Tal Elad

Distance-Decay Relationships Partially Determine Diversity Patterns of Phyllosphere Bacteria on Tamrix Trees across the Sonoran Desert

ABSTRACT Dispersal limitation in phyllosphere communities was measured on the leaf surfaces of salt-excreting Tamarix trees, which offer unique, discrete habitats for microbial assemblages. We employed 16S rRNA gene pyrosequencing to measure bacterial community dissimilarity on leaves of spatially dispersed Tamarix specimens in sites with uniform climatic conditions across the Sonoran Desert in the Southwestern United States. Our analyses revealed diverse bacterial communities with four dominant phyla that exhibited differential effects of environmental and geographic variables. Geographical distance was the most important parameter that affected community composition, particularly that of betaproteobacteria, which displayed a statistically significant, distance-decay relationship.

Microbial sensor cell arrays

Motivated by the advantages endowed by high-throughput analysis, researchers have succeeded in incorporating multiple reporter cells into a single platform; the technology now allows the simultaneous scrutiny of a large collection of sensor strains. We review current aspects in cell array technology with emphasis on microbial sensor arrays. We consider various techniques for patterning live cells on solid surfaces, describe different array-based applications and devices, and highlight recent efforts for live cell storage. We review mathematical approaches for deciphering the data emanating from bioreporter collections, and discuss the future of single cell arrays. Innovative technologies for cell patterning, preservation and interpretation are continuously being developed; when they all mature, cell arrays may become an efficient analytical tool, in a scope resembling that of DNA microarray biochips.

Online Monitoring of Water Toxicity by Use of Bioluminescent Reporter Bacterial Biochips

We describe a flow-through biosensor for online continuous water toxicity monitoring. At the heart of the device are disposable modular biochips incorporating agar-immobilized bioluminescent recombinant reporter bacteria, the responses of which are probed by single-photon avalanche diode detectors. To demonstrate the biosensor capabilities, we equipped it with biochips harboring both inducible and constitutive reporter strains and exposed it to a continuous water flow for up to 10 days. During these periods we challenged the biosensor with 2-h pulses of water spiked with model compounds representing different classes of potential water pollutants, as well as with a sample of industrial wastewater. The biosensor reporter panel detected all simulated contamination events within 0.5-2.5 h, and its response was indicative of the nature of the contaminating chemicals. We believe that a biosensor of the proposed design can be integrated into future water safety and security networks, as part of an early warning system against accidental or intentional water pollution by toxic chemicals.

Microbial whole-cell arrays.

The coming of age of whole‐cell biosensors, combined with the continuing advances in array technologies, has prepared the ground for the next step in the evolution of both disciplines – the whole‐cell array. In the present review, we highlight the state‐of‐the‐art in the different disciplines essential for a functional bacterial array. These include the genetic engineering of the biological components, their immobilization in different polymers, technologies for live cell deposition and patterning on different types of solid surfaces, and cellular viability maintenance. Also reviewed are the types of signals emitted by the reporter cell arrays, some of the transduction methodologies for reading these signals and the mathematical approaches proposed for their analysis. Finally, we review some of the potential applications for bacterial cell arrays, and list the future needs for their maturation: a richer arsenal of high‐performance reporter strains, better methodologies for their incorporation into hardware platforms, design of appropriate detection circuits, the continuing development of dedicated algorithms for multiplex signal analysis and – most importantly – enhanced long‐term maintenance of viability and activity on the fabricated biochips.

A bacterial reporter panel for the detection and classification of antibiotic substances

The ever‐growing use of pharmaceutical compounds, including antibacterial substances, poses a substantial pollution load on the environment. Such compounds can compromise water quality, contaminate soils, livestock and crops, enhance resistance of microorganisms to antibiotic substances, and hamper human health. We report the construction of a novel panel of genetically engineered Escherichia coli reporter strains for the detection and classification of antibiotic substances. Each of these strains harbours a plasmid that carries a fusion of a selected gene promoter to bioluminescence (luxCDABE) reporter genes and an alternative tryptophan auxotrophy‐based non‐antibiotic selection system. The bioreporter panel was tested for sensitivity and responsiveness to diverse antibiotic substances by monitoring bioluminescence as a function of time and of antibiotic concentrations. All of the tested antibiotics were detected by the panel, which displayed different response patterns for each substance. These unique responses were analysed by several algorithms that enabled clustering the compounds according to their functional properties, and allowed the classification of unknown antibiotic substances with a high degree of accuracy and confidence.

Microbial Cell Arrays

The coming of age of whole-cell biosensors, combined with the continuing advances in array technologies, has prepared the ground for the next step in the evolution of both disciplines - the whole cell array. In the present chapter, we highlight the state-of-the-art in the different disciplines essential for a functional bacterial array. These include the genetic engineering of the biological components, their immobilization in different polymers, technologies for live cell deposition and patterning on different types of solid surfaces, and cellular viability maintenance. Also reviewed are the types of signals emitted by the reporter cell arrays, some of the transduction methodologies for reading these signals, and the mathematical approaches proposed for their analysis. Finally, we review some of the potential applications for bacterial cell arrays, and list the future needs for their maturation: a richer arsenal of high-performance reporter strains, better methodologies for their incorporation into hardware platforms, design of appropriate detection circuits, the continuing development of dedicated algorithms for multiplex signal analysis, and - most importantly - enhanced long term maintenance of viability and activity on the fabricated biochips.

Optical modeling of bioluminescence in whole cell biosensors.

Bioluminescence-based whole cell biosensors are devices that can be very useful for environmental monitoring applications. The advantages of these devices are that they can be produced as a single-chip, low-power, rugged, inexpensive component, and can be deployed in a variety of non-laboratory settings. However, such biosensors encounter inherent problems in overall system light collection efficiency. The light emitted from the bioluminescent microbial cells is isotropic and passes through various media before it reaches the photon detectors. We studied the bioluminescence distribution and propagation in microbial whole cell biochips. Optical emission and detection were modeled and simulated using an optical ray tracing method. Light emission, transfer and detection were simulated and optimized with respect to two fundamental system parameters: system geometry and bacterial concentration. Optimization elucidated some of the optical aspects of the biochip, e.g. detector radius values between 300 and 750 microm, and bacterial fixation radius values between 800 and 1200 microm. Understanding theses aspects may establish a basis for future optimization of similar chips.

Broad spectrum detection and “barcoding” of water pollutants by a genome-wide bacterial sensor array

An approach for the rapid detection and classification of a broad spectrum of water pollutants, based on a genome-wide reporter bacterial live cell array, is proposed and demonstrated. An array of ca. 2000 Escherichia coli fluorescent transcriptional reporters was exposed to 25 toxic compounds as well as to unpolluted water, and its responses were recorded after 3 h. The 25 toxic compounds represented 5 pollutant classes: genotoxicants, metals, detergents, alcohols, and monoaromatic hydrocarbons. Identifying unique gene expression patterns, a nearest neighbour-based model detected pollutant presence and predicted class attribution with an estimated accuracy of 87%. Sensitivity and positive predictive values varied among classes, being higher for pollutant classes that were defined by mode of action than for those defined by structure only. Sensitivity for unpolluted water was 0.90 and the positive predictive value was 0.79. All pollutant classes induced the transcription of a statistically significant proportion of membrane associated genes; in addition, the sets of genes responsive to genotoxicants, detergents and alcohols were enriched with genes involved in DNA repair, iron utilization and the translation machinery, respectively. Following further development, a methodology of the type described herein may be suitable for integration in water monitoring schemes in conjunction with existing analytical and biological detection techniques.

High-throughput prescreening of pharmaceuticals using a genome-wide bacterial bioreporter array

We assessed the applicability of multi-strain bacterial bioreporter bioassays to drug screening. To this end, we investigated the reactions of a panel of 15 luminescent recombinant Escherichia coli bacterial bioreporters to a library of 420 pharmaceuticals. The panel included bacterial bioreporters associated with oxidative stress, DNA damage, heat shock, and efflux of excess metals. Eighty nine drugs elicited a response from at least one of the panel members and formed distinctive clusters, some of which contained closely related drugs. In addition, we tested a group of selected nine drugs against a collection of about 2000 different fluorescent transcriptional reporters that covers the great majority of gene promoters in E. coli. The sets of induced genes were in accord with the in vitro toxicity of the tested drugs, as reflected by the response patterns of the 15-member panel, and provided more insights into their toxicity mechanisms. Facilitated by microplates and robotic systems, all assays were conducted in high-throughput. Our results thus suggest that multi-strain assemblages of bacterial bioreporters have the potential for playing a significant role in drug development alongside current in vitro toxicity tests.

Whole-cell biochips for online water monitoring

Chip-integrated luminescent recombinant reporter bacteria were combined with fluidics and light detection systems to form a real-time water biomonitor. The biomonitor was exposed to a continuous water flow for up to ten days, in the course of which it was challenged with spikes of both model toxic compounds and toxic environmental samples. All simulated contamination events were reported within 0.5–2.5 h. Furthermore, the response pattern of the reporter bacteria was indicative of the nature of the contaminating chemicals. Efforts were aimed at improving signal quality and at the development of an alarm management software. Following further research, a device of the proposed design could be implemented in monitoring networks as an early warning system against water pollution by toxic chemicals.