Rachel Rosen

Sol-gel luminescence biosensors: Encapsulation of recombinant E. coli reporters in thick silicate films

A class of sensing elements based on the encapsulation of genetically engineered bioluminescent Escherichia coli(E. coli) reporter strains in sol–gel derived silicates is described. We demonstrate the concept by the immobilization of these bacterial cells in thick silicate films. Heat shock, oxidative stress, fatty acids, peroxides, and genotoxicity reporting bacteria were incorporated in the sol–gel silicates and their luminescence response was compared to that of the non-immobilized culture. All the immobilized bacteria maintained viability and luminescence activity for several months. The bacteria–silicate hybrids can be used either as disposable sensors or in multiple use sensing test-kits, and they can be also integrated in early warning devices operated in continuous flow conditions.

Fluorescence and bioluminescence reporter functions in genetically modified bacterial sensor strains

Genetically modified bacteria, engineered to generate a quantifiable signal in response to pre-determined sets of environmental conditions, may serve as combined sensing/reporting elements in whole-cell biosensors. We have compared two of the several available reporter genes in such cells: green fluorescent proteins (GFPs) (Aquorea victoria gfp) and bioluminescence (Vibrio fischeri luxCDABE) genes, fused to either SOS (recA) or heat shock (grpE) promoters. In both cases, bacterial bioluminescence allowed faster and more sensitive detection of the model toxicants; the fluorescent reporter proteins were much more stable, and following long-term exposure allowed detection at levels similar to that of the bioluminescent sensors. From the two green fluorescent proteins tested, enhanced GFP (EGFP) displayed a more rapid response and higher signal intensity than GFPuv. To combine the advantages of both reporter functions, representatives of both types were jointly encapsulated in a sol‐gel matrix and immobilized onto a glass surface, to generate a bioluminescent toxicity and a fluorescent genotoxicity sensor. The dual-function sensor detected both toxic and genotoxic model compounds with no interference from the co-immobilized member. # 2003 Elsevier Science B.V. All rights reserved.

Antibody-based immobilization of bioluminescent bacterial sensor cells.

Whole-cell luminescent bioreporter sensors based on immobilized recombinant Escherichia coli are described and evaluated. The sensors were prepared by glutaraldehyde-anchoring of nonspecific anti-E. coli antibodies on aminosylilated gold or silica glass surfaces with subsequent attachment of the probe bacteria. We demonstrate the generality of the concept by attachment of several E. coli strains that express luciferase in response to different physiological stress conditions including heat shock, DNA damage (SOS), fatty acid availability, peroxide and oxidative stress. The sensors can be used either as single- or multiple-use disposable sensing elements or for continuous operation. We show compatibility with optical fiber technology. Storage stability of the sensors exceeded 5 months with no measurable deterioration of the signal. Repeatability on exposure in successive days was <15%, as was sensor to sensor reproducibility. Sensitivity and detection limits of the immobilized cells were comparable to that of non-immobilized bacteria.

Characterization of Arabidopsis NEET reveals an ancient role for NEET proteins in iron metabolism.

This work describes biochemical, biophysical, structural, and genetic analyses of an Arabidopsis homolog of mammalian NEET proteins, which are involved in a wide range of cellular processes. It finds that At-NEET plays a key role in plant development, senescence, reactive oxygen species homeostasis, and iron metabolism. The NEET family is a newly discovered group of proteins involved in a diverse array of biological processes, including autophagy, apoptosis, aging, diabetes, and reactive oxygen homeostasis. They form a novel structure, the NEET fold, in which two protomers intertwine to form a two-domain motif, a cap, and a unique redox-active labile 2Fe-2S cluster binding domain. To accelerate the functional study of NEET proteins, as well as to examine whether they have an evolutionarily conserved role, we identified and characterized a plant NEET protein. Here, we show that the Arabidopsis thaliana At5g51720 protein (At-NEET) displays biochemical, structural, and biophysical characteristics of a NEET protein. Phenotypic characterization of At-NEET revealed a key role for this protein in plant development, senescence, reactive oxygen homeostasis, and Fe metabolism. A role in Fe metabolism was further supported by biochemical and cell biology studies of At-NEET in plant and mammalian cells, as well as mutational analysis of its cluster binding domain. Our findings support the hypothesis that NEET proteins have an ancient role in cells associated with Fe metabolism.

Escherichia coli bioreporters for the detection of 2,4-dinitrotoluene and 2,4,6-trinitrotoluene

The primary explosive found in most land mines, 2,4,6-trinitrotoluene (2,4,6-TNT), is often accompanied by 2,4-dinitrotoluene (2,4-DNT) and 1,3-dinitrobenzene (1,3-DNB) impurities. The latter two compounds, being more volatile, have been reported to slowly leak through land mine covers and permeate the soil under which they are located, thus serving as potential indicators for buried land mines. We report on the construction of genetically engineered Escherichia coli bioreporter strains for the detection of these compounds, based on a genetic fusion between two gene promoters, yqjF and ybiJ, to either the green fluorescent protein gene GFPmut2 or to Photorhabdus luminescens bioluminescence luxCDABE genes. These two gene promoters were identified by exposing to 2,4-DNT a comprehensive library of about 2,000 E. coli reporter strains, each harboring a different E. coli gene promoter controlling a fluorescent protein reporter gene. Both reporter strains detected 2,4-DNT in an aqueous solution as well as in vapor form or when buried in soil. Performance of the yqjF-based sensor was significantly improved in terms of detection threshold, response time, and signal intensity, following two rounds of random mutagenesis in the promoter region. Both yqjF-based and ybiJ-based reporters were also induced by 2,4,6-TNT and 1,3-DNB. It was further demonstrated that both 2,4,6-TNT and 2,4-DNT are metabolized by E. coli and that the actual induction of both yqjF and ybiJ is caused by yet unidentified degradation products. This is the first demonstration of an E. coli whole-cell sensor strain for 2,4-DNT and 2,4,6-TNT, constructed using its own endogenous sensing elements.

Microbial sensors of ultraviolet radiation based on recA'::lux fusions.

Escherichia coli strains containing plasmid-borne fusions of the recA promoter-operator region to the Vibrio fischeri lux genes were previously shown to increase their luminescence in the presence of DNA damage hazards, and thus to be useful for genotoxicant detection. The present study expands previous work by demonstrating and investigating the luminescent response of these strains to ultraviolet radiation. Several genetic variants of the basic recA’::lux design were examined, including a tolC modification of membrane efflux capacity, a chromosomal integration of the recA’::lux fusion, a different lux reporter (Photorhabdus luminescens instead of V. fischeri, allowing the assay to be run at 37°C), and a different host bacterium (Salmonella typhimurium instead of E. coli). Generally, two modifications provided the fastest responses: the use of the S. typhimurium host or the P. luminescens lux reporter. Highest sensitivity, however, was demonstrated in an E. coli strain in which a single copy of the V. fischeri lux fusion was integrated into the bacterial chromosome.

Towards toxicity detection using a lab-on-chip based on the integration of MOEMS and whole-cell sensors

A lab-on-chip consisting of a unique integration of whole-cell sensors, a MOEMS (Micro-Opto-Electro-Mechanical-System) modulator, and solid-state photo-detectors was implemented for the first time. Whole-cell sensors were genetically engineered to express a bioluminescent reporter (lux) as a function of the lac promoter. The MOEMS modulator was designed to overcome the inherent low frequency noise of solid-state photo-detectors by means of a previously reported modulation technique, named IHOS (Integrated Heterodyne Optical System). The bio-reporter signals were modulated prior to photo-detection, increasing the SNR of solid-state photo-detectors at least by three orders of magnitude. Experiments were performed using isopropyl-beta-d-thiogalactopyranoside (IPTG) as a preliminary step towards testing environmental toxicity. The inducer was used to trigger the expression response of the whole-cell sensors testing the sensitivity of the lab-on-chip. Low intensity bio-reporter optical signals were measured after the whole-cell sensors were exposed to IPTG concentrations of 0.1, 0.05, and 0.02mM. The experimental results reveal the potential of this technology for future implementation as an inexpensive massive method for rapid environmental toxicity detection.

CdSe quantum dots induce superoxide stress in engineered biosensor bacteria

A panel of genetically-engineered Escherichia coli biosensor bacteria, responsive to chemical stressors inducing heat shock, DNA damage, oxidative stress (peroxide- and superoxide-type) and fatty acid metabolism interference, was used to characterize the chemical effect of CdSe quantum dot exposure. Results implicate a primary mode of superoxide-type stress toxicity that is dependent upon quantum dot dose, size, and chain length of capping agent, and are consistent with results from other reports employing mammalian cell lines. Imaging studies confirm association of quantum dots with lipophilic regions of the biosensor bacteria (i.e., inner/outer membranes, cell wall, periplasmic space), and demonstrate intracellular alteration of the quantum dot lattice. Corroboratory experiments with superoxide stress mutants and antioxidant amendments confirm oxidative stress while also implicating destabilization of quantum dots.

Whole-cell biodetection of halogenated organic acids

A whole-cell bacterial sensor system for short-chain halo-organic acids was constructed, using 2-chloropropionic acid (2-CPA) as a model pollutant. An Escherichia coli host was transformed with a moderate-copy plasmid containing a fusion of two foreign genetic elements: (a) a promoter-containing segment of the Pseudomonas DL-DEX (DL-2-haloacid dehalogenase) encoding gene and (b) bioluminescence (luxCDABE) genes of Photorhabdus luminescens. The resulting construct, named MT1, responded to the presence of 2-CPA by dose-dependent light emission, in a highly specific albeit a very insensitive manner. Thus, while the desired concept was successfully demonstrated, further genetic work is needed in order to make such a construct practical for environmental monitoring purposes.

SOS gene induction and possible mutagenic effects of freeze-drying in Escherichia coli and Salmonella typhimurium

We report the results of a study of the potential negative effects of the freeze-drying process, normally considered a benign means for long-term conservation of living cells and the golden standard in bacterial preservation. By monitoring gene induction using a whole-cell Escherichia coli bioreporter panel, in which diverse stress-responsive gene promoters are fused to luminescent or fluorescent reporting systems, we have demonstrated that DNA repair genes belonging to the SOS operon (recA, sulA, uvrA, umuD, and lexA) were induced upon resuscitation from the freeze-dried state, whereas other stress-responsive promoters such as grpE, katG, phoA, soxS, and sodA were not affected. This observation was confirmed by the UMU-chromotest (activation of the umuD gene promoter) in Salmonella typhimurium, as well as by real-time PCR analyses of selected E. coli SOS genes. We further show that a functional SOS operon is important in viability maintenance following resuscitation, but that at the same time, this repair system may introduce significantly higher mutation rates, comparable to those induced by high concentrations of a known mutagen. Our results also indicate that the entire freeze-drying process, rather than either freezing or drying separately, is instrumental in the induction of DNA damage.