Fig. 1: Construction and characterization of a fluoride-responsive gene circuit.

a Constructs bearing the F⁻ responsive riboswitch (FRS) with different combinations of the native eriC^F promoter (P_eriC^F, orange arrows) or a constitutive, synthetic promoter (P_EM7, dark green arrows) and the sequence encoding the first eight amino acids of EriC^F (purple box) were tested. The msfGFP sequence and transcriptional terminators are indicated in light green and T symbols, respectively. b Normalized fluorescence of P. putida KT2440 transformed with the FRS constructs shown in (a) in response to different NaF concentrations. The fluorescence baseline (empty vector) is marked with a dashed line; data are presented as mean values and error bars represent standard deviations of three different biological replicates. (c) Synthetic circuit based on the FRS and the T7 RNA polymerase. FRSv1 regulates the expression of the T7 RNA polymerase gene (dark green), encoded in a mini-Tn7 module (left and right sites of mini-Tn7 depicted as dark gray boxes), along with a gentamycin resistance cassette (orange). This module is genome-integrated in P. putida; a plasmid module containing P_T7 → msfGFP (pS231T7::msfGFP) was transformed in the same strain. d Normalized fluorescence of P. putida KT2440::FRS-T7RNAP/pS231T7::msfGFP exposed to increasing NaF concentrations. Data are presented as mean values and error bars correspond to standard deviations from three biological replicates. e Phylogenetic tree for CrcB proteins in Pseudomonas species. f Toxicity of F⁻ in P. putida KT2440 grown in M9 minimal medium with different NaF concentrations. Bacterial growth (optical density at 600 nm, OD₆₀₀) is shown after 20 h of incubation. Error bars represent standard deviations of three biological replicates. g Same as in (f), but with P. putida KT2440ΔcrcB. Error bars represent standard deviations of at least n = 2 different biological replicates h Tolerance of P. putida KT2440 and P. putida KT2440ΔcrcB to NaF exposed by spotting culture dilutions onto LB medium plates containing 0, 0.1, and 10 mM NaF. i Same as in (d), but with P. putida KT2440ΔcrcB::FRS-T7RNAP. Data are presented as mean values and error bars correspond to standard deviations from three biological replicates. Source data underlying b, d, f, g, i are provided as a Source Data file.