Analysis of of Pu and Ps activation dynamics by XylRa. (a) Proposed single-input module for XylR (SIMXylR). In this motif, XylRa controls negatively its own expression and activates XylS and the upper pathway. While no other target is known for XylR, it cannot be excluded that this regulator controls additional genes (represented as X). (b) Simulations for upper and xylS expression under inducing condition show the synchrony of gene activation. (c) Genetic constructs used to analyze promoter kinetics. The architecture of Pu, Ps and Pm are sketched. The UAS (for upstream activator sequences) for XylR in Pu and Ps, and the XylS binding sites of Pm are shown, with an indication of the boxes for σ54-RNAP (-12/-24) and σ70-RNAP (-10/-35) recognition. Below, the main features of the broad host range lux reporter vector pSEVA226 were each of the promoters was cloned are indicated. (d) Light emission of reporter strains P. putida mt-2 (pSEVA226Pu), P. putida mt-2 (pSEVA226Ps) and P. putida mt-2 (pSEVA226Pm). Each of the strains was cultured in minimal medium with succinate and then added with 5 mM 3MBA as described in the Methods section. Light emission was recorded after 4 h and the figures of bioluminescence/OD600 converted into arbitrary promoter activity units, A.U. (e) Induction kinetics of Pu and Ps assayed in minimal/succinate medium and 1 mM of 3MBA. Reporter strains P. putida mt-2 (pSEVA226Pu) and P. putida mt-2 (pSEVA226Ps) were treated as before but the aromatic inducer was present throughout the entire growth. (f) Relative induction kinetics of Pu, Ps and Pm in minimal medium with 5.0 mM 3MBA as the sole carbon source. Promoter activities were normalized in all cases in respect to their respective maximum values. Note the virtual identity between Pu and Ps promoters and the delay of Pm.