MUTATION ANALYSIS OF POBR AND PCAU, CLOSELY-RELATED TRANSCRIPTIONAL ACTIVATORS IN ACINETOBACTER

Acinetobacter PobR and PcaU are transcriptional activators that closel y resemble each other in primary structure, DNA-binding sites, metabol ic modulators, and physiological function. PobR responds to the induce r-metabolite p-hydroxybenzoate and activates transcription of pobA, th e structural gene for the enzyme that converts p-hydroxybenzoate to pr otocatechuate. This compound, differing from p-hydroxybenzoate only in that it contains an additional oxygen atom, binds to PcaU and thereby specifically activates transcription of the full set of genes for pro tocatechuate catabolism. Particular experimental attention has been pa id to PobR and PcaU from Acinetobacter strain ADP1, which exhibits exc eptional competence for natural transformation. This trait allowed sel ection of mutant strains in which pobR function had been impaired by n ucleotide substitutions introduced by PCR replication errors. Contrary to expectation, the spectrum of amino acids whose substitution led to loss of function in PobR shows no marked similarity to the spectrum o f amino acids conserved by the demand for continued function during ev olutionary divergence of PobR, PcaU, and related proteins. Surface pla smon resonance was used to determine the ability of mutant PobR protei ns to bind to DNA in the pobA-pobR intergenic region. Deleterious muta tions that strongly affect DNA binding all cluster in and around the P obR region that contains a helix-turn-helix motif, whereas mutations c ausing defects in the central portion of the PobR primary sequence do not seem to have a significant effect on operator binding. PCR-generat ed mutations allowing PobR to mimic PcaU function invariably caused a T57A amino acid substitution, making the helix-turn-helix sequence of PobR more like that of PcaU. The mutant PobR depended on p-hydroxybenz oate for its activity, but this dependence could be relieved by any of six amino acid substitutions in the center of the PobR primary sequen ce. Independent mutations allowing PcaU to mimic PobR activity were sh own to be G222V amino acid substitutions in the C terminus of the 274- residue protein. Together, the analyses suggest that PobR and PcaU pos sess a linear domain structure similar to that of LysR transcriptional activators which largely differ in primary structure.