Expression of the TOL plasmid xylS gene in Pseudomonas putida occurs from a sigma(70)-dependent promoter or from sigma(70)- and sigma(54)-dependent tandem promoters according to the compound used for growth

Growth of Pseudomonas putida (pWW0) on alkylbenzoates requires the expression of the meta pathway operon, which is mediated by the XylS protein after binding of a benzoate effector. Alternatively, in cells growing on toluene or its aromatic alcohols, overexpression of xylS mediated by XylR activated by these compounds leads to overproduction of the XylS regulator, which even in the absence of benzoate effecters stimulates transcription from the meta cleavage pathway operon promoter. We show here that in bacteria growing on glycerol or alkylbenzoates, the xylS gene is expressed at a low but constitutive level from a newly found sigma(70)-dependent promoter called Ps2, The amount of XylS protein made from the transcript originated from Ps2 was sufficient to allow high levels of expression from the meta cleavage pathway operon promoter when the cells were grown in the presence of 3-methylbenzoate. The transcription initiation point of the transcript generated from Ps2 mapped 9 bp upstream from the proposed ATG of the xylS gene; this transcript contains the ribosome-binding site. The Ps2 promoter was located 110 bp downstream from a previously described sigma(54)-dependent promoter located upstream from the xylS open reading frame, now railed Ps1, In cells growing on toluene or benzyl alcohols, the XylS regulator is overproduced as a consequence of increased expression of the gent: through the effect of the two promoters working in tandem: the newly found (sigma(70)-dependent promoter, whose expression is XylR and toluene independent, and the sigma(54)-dependent promoter, whose expression is dependent on XylR activated by its effecters, This expression pathway of the xylS gene explains why sigma(54)-deficient P. putida bearing the wild-type TOL plasmid, or the wild-type P. putida strain bearing a TOL plasmid with a knocked-out xylR gene, can grow on alkylbenzoates. Until now this has been one of the unresolved paradoxes in the transcriptional control of the TOL meta cleavage pathway.