Unusual location of two nearby pairs of upstream activating sequences for HbpR, the main regulatory protein for the 2-hydroxybiphenyl degradation pathway of "Pseudomonas azelaica" HBP1.

Abstract

"Pseudomonas azelaica" HBP1 degrades 2-hydroxybiphenyl (2-HBP) and 2,2'-diHBP by employing a meta-cleavage pathway encoded by the hbpCAD genes. The regulatory gene hbpR, located directly upstream of the hbpCAD genes and oriented in the opposite direction, encodes a transcription activator protein belonging to the so-called XylR/DmpR subclass within the NtrC family. HbpR activates transcription from two separate sigma(54)-dependent promoters upstream of the hbpC and the hbpD genes, in the presence of the pathway substrates 2-HBP and 2,2'-diHBP. The DNA region upstream of the hbpC gene displays an unusual organization, containing two adjacent 0.3 kb regions that share 71% sequence identity. The DNA region most proximal to the hbpC promoter harbours one pair of putative upstream activating sequences (UASs C-1/C-2) and a small cryptic ORF that shows homology to hbpR itself. The second, more distal, region contains a second pair of putative UASs (UASs C-3/4) and the 5'-part of the hbpR gene. Transcriptional fusions in Escherichia coli between different deletions of the hbpR-hbpC intergenic region and the genes for bacterial luciferase revealed that most if not all of the transcriptional output from the hbpC promoter is mediated from the proximal UASs C-1/C-2. However, when the UASs C-1/C-2 were deleted and UASs C-3/C-4 were placed in an appropriate position with respect to the promoter region, the hbpC promoter was still inducible with 2-HBP, albeit at a lower level. Transcription studies in E. coli and "P. azelaica" revealed that the divergently oriented hbpR gene is expressed constitutively from a sigma(70)-dependent promoter situated within the cryptic ORF. The presence of UAS pair C-3/C-4 mediated a slightly higher promoter activity for transcription of hbpR.