Upstream binding sequences of the XylR activator protein and integration host factor in the xylS gene promoter region of the Pseudomonas TOL plasmid

Abstract

The xyIR and xylS genes, which encode the positive regulators of the TOL plasmid catabolic pathways, are adjacent genes on the TOL plasmid and are transcribed from divergent promoters. Transcription from the xylS gene promoter, Ps, is positively regulated by effectoractivated XyIR protein and requires the specific RNA polymerase sigma54 subunit (RpoN). Deletions and point mutations in the Ps upstream region localized the site of XyIR interaction to the region between - 133 bp and - 207 bp (with respect to the transcriptional start of the xylS messenger), which contains an inverted sequence repeat largely homologous to the motif recognised by XyIR in the XyIR-regulated 'upper' catabolic pathway promoter, Pu. Gel retardation experiments showed binding of IHF to the Ps promoter region. Corresponding sequences showing good homology to the IHF-binding consensus were identified close to the Ps Promoter (between - 35 bp and - 47 bp, Ps proximal site) and further upstream overlapping the XyIR recognition sequence (Ps distal site). In the latter case IHF recognition motifs were found well conserved on both strands at nearly the same position (between -140 bp and -152 bp on the upper and between - 141 bp and - 153 bp on the lower strand). Expression from Ps, either under inducing or noninducing conditions, was, however, only slightly influenced by the absence of IHF in an IHF-deficient mutant and thus activation of Ps, like that of other sigma54-dependent promoters which are rich in Ts, does not absolutely require IHF protein.