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Identification and physical characterization of the HbpR binding sites of the hbpC and hbpD promoters.

  • Tropel D Process of Environmental Microbiology and Molecular Ecotoxicology, Swiss Federal Institute for Environmental Science and Technology (EAWAG), CH-8600 Dübendorf, Switzerland.
  • van der Meer JR
  • 2002-05-11
Published in:
  • Journal of bacteriology. - 2002
Adenosine Triphosphate
Bacterial Proteins
Base Sequence
Binding Sites
Calmodulin-Binding Proteins
DNA Footprinting
DNA-Binding Proteins
Deoxyribonuclease I
Electrophoretic Mobility Shift Assay
Enhancer Elements, Genetic
Escherichia coli
Molecular Sequence Data
Promoter Regions, Genetic
Pseudomonas
Recombinant Fusion Proteins
Trans-Activators
Transcription Factors
English Pseudomonas azelaica HBP1 can use 2-hydroxybiphenyl (2-HBP) and 2,2'-dihydroxybiphenyl as sole carbon and energy sources by means of the hbp regulon. This regulon is composed of three genes, hbpCA and hbpD, coding for enzymes of a meta-cleavage pathway and the hbpR gene, which codes for a XylR/DmpR-type transcription regulator. It was previously shown that HbpR activates transcription from two sigma(54)-dependent promoters, P(hbpC) and P(hbpD), in the presence of 2-HBP. In this study, by using gel mobility shift assays with a purified fusion protein containing calmodulin binding protein (CBP) and HbpR, we detected two binding regions for HbpR in P(hbpC) and one binding region in P(hbpD). DNase I footprints of the proximal binding region of P(hbpC) and of the binding region in P(hbpD) showed that CBP-HbpR protected a region composed of two inverted repeat sequences which were homologous to the binding sites identified for XylR. Unlike the situation in the XylR/P(u) system, we observed simultaneous binding of CBP-HbpR on the two upstream activating sequences (UASs). Fragments with only one UAS did not show an interaction with HbpR, indicating that both pairs of UASs are needed for HbpR binding. The addition of both ATP and 2-HBP increased the DNA binding affinity of HbpR. These results showed for the first time that, for regulators of the XylR/DmpR type, the effector positively affects the recruitment of the regulatory protein on the enhancer DNA.
Language
  • English
Open access status
bronze
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Persistent URL
https://sonar.ch/global/documents/17343
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