Reduction of Protein Bound Methionine Sulfoxide by a Periplasmic Dimethyl Sulfoxide Reductase.

In proteins, methionine (Met) can be oxidized into Met sulfoxide (MetO). The ubiquitous methionine sulfoxide reductases (Msr) A and B are thiol-oxidoreductases reducing MetO. Reversible Met oxidation has a wide range of consequences, from protection against oxidative stress to fine-tuned regulation of protein functions. Bacteria distinguish themselves by the production of molybdenum-containing enzymes reducing MetO, such as the periplasmic MsrP which protects proteins during acute oxidative stress. The versatile dimethyl sulfoxide (DMSO) reductases were shown to reduce the free amino acid MetO, but their ability to reduce MetO within proteins was never evaluated. Here, using model oxidized proteins and peptides, enzymatic and mass spectrometry approaches, we showed that the Rhodobacter sphaeroides periplasmic DorA-type DMSO reductase reduces protein bound MetO as efficiently as the free amino acid L-MetO and with catalytic values in the range of those described for the canonical Msrs. The identification of this fourth type of enzyme able to reduce MetO in proteins, conserved across proteobacteria and actinobacteria, suggests that organisms employ enzymatic systems yet undiscovered to regulate protein oxidation states.

A Sensitive Magnetic Arsenite-Specific Biosensor Hosted in Magnetotactic Bacteria.

According to the World Health Organization, arsenic is the water contaminant that affects the largest number of people worldwide. To limit its impact on the population, inexpensive, quick, and easy-to-use systems of detection are required. One promising solution could be the use of whole-cell biosensors, which have been extensively studied and could meet all these criteria even though they often lack sensitivity. Here, we investigated the benefit of using magnetotactic bacteria as cellular chassis to design and build sensitive magnetic bacterial biosensors. Promoters potentially inducible by arsenic were first identified in silico within the genomes of two magnetotactic bacteria strains, Magnetospirillum magneticum AMB-1 and Magnetospirillum gryphiswaldense MSR-1. The ArsR-dependent regulation was confirmed by reverse transcription-PCR experiments. Biosensors built by transcriptional fusion between the arsenic-inducible promoters and the bacterial luciferase luxCDABE operon gave an element-specific response in 30 min with an arsenite detection limit of 0.5 µM. After magnetic concentration, we improved the sensitivity of the biosensor by a factor of 50 to reach 10 nM, more than 1 order of magnitude below the recommended guidelines for arsenic in drinking water (0.13 µM). Finally, we demonstrated the successful preservation of the magnetic bacterium biosensors by freeze-drying.IMPORTANCE Whole-cell biosensors based on reporter genes can be designed for heavy metal detection but often require the optimization of their sensitivity and specific adaptations for practical use in the field. Magnetotactic bacteria as cellular hosts for biosensors are interesting models, as their intrinsic magnetism permits them to be easily concentrated and entrapped to increase the arsenic-response signal. This paves the way for the development of sensitive and immobilized whole-cell biosensors tailored for use in the field.

Repeated horizontal gene transfers triggered parallel evolution of magnetotaxis in two evolutionary divergent lineages of magnetotactic bacteria.

Under the same selection pressures, two genetically divergent populations may evolve in parallel toward the same adaptive solutions. Here, we hypothesized that magnetotaxis (i.e., magnetically guided chemotaxis) represents a key adaptation to micro-oxic habitats in aquatic sediments and that its parallel evolution homogenized the phenotypes of two evolutionary divergent clusters of freshwater spirilla. All magnetotactic bacteria affiliated to the Magnetospirillum genus (Alphaproteobacteria class) biomineralize the same magnetic particle chains and share highly similar physiological and ultrastructural features. We looked for the processes that could have contributed at shaping such an evolutionary pattern by reconciling species and gene trees using newly sequenced genomes of Magnetospirillum related bacteria. We showed that repeated horizontal gene transfers and homologous recombination of entire operons contributed to the parallel evolution of magnetotaxis. We propose that such processes could represent a more parsimonious and rapid solution for adaptation compared with independent and repeated de novo mutations, especially in the case of traits as complex as magnetotaxis involving tens of interacting proteins. Besides strengthening the idea about the importance of such a function in micro-oxic habitats, these results reinforce previous observations in experimental evolution suggesting that gene flow could alleviate clonal interference and speed up adaptation under some circumstances.

Transformation Cycle of Magnetosomes in Human Stem Cells: From Degradation to Biosynthesis of Magnetic Nanoparticles Anew.

The nanoparticles produced by magnetotactic bacteria, called magnetosomes, are made of a magnetite core with high levels of crystallinity surrounded by a lipid bilayer. This organized structure has been developed during the course of evolution of these organisms to adapt to their specific habitat and is assumed to resist degradation and to be able to withstand the demanding biological environment. Herein, we investigated magnetosomes' structural fate upon internalization in human stem cells using magnetic and photothermal measurements, electron microscopy, and X-ray absorption spectroscopy. All measurements first converge to the demonstration that intracellular magnetosomes can experience an important biodegradation, with up to 70% of their initial content degraded, which is associated with the progressive storage of the released iron in the ferritin protein. It correlates with an extensive magnetite to ferrihydrite phase transition. The ionic species delivered by this degradation could then be used by the cells to biosynthesize magnetic nanoparticles anew. In this case, cell magnetism first decreased with magnetosomes being dissolved, but then cells remagnetized entirely, evidencing the neo-synthesis of biogenic magnetic nanoparticles. Bacteria-made biogenic magnetosomes can thus be totally remodeled by human stem cells, into human cells-made magnetic nanoparticles.

RGD-functionalized magnetosomes are efficient tumor radioenhancers for X-rays and protons.

Although chemically synthesized ferro/ferrimagnetic nanoparticles have attracted great attention in cancer theranostics, they lack radio-enhancement efficacy due to low targeting and internalization ability. Herein, we investigated the potential of RGD-tagged magnetosomes, bacterial biogenic magnetic nanoparticles naturally coated with a biological membrane and genetically engineered to express an RGD peptide, as tumor radioenhancers for conventional radiotherapy and proton therapy. Although native and RGD-magnetosomes similarly enhanced radiation-induced damage to plasmid DNA, RGD-magnetoprobes were able to boost the efficacy of radiotherapy to a much larger extent than native magnetosomes both on cancer cells and in tumors. Combined to magnetosomes@RGD, proton therapy exceeded the efficacy of X-rays at equivalent doses. Also, increased secondary emissions were measured after irradiation of magnetosomes with protons versus photons. Our results indicate the therapeutic advantage of using functionalized magnetoparticles to sensitize tumors to both X-rays and protons and strengthen the case for developing biogenic magnetoparticles for multimodal nanomedicine in cancer therapy.

Crystal structure of the transcriptional repressor DdrO: insight into the metalloprotease/repressor-controlled radiation response in Deinococcus.

Exposure to harmful conditions such as radiation and desiccation induce oxidative stress and DNA damage. In radiation-resistant Deinococcus bacteria, the radiation/desiccation response is controlled by two proteins: the XRE family transcriptional repressor DdrO and the COG2856 metalloprotease IrrE. The latter cleaves and inactivates DdrO. Here, we report the biochemical characterization and crystal structure of DdrO, which is the first structure of a XRE protein targeted by a COG2856 protein. DdrO is composed of two domains that fold independently and are separated by a flexible linker. The N-terminal domain corresponds to the DNA-binding domain. The C-terminal domain, containing three alpha helices arranged in a novel fold, is required for DdrO dimerization. Cleavage by IrrE occurs in the loop between the last two helices of DdrO and abolishes dimerization and DNA binding. The cleavage site is hidden in the DdrO dimer structure, indicating that IrrE cleaves DdrO monomers or that the interaction with IrrE induces a structural change rendering accessible the cleavage site. Predicted COG2856/XRE regulatory protein pairs are found in many bacteria, and available data suggest two different molecular mechanisms for stress-induced gene expression: COG2856 protein-mediated cleavage or inhibition of oligomerization without cleavage of the XRE repressor.

Ectosymbiotic bacteria at the origin of magnetoreception in a marine protist.

Mutualistic symbioses are often a source of evolutionary innovation and drivers of biological diversification1. Widely distributed in the microbial world, particularly in anoxic settings2,3, they often rely on metabolic exchanges and syntrophy2,4. Here, we report a mutualistic symbiosis observed in marine anoxic sediments between excavate protists (Symbiontida, Euglenozoa)5 and ectosymbiotic Deltaproteobacteria biomineralizing ferrimagnetic nanoparticles. Light and electron microscopy observations as well as genomic data support a multi-layered mutualism based on collective magnetotactic motility with division of labour and interspecies hydrogen-transfer-based syntrophy6. The guided motility of the consortia along the geomagnetic field is allowed by the magnetic moment of the non-motile ectosymbiotic bacteria combined with the protist motor activity, which is a unique example of eukaryotic magnetoreception7 acquired by symbiosis. The nearly complete deltaproteobacterial genome assembled from a single consortium contains a full magnetosome gene set8, but shows signs of reduction, with the probable loss of flagellar genes. Based on the metabolic gene content, the ectosymbiotic bacteria are anaerobic sulfate-reducing chemolithoautotrophs that likely reduce sulfate with hydrogen produced by hydrogenosome-like organelles6 underlying the plasma membrane of the protist. In addition to being necessary hydrogen sinks, ectosymbionts may provide organics to the protist by diffusion and predation, as shown by magnetosome-containing digestive vacuoles. Phylogenetic analyses of 16S and 18S ribosomal RNA genes from magnetotactic consortia in marine sediments across the Northern and Southern hemispheres indicate a host-ectosymbiont specificity and co-evolution. This suggests a historical acquisition of magnetoreception by a euglenozoan ancestor from Deltaproteobacteria followed by subsequent diversification. It also supports the cosmopolitan nature of this type of symbiosis in marine anoxic sediments.

Magnetosomes: biogenic iron nanoparticles produced by environmental bacteria.

The scientific community's interest in magnetotactic bacteria has increased substantially in recent decades. These prokaryotes have the particularity of synthesizing nanomagnets, called magnetosomes. The majority of research is based on several scientific questions. Where do magnetotactic bacteria live, what are their characteristics, and why are they magnetic? What are the molecular phenomena of magnetosome biomineralization and what are the physical characteristics of magnetosomes? In addition to scientific curiosity to better understand these stunning organisms, there are biotechnological opportunities to consider. Magnetotactic bacteria, as well as magnetosomes, are used in medical applications, for example cancer treatment, or in environmental ones, for example bioremediation. In this mini-review, we investigated all the aspects mentioned above and summarized the currently available knowledge.

Control by Metals of Staphylopine Dehydrogenase Activity during Metallophore Biosynthesis.

Enzymatic regulations are central processes for the adaptation to changing environments. In the particular case of metallophore-dependent metal uptake, there is a need to quickly adjust the production of these metallophores to the metal level outside the cell, to avoid metal shortage or overload, as well as waste of metallophores. In Staphylococcus aureus, CntM catalyzes the last biosynthetic step in the production of staphylopine, a broad-spectrum metallophore, through the reductive condensation of a pathway intermediate (xNA) with pyruvate. Here, we describe the chemical synthesis of this intermediate, which was instrumental in the structural and functional characterization of CntM and confirmed its opine synthase properties. The three-dimensional structure of CntM was obtained in an "open" form, in the apo state or as a complex with substrate or product. The xNA substrate appears mainly stabilized by its imidazole ring through a π-π interaction with the side chain of Tyr240. Intriguingly, we found that metals exerted various and sometime antagonistic effects on the reaction catalyzed by CntM: zinc and copper are moderate activators at low concentration and then total inhibitors at higher concentration, whereas manganese is only an activator and cobalt and nickel are only inhibitors. We propose a model in which the relative affinity of a metal toward xNA and an inhibitory binding site on the enzyme controls activation, inhibition, or both as a function of metal concentration. This metal-dependent regulation of a metallophore-producing enzyme might also take place in vivo, which could contribute to the adjustment of metallophore production to the internal metal level.

Tuning the redox properties of a [4Fe-4S] center to modulate the activity of Mo-bisPGD periplasmic nitrate reductase.

Molybdoenzymes are ubiquitous in living organisms and catalyze, for most of them, oxidation-reduction reactions using a large range of substrates. Periplasmic nitrate reductase (NapAB) from Rhodobacter sphaeroides catalyzes the 2-electron reduction of nitrate into nitrite. Its active site is a Mo bis-(pyranopterin guanine dinucleotide), or Mo-bisPGD, found in most prokaryotic molybdoenzymes. A [4Fe-4S] cluster and two c-type hemes form an intramolecular electron transfer chain that deliver electrons to the active site. Lysine 56 is a highly conserved amino acid which connects, through hydrogen-bonds, the [4Fe-4S] center to one of the pyranopterin ligands of the Mo-cofactor. This residue was proposed to be involved in the intramolecular electron transfer, either defining an electron transfer pathway between the two redox cofactors, and/or modulating their redox properties. In this work, we investigated the role of this lysine by combining site-directed mutagenesis, activity assays, redox titrations, EPR and HYSCORE spectroscopies. Removal of a positively-charged residue at position 56 strongly decreased the redox potential of the [4Fe-4S] cluster at pH 8 by 230 mV to 400 mV in the K56H and K56M mutants, respectively, thus affecting the kinetics of electron transfer from the hemes to the [4Fe-4S] center up to 5 orders of magnitude. This effect was partly reversed at acidic pH in the K56H mutant likely due to protonation of the imidazole ring of the histidine. Overall, our study demonstrates the critical role of a charged residue from the second coordination sphere in tuning the reduction potential of the [4Fe-4S] cluster in RsNapAB and related molybdoenzymes.

Rhodobactersphaeroides methionine sulfoxide reductase P reduces R- and S-diastereomers of methionine sulfoxide from a broad-spectrum of protein substrates.

Methionine (Met) is prone to oxidation and can be converted to Met sulfoxide (MetO), which exists as R- and S-diastereomers. MetO can be reduced back to Met by the ubiquitous methionine sulfoxide reductase (Msr) enzymes. Canonical MsrA and MsrB were shown to be absolutely stereospecific for the reduction of S-diastereomer and R-diastereomer, respectively. Recently, a new enzymatic system, MsrQ/MsrP which is conserved in all gram-negative bacteria, was identified as a key actor for the reduction of oxidized periplasmic proteins. The haem-binding membrane protein MsrQ transmits reducing power from the electron transport chains to the molybdoenzyme MsrP, which acts as a protein-MetO reductase. The MsrQ/MsrP function was well established genetically, but the identity and biochemical properties of MsrP substrates remain unknown. In this work, using the purified MsrP enzyme from the photosynthetic bacteria Rhodobacter sphaeroides as a model, we show that it can reduce a broad spectrum of protein substrates. The most efficiently reduced MetO is found in clusters, in amino acid sequences devoid of threonine and proline on the C-terminal side. Moreover, R. sphaeroides MsrP lacks stereospecificity as it can reduce both R- and S-diastereomers of MetO, similarly to its Escherichia coli homolog, and preferentially acts on unfolded oxidized proteins. Overall, these results provide important insights into the function of a bacterial envelop protecting system, which should help understand how bacteria cope in harmful environments.

Genomic study of a novel magnetotactic Alphaproteobacteria uncovers the multiple ancestry of magnetotaxis.

Ecological and evolutionary processes involved in magnetotactic bacteria (MTB) adaptation to their environment have been a matter of debate for many years. Ongoing efforts for their characterization are progressively contributing to understand these processes, including the genetic and molecular mechanisms responsible for biomineralization. Despite numerous culture-independent MTB characterizations, essentially within the Proteobacteria phylum, only few species have been isolated in culture because of their complex growth conditions. Here, we report a newly cultivated magnetotactic, microaerophilic and chemoorganoheterotrophic bacterium isolated from the Mediterranean Sea in Marseille, France: Candidatus Terasakiella magnetica strain PR-1 that belongs to an Alphaproteobacteria genus with no magnetotactic relative. By comparing the morphology and the whole genome shotgun sequence of this MTB with those of closer relatives, we brought further evidence that the apparent vertical ancestry of magnetosome genes suggested by previous studies within Alphaproteobacteria hides a more complex evolutionary history involving horizontal gene transfers and/or duplication events before and after the emergence of Magnetospirillum, Magnetovibrio and Magnetospira genera. A genome-scale comparative genomics analysis identified several additional candidate functions and genes that could be specifically associated to MTB lifestyle in this class of bacteria.

Targeted thermal therapy with genetically engineered magnetite magnetosomes@RGD: Photothermia is far more efficient than magnetic hyperthermia.

Providing appropriate means for heat generation by low intratumoral nanoparticle concentrations is a major challenge for cancer nanotherapy. Here we propose RGD-tagged magnetosomes (magnetosomes@RGD) as a biogenic, genetically engineered, inorganic platform for multivalent thermal cancer treatment. Magnetosomes@RGD are biomagnetite nanoparticles synthesized by genetically modified magnetotactic bacteria thanks to a translational fusion of the RGD peptide with the magnetosomal protein MamC. Magnetosomes@RGD thus combine the high crystallinity of their magnetite core with efficient surface functionalization. The specific affinity of RGD was first quantified by single-cell magnetophoresis with a variety of cell types, including immune, muscle, endothelial, stem and cancer cells. The highest affinity and cellular uptake was observed with PC3 prostatic and HeLa uterine cancer cells. The efficiency of photothermia and magnetic hyperthermia was then compared on PC3 cells. Unexpectedly, photothermia was far more efficient than magnetic hyperthermia, which was almost totally inhibited by the cellular environment. RGD targeting was then assessed in vivo at tumor site, in mice bearing PC3 tumors. As a result, we demonstrate that targeted magnetic nanoparticles could generate heat on a therapeutic level after systemic administration, but only under laser excitation, and successfully inhibit tumor progression.

Accumulation and Dissolution of Magnetite Crystals in a Magnetically Responsive Ciliate.

Magnetotactic bacteria (MTB) represent a group of microorganisms that are widespread in aquatic habitats and thrive at oxic-anoxic interfaces. They are able to scavenge high concentrations of iron thanks to the biomineralization of magnetic crystals in their unique organelles, the so-called magnetosome chains. Although their biodiversity has been intensively studied, their ecology and impact on iron cycling remain largely unexplored. Predation by protozoa was suggested as one of the ecological processes that could be involved in the release of iron back into the ecosystem. Magnetic protozoa were previously observed in aquatic environments, but their diversity and the fate of particulate iron during grazing are poorly documented. In this study, we report the morphological and molecular characterizations of a magnetically responsive MTB-grazing protozoan able to ingest high quantities of MTB. This protozoan is tentatively identified as Uronema marinum, a ciliate known to be a predator of bacteria. Using light and electron microscopy, we investigated in detail the vacuoles in which the lysis of phagocytized prokaryotes occurs. We carried out high-resolution observations of aligned magnetosome chains and ongoing dissolution of crystals. Particulate iron in the ciliate represented approximately 0.01% of its total volume. We show the ubiquity of this interaction in other types of environments and describe different grazing strategies. These data contribute to the mounting evidence that the interactions between MTB and protozoa might play a significant role in iron turnover in microaerophilic habitats.IMPORTANCE Identifying participants of each biogeochemical cycle is a prerequisite to our understanding of ecosystem functioning. Magnetotactic bacteria (MTB) participate in iron cycling by concentrating large amounts of biomineralized iron minerals in their cells, which impacts their chemical environment at, or below, the oxic-anoxic transition zone in aquatic habitats. It was shown that some protozoa inhabiting this niche could become magnetic by the ingestion of magnetic crystals biomineralized by grazed MTB. In this study, we show that magnetic MTB grazers are commonly observed in marine and freshwater sediments and can sometimes accumulate very large amounts of particulate iron. We describe here different phagocytosis strategies, determined using magnetic particles from MTB as tracers after their ingestion by the protozoa. This study paves the way for potential scientific or medical applications using MTB grazers as magnetosome hyperaccumulators.

Pseudomonas aeruginosa zinc uptake in chelating environment is primarily mediated by the metallophore pseudopaline.

Metal uptake is vital for all living organisms. In metal scarce conditions a common bacterial strategy consists in the biosynthesis of metallophores, their export in the extracellular medium and the recovery of a metal-metallophore complex through dedicated membrane transporters. Staphylopine is a recently described metallophore distantly related to plant nicotianamine that contributes to the broad-spectrum metal uptake capabilities of Staphylococcus aureus. Here we characterize a four-gene operon (PA4837-PA4834) in Pseudomonas aeruginosa involved in the biosynthesis and trafficking of a staphylopine-like metallophore named pseudopaline. Pseudopaline differs from staphylopine with regard to the stereochemistry of its histidine moiety associated with an alpha ketoglutarate moiety instead of pyruvate. In vivo, the pseudopaline operon is regulated by zinc through the Zur repressor. The pseudopaline system is involved in nickel uptake in poor media, and, most importantly, in zinc uptake in metal scarce conditions mimicking a chelating environment, thus reconciling the regulation of the cnt operon by zinc with its function as the main zinc importer under these metal scarce conditions.

An algal photoenzyme converts fatty acids to hydrocarbons.

Although many organisms capture or respond to sunlight, few enzymes are known to be driven by light. Among these are DNA photolyases and the photosynthetic reaction centers. Here, we show that the microalga Chlorella variabilis NC64A harbors a photoenzyme that acts in lipid metabolism. This enzyme belongs to an algae-specific clade of the glucose-methanol-choline oxidoreductase family and catalyzes the decarboxylation of free fatty acids to n-alkanes or -alkenes in response to blue light. Crystal structure of the protein reveals a fatty acid-binding site in a hydrophobic tunnel leading to the light-capturing flavin adenine dinucleotide (FAD) cofactor. The decarboxylation is initiated through electron abstraction from the fatty acid by the photoexcited FAD with a quantum yield >80%. This photoenzyme, which we name fatty acid photodecarboxylase, may be useful in light-driven, bio-based production of hydrocarbons.

Desulfamplus magnetovallimortis gen. nov., sp. nov., a magnetotactic bacterium from a brackish desert spring able to biomineralize greigite and magnetite, that represents a novel lineage in the Desulfobacteraceae.

A magnetotactic bacterium, designated strain BW-1T, was isolated from a brackish spring in Death Valley National Park (California, USA) and cultivated in axenic culture. The Gram-negative cells of strain BW-1T are relatively large and rod-shaped and possess a single polar flagellum (monotrichous). This strain is the first magnetotactic bacterium isolated in axenic culture capable of producing greigite and/or magnetite nanocrystals aligned in one or more chains per cell. Strain BW-1T is an obligate anaerobe that grows chemoorganoheterotrophically while reducing sulfate as a terminal electron acceptor. Optimal growth occurred at pH 7.0 and 28°C with fumarate as electron donor and carbon source. Based on its genome sequence, the G+C content is 40.72mol %. Phylogenomic and phylogenetic analyses indicate that strain BW-1T belongs to the Desulfobacteraceae family within the Deltaproteobacteria class. Based on average amino acid identity, strain BW-1T can be considered as a novel species of a new genus, for which the name Desulfamplus magnetovallimortis is proposed. The type strain of D. magnetovallimortis is BW-1T (JCM 18010T-DSM 103535T).

Conservation and diversity of the IrrE/DdrO-controlled radiation response in radiation-resistant Deinococcus bacteria.

The extreme radiation resistance of Deinococcus bacteria requires the radiation-stimulated cleavage of protein DdrO by a specific metalloprotease called IrrE. DdrO is the repressor of a predicted radiation/desiccation response (RDR) regulon, composed of radiation-induced genes having a conserved DNA motif (RDRM) in their promoter regions. Here, we showed that addition of zinc ions to purified apo-IrrE, and short exposure of Deinococcus cells to zinc ions, resulted in cleavage of DdrO in vitro and in vivo, respectively. Binding of IrrE to RDRM-containing DNA or interaction of IrrE with DNA-bound DdrO was not observed. The data are in line with IrrE being a zinc peptidase, and indicate that increased zinc availability, caused by oxidative stress, triggers the in vivo cleavage of DdrO unbound to DNA. Transcriptomics and proteomics of Deinococcus deserti confirmed the IrrE-dependent regulation of predicted RDR regulon genes and also revealed additional members of this regulon. Comparative analysis showed that the RDR regulon is largely well conserved in Deinococcus species, but also showed diversity in the regulon composition. Notably, several RDR genes with an important role in radiation resistance in Deinococcus radiodurans, for example pprA, are not conserved in some other radiation-resistant Deinococcus species.

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.

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.