Cloning and Sequence Analysis of the 4-Hydroxybenzoate 3-Hydroxylase Gene from a Cyclohexanecarboxylate-degrading Gram-positive Bacterium, "Corynebacterium cyclohexanicum" Strain ATCC 51369

Abstract

A DNA fragment that carries the gene encoding 4-hydroxybenzoate 3-hydroxylase (pobA) together with genes encoding a potential regulator (pobR) and a potential transporter (pobK) was cloned from Corynebacterium cyclohexanicum strain ATCC 51369, which is herein reclassified as belonging to the genus Arthrobacter. Nucleotide sequencing revealed that the deduced amino acid sequence encoded by the Arthrobacter pobA gene exhibits 42.0-46.7% identity with that of gram-negative bacteria. The gene organization of the pob cluster differs from that of gram-negative bacteria. The pobA gene product (PobA), expressed in Escherichia coli, preferred NADH over NADPH similar to 4-hydroxybenzoate 3-hydroxylase of ATCC 51369. The Arthrobacter pobA gene was inactivated by insertion of pK19mob. The resultant mutant strain, POBA1, grew on neither cyclohexanecarboxylate nor 4-hydroxybenzoate. These results strongly suggest that the cloned pobA gene plays an essential role in the catabolism not only of 4-hydroxybenzoate but also of cyclohexanecarboxylate in strain ATCC 51369.