Main content area

Characterization of a Polycyclic Aromatic Hydrocarbon Degradation Gene Cluster in a Phenanthrene-Degrading Acidovorax Strain

Singleton, David R., Guzmán Ramirez, Liza, Aitken, Michael D.
Applied and environmental microbiology 2009 v.75 no.9 pp. 2613-2620
Acidovorax, Alcaligenes faecalis, Pseudomonas, Southern blotting, carbon, chromosomes, complementary DNA, energy, enzymes, gene expression, gene induction, genomic libraries, metabolites, mineralization, multigene family, naphthalene, nucleotide sequences, phenanthrene, polycyclic aromatic hydrocarbons, polymerase chain reaction, ribosomal RNA, soil, stable isotopes
Acidovorax sp. strain NA3 was isolated from polycyclic aromatic hydrocarbon (PAH)-contaminated soil that had been treated in a bioreactor and enriched with phenanthrene. The 16S rRNA gene of the isolate possessed 99.8 to 99.9% similarity to the dominant sequences recovered during a previous stable-isotope probing experiment with [U-¹³C]phenanthrene on the same soil (D. R. Singleton, S. N. Powell, R. Sangaiah, A. Gold, L. M. Ball, and M. D. Aitken, Appl. Environ. Microbiol. 71:1202-1209, 2005). The strain grew on phenanthrene as a sole carbon and energy source and could mineralize ¹⁴C from a number of partially labeled PAHs, including naphthalene, phenanthrene, chrysene, benz[a]anthracene, and benzo[a]pyrene, but not pyrene or fluoranthene. Southern hybridizations of a genomic fosmid library with a fragment of the large subunit of the ring-hydroxylating dioxygenase gene from a naphthalene-degrading Pseudomonas strain detected the presence of PAH degradation genes subsequently determined to be highly similar in both nucleotide sequence and gene organization to an uncharacterized Alcaligenes faecalis gene cluster. The genes were localized to the chromosome of strain NA3. To test for gene induction by selected compounds, RNA was extracted from amended cultures and reverse transcribed, and cDNA associated with the enzymes involved in the first three steps of phenanthrene degradation was quantified by quantitative real-time PCR. Expression of each of the genes was induced most strongly by phenanthene and to a lesser extent by naphthalene, but other tested PAHs and PAH metabolites had negligible effects on gene transcript levels.