Regulation of p-hydroxybenzoate hydroxylase synthesis by PobR bound to an operator in Acinetobacter calcoaceticus

Abstract

PobR is a transcriptional activator required for the expression of pobA, the structural gene for p-hydroxybenzoate hydroxylase. The pobA and pobR genes are divergently transcribed and separated by 134 bp in the Acinetobacter calcoaceticus chromosome. Primer extension analysis revealed that the pobA transcript begins 22 bp upstream from the structural gene and the pobR transcript begins 69 bp upstream from the regulatory gene. This arrangement requires superimposition of the -10 base pair and -35 base pair RNA polymerase-binding sites for the respective genes. Expression of a pobR-lacZ fusion was found to be repressed three- to fourfold by pobR when the functional gene was carried in trans on a plasmid. The pobR gene was placed under control of a lac promoter in an expression vector, and the recombinant plasmid inducibly expressed high levels of PobR in Escherichia coli. Cell extracts containing this protein were used to conduct gel mobility shift analyses. PobR binds specifically to DNA in the pobA-pobR intergenic region, and this binding does not appear to be influenced by p-hydroxybenzoate, the inducer of pobA expression. DNase I footprinting indicates that the DNA-binding site for PobR extends from about 10 bp to about 45 bp downstream from the site of the beginning of the pobR transcript. Within this putative operator is a region of inverted symmetry. Evidently, interaction of the inducer with the PobR-operator complex triggers elevated expression of pobA, beginning at a position separated by 55 bp of DNA. The general mechanisms by which PobR exerts transcriptional control resemble those that typify the LysR family of transcriptional activators, a group from which PobR is evolutionarily remote.