Fig. 2

Parsed PYO synthesis enables context dependent activation of soxRS induced gene expression. a NEB10β, LW7, and PH04 host strains containing pZE-phzMS, pZE-lacZ, or no plasmid (as indicated) were grown in M9 media with 30 µM PCA. Cultures were inoculated in M9 1% (to approximately 0.05 OD₆₀₀) from overnight cultures and PCA was added 1.5 h after inoculation. After overnight growth, CM was collected and tested in a PYO reporter assay using fluorescent reporter cells (SW101 pCT10 pET-DsRedExpress2). The reporter cells were grown to approximately OD₆₀₀ 0.2 in LB media, and 20 µL CM were added to 180 µL reporter cells. Fluorescence was recorded after approximately 4 h. The values reported are the relative fluorescence units divided by the cell density (OD₆₀₀) of the reporter cells. Error bars represent s.d. of technical duplicates. b PH04 pZE-phzMS was inoculated 1.5% from overnight cultures. After 1 h, varying amounts of PCA (ranging 0–40 µM) were added. After overnight growth, CM samples were collected. Samples were diluted 5 × in LB media then measured for PYO activity using the reporter bioassay as in a. The values reported in a and b are the RFUs divided by the reporter cells’ OD₆₀₀. Error bars represent s.d. of technical duplicates. c PH04 pCT10 pET-eGFP was grown for 4 h during log-phase in either LB or M9 with PYO or PCA at the concentrations indicated (ranging 0–1 µM), then eGFP expression was measured. Fluorescence values were normalized by subtracting out background from reporter cells, then taking its fold over blank. Error bars represent s.d. of technical triplicates. d NEB10β pZE-phzAG and NEB10β pZE-phzMS were cultured alone or together (9:1 ratio). Similarly, PH04 pZE-phzAG and PH04 pZE-phzMS were cultured alone or together (9:1 ratio). After overnight growth in M9 media, CM media was collected and assayed for PYO activity in LB media. Error bars represent s.d. of technical duplicates