Glutamine (Gln) is a critical intermediate in nitrogen metabolism in all organisms. Here, a whole cell biosensor (GlnLux) for Gln was constructed by transforming a bacterial Gln auxotroph with a constitutive lux reporter. The biosensor was optimized for sensitivity, linearity, efficiency, specificity and robustness to permit detection of Gln in vitro and in vivo. The optimized GlnLux biosensor achieved nanomolar sensitivity with Gln standards. Extracts from only 1 mg of maize (Zea mays L.) leaf tissue were sufficient for Gln detection by GlnLux. Measurements of Gln in leaf extracts by GlnLux correlated with quantification by high performance liquid chromatography (Spearman r = 0.95). GlnLux permitted indirect in planta imaging of Gln using a CCD camera, enabling identification of plants that had been fertilized with nitrogen. Imaging using GlnLux also resolved predicted spatial differences in leaf Gln concentration. In a second application, it was demonstrated that GlnLux embedded into agar permits non-destructive screening of co-inoculated bacterial colonies for biological nitrogen fixation (BNF). GlnLux agar was able to distinguish a Bradyrhizobium japonicum wild type strain (nif+) from a mutant strain defective in nitrogenase (nif-) following ≥8 h of co-incubation. The technology was used to screen a bacterial endophyte diversity library cultured from Zea mays (L.) seeds for biological nitrogen fixation. / OMAFRA

Detection and quantification of small peptides, such as yeast pheromones, are often challenging. We developed a highly sensitive and robust affinity-assay for the quantification of the α-factor pheromone of Saccharomyces cerevisiae based on recombinant hydrophobins. These small, amphipathic proteins self-assemble into highly stable monolayers at hydrophilic-hydrophobic interfaces. Upon functionalization of solid supports with a combination of hydrophobins either lacking or exposing the α-factor, pheromone-specific antibodies were bound to the surface. Increasing concentrations of the pheromone competitively detached the antibodies, thus allowing for quantification of the pheromone. By adjusting the percentage of pheromone-exposing hydrophobins, the sensitivity of the assay could be precisely predefined. The assay proved to be highly robust against changes in sample matrix composition. Due to the high stability of hydrophobin layers, the functionalized surfaces could be repeatedly used without affecting the sensitivity. Furthermore, by using an inverse setup, the sensitivity was increased by three orders of magnitude, yielding a novel kind of biosensor for the yeast pheromone with the lowest limit of detection reported so far. This assay was applied to study the pheromone secretion of diverse yeast strains including a whole-cell biosensor strain of Schizosaccharomyces pombe modulating α-factor secretion in response to an environmental signal.

Detection and quantification of small peptides, such as yeast pheromones, are often challenging. We developed a highly sensitive and robust affinity-assay for the quantification of the α-factor pheromone of Saccharomyces cerevisiae based on recombinant hydrophobins. These small, amphipathic proteins self-assemble into highly stable monolayers at hydrophilic-hydrophobic interfaces. Upon functionalization of solid supports with a combination of hydrophobins either lacking or exposing the α-factor, pheromone-specific antibodies were bound to the surface. Increasing concentrations of the pheromone competitively detached the antibodies, thus allowing for quantification of the pheromone. By adjusting the percentage of pheromone-exposing hydrophobins, the sensitivity of the assay could be precisely predefined. The assay proved to be highly robust against changes in sample matrix composition. Due to the high stability of hydrophobin layers, the functionalized surfaces could be repeatedly used without affecting the sensitivity. Furthermore, by using an inverse setup, the sensitivity was increased by three orders of magnitude, yielding a novel kind of biosensor for the yeast pheromone with the lowest limit of detection reported so far. This assay was applied to study the pheromone secretion of diverse yeast strains including a whole-cell biosensor strain of Schizosaccharomyces pombe modulating α-factor secretion in response to an environmental signal.