Bacterial Bioreporters Research Articles

Where microbiology meets microengineering: design and applications of reporter bacteria

Bacteria have long been the targets for genetic manipulation, but more recently they have been synthetically designed to carry out specific tasks. Among the simplest of these tasks is chemical compound and toxicity detection coupled to the production of a quantifiable reporter signal. In this Review, we describe the current design of bacterial bioreporters and their use in a range of assays to measure the presence of harmful chemicals in water, air, soil, food or biological specimens. New trends for integrating synthetic biology and microengineering into the design of bacterial bioreporter platforms are also highlighted.

Improving the sensitivity of bacterial bioreporters for heavy metals

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

Development of a Multistrain Bacterial Bioreporter Platform for the Monitoring of Hydrocarbon Contaminants in Marine Environments

Petroleum hydrocarbons are common contaminants in marine and freshwater aquatic habitats, often occurring as a result of oil spillage. Rapid and reliable on-site tools for measuring the bioavailable hydrocarbon fractions, i.e., those that are most likely to cause toxic effects or are available for biodegradation, would assist in assessing potential ecological damage and following the progress of cleanup operations. Here we examined the suitability of a set of different rapid bioassays (2-3 h) using bacteria expressing the LuxAB luciferase to measure the presence of short-chain linear alkanes, monoaromatic and polyaromatic compounds, biphenyls, and DNA-damaging agents in seawater after a laboratory-scale oil spill. Five independent spills of 20 mL of NSO-1 crude oil with 2 L of seawater (North Sea or Mediterranean Sea) were carried out in 5 L glass flasks for periods of up to 10 days. Bioassays readily detected ephemeral concentrations of short-chain alkanes and BTEX (i.e., benzene, toluene, ethylbenzene, and xylenes) in the seawater within minutes to hours after the spill, increasing to a maximum of up to 80 muM within 6-24 h, after which they decreased to low or undetectable levels. The strong decrease in short-chain alkanes and BTEX may have been due to their volatilization or biodegradation, which was supported by changes in the microbial community composition. Two- and three-ring PAHs appeared in the seawater phase after 24 h with a concentration up to 1 muM naphthalene equivalents and remained above 0.5 muM for the duration of the experiment. DNA-damage-sensitive bioreporters did not produce any signal with the oil-spilled aqueous-phase samples, whereas bioassays for (hydroxy)biphenyls showed occasional responses. Chemical analysis for alkanes and PAHs in contaminated seawater samples supported the bioassay data, but did not show the typical ephemeral peaks observed with the bioassays. We conclude that bacterium-based bioassays can be a suitable alternative for rapid on-site quantitative measurement of hydrocarbons in seawater.

Effect of two types of biosurfactants on phenanthrene availability to the bacterial bioreporter Burkholderia sartisoli strain RP037

Biosurfactants are tensio-active agents that have often been proposed as a means to enhance the aqueous solubility of hydrophobic organic contaminants, such as polycyclic aromatic hydrocarbons (PAHs). Biosurfactant-producing bacteria such as those belonging to the genus Pseudomonas might therefore enhance PAH availability to PAH-degrading bacteria. We tested the effects of two types of biosurfactants produced by Pseudomonas sp., cyclic lipopeptides and rhamnolipids, on phenanthrene bioavailability. Bioavailability was judged from growth rates on phenanthrene and from specific induction of a phenanthrene-responsive GFP-reporter in Burkholderia sartisoli strain RP037. Co-culturing of strain RP037 with the lipopeptide-producing bacterium Pseudomonas putida strain PCL1445 enhanced GFP expression compared to a single culture, but this effect was not significantly different when strain RP037 was co-cultivated with a non-lipopeptide-producing mutant of P. putida. The addition of partially purified supernatant extracts from the P. putida lipopeptide producer equally did not unequivocally enhance phenanthrene bioavailability to strain RP037 compared to controls. In contrast, a 0.1% rhamnolipid solution strongly augmented RP037 growth rates on phenanthrene and led to a significantly larger proportion of cells in culture with high GFP expression. Our data therefore suggest that biosurfactant effects may be strongly dependent on the strain and type of biosurfactant.

Biochip with E. coli bacteria for detection of arsenic in drinking water

Arsenic and other pollutants are often present in trace amounts in drinking water in a number of countries. In aqueous solutions the sensitivity of arsenic-responsive bacterial bioreporters is far better than the performance of chemical field test kits. However, biological detection currently requires extensive handling and expensive fluorescence microscopy. We fabricated and tested microfluidic chips with fluorescent (GFP) E. coli bacteria that respond to arsenic. Measurement results of fluorescence intensity as function of arsenic concentration and exposure time are promising, showing a clear response to concentrations of 50 μg/l of arsenite.

Novel Use of a Whole Cell E. coli Bioreporter as a Urinary Exposure Biomarker

Bacterial bioreporters have substantial potential for contaminant assessment but their real world application is currently impaired by a lack of sensitivity. Here, we exploit the bioconcentration of chemicals in the urine of animals to facilitate pollutant detection. The shore crab Carcinus maenas was exposed to the organic contaminant 2-hydroxybiphenyl, and urine was screened using an Escherichia coli-based luciferase gene (luxAB) reporter assay specific to this compound. Bioassay measurements differentiated between the original contaminant and its metabolites, quantifying bioconcentration factors of up to one hundred-fold in crab urine. Our results reveal the substantial potential of using bacterial bioreporter assays in real-time monitoring of biological matricesto determine exposure histories, with wide ranging potential for the in situ measurement of xenobiotics in risk assessments and epidemiology.

Toxicant Identification by a Luminescent Bacterial Bioreporter Panel: Application of Pattern Classification Algorithms

Genetically engineered microorganisms, tailored to respond by a dose-dependent signal to the presence of toxic chemicals, are a potentially useful tool for environmental monitoring. One manifestation of this approach is based on a panel of luminescent bacterial bioreporters, harboring fusions of the luxCDABE operon to various stress-responsive gene promoters. Such sensors can report by a dose-dependent luminescent signal on the stress sensed by the cells and thus on the presence of toxic compound(s), but they lack the ability to identify the chemicals involved. Here, we demonstrate how the use of a panel of such sensors might offer a solution to this drawback. Five selected Escherichia coli reporter strains harboring fusions of selected gene promoters (grpE, nhoA, oraA, lacZ, and mipA) to luxCDABE were exposed to five model toxicants and to a toxicant-free control in a 40-repetition format. Each of the six treatments activated different promoters to different extents, producing its own unique fingerprint. Two machine learning schemes were challenged with the obtained data set: Bayesian decision theory and the nonparametric nearest-neighbor technique. The Bayesian classifiers performed better and were able to identify the sample's contents within 30 min with an error rate estimate that did not exceed 3% at a 95% confidence level and with zero false negatives. Performance in tap water and wastewater samples was similar. Given the coming of age of whole-cell sensing devices, pattern classification algorithms such as the ones described here offer a step toward the incorporation of reporter cells into future biosensor formats, including whole-cell arrays.

Artificial Neural Network Study of Whole-Cell Bacterial Bioreporter Response Determined Using Fluorescence Flow Cytometry

Genetically engineered bioreporters are an excellent complement to traditional methods of chemical analysis. The application of fluorescence flow cytometry to detection of bioreporter response enables rapid and efficient characterization of bacterial bioreporter population response on a single-cell basis. In the present study, intrapopulation response variability was used to obtain higher analytical sensitivity and precision. We have analyzed flow cytometric data for an arsenic-sensitive bacterial bioreporter using an artificial neural network-based adaptive clustering approach (a single-layer perceptron model). Results for this approach are far superior to other methods that we have applied to this fluorescent bioreporter (e.g., the arsenic detection limit is 0.01 microM, substantially lower than for other detection methods/algorithms). The approach is highly efficient computationally and can be implemented on a real-time basis, thus having potential for future development of high-throughput screening applications.

Variability in the in situ bioavailability of Fe to bacterioplankton communities in the eastern subtropical Pacific Ocean

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 46:239-251 (2007) - doi:10.3354/ame046239 Variability in the in situ bioavailability of Fe to bacterioplankton communities in the eastern subtropical Pacific Ocean Cécile E. Mioni1,2, J. Dean Pakulski3, Leo Poorvin1, Amy Baldwin3, Michael R. Twiss4, Wade H. Jeffrey3, Steven W. Wilhelm1,* 1Department of Microbiology, and 2Center for Environmental Biotechnology, 1414 West Cumberland, The University of Tennessee, Knoxville, Tennessee 37996, USA 3Center for Environmental Diagnostics and Bioremediation, University of West Florida, 11000 University Parkway, Pensacola, Florida 32514, USA 4Department of Biology, Clarkson University, Potsdam, New York 13699, USA *Corresponding author. Email: wilhelm@utk.edu ABSTRACT: It is now established that iron (Fe) availability controls phytoplankton productivity and community structure in ca. 50% of the Pacific Ocean’s surface waters and that heterotrophic bacterioplankton may also be either directly or indirectly Fe-limited. Proxy indicators of Fe-stress are available for the phototrophic community (e.g. ferredoxin/flavodoxin ratios) but are lacking for the heterotrophic bacterioplankton. While current analytical tools provide valuable information with regard to micronutrient chemistry and speciation, they do not provide insight into the relative bioavailability of different Fe sources. We present the results of a field trial in an oceanic system of a tool that allows for the assessment of Fe bioavailability in natural systems: the Fe-responsive bioluminescent heterotrophic bacterial reporter. Fe bioavailability was monitored with this tool at the scale of the Eastern Pacific Basin during the mature phase of the El Niño event of 2002. The results demonstrate significant spatial variance, highlighted by regions of decreased Fe availability at equatorial stations along the transect. Using this tool in combination with radiotracer studies of bacterial growth and community Fe uptake, we provide insight into system Fe chemistry and the status of the heterotrophic bacterial community. Our results indicate that different environments with similar concentrations of total Fe can demonstrate different Fe bioavailabilities. Moreover, the small particulate size fraction (0.2 to 0.8 µm) appears to buffer artificially induced variations in Fe bioavailability, implying that studies of Fe bioavailability need to be extended beyond those in the dissolved (<0.2 µm) size class. KEY WORDS: Luminescent bacterial bioreporter · Iron bioavailability · HNLC · El Niño · Pacific Ocean Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 46, No. 3. Online publication date: March 13, 2007 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2007 Inter-Research.

Prokaryotic Gene Profiling Assays to Detect Sediment Toxicity: Evaluating the Ecotoxicological Relevance of a Cell-Based Assay

Despite their complexity, ecotoxicological measurements using higher level responses remain a major tool in the assessment of ecosystem integrity. Nevertheless, the past decade saw an increasing number of cell based testing systems have found widespread application in ecotoxicology. One such test is bacterial bioreporters carrying a stress sensitive promoter fused to an easily detectable reporter gene. In the presence of a specific toxic stress,the expression cassette is switched on and the reporter gene is produced. This study evaluated the use of 14 different Escherichia coli bioreporter strains sensitive to different types of toxicity in the assessment of the ecological status of a small river basin in Flanders, Belgium. The river is fed at two geographically separate locations by two distinct and well-described effluents, one from a household sewage treatment facility and one from the discharge of the wastewater treatment facility of a large chemical plant. The results of the bacterial gene profiling assay were related to active biomonitoring results obtained through higher-level responses of caged Dreissena polymorpha, Chironomus riparius, and Cyprinus carpio deployed at the locations sampled for the bacterial assay. The results of the gene induction assay and the active biomonitoring data correlated well and corresponded to the flow dilution data, which is used here as a surrogate forthe chemical pollution gradient present in the river basin.

Genetically engineered naphthalene sensitive whole‐cell bacterial bioreporters for the study of bioavailability

Naphthalene is an important contaminant, and the primary ingredient in coal tar. It is poorly soluble, and a good model for other hydrophobic organic contaminants. The objective of this research was to study the role of complex matrices in mitigating bioavailable naphthalene. Biosensor bacteria were exposed to samples of sediments, chosen by the E.U. Abacus study on bioavailability for having various chemical properties, and controls all of which were subjected to various levels of naphthalene. Results were obtained using epi-fluorescence microscopy and computer analysis. The data obtained showed a good correlation between the projected results as determined by chemical analysis, and expectations based on the chemical nature of the matrices. This project demonstrates the ability of these bioreporters to be used in a quantitative manner to study bioavailability of naphthalene in a vast array of complex matrix samples, and could be extended to samples exhibiting active biological dynamics impacting bioavailability.

Analytics with engineered bacterial bioreporter strains and systems

A dynamic atmosphere generator with a naphthalene emission source has been constructed and used for the development and evaluation of a bioluminescence sensor based on the bacteria Pseudomonas fluorescens HK44 immobilized in 2% agar gel (107 cell mL−1) placed in sampling tubes. A steady naphthalene emission rate (around 7.3 nmol min−1 at 27 °C and 7.4 mL min−1 of purified air) was obtained by covering the diffusion unit containing solid naphthalene with a PTFE filter membrane. The time elapsed from gelation of the agar matrix to analyte exposure (“maturation time”) was found relevant for the bioluminescence assays, being most favorable between 1.5 and 3 h. The maximum light emission, observed after 80 min, is dependent on the analyte concentration and the exposure time (evaluated between 5 and 20 min), but not on the flow rate of naphthalene in the sampling tube, over the range of 1.8–7.4 nmol min−1. A good linear response was obtained between 50 and 260 nmol L−1 with a limit of detection estimated in 20 nmol L−1 far below the recommended threshold limit value for naphthalene in air.

Tracking changes in bioavailable Fe within high‐nitrate low‐chlorophyll oceanic waters: A first estimate using a heterotrophic bacterial bioreporter

It is conventional knowledge that heterotrophic bacteria play a key role in the biogeochemical cycling of oceanic carbon. However, only recently has their role in marine iron (Fe) biogeochemical cycles been examined. Research during this past decade has demonstrated an inextricable link between Fe chemistry and the biota, as &gt;99% of Fe in marine systems is complexed to organic chelates of unknown but obviously biotic origin. Here we present a novel approach to assess and compare Fe bioavailability in low Fe HNLC waters using a bioluminescent bacterial reporter that quantitatively responds to the concentration of bioavailable Fe by producing light. Originally tested in freshwater environments, this study presents the first characterization of this halotolerant reporter organism in a defined seawater medium and then subsequently in marine surface waters. Laboratory characterizations demonstrate that this reporter displays a dose‐dependent response to Fe availability in our defined marine medium. Field tests were performed during the 10‐day mesoscale FeCycle experiment (February 2003) in the Pacific sub‐Antarctic high‐nitrate low‐chlorophyll region. Data from both biogeochemical measures and bioreporter assays are provided which describe how the bioreporter detected changes in Fe bioavailability that occurred during a natural shift in ambient dissolved Fe concentrations (∼40 pM). Our data explore the use of heterotrophic bioluminescent reporters as a comparable tool for marine ecosystems and demonstrate the potential utility of this tool in elucidating the relationship between Fe bioavailability and Fe chemistry in complex marine systems.

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

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

Selectivity of whole cell optical biosensor with immobilized bioreporter Pseudomonas fluorescens HK44

Pseudomonas fluorescens HK44, a whole-cell bacterial bioreporter that responds to naphthalene and salicylate exposure by production of visible light, was immobilized to thick (1 mm) silica films prepared from pre-polymerized tetramethoxysilane. Bioluminescence was first detected 50 min after induction; maximum was reached after 4.5 h and followed saturation dependence on the inducer concentration. Higher cell concentration resulted in higher bioluminescence and in lower mechanical stability. Minimal concentration inducing detectable bioluminescence was 1.2 and 0.5 mg/l for naphthalene and salicylate, respectively. From 32 possible inducers of bioluminescence (11 substituted naphthalenes, 7 substituted salicylic acids, 6 o-substituted benzoic acids, 7 substituted phenols) only salicylaldehyde, 2-aminobenzoic acid, 4-methylsalicylic acid and 4-chlorosalicylic acid induced bioluminescence, however the minimal inducing concentration was two to three orders of magnitude higher compared to salicylate.

Illuminating the detection chain of bacterial bioreporters.

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

The Use of a Genetically Engineered Pseudomonas Species (Shk1) as a Bioluminescent Reporter for Heavy Metal Toxicity Screening in Wastewater Treatment Plant Influent

Heavy metals are known to be inhibitory and toxic to the activated-sludge microbial community in biological wastewater treatment plants. Toxicity screening of aqueous mixtures of these heavy metal ions in plant influent could use both chemical and biological methods. As a biological method, luminescent bacterial bioreporters offer the advantages of a simple test procedure and rapid response. Current biologically based methods for screening aqueous streams for toxicity are labor-intensive, inaccurate, or difficult to use in continuous monitoring applications. In the present study, a system was developed that is simple and easily automated. This system is based on the bacterium Shk1, a genetically engineered bioluminescent Pseudomonad whose parent strain was originally isolated from activated sludge. Compared with other bioluminescence-based systems (specifically, the Microtox assay), the system of the present study more accurately reflects the effects of the toxicity of common metal ions on activated-sludge respirometry without being overly sensitive to typical constituents of wastewater. The use of Shk1 as a bioluminescent reporter for heavy metal toxicity testing for the application of wastewater treatment influent toxicity screening is presented in this study.

Appetite of an epiphyte: Quantitative monitoring of bacterial sugar consumption in the phyllosphere

We report here the construction, characterization, and application of a bacterial bioreporter for fructose and sucrose that was designed to monitor the availability of these sugars to microbial colonizers of the phyllosphere. Plasmid pP(fruB)-gfp[AAV] carries the Escherichia coli fruB promoter upstream from the gfp[AAV] allele that codes for an unstable variant of green fluorescent protein (GFP). In Erwinia herbicola, this plasmid brings about the accumulation of GFP fluorescence in response to both fructose and sucrose. Cells of E. herbicola (pP(fruB)-gfp[AAV]) were sprayed onto bean plants, recovered from leaves at various time intervals after inoculation, and analyzed individually for GFP content by quantitative analysis of digital microscope images. We observed a positive correlation between single-cell GFP accumulation and ribosomal content as determined by fluorescence in situ hybridization, indicating that foliar growth of E. herbicola occurred at the expense of fructose and/or sucrose. One hour after inoculation, nearly all bioreporter cells appeared to be actively engaged in fructose consumption. This fraction dropped to approximately 11% after 7 h and to approximately 1% a day after inoculation. This pattern suggests a highly heterogeneous availability of fructose to individual E. herbicola cells as they colonize the phyllosphere. We estimated that individual cells were exposed to local initial fructose abundances ranging from less than 0.15 pg fructose to more than 4.6 pg.