single time point data snapshots. To overcome this disadvantage, we have demonstrated substrate-independent bioluminescent imaging using an optimized bacterial bioluminescence ( lux ) system. The lux reporter produces bioluminescence autonomously using components found naturally within the cell, thereby allowing imaging to occur continuously and in real-time over the lifetime of the host. We have validated this technology in human cells with demonstrated chemical toxicological profiling against exotoxin exposures at signal strengths comparable to existing luc systems (~1.33 × 10 7 photons/second). As a proof-in-principle demonstration, we have engineered breast carcinoma cells to express bioluminescence for real-time screening of endocrine disrupting chemicals and validated detection of 17β-estradiol (EC 50 = ~ 10 pM). These and other applications of this new reporter technology will be discussed as potential new pathways towards improved models of target chemical bioavailability, toxicology, efficacy, and human safety.

Living whole-cell bioreporters serve as environmental biosentinels that survey their ecosystems for harmful pollutants and chemical toxicants, and in the process act as human and other higher animal proxies to pre-alert for unfavorable, damaging, or toxic conditions. Endowed with bioluminescent, fluorescent, or colorimetric signaling elements, bioreporters can provide a fast, easily measured link to chemical contaminant presence, bioavailability, and toxicity relative to a living system. Though well tested in the confines of the laboratory, real-world applications of bioreporters are limited. In this review, we will consider bioreporter technologies that have evolved from the laboratory towards true environmental applications, and discuss their merits as well as crucial advancements that still require adoption for more widespread utilization. Although the vast majority of environmental monitoring strategies rely upon bioreporters constructed from bacteria, we will also examine environmental biosensing through the use of less conventional eukaryotic-based bioreporters, whose chemical signaling capacity facilitates a more human-relevant link to toxicity and health-related consequences.

The bacterial luciferase gene cassette (lux) is unique among bioluminescent bioreporter systems due to its ability to synthesize and/or scavenge all of the substrate compounds required for its production of light. As a result, the lux system has the unique ability to autonomously produce a luminescent signal, either continuously or in response to the presence of a specific trigger, across a wide array of organismal hosts. While originally employed extensively as a bacterial bioreporter system for the detection of specific chemical signals in environmental samples, the use of lux as a bioreporter technology has continuously expanded over the last 30 years to include expression in eukaryotic cells such as Saccharomyces cerevisiae and even human cell lines as well. Under these conditions, the lux system has been developed for use as a biomedical detection tool for toxicity screening and visualization of tumors in small animal models. As the technologies for lux signal detection continue to improve, it is poised to become one of the first fully implantable detection systems for intra-organismal optical detection through direct marriage to an implantable photon-detecting digital chip. This review presents the basic biochemical background that allows the lux system to continuously autobioluminesce and highlights the important milestones in the use of lux-based bioreporters as they have evolved from chemical detection platforms in prokaryotic bacteria to rodent-based tumorigenesis study targets. In addition, the future of lux imaging using integrated circuit microluminometry to image directly within a living host in real-time will be introduced and its role in the development of dose/response therapeutic systems will be highlighted.