Isolation and characterization of a gene cluster involved in PAH degradation in Mycobacterium sp. strain SNP11: Expression in Mycobacterium smegmatis mc 2155

Abstract

Mycobacterium sp. strain SNP11 is able to grow with pyrene, fluoranthene, phenanthrene and fluorene the sole carbon and energy sources. A probe based on the previously described gene pdoA2, which encodes the α subunit of a PAH ring-hydroxylating dioxygenase in Mycobacterium sp. strain 6PY1 [S. Krivobok et al., Identification of pyrene-induced proteins in Mycobacterium sp. strain 6PY1: evidence for two ring-hydroxylating dioxygenases, J. Bacteriol. 185(13) (2003) 3828–3841], was used to isolate a 14 kb DNA fragment from strain SNP11. Twelve putative open reading frames (ORFs), divided into two groups by a promoter intergenic region, were detected in this DNA sequence. The first gene cluster, located upstream of the promoter region, showed low but significant deduced amino acid sequence homologies with enzymes involved in aromatic degradation. The second gene cluster, under control of the promoter, contained pdoA2 (designated phdA in this study) and several other ORFs with deduced amino acid sequences closely related to enzymes involved in the phenanthrene-degrading pathway of Nocardioides sp. strain KP7. Gene expression analysis in Mycobacterium smegmatis mc 2155 revealed broad substrate specificity of the ring-hydroxylating dioxygenase, since transformant cells containing phdAB strongly oxidized fluoranthene, phenanthrene, anthracene, fluorine and dibenzofuran. Laser desorption/ionization time-of-flight mass spectrometry (LDI-ToF MS) analyses of culture media after PAH degradation by M. smegmatis transformants also revealed that the second gene cluster, located downstream of the promoter, takes an active share in initial phenanthrene and anthracene degradation by allowing transformation of these two PAHs in aromatic ring-cleaved metabolites.