Whole-cell Bacterial Biosensors Research Articles

Whole-cell bacterial biosensor applied to identify the presence of Thaumatotibia leucotreta larva in citrus fruits by volatile sensing

The false codling moth (FCM), Thaumatotibia leucotreta (Meyrick), is a major pest of citrus fruits and other plants and is defined as a quarantine pest in several countries. Supervision and visual monitoring are crucial for the detection of eggs and larvae. There is a growing need to implement alternative pest control strategies and early detection methodologies in the packing house before shipping. Biosensors may provide a better-suited solution, as they are portable, do not require expensive equipment, and may be used with simple methodologies. In this study, a whole-cell bacterial biosensor was developed to identify the presence of Thaumatotibia leucotreta larvae in citrus fruits. The volatile organic compounds (VOCs) released by the larvae were identified (n = 11), which were not previously tested. The change in the VOCs composition in infected and uninfected mandarin (Citrus reticulata) was examined using gas chromatography-mass spectrometry (GC-MS) and the bioluminescent bacterial panel. During this work, two fixation methods (alginate beads and tablets) were tested for the bioluminescent bacteria cells, and tablets were preferred due to their higher uniformity and consistency of the light signal from the cells. Furthermore, the response of the bacteria to the changes in the concentration of VOCs as a result of a change in the amount of the larvae was examined (larvae n = 3, 6, or 9). In addition, in order to demonstrate the ability of the whole-cell biosensor to detect the presence of larva within the fruit, infected and uninfected mandarins were tested on the 14th day of larval hatching. The results showed that the bioluminescent bacteria could differentiate between mandarins with and without larva. The differences in the light signals (Induction Factor (IF)) between the mandarin fruits can be seen in the results with the larva (cytotoxicity IF = 3.046, genotoxicity IF = 2.643, and quorum sensing IF = 1.954) and without (cytotoxicity IF = 1.589, genotoxicity IF = 1.149 and quorum sensing IF = 1.205). The findings of this study make it possible to further develop a prototype using bioluminescent bacteria in order to detect pests of citrus fruits. The early detection of infected fruits will allow their filtering from the crop and therefore reduce the damage to additional fruits. Even one larva can invalidate the whole stock and the plot for future export.

Bioaccessible PAH influence on distribution of antibiotic resistance genes and soil toxicity of different types of land use.

For a better understanding of the dissemination of antibiotic resistance genes (ARGs) in natural microbial communities, it is necessary to study the factors influencing it. There are not enough studies showing the connection of some pollutants with the dissemination of ARGs and especially few works on the effect of polycyclic aromatic compounds (PAHs) on the spread of resistance in microbiocenosis. In this respect, the aim of the study was to determine the effect of bioaccessible PAHs on soil resistome. The toxicity and the content of bioaccessible PAHs and ARGs were studied in 64 samples of soils of different types of land use in the Rostov Region of Russia. In most soils, a close positive correlation was demonstrated between different ARGs and bioaccessible PAHs with different content of rings in the structure. Six of the seven studied ARGs correlated with the content of 2-, 3-, 4-, 5- or 6-ring PAHs. The greatest number of close correlations was found between the content of PAHs and ARGs in the soils of protected areas, for agricultural purposes, and in soils of hospitals. The diverse composition of microbial communities in these soils might greatly facilitate this process. A close correlation between various toxic effects identified with a battery of whole-cell bacterial biosensors and bioaccessible PAHs of various compositions was established. This correlation showed possible mechanisms of PAHs' influence on microorganisms (DNA damage, oxidative stress, etc.), which led to a significant increase in horizontal gene transfer and spread of some ARGs in soil microbial communities. All this information, taken together, suggests that bioaccessible PAHs can enhance the spread of antibiotic resistance genes.

Whole-cell bacterial biosensor with the capability to detect red palm weevil, Rhynchophorus ferrugineus, in date palm trees, Phoenix dactylifera: a proof of concept study

The red palm weevil (RPW), Rhynchophorus ferrugineus, is considered a severe pest of palms. Usually, the early stages of infection are without visible signs. An attractive early sensing approach of non-visible infections is based on volatile organic compounds (VOCs). In this study, a whole-cell bacterial biosensor was used for the identification of RPW in date palm (Phoenix dactylifera). The cells are genetically modified to produce light in the presence of general stresses. The bioluminescent bacterial panel is based on three genetically engineered Escherichia coli strains that are sensitive to cytotoxicity (TV1061), genotoxicity (DPD2794), or quorum-sensing (K802NR). The bioluminescent bacterial panel detects the presence of VOCs and a change in the light signal is then generated, reflecting the health status of the date palm tree. The bioreporter bacteria cells are immobilized in calcium alginate tablets and placed in a sealed jar without direct contact with the tested sample, thereby exposing them only to the VOCs in the surrounding air. The immobilized bacteria cells were exposed to the air near infected by RPW or uninfected sugar canes, date palm tree pieces, and on date palm trees. Commercial plate reader was used for signal measurement. The findings show that quorum-sensing was induced by all the tested samples of infected sugar canes, date palm tree pieces, and date palm trees. While, cytotoxicity was induced only by infected date palm tree pieces, and genotoxicity was induced only by infected date palm trees. The bacterial monitoring results enable the identification of specific signatures that will allow a quick and accurate diagnosis.

Whole-cell bacterial biosensor for volatile detection from Pectobacterium-infected potatoes enables early identification of potato tuber soft rot disease

Half of the harvested food is lost due to rots caused by microorganisms. Plants emit various volatile organic compounds (VOCs) into their surrounding environment, and the VOC profiles of healthy crops are altered upon infection. In this study, a whole-cell bacterial biosensor was used for the early identification of potato tuber soft rot disease caused by the pectinolytic bacteria Pectobacterium in potato tubers. The detection is based on monitoring the luminescent responses of the bacteria panel to changes in the VOC profile following inoculation. First, gas chromatography-mass spectrometry (GC-MS) was used to specify the differences between the VOC patterns of the inoculated and non-inoculated potato tubers during early infection. Five VOCs were identified, 1-octanol, phenylethyl alcohol, 2-ethyl hexanol, nonanal, and 1-octen-3-ol. Then, the infection was detected by the bioreporter bacterial panel, firstly measured in a 96-well plate in solution, and then also tested in potato plugs and validated in whole tubers. Examination of the bacterial panel responses showed an extensive cytotoxic effect over the testing period, as seen by the elevated induction factor (IF) values in the bacterial strain TV1061 after exposure to both potato plugs and whole tubers. Moreover, quorum sensing influences were also observed by the elevated IF values in the bacterial strain K802NR. The developed whole-cell biosensor system based on bacterial detection will allow more efficient crop management during postharvest, storage, and transport of crops, to reduce food losses.

Method for acrylic acid monomer detection with recombinant biosensor cells for enhanced plastic degradation monitoring from water environments

Plastic debris degrades in the water environments due to various factors such as mechanical stress. Small-sized degradation products, including plastic monomers, are currently monitored using equipment which might be unsuitable for screening. Here, we developed a recombinant whole-cell bacterial biosensor, which could be used for this type of monitoring. The Escherichia coli pBAV1K-ACU-lucFF cells contain a luciferase-based reporter system under the control of acrylic acid specific promoter. The biosensor cells were used to detect acrylic acid monomers from both sterile water and spiked lake water samples, indicating usability with environmental samples. Furthermore, poly(acrylic acid) was incubated in salt water, and the biosensor cells could identify acrylic acid monomers originating from it. Thus, the cells could be used to observe similar processes in the environment. The results show that the bacterial biosensors could complement the current research methods of plastic monomer monitoring in water environments with a potential for higher throughputs.

The Integration of Whole-Cell Biosensors for the Field-Ready Electrochemical Detection of Arsenic

Rapid on-site measurements of arsenic (As) are essential for the timely remediation of As-contaminated groundwater for both municipal and emergency response applications. Current field tests suffer from either complicated end-user instructions or a lack of accuracy and specificity. The system presented here combines a whole-cell bacterial biosensor with an electrochemical measurement that provides enhanced accuracy and signal intensity compared to traditional bacterial-detection approaches. When integrated within a customized hardware system, this whole-cell sensor demonstrated excellent specificity and sensitivity. This fast, sensitive, and easy-to-use approach is a viable alternative for on-site arsenic testing.

A Whole-Cell Bacterial Biosensor for Blood Markers Detection in Urine

The early detection of blood in urine (hematuria) can play a crucial role in the treatment of serious diseases (e.g., infections, kidney disease, schistosomiasis, and cancer). Therefore, the development of low-cost portable biosensors for blood detection in urine has become necessary. Here, we designed an ultrasensitive whole-cell bacterial biosensor interfaced with an optoelectronic measurement module for heme detection in urine. Heme is a red blood cells (RBCs) component that is liberated from lysed cells. The bacterial biosensor includes Escherichia coli cells carrying a heme-sensitive synthetic promoter integrated with a luciferase reporter (luxCDABE) from Photorhabdus luminescens. To improve the bacterial biosensor performance, we re-engineered the genetic structure of luxCDABE operon by splitting it into two parts (luxCDE and luxAB). The luxCDE genes were regulated by the heme-sensitive promoter, and the luxAB genes were regulated by either constitutive or inducible promoters. We examined the genetic circuit's performance in synthetic urine diluent supplied with heme and in human urine supplied with lysed blood. Finally, we interfaced the bacterial biosensor with a light detection setup based on a commercial optical measurement single-photon avalanche photodiode (SPAD). The whole-cell biosensor was tested in human urine with lysed blood, demonstrating a low-cost, portable, and easy-to-use hematuria detection with an ON-to-OFF ratio of 6.5-fold for blood levels from 5 × 104 to 5 × 105 RBC per mL of human urine.

A panel of bioluminescent whole-cell bacterial biosensors for the screening for new antibacterial substances from natural extracts

Whole-cell bacterial biosensors can be applied for the screening of antibacterial properties of extracts. We constructed a biosensor panel consisting of four different bacterial biosensor strains: Escherichia, Staphylococcus, Acinetobacter and Pseudomonas for expanded screening potential. The functionality of the panel was first evaluated with known antibacterial compounds: ethanol, naphthoquinones (juglone, lawsone, plumbagin) and a flavonoid (quercetin). Natural extracts comprise a vast source of potential new antibacterials for diverse functional purposes. To demonstrate the utilization of the panel for screening of a demanding sample material, round-leaved sundew (Drosera rotundifolia) extracts were used as an example. Differences between field- and laboratory originating sundew extracts could be detected. This demonstrates the efficiency of the developed biosensor panel in the rapid screening of the antibacterial properties of plant extracts.

Development of whole-cell and cell-free biosensors for the detection and differentiation of organic and inorganic forms of copper.

The modern world has seen exposure of bacterial communities to toxic metals at selective levels. This manifests itself both intentionally, through medicines and un-intentionally through waste streams. There is growing concern that selective exposure to metals may be linked to microbial resistance to antibiotics. For a microbe to become resistant to a specific metal it must first come in contact with it. The transition metal copper has the ability to enter bacterial cells without need for a copper specific uptake mechanism. Copper is commonly used as an antimicrobial in the healthcare industry, consumer products and as a growth promoter of livestock in the agricultural sector. Here we report a study into the uptake of different organic and inorganic sources of copper. A whole-cell bacterial biosensor was developed to quantify the specific uptake of copper from various sources. Furthermore, a cell-free sensor was utilized to investigate the response to copper sources when uptake is eliminated as a factor. The data within suggest inorganic copper to have greatly reduced uptake compared to organic sources and that there is significant difference between copper oxides, Cu2O and CuO.

Immobilization of fluorescent bacterial bioreporter for arsenic detection.

Whole-cell bacterial biosensors hold great promise as a practical complementary approach for in-field detection of arsenic. Although there are various bacterial bioreporter systems for arsenic detection, fewer studies reported the immobilization of arsenic bioreporters. This study aimed at determining immobilization of specific bacterial bioreporter in agar and alginate biopolymers to measure level of arsenite and/or arsenate. To achieve sensitive detection, immobilization parameters of polymer concentration and cell density were evaluated. Moreover, by changing the culture medium, immobilized bioreporter cells in minimal medium can detect arsenite while they can detect both arsenite and arsenate in phosphate-limited minimal medium. When optimal parameters were applied, agar and alginate immobilized bioreporter systems can detect arsenite and arsenate concentrations of 10μg/l and 200μg/l within 5h and 2h, respectively. The results showed that the immobilized bacterial bioreporter systems are able to determine the concentrations of the two abundant species of arsenic; arsenite and arsenate, as opposed to other studies which reported only arsenite detection. This is the first study describe agar hydrogel and alginate bead immobilization of fluorescent arsenic bacterial bioreporter that can detect both arsenite and arsenate at the safe drinking water limit. Thus, this study will enable further steps to be taken towards developing sensitive and selective portable devices to assess environmental arsenic contamination and prevent acute arsenic toxicity.

Detecting Changes in the Caenorhabditis elegans Intestinal Environment Using an Engineered Bacterial Biosensor.

Caenorhabditis elegans has become a key model organism within biology. In particular, the transparent gut, rapid growing time, and ability to create a defined gut microbiota make it an ideal candidate organism for understanding and engineering the host microbiota. Here we present the development of an experimental model that can be used to characterize whole-cell bacterial biosensors in vivo. A dual-plasmid sensor system responding to isopropyl β-d-1-thiogalactopyranoside was developed and fully characterized in vitro. Subsequently, we show that the sensor was capable of detecting and reporting on changes in the intestinal environment of C. elegans after introducing an exogenous inducer into the environment. The protocols presented here may be used to aid the rational design of engineered bacterial circuits, primarily for diagnostic applications. In addition, the model system may serve to reduce the use of current animal models and aid in the exploration of complex questions within general nematode and host–microbe biology.

Two plant-hosted whole-cell bacterial biosensors for detection of bioavailable Cr(VI).

Metal whole-cell biosensors (WCBs) have been reported as very useful tools to detect and quantify the presence of bioavailable fractions of certain metals in water and soil samples. In the current work, two bacterial WCBs able to report Cr(VI) presence and plants growing on Cr(VI)-enriched soil/medium were used to assess the potential transfer of this metal to organisms of higher trophic levels, and the risk of transfer to the food chain. To do it, the functionality of the WCBs within tissues of inoculated plants in contact with Cr(VI)-contaminated soil and water was studied in vitro and in a controlled greenhouse environment. One WCB was the previously described Ochrobactrum tritici pCHRGFP2 and the second, Nitrospirillum amazonense pCHRGFP2, is a newly engineered naturally-occurring endophytic microorganism. Three rice varieties (IAC 4440, BRS 6 CHUÍ, IRGA 425) and one maize variety (1060) were tested as hosts and subjected to Cr(VI) treatments (25μM), with different results obtained. Inoculation of each WCB into plants exposed to Cr(VI) showed GFP expression within plant tissues. WCBs penetrated the root tissues and later colonized the shoots and leaves. In general, a higher fluorescence signal was detected in roots, together with a higher Cr content and denser WCB colonization. Best fluorescence intensities per plant biomass of shoots were obtained for plant host IRGA 425. Therefore, by analyzing colonized tissues, both WCBs allowed the detection of Cr(VI) contamination in soils and its transfer to plants commonly used in crops for human diet.

Evaluation of pH-Dependent Metal Speciation Artifacts in Whole-Cell Bioreporter Analysis.

Whole-cell bacterial biosensors (bioreporters) are commonly applied for determination of metal toxicity and bioavailability in environmental samples. This is accomplished using a standard procedure whereby the sample is mixed with bioreporter cells suspended in a buffered medium at a fixed pH (set-point pH assay). This experimental approach can alter the sample pH. We therefore hypothesized that metal speciation artifacts compromising our ability to use bioreporters for determination of the "true" metal bioavailability in environmental samples may be introduced. Using the copper-specific bioreporter DF57-Cu15 as a model, we compared the conventional set-point pH assay to a flexible pH assay allowing for bioavailability determination at in situ sample pH. Our results demonstrate that pH-dependent metal speciation bias may occur when using the conventional set-point pH assay, and we recommend performing bioreporter measurements and calibrations at in situ sample pH. Although we only studied copper bioavailability, our results also have implications for bioreporter determination of other analytes displaying pH-dependent speciation such as other metals and some organics. We call for additional bioreporter studies of chemical speciation artifacts as this represents a problem hitherto overlooked in bioreporter literature. We thus conclude that there may be considerable scope for optimization of existing bioreporter assays for assessment of environmental pollutant bioavailability.

Development and Application of a Synthetically-Derived Lead Biosensor Construct for Use in Gram-Negative Bacteria.

The use of lead in manufacturing has decreased significantly over the last few decades. However, previous widespread use of lead-containing products and their incorrect disposal has resulted in environmental contamination. Accumulation of harmful quantities of lead pose a threat to all living organisms, through inhalation, ingestion, or direct contact, resulting in lead poisoning. This study utilized synthetic biology principles to develop plasmid-based whole-cell bacterial biosensors for detection of lead. The genetic element of the lead biosensor construct consists of pbrR, which encodes the regulatory protein, together with its divergent promoter region and a promoterless gfp. GFP expression is controlled by PbrR in response to the presence of lead. The lead biosensor genetic element was cloned onto a low-copy number broad host range plasmid, which can stably exist in a range of laboratory and environmental isolates, including Pseudomonas, Shewanella, and Enterobacter. The biosensors constructed were found to be sensitive, rapid, and specific and could, as such, serve as monitoring tools for lead-contaminated water.

Whole-cell bacterial biosensors for the detection of aromatic hydrocarbons and their chlorinated derivatives (Review)

The review summarizes the data on new directions in biosensor technologies based on whole bacterial cells. Biosensors for the monitoring of mono(poly)aromatic hydrocarbons and their chlorinated derivatives, which are constructed with genetically modified bacterial cells bearing a reporter gene fusion, are considered. The operating principle of these biosensors is based on the expression of reporter genes (luc, lux, gfp, rfp) under the control of a promoter and a regulator that specifically respond to a detected compound.

Designing a bacterial biosensor for detection of mercury in water solutions

Due to increasing advances in physical and chemical techniques for the assessment of pollutants in the environment, there is an immediate demand for a bioassay that can report both the presence of an analyte and its biological effects. In accordance with this need, there has been a fast growth in whole-cell biosensor technology. In this study we aimed to design a whole-cell bacterial biosensor to detect mercury in liquid solutions. The Pseudomonas pBS228 merR gene and its related promoter/operator was synthesized by Bioneer. The green fluorescence protein (GFP) gene was used as a reporter. GFP was cloned downstream of the merR gene. The construct, including the merR promoter, gene, and GFP, was cloned in a pUC19 vector and transferred into E. coli BL21 (DE3) bacteria. Transformed bacteria were used as whole-cell biosensors for detecting mercury. Mercury detection was monitored by means of microscopy and fluorometry techniques. Transformed BL21 (DE3) biosensors responded mainly to Hg(II), with the lowest detectable concentration being 10-8 M during a 3-h exposure induction period. Our results demonstrated that the noninfectious bacterial biosensors developed in the present study could be beneficial and enforceable in detection of mercury in contaminated water samples at concentrations as low as 10-8 M.

Biosensors for inorganic and organic arsenicals.

Arsenic is a natural environmental contaminant to which humans are routinely exposed and is strongly associated with human health problems, including cancer, cardiovascular and neurological diseases. To date, a number of biosensors for the detection of arsenic involving the coupling of biological engineering and electrochemical techniques has been developed. The properties of whole-cell bacterial or cell-free biosensors are summarized in the present review with emphasis on their sensitivity and selectivity. Their limitations and future challenges are highlighted.

Construction and evaluation of a genetic construct for specific detection and measurement of propionate by whole-cell bacteria.

Anaerobic digestion is a microbiological technology that converts biomass wastes into biogas, achieving both waste treatment and bioenergy production. Accumulation of volatile fatty acids (VFA) during acidogenesis, particularly propionate, often causes upset or failure of digesters. Early detection and monitoring of propionate concentration in digesters allow for just-in-time interventions to prevent irreversible costly process breakdown. In an attempt to develop a rapid method of measuring propionate concentration and bioavailability, we constructed a genetic construct for specific detection of bioavailable propionate. The genetic construct was constructed by transcriptional fusion of the regulatory gene (prpR) and the promoter of the prp operon (PprpB ) of Escherichia coli W3110 with the reporter gene cassette luxCDABE. When the genetic construct was carried on a plasmid and transformed into E. coli (referred to as plasmid-based biosensor), it resulted in stronger emission of luminescence than when it was inserted into the chromosome of E. coli (referred to as chromosome-based biosensor). The biosensor responded specifically to propionate. The luminescence signal increased linearly with increasing concentration of propionate from 1 to 10 mM. The utility of the biosensor was evaluated using samples collected from anaerobic digesters. Once instrumented in future studies, the whole-cell bacterial biosensor developed in this study may provide an alternative technology for real-time detection and measurement of propionate in digesters.

Evaluation of the leucine incorporation technique for detection of pollution-induced community tolerance to copper in a long-term agricultural field trial with urban waste fertilizers

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.

Synthesis, characterization and antimicrobial activity of arylhydrazones of methylene active compounds

Novel arylhydrazones of methylene active compounds – 3-(2-(1-ethoxy-1,3-dioxobutan-2-ylidene)hydrazinyl)-2-hydroxy-5-nitrobenzenesulfonic acid (1), 2-(2-(2-hydroxy-4-nitrophenyl)hydrazono)-2 H-indene-1,3-dione (2), 5-chloro-2-hydroxy-3-(2-(4,4,4-trifluoro-1,3-dioxo-1-(thiophen-2-yl)butan-2-ylidene)hydrazinyl) benzenesulfonic acid (3), 5-chloro-2-hydroxy-3-(2-(2,4,6-trioxo-tetrahydropyrimidin-5(6 H)-ylidene)-hydrazinyl)benzenesulfonic acid (4), 5-(2-(2-hydroxyphenyl)hydrazono)pyrimidine-2,4,6(1 H,3 H,5 H)-trione (5), 4-hydroxy-5-(2-(2,4,6-trioxo-tetrahydro-pyrimidin-5(6 H)-ylidene)hydrazinyl)benzene-1,3-disulfonic acid (6), 5-(2-(2-hydroxy-4-nitrophenyl)hydrazono)pyrimidine-2,4,6(1 H,3 H,5 H)-trione (7) and 5-chloro-3-(2-(4,4-dimethyl-2,6-dioxocyclohexylidene)hydrazinyl)-2-hydroxybenzenesulfonic acid (8) have been synthesized by a reaction between the corresponding methylene active compounds and aryldiazonium salts, and characterized by IR, 1 H and 13 C NMR spectroscopies and element analysis. The collected information confirms that 2, and 4 – 8 exist in DMSO solution in hydrazone form, being stabilized by an intramolecular hydrogen bond, while 1 and 3 exist in mixed enol-azo and hydrazo tautomeric forms. The antimicrobial activity of 1 – 8, as well as that of known analogs 5-(2-(4,4-dimethyl-2,6-dioxocyclohexylidene)hydrazinyl)-4-hydroxybenzene-1,3-disulfonic acid (9), 2-(2-sulfophenylhydrazo)malononitrile (10), 2-(2-carboxyphenylhydrazo) malononitrile (11), 2-(2-(2,4-dioxopentan-3-ylidene)hydrazinyl)phenylarsonic acid (12) and 5-(2-(2,4-dioxopentan-3-ylidene)hydrazinyl)-2,3-dihydrophthalazine-1,4-dione (13), have been studied. It was found that arylhydrazones of barbituric acid are more active than other methylene active compounds. A whole-cell bacterial luminescence biosensor method was used to detect the antimicrobial activity of the synthesized compounds. The results show that compounds 5 and 9 effectively inhibit the luminescence of Staphylococcus aureus at a minimum inhibitory concentration (MIC50) of 10 and 9 μg/ml, respectively. For Staphylococcus epidermidis, compound 11 showed the most potent activity with MIC50 of 17 μg/mL; for Pseudomonas aeruginosa, compounds 11 showed the most potent activity at MIC50 of 10 μg/mL. Observed results have been verified to be statistically significant using the two-tailed test.