Identification and Physical Characterization of the HbpR Binding Sites of the hbpC and hbpD Promoters

AUTOR(ES)

Tropel, David

FONTE

American Society for Microbiology

RESUMO

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 σ54-dependent promoters, PhbpC and PhbpD, 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 PhbpC and one binding region in PhbpD. DNase I footprints of the proximal binding region of PhbpC and of the binding region in PhbpD 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/Pu 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.

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