Characterization of a polycyclic aromatic hydrocarbon degradation gene cluster in a phenanthrene-degrading Acidovorax strain.

Increasingly, stable-isotope probing (SIP) has been used to identify environmentally relevant bacteria capable of expressing a particular phenotype prior to directed efforts to cultivate those organisms. For instance, RNA-based SIP with [13C6]benzene was used to identify Azoarcus strains in gasoline-contaminated groundwater before targeted cultivation attempts (19). In another example, a Burkholderia strain isolated from soil was matched to the dominant terminal restriction fragment length polymorphism band from heavy DNA resulting from incubation with [13C]benzoic acid (40). The bacterium Polaromonas naphthalenivorans CJ2 was isolated from coal-tar-contaminated sediments after having been identified as a dominant member of clone libraries in SIP experiments with 13C-labeled naphthalene (17). That strain has since been the focus of additional studies, one of which resulted in the discovery of a unique ring-hydroxylating dioxygenase (RHD) (18). In prior research on polycyclic aromatic hydrocarbon (PAH) degradation, we performed DNA-SIP with uniformly labeled naphthalene, phenanthrene, and pyrene to identify the dominant degraders of those compounds in a bioreactor treating PAH-contaminated soil (45, 46). In those experiments, the most frequently encountered sequences in 13C-enriched DNA fractions from incubations with labeled phenanthrene were associated with the Acidovorax genus (45). Members of that genus have previously been found in high abundance in soils containing PAHs (9, 22) or have been implicated in PAH degradation (12), particularly phenanthrene degradation (7, 35, 42, 45). However, little is known about the genetic determinants for PAH degradation among Acidovorax strains. There exists wide genetic diversity among organisms capable of PAH degradation. Members of the Pseudomonas genus generally contain the well-studied and commonly encountered nah-type genes and are typically associated with the degradation of naphthalene (14, 56). Other organisms contain homologous but significantly different genes. Examples of these include the nag genes from Polaromonas naphthalenivorans (16) and Ralstonia sp. strain U2 (58), the phn genes of Burkholderia sp. strain RP007 (29), and the nid genes of Mycobacterium vanbaalenii (20). Multiple attempts have been made by researchers to design specific PCR primer sets for the detection of the RHD (or initial dioxygenase) gene, whose product catalyzes the first reaction in the aerobic bacterial metabolism of PAHs (5, 30, 33, 36, 54). More recently, degenerate primers based on the known diversity of dioxygenase sequences from both gram-negative and gram-positive organisms have been designed (8). Yet, despite their frequent association with PAH degradation, Acidovorax sequences do not appear in environmental surveys using these primer sets due to the lack of reference sequences from isolates in the genus. In this study we describe the isolation and PAH-degradative properties of a phenanthrene-degrading Acidovorax isolate representative of those members of the genus previously detected by SIP in the same soil. We identified and sequenced the putative upper-pathway genes of phenanthrene degradation and determined compounds that induce expression of those genes.