Burk’s GlnLux Gln Secretion Assay

From a glycerol stock, single streaked GlnLux colonies grown on LB agar + carbenicillin (100 mg/ml) and kanamycin (50 mg/ml) at 37°C were used to inoculate 25 ml of LB in 50 ml tubes. LB media was supplemented with 50 μL 2 M glucose, 25 μL 0.2 M Gln, and 25 μL kanamycin and 25 μL carbenicillin. These antibiotics at these concentrations were used to supplement all GlnLux media in this report. Liquid cultures were grown overnight at 37°C with shaking at 200 rpm in Burk’s nitrogen free Burk’s media to deplete endogenous nitrogen. The cultures were centrifuged at 2,500 × g at 20°C for 10 min, then washed 2× in nitrogen free Burk’s media. Cells were resuspended to an OD₅₉₅ of 1.0 in the same media.

The protocol was adapted from Tessaro et al. (2012). Glycerol stocks of endophytes stored in 96 well plates at −80°C were spotted on Burk’s GlnLux plates using a 96-pin replicator. Burk’s GlnLux plates were made as follows: per L, 800 ml of M9 medium (no NH₄, pre-warmed to 42°C) was mixed with 100 ml of molten agar (100 g/L), cooled to 42°C, to which 100 ml of GlnLux Burk’s liquid culture (OD595 = 1.0) was added. Subsequently, 75 ml of this mixture was poured into Petri dishes (150 × 15 mm), cooled at room temperature, and stored at 4°C. Molten agar was mixed with nitrogen-free Burk’s media to a final concentration of 10 g/L and cooled to 42°C. Washed GlnLux culture in nitrogen free Burk’s media was added to the molten agar media to make 10% of the final volume. 75 ml of this mixture was poured in large Petri dishes, cooled at room temperature, and stored at 4°C. Inoculated plates were incubated at 30°C for 3 days to allow endophytes to fix nitrogen and grow. After 3 days, GlnLux plates were moved to 37°C incubator to allow the GlnLux biosensor cells to grow for 24 h. These plates were then imaged using a ChemiProHT Luminescence Imaging System (Roper, United States) with Winview 32 software with a 10-min exposure. The CCD chip was cooled using liquid nitrogen for 1 h prior to imaging to reduce background dark noise. Treatments and replicates were normalized by equating minimum and maximum light intensities across plates in the Winview 32 software. Each endophyte plate had three replicates.