Characterization of the Diversity of Naphthalene Degradation Pathways Among Soil Bacteria.

Soil contamination with polycyclic aromatic hydrocarbons (PAHs) can result from direct exposure to fossil fuels or the incomplete combustion of organic matter and natural vegetation.  Naphthalene is the simplest PAH (two fused benzene rings) and has been shown to be a component of both fossil fuels and wood char.  The Actinobacteria are ubiquitous Gram-positive, high G+C DNA, bacteria that have been shown to degrade a variety of PAHs and potentially play a significant role in the long-term degradation of PAHs.  We are currently investigating the presence and diversity of both Actinobacterial- and Pseudomonad-like naphthalene dioxygenases (NDOs) in a variety of soils.  These soils represent a range of exposures to naphthalene, including forested versus urban, burned forest versus unburned, and anthropogenically-contaminated versus those with no direct exposure to naphthalene.  We designed degenerate PCR primers that target Actinobacterial naphthalene dioxygenases (NDOs) and related enzymes (such as phthalate dioxygenases; PhtDOs) based on the large subunit of Rhodococcus sp. NCIMB12038 naphthalene dioxygenase narAa (gb|AF082663.3).  Previously published PCR primers targeting Pseudomonad-like NDOs (nahAc) are being employed for comparison.  Eight Actinobacterial clone libraries have been completed.  We have not detected narAa-like sequences in non-contaminated soils.  The remaining phylogenetic distribution of Actinobacterial soil clone sequences shows significant associations with published phthalate-related dioxygenases, likely because Phthalate can be an alternative to salicylate as an intermediate in the degradation of naphthalene by some soil Actinobacteria.  This work will provide insight into the distribution of Actinobacterial dioxygenases and environmental factors controlling their abundance and distribution across a variety of soils.