ABSTRACT: The quinoline-degradative gene cluster (oxoO, open reading frames 1 to 6 [ORF1 to -6], qorMSL, ORF7 to -9, oxoR) of Pseudomonas putida 86 consists of several overlapping operons controlled in response to quinoline by the master promoter PoxoO and internal promoters Porf3, PqorM, and PoxoR. ORF7 to -9, presumed to be important for maturation of the molybdenum hydroxylase quinoline 2-oxidoreductase, are also weakly transcribed independently of quinoline. Expression of the oxoS gene, located upstream of oxoO, is not influenced by the carbon source. OxoS shows 26% amino acid sequence identity to XylS, the transcriptional regulator of the meta pathway promoter Pm of TOL plasmid pWW0, and is required for quinoline-dependent transcription from PoxoO, Porf3, PqorM, and PoxoR. 5' deletion analysis of PoxoO and PqorM suggested that a 5'-TGCPuCT-N3-GGGATA-3' motif, which resembles the distal 5'-TGCA-N6-GGNTA-3' half-site of the tandem XylS binding site, is essential for oxoS-dependent transcriptional activation. PqorM, which shows similarity to the tandem XylS recognition site of Pm, was cross-activated by the xylS gene product in response to benzoate. The distal half-site of PqorM is necessary, but probably not sufficient, for transcriptional activation by XylS. Despite conservation in PoxoO of a distal 5'-TGCA-N6-GGNTA-3' sequence, cross-activation of PoxoO by XylS and benzoate was not observed. The oxoS gene product in the presence of quinoline weakly stimulated transcription from the Pm promoter. Involvement of an XylS-type protein in the regulation of genes encoding synthesis of a molybdenum hydroxylase is without precedent and may reflect the evolutionary origin of this pathway in the metabolism of aromatic compounds.