A Whole Cell Biosensor (GlnLux) for Imaging of Active Sites of Symbiotic Nitrogen Fixation in Legumes.

Within a single legume root system, nodules are reported to vary in their rates of symbiotic nitrogen fixation (SNF), but this variation is poorly characterized. Localized detection of SNF activity in nodules is a challenge. Here we present a biosensor strategy to locate and image active sites of SNF in legume nodules. A whole cell biosensor (GlnLux) for glutamine (Gln) was constructed by transforming a bacterial Gln auxotroph with a constitutive lux reporter. For imaging of Gln in whole plant organs, tissues were freeze-thawed to cause Gln leakage, placed on agar pre-embedded with GlnLux cells (GlnLux agar), and then imaged using a photon capture camera. This method located active sites of nitrogen fixation in amide exporting legumes (lentil, alfalfa), and surprisingly, in ureide exporting legumes (common bean, soybean). Furthermore, GlnLux was shown to be a new method for relative measurements of SNF in legumes growing in growth pouches, using extracts from only a single leaf punch. Leaf punch extracts were incubated with GlnLux and luminescence was measured using a luminometer. This method allowed screening SNF using a large number of plant samples within a short period of time at a very low cost. GlnLux was used to identify the effect of different rhizobia strains and plant genotypes on SNF.