Downloaded from www.microbiologyresearch.org by IP: 54.242.161.225 On: Thu, 12 May 2016 05:39:11 Microbiology (2001), 147, 2183–2194 Printed in Great Britain 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 Marco C. M. Jaspers, Mark Sturme† and Jan Roelof van der Meer Author for correspondence : Jan Roelof van der Meer. Tel : 41 1 823 5438. Fax : 41 1 823 5547. e-mail : vdmeereawag.ch Process of Environmental Microbiology and Molecular Ecotoxicology, Swiss Federal Institute for Environmental Science and Technology (EAWAG), Postbox 611, CH-8600 Du  bendorf, Switzerland ‘ 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 σ 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 σ 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. Keywords : NtrC-type transcriptional activator, regulation, evolution INTRODUCTION The hbp system allows ‘ Pseudomonas azelaica ’ HBP1 to metabolize 2-hydroxybiphenyl (2-HBP) and 2,2- diHBP by employing a meta-cleavage pathway (Kohler et al., 1988, 1993 ; Schmid, 1997). The hbp system consists of the two transcriptional units, hbpCA and ................................................................................................................................................. † Present address : Department of Microbiology, Wageningen Agri- cultural University, Wageningen, The Netherlands. Abbreviations : 2-HBP, 2-hydroxybiphenyl ; UAS, upstream activating sequence ; RNAP, RNA polymerase ; IHF, integration host factor. hbpD, encoding the enzymes for the first steps of 2-HBP degradation, plus the regulatory gene hbpR, which encodes the transcriptional activator (Fig. 1). HbpR belongs to the XylRDmpR subclass of the NtrC family (Jaspers et al., 2000). Members of this family activate gene expression in concert with RNA polymerase core enzyme (RNAP) containing a σ subunit (RNAP-σ) (reviewed by Kustu et al., 1991; Morett & Segovia, 1993). Activation of XylRDmpR-type transcription activators occurs through direct interaction with aro- matic effector compounds, without the need for a sensor kinase protein (reviewed by Shingler, 1996). To mediate transcription activation, they bind specifically to 0002-4781  2001 SGM 2183