SummaryMedically important yeasts of the genus Candida secrete aspartyl proteinases (Sap), which are of particular interest as virulence factors. Six closely related gene sequences, SAP1 to SAP6, for secreted proteinases are present in Candida albicans. The methylotrophic yeast Pichia pastoris was chosen as an expression system for preparing substantial amounts of each Sap isoenzyme. Interestingly, Sap4, Sap5 and Sap6, which have not yet been detected in C. albicans cultures in vitro, were produced as active recombinant enzymes. Different Sap polyclonal antibodies were raised in rabbits and tested before further application by enzyme-linked immunosorbent assay (ELISA) against each recombinant Sap. Two antisera recognized only Sap4 to Sap6. Using these antisera, together with sap null mutants obtained by targeted mutagenesis, we could demonstrate a high production of Sap4, Sap5 and Sap6 by C. albicans cells after phagocytosis by murine peritoneal macrophages. Furthermore, a ⌬sap4,5,6 null mutant was killed 53% more effectively after contact with macrophages than the wild-type strain. These results support a role for Sap4 to Sap6 in pathogenicity.

Contamination with arsenic is a recurring problem in both industrialized and developing countries. Drinking water supplies for large populations can have concentrations much higher than the permissible levels (for most European countries and the United States, 10 μg As per L; elsewhere, 50 μg As per L). Arsenic analysis requires high-end instruments, which are largely unavailable in developing countries. Bioassays based on genetically engineered bacteria have been proposed as suitable alternatives but such tests would profit from better standardization and direct incorporation into sensing devices. The goal of this work was to develop and test microfluidic devices in which bacterial bioreporters could be embedded, exposed and reporter signals detected, as a further step towards a complete miniaturized bacterial biosensor. The signal element in the biosensor is a nonpathogenic laboratory strain of Escherichia coli, which produces a variant of the green fluorescent protein after contact to arsenite and arsenate. E. coli bioreporter cells were encapsulated in agarose beads and incorporated into a microfluidic device where they were captured in 500 × 500 μm(2) cages and exposed to aqueous samples containing arsenic. Cell-beads frozen at -20 °C in the microfluidic chip retained inducibility for up to a month and arsenic samples with 10 or 50 μg L(-1) could be reproducibly discriminated from the blank. In the 0-50 μg L(-1) range and with an exposure time of 200 minutes, the rate of signal increase was linearly proportional to the arsenic concentration. The time needed to reliably and reproducibly detect a concentration of 50 μg L(-1) was 75-120 minutes, and 120-180 minutes for a concentration of 10 μg L(-1).

Marine environments are frequently exposed to oil spills as a result of transportation, oil drilling or fuel usage. Whereas large oil spills and their effects have been widely documented, more common and recurrent small spills typically escape attention. To fill this important gap in the assessment of oil-spill effects, we performed two independent supervised full sea releases of 5 m3 of crude oil, complemented by on-board mesocosm studies and sampling of accidentally encountered slicks. Using rapid on-board biological assays, we detect high bioavailability and toxicity of dissolved and dispersed oil within 24 h after the spills, occurring fairly deep (8 m) below the slicks. Selective decline of marine plankton is observed, equally relevant for early stages of larger spills. Our results demonstrate that, contrary to common thinking, even small spills have immediate adverse biological effects and their recurrent nature is likely to affect marine ecosystem functioning.

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a b s t r a c tThe development of a whole-cell based sensor for arsenite detection coupling biological engineering and electrochemical techniques is presented. This strategy takes advantage of the natural Escherichia coli resistance mechanism against toxic arsenic species, such as arsenite, which consists of the selective intracellular recognition of arsenite and its pumping out from the cell. A whole-cell based biosensor can be produced by coupling the intracellular recognition of arsenite to the generation of an electrochemical signal. Hereto, E. coli was equipped with a genetic circuit in which synthesis of beta-galactosidase is under control of the arsenite-derepressable arsR-promoter. The E. coli reporter strain was filled in a microchip containing 16 independent electrochemical cells (i.e. two-electrode cell), which was then employed for analysis of tap and groundwater samples. The developed arsenic-sensitive electrochemical biochip is easy to use and outperforms state-of-the-art bacterial bioreporters assays specifically in its simplicity and response time, while keeping a very good limit of detection in tap water, i.e. 0.8 ppb. Additionally, a very good linear response in the ranges of concentration tested (0.94 ppb to 3.75 ppb, R 2 ¼0.9975 and 3.75 ppb to 30 ppb, R 2 ¼0.9991) was obtained, complying perfectly with the acceptable arsenic concentration limits defined by the World Health Organization for drinking water samples (i.e. 10 ppb). Therefore, the proposed assay provides a very good alternative for the portable quantification of As (III) in water as corroborated by the analysis of natural groundwater samples from Swiss mountains, which showed a very good agreement with the results obtained by atomic absorption spectroscopy