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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.

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

Water safety is a major concern for public health and for natural environment preservation. We propose to use bacteriophages to develop biosensor tools able to detect human and animal pathogens present in water. For this purpose, we take advantage of the highly discriminating properties of the bacteriophages, which specifically infect their bacterial hosts. The challenge is to use a fluorescent reporter protein that will be synthesized, and thus detected, only once the specific recognition step between a genetically modified temperate bacteriophage and its bacterial host has occurred. To ensure the accuracy and the execution speed of our system, we developed a test that does not require bacterial growth, since a simple 1-hour infection step is required. To ensure a high sensitivity of our tool and in order to detect up to a single bacterium, fluorescence is measured using a portable flow cytometer, also allowing on-site detection. In this study, we have constructed and characterized several "phagosensor" prototypes using the HK620 bacteriophage and its host Escherichia coli TD2158 and we successfully adapted this method to Salmonella detection. We show that the method is fast, robust and sensitive, allowing the detection of as few as 10 bacteria per ml with no concentration nor enrichment step. Moreover, the test is functional in sea water and allows the detection of alive bacteria. Further development will aim to develop phagosensors adapted on demand to the detection of any human or animal pathogen that may be present in water.

Cobalt can be toxic and the way cells adapt to its presence is largely unknown. Here we carried out a transcriptomic analysis of Escherichia coli exposed to cobalt. A limited number of genes were either up- or downregulated. Upregulated genes include the isc and the nfuA genes encoding Fe/S biogenesis assisting factors, and the rcnA gene encoding a cobalt efflux system. Downregulated genes are implicated in anaerobic metabolism (narK, nirB, hybO, grcA), metal transport (feoB, nikA), sulfate/thiosulfate import (cysP), and one is of unknown function (yeeE). Cobalt regulation of isc, nfuA, hybO, cysP and yeeE genes was found to involve IscR, a Fe/S transcriptional regulator. Previously, the Suf Fe/S biogenesis machinery was found to be important for cobalt stress adaptation, but suf genes did not show up in the microarray analysis. Therefore, we used qRT-PCR analysis and found that cobalt induced the suf operon expression. Moreover, kinetic analysis of the cobalt-mediated induction of the suf operon expression allowed us to propose that cobalt toxicity is caused first by impaired Fe/S biogenesis, followed by decreased iron bioavailability and eventually oxidative stress., (© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.)

ABC (ATP-binding cassette) transporters form the largest family of membrane proteins in micro-organisms where they are able to transport a wide variety of substrates against a concentration gradient, in an ATP-dependent process. Two genes from the same putative Bacillus subtilis operon, yheI and yheH, encoding possibly two different ABC transporters, were overexpressed in Escherichia coli in high yield, either separately or jointly. Using membrane vesicles, it is shown here that both subunits were required to detect, (i) the transport of four structurally unrelated drugs, and (ii) a vanadate-sensitive ATPase activity. Mutation of the invariant Walker-A lysine to an alanine residue in both subunits led to an inactive transporter. Moreover, after membrane solubilization by detergent, both wild-type subunits co-purified on a Ni-Agarose affinity column while only the YheH subunit contained a hexa-histidine tag. This shows that YheI and YheH are indeed able to interact together to form a heterodimer. Importantly, expression of both yheI and yheH genes in B. subtilis could be strongly stimulated by addition of sub-inhibitory concentrations of various unrelated antibiotics. Therefore, B. subtilis YheI/YheH forms a new heterodimeric multidrug ABC transporter possibly involved in multiple antibiotic resistance in vivo.

The uncharacterized protein family UPF0042 of the Swiss-Prot database is predicted to be a member of the conserved group of bacterium-specific P-loop-containing proteins. Here we show that two of its members, YvcJ from Bacillus subtilis and YhbJ, its homologue from Escherichia coli, indeed bind and hydrolyze nucleotides. The cellular function of yvcJ was then addressed. In contrast to results recently obtained for E. coli, which indicated that yhbJ mutants strongly overproduced glucosamine-6-phosphate synthase (GlmS), comparison of the wild type with the yvcJ mutant of B. subtilis showed that GlmS expression was quite similar in the two strains. However, in mutants defective in yvcJ, the transformation efficiency and the fraction of cells that expressed competence were reduced. Furthermore, our data show that YvcJ positively controls the expression of late competence genes. The overexpression of comK or comS compensates for the decrease in competence of the yvcJ mutant. Our results show that even if YvcJ and YhbJ belong to the same family of P-loop-containing proteins, the deletion of corresponding genes has different consequences in B. subtilis and in E. coli.

A generic whole-cell bacterial sensor called sposensor was developed with immobilized spores from engineered Bacillus subtilis. Sposensor contains two different types of spores: reporting spores that contain a reporter gene fused to a promoter responding to a compound to be detected, and control spores use to monitor cell germination and viability. A one-step incubation/detection process was developed to meet the constraints of on-site analysis. Spores were directly incubated with culture medium containing the compound to be detected. beta-Galactosidase was chosen as a reporter protein in both cases and its activity followed by a colorimetric assay. Results showed that sposensor was efficient in detecting two different compounds, a metal (Zn(2+)) and a peptidic antibiotic (bacitracin). Owing to the stability and robustness of spores, sposensor is a very efficient and easy tool to manipulate for analyzing the presence of toxic compounds in natural settings.

The involvement of transporters in multidrug resistance of bacteria is an increasingly challenging problem, and most of the pumps identified so far use the protonmotive gradient as the energy source. A new member of the ATP-binding cassette (ABC) family, known in Bacillus subtilis as YvcC and homologous to each half of mammalian P-glycoprotein and to LmrA of Lactococcus lactis, has been studied here. The yvcC gene was constitutively expressed in B. subtilis throughout its growth, and a knockout mutant showed a lower rate of ethidium efflux than the wild-type strain. Overexpression of yvcC in Escherichia coli allowed the preparation of highly enriched inverted-membrane vesicles that exhibited high transport activities of three fluorescent drugs, namely, Hoechst 33342, doxorubicin, and 7-aminoactinomycin D. After solubilization with n-dodecyl beta-D-maltoside, the hexahistidine-tagged YvcC was purified by a one-step affinity chromatography, and its ability to bind many P-glycoprotein effectors was evidenced by fluorescence spectroscopy experiments. Collectively, these results showed that YvcC is a multidrug ABC transporter functionally active in wild-type B. subtilis, and YvcC was therefore renamed BmrA for Bacillus multidrug resistance ATP. Besides, reconstitution of YvcC into liposomes led to the highest, vanadate-sensitive, ATPase activity reported so far for an ABC transporter. Interestingly, such a high ATP hydrolysis proceeds with a positive cooperativity mechanism, a property only found so far with ABC importers.

An expression vector for systematic protein overproduction in Bacillus subtilis has been constructed. It derives from pDG148 and combines the main property of this vector, i.e. conditional expression of the gene in response to isopropylbeta-D-thiogalactopyranoside, with (i) rapid orientated cloning by a ligation independent procedure and (ii) a ribosome binding site of high translational efficiency. When used for overproduction of several proteins in B. subtilis, this vector gave good levels of protein synthesis.