Experimental strategy for quantification of XylSa-dependent and XylSh-dependent Pm/meta activation. (a) Default scenario, i.e. Pm is activated by both XylSa and XylSh. The inducer employed in this case is m-xylene (or its proxy 3MBA), which both activates XylR (and thus triggers the XylSh loop and is metabolized by upper to produce 3 MBz, necessary for XylSa formation. (b) XylSa alone i.e. no XylSh. The added inducer is 3 MBz, which is specific for XylS. (c) XylSh alone i.e., no XylSa. The inducer employed is ortho-xylene (o-xylene), which fully activates XylR (thus generating high levels of XylS = XylSh) but cannot be converted into 3 MBz and therefore XylSa cannot be formed. (d) Pu activation by m-xylene and o-xylene. Reporter strain P. putida mt-2 (pSEVA226Pu) was patched on the surface of minimal-succinate agar plates, grown overnight and then exposed to saturating vapors of either inducer as indicated. Bioluminescence was captured along time and the figures in arbitrary units represented with a color code according to the signal intensity (bar on the right represents the scale). Nil: Control with no inducer. (e) Promoter activities on the basis of the densitometry of the images of panel (d). Values were normalized in respect to maximum activity as above.