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Applied and Environmental Microbiology, May 2009, p. 2613-2620, Vol. 75, No. 9
0099-2240/09/$08.00+0     doi:10.1128/AEM.01955-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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

David R. Singleton,* Liza Guzmán Ramirez, and Michael D. Aitken

Department of Environmental Sciences and Engineering, School of Public Health, University of North Carolina, Chapel Hill, North Carolina 27599-7431

Received 21 August 2008/ Accepted 21 February 2009

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.

* Corresponding author. Mailing address: 1108 Michael Hooker Research Center, CB no. 7431, University of North Carolina, Chapel Hill, NC 27599. Phone: (919) 966-3860. Fax: (919) 966-7911. E-mail: drsingle{at}unc.edu

{triangledown} Published ahead of print on 6 March 2009.

Applied and Environmental Microbiology, May 2009, p. 2613-2620, Vol. 75, No. 9
0099-2240/09/$08.00+0     doi:10.1128/AEM.01955-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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