Genes for Mn(II)-dependent NahC and Fe(II)-dependent NahH located in close proximity in the thermophilic naphthalene and PCB degrader, Bacillus sp. JF8: cloning and characterization

Abstract

A 10 kb DNA fragment was isolated using a DNA probe derived from the N-terminal amino acid sequence of the extradiol dioxygenase purified from naphthalene-grown Bacillus sp. JF8, a thermophilic naphthalene and polychlorinated biphenyl degrader. The cloned DNA fragment had six open reading frames, designated nahHLOMmocBnahC based on sequence homology, of which the products NahH_JF8 and NahC_JF8 were extradiol dioxygenases. Although NahC_JF8 and NahH_JF8 exhibit low homology to known extradiol dioxygenases, the active-site residues and metal ion ligands are conserved. The presence of Mn(II) in culture medium was found to be essential for production of active recombinant NahC_JF8, while Fe(II) was necessary for active recombinant NahH_JF8. Inductively coupled plasma mass spectrometry analysis of active NahC_JF8 identified the cofactor to be manganese, indicating a Mn(II)-dependent extradiol dioxygenase. NahC_JF8 exhibited K(m) values of 32+/-5 microM for 1,2-dihydroxynaphthalene and 510+/-90 microM for 2,3-dihydroxybiphenyl at 60 degrees C. In cell-free extracts, NahH_JF8 exhibited a broad substrate range for 2,3-dihydroxybiphenyl, catechol, and 3- and 4-methylcatechol at 25 degrees C. Stability studies on the Mn(II)-dependent NahC_JF8 indicated that it was thermostable, retaining 50 % activity after incubation at 80 degrees C for 20 min, and it exhibited resistance to EDTA and H(2)O(2). Northern hybridization studies clarified that both NahC_JF8 and NahH_JF8 were induced by naphthalene; RT-PCR showed that nahHLOMmocBnahC is expressed as a single transcript.