Analysis of the pobA and pobR genes controlling expression of p-hydroxybenzoate hydroxylase in Azotobacter chroococcum

We report the cloning and analysis of a gene and its cognate regulatory ele ment from a member of the Azotobacteriaceae which are involved in the break down of an aromatic compound. The genes from Azotobacter chroococcum encodi ng p-hydroxybenzoate hydroxylase (pobA) and its regulatory protein (pobR) w ere cloned from a genomic library and sequenced. Sequence analysis of pobA revealed homology with other bacterial p-hydroxybenzoate hydroxylase enzyme s. Residues essential to the structure and function of the enzyme have been conserved. The pobR gene encodes a DNA binding regulatory protein with sim ilarity to proteins from the AraC/XylS family of transcriptional activators . A fragment containing both pobA and pohR was cloned into pUC19 and p-hydr oxybenzoate hydroxylase activity was induced in Escherichia coli by the add ition of p-hydroxybenzoate. A frame-shift mutation introduced into the pobR gene prevented expression of p-hydroxybenzoate hydroxylase, indicating tha t PobR is the protein required for transcription of pobA. Interestingly, A. chroococcum PobR has no homology to the PobR protein that is the transcrip tional activator of pobA in Acinetobacter strain ADP1, a protein that is ho mologous to the IclR family of transcriptional regulators. However, PobR fr om A. chroococcum is homologous to several other proteins, suggesting that these proteins will also function as transcriptional activators of pobA. (C ) 2001 Elsevier Science B.V. All rights reserved.