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nahR, encoding a LysR-type transcriptional regulator, is highly conserved among naphthalene-degrading bacteria isolated from a coal tar waste-contaminated site and in extracted community DNA^b

Department of Microbiology, Cornell University, Ithaca, NY, 14853-8101, USA¹
Biotechnology Center For Agriculture and the Environment, Cook College, Rutgers University, New Brunswick, NJ, 08901-8520, USA²

Author for correspondence: Eugene L. Madsen. Tel: +1 607 255 3086. Fax: +1 607 255 3904. e-mail: elm3{at}cornell.edu

In Pseudomonas putida strain G7, a LysR-type positive transcriptional activator protein encoded by nahR is necessary for activation of two operons involved in naphthalene catabolism [Schell, M. A. & Poser, E. F. (1989) . J Bacteriol 171, 837–846]. The role of an nahR homologue, NCIB-nahR, in another naphthalene-metabolizing bacterium, P. putida NCIB 9816-4 was verified. Targeted disruption of NCIB-nahR by homologous recombination resulted in a growth defect in the presence of naphthalene or salicylate as sole carbon and energy source. The nahR homologues and intergenic regions between nahR-like and nahG-like genes from P. putida NCIB 9816-4 and seven bacteria native to a naphthalene-rich coal tar contaminated site were amplified by PCR using degenerate primers. The amplified nahR homologues and the intergenic regions were cloned and sequenced. Alignment of the deduced amino acid sequences from NahR homologues revealed that NahR-like proteins showed only minor variations in all investigated naphthalene-degrading isolates. The intergenic regions, together with known NahR-binding sites showed the consensus NahR-protein-binding sites (5'-ATTCACGCTN₂TGAT-3'). Surprisingly, amplified intergenic regions from naphthalene-degrading micro-organisms native to this study site were 100% identical to that of the pDTG1 plasmid (an archetypal naphthalene-catabolic plasmid from Pseudomonas putida NCIB 9816-4), but the nahR coding regions were not. DNA representing the uncultured microbial community was extracted from six sediment samples with varying coal tar exposure histories. PCR amplification of nahR from sediment DNA was observed in contaminated samples, but in uncontaminated samples only following laboratory incubation with naphthalene. The sediment-derived PCR products were sequenced and also found to be almost identical to known nahR genes. Thus, the structure and function of nahR-nahG regulatory genes appear to be highly conserved.

Keywords: microbial community, sediment, biodegradation, diversity, nahG, salA

^b The GenBank accession number for the sequences of the tnpA-like gene, nahG and nahR of P. putida NCIB 9816-4 is AF491307. The GenBank accession numbers for the sequences of the nahR–nahG intergenic region and the nahR homologue genes of strains Cg1, Cg2, Cg5, Cg7, Cg9, Cg11, Hg8 and N1 are AF491308–AF491315, respectively. The GenBank accession numbers for the sequences of nahR from sediment-extracted DNA are AF491316–AF491324.

^a Present address: National Environmental Engineering Research Institute (NEERI), Nehru Marg, Nagpur – 440 020, India.

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