Pushing the limits of nickel detection to nanomolar range using a set of engineered bioluminescent Escherichia coli.

The detection of nickel in water is of great importance due to its harmfulness for living organism. A way to detect Ni is the use of whole-cell biosensors. The aim of the present work was to build a light-emitting bacterial biosensor for the detection of Ni with high specificity and low detection limit properties. For that purpose, the regulatory circuit implemented relied on the RcnR Ni/Co metallo-regulator and its rcnA natural target promoter fused to the lux reporter genes. To convert RcnR to specifically detect Ni, several mutations were tested and the C35A retained. Deleting the Ni efflux pump rcnA and introducing genes encoding several Ni-uptake systems lowered the detection thresholds. When these constructs were assayed in several Escherichia coli strains, it appeared that the detection thresholds were highly variable. The TD2158 wild-type E. coli gave rise to a biosensor ten times more active and sensitive than its W3110 E. coli K12 equivalent. This biosensor was able to confidently detect Ni concentrations as little as 80 nM (4.7 µg l-1), which makes its use compatible with the norms governing the drinking water quality.

A bioluminescent arsenite biosensor designed for inline water analyzer.

Whole-cell biosensors based on the reporter gene system can offer rapid detection of trace levels of organic or metallic compounds in water. They are well characterized in laboratory conditions, but their transfer into technological devices for the surveillance of water networks remains at a conceptual level. The development of a semi-autonomous inline water analyzer stumbles across the conservation of the bacterial biosensors over a period of time compatible with the autonomy requested by the end-user while maintaining a satisfactory sensitivity, specificity, and time response. We focused here on assessing the effect of lyophilization on two biosensors based on the reporter gene system and hosted in Escherichia coli. The reporter gene used here is the entire bacterial luciferase lux operon (luxCDABE) for an autonomous bioluminescence emission without the need to add any substrate. In the cell-survival biosensor that is used to determine the overall fitness of the bacteria when mixed with the water sample, lux expression is driven by a constitutive E. coli promoter PrpoD. In the arsenite biosensor, the arsenite-inducible promoter P ars involved in arsenite resistance in E. coli controls lux expression. Evaluation of the shelf life of these lyophilized biosensors kept at 4 °C over a year evidenced that about 40 % of the lyophilized cells can be revived in such storage conditions. The performances of the lyophilized biosensor after 7 months in storage are maintained, with a detection limit of 0.2 µM arsenite for a response in about an hour with good reproducibility. These results pave the way to the use in tandem of both biosensors (one for general toxicity and one for arsenite contamination) as consumables of an autonomous analyzer in the field.

Bacterial host and reporter gene optimization for genetically encoded whole cell biosensors.

Whole-cell biosensors based on reporter genes allow detection of toxic metals in water with high selectivity and sensitivity under laboratory conditions; nevertheless, their transfer to a commercial inline water analyzer requires specific adaptation and optimization to field conditions as well as economical considerations. We focused here on both the influence of the bacterial host and the choice of the reporter gene by following the responses of global toxicity biosensors based on constitutive bacterial promoters as well as arsenite biosensors based on the arsenite-inducible Pars promoter. We observed important variations of the bioluminescence emission levels in five different Escherichia coli strains harboring two different lux-based biosensors, suggesting that the best host strain has to be empirically selected for each new biosensor under construction. We also investigated the bioluminescence reporter gene system transferred into Deinococcus deserti, an environmental, desiccation- and radiation-tolerant bacterium that would reduce the manufacturing costs of bacterial biosensors for commercial water analyzers and open the field of biodetection in radioactive environments. We thus successfully obtained a cell survival biosensor and a metal biosensor able to detect a concentration as low as 100 nM of arsenite in D. deserti. We demonstrated that the arsenite biosensor resisted desiccation and remained functional after 7 days stored in air-dried D. deserti cells. We also report here the use of a new near-infrared (NIR) fluorescent reporter candidate, a bacteriophytochrome from the magnetotactic bacterium Magnetospirillum magneticum AMB-1, which showed a NIR fluorescent signal that remained optimal despite increasing sample turbidity, while in similar conditions, a drastic loss of the lux-based biosensors signal was observed.

Semi-autonomous inline water analyzer: design of a common light detector for bacterial, phage, and immunological biosensors.

The use of biosensors as sensitive and rapid alert systems is a promising perspective to monitor accidental or intentional environmental pollution, but their implementation in the field is limited by the lack of adapted inline water monitoring devices. We describe here the design and initial qualification of an analyzer prototype able to accommodate three types of biosensors based on entirely different methodologies (immunological, whole-cell, and bacteriophage biosensors), but whose responses rely on the emission of light. We developed a custom light detector and a reaction chamber compatible with the specificities of the three systems and resulting in statutory detection limits. The water analyzer prototype resulting from the COMBITOX project can be situated at level 4 on the Technology Readiness Level (TRL) scale and this technical advance paves the way to the use of biosensors on-site.

A novel pathway for sesquiterpene biosynthesis from Z,Z-farnesyl pyrophosphate in the wild tomato Solanum habrochaites.

In the wild tomato Solanum habrochaites, the Sst2 locus on chromosome 8 is responsible for the biosynthesis of several class II sesquiterpene olefins by glandular trichomes. Analysis of a trichome-specific EST collection from S. habrochaites revealed two candidate genes for the synthesis of Sst2-associated sesquiterpenes. zFPS encodes a protein with homology to Z-isoprenyl pyrophosphate synthases and SBS (for Santalene and Bergamotene Synthase) encodes a terpene synthase with homology to kaurene synthases. Both genes were found to cosegregate with the Sst2 locus. Recombinant zFPS protein catalyzed the synthesis of Z,Z-FPP from isopentenylpyrophosphate (IPP) and dimethylallylpyrophosphate (DMAPP), while coincubation of zFPS and SBS with the same substrates yielded a mixture of olefins identical to the Sst2-associated sesquiterpenes, including (+)-alpha-santalene, (+)-endo-beta-bergamotene, and (-)-endo-alpha-bergamotene. In addition, headspace analysis of tobacco (Nicotiana sylvestris) plants expressing zFPS and SBS in glandular trichomes afforded the same mix of sesquiterpenes. Each of these proteins contains a putative plastid targeting sequence that mediates transport of a fused green fluorescent protein to the chloroplasts, suggesting that the biosynthesis of these sesquiterpenes uses IPP and DMAPP from the plastidic DXP pathway. These results provide novel insights into sesquiterpene biosynthesis and have general implications concerning sesquiterpene engineering in plants.