The naphthalene catabolic (nag) genes of Polaromonas naphthalenivorans CJ2: evolutionary implications for two gene clusters and novel regulatory control

Abstract

Polaromonas naphthalenivorans CJ2, found to be responsible for the degradation of naphthalene in situ at a coal tar waste-contaminated site (C.-O. Jeon et al., Proc. Natl. Acad. Sci. USA 100:13591-13596, 2003), is able to grow on mineral salts agar media with naphthalene as the sole carbon source. Beginning from a 484-bp nagAc-like region, we used a genome walking strategy to sequence genes encoding the entire naphthalene degradation pathway andadditional flanking regions. We found that the naphthalene catabolic genes in P. naphthalenivorans CJ2 were divided into one large and one small gene cluster, separated by an unknown distance. The large gene cluster (nagRAaGHAbAcAdBFCQEDJI'ORF1tnpA) is bounded by a LysR-type regulator (nagR). The small cluster (nagR2ORF2I"KL) is bounded by a MarR-type regulator (nagR2). The catabolic genes of P. naphthalenivorans CJ2 were homologous to many of those of Ralstonia U2, which uses the gentisate pathway to convert naphthalene to central metabolites. However, three open reading frames (nagY, nagM, and nagN), present in Ralstonia U2, were absent. Also, P. naphthalenivorans carries two copies of gentisate dioxygenase (nagI) with 77.4% DNA sequence identity to one another and 82% amino acid identity to their homologue in Ralstonia sp. strain U2. Investigation of the operons using reverse transcription PCR showed that each cluster was controlled independently by its respective promoter. Insertional inactivation and lacZ reporter assays showed that nagR2 is a negative regulator and that expression of the small cluster is not induced by naphthalene, salicylate, or gentisate. Association of two putative Azoarcus-related transposases with the large cluster and one Azoarcus-related putative salicylate 5-hydroxylase gene (ORF2) in the small cluster suggests that mobile genetic elements were likely involved in creating the novel arrangement of catabolic and regulatory genes in P. naphthalenivorans.

FIG. 1.

Construction of mutants CJM110 and CJM112 via Campbell-type homologous recombination in the small cluster of P. naphthalenivorans strain CJ2 naphthalene degradation genes. (A) The mutant CJM110 was designed to create a polar knock out of regulator nagR2. (B) The mutant CJM112 reported transcriptional activity in the small cluster of the naphthalene degradation pathway.

FIG. 2.

Localization of naphthalene degradation genes in P. naphthalenivorans strain CJ2. (A) Plasmids retrieved from wild-type CJ2 and two positive-control pseudomonads carrying 80-kb naphthalene catabolic plasmids, P. putida NCIB 9816-4 and P. putida G7; (B) Southern hybridization of plasmids with nahAc probes; (C) the genomic DNA from strain CJ2 digested with HindIII, EcoRI, KpnI, and XbaI; (D) Southern hybridization of the digested genomic DNA with nahAc probes; M, molecular size marker, λ-DNA digested with HindIII.

FIG. 3.

Physical maps of naphthalene degradation genes from P. naphthalenivorans strain CJ2 and of RT-PCR analysis of expressed genes. (A) Gene order in large and small naphthalene catabolic clusters. Bold solid lines show locations of 13 primer pairs used in the RT-PCR assays. (B) Agarose gel electrophoresis of 13 RT-PCR products amplified from strain CJ2 grown on naphthalene. Numbers refer to the locations of PCR fragments shown in Fig. 3A. Lowercase letters refer to amplification conditions: a, RT-PCR products from total RNA; b, PCR products from total RNA without RT; c, PCR products from genomic DNA. M, molecular size marker (100-bp ladder).

FIG. 4.

Comparative sequence analysis of regulatory promoters. (A) Promoters and NagR binding regions of the nag promoter region from strains U2 and CJ2. The arrows indicate the start of translation. The putative −35 and −10 motifs and the putative NagR binding motif are underlined. (B) Alignment of the conserved upstream regions controlled by NahR and NagR regulators. (C) The putative promoter region of nagR2 ORF2 in the small cluster of naphthalene degradation genes in strain CJ2. Conserved regions are enclosed in boxes.

FIG. 5.

Characterization of regulatory mutants. (A) Growth of wild-type strain CJ2, the nagR mutant CJN110, and nagR2 mutant CJM110 in MSB-N liquid culture. The growth of each strain was monitored by measuring the optical density (O.D.) at 600 nm of the cultures. The mutant strain CJM110 caused the medium to darken after 45 h. For location of nagR and nagR2, see Fig. 3.

FIG. 6.

Reporter assays examining transcriptional activation of nagR2 in the small gene cluster of P. naphthalenivorans strain CJ2. Shown are β-galactosidase activities (Miller units) from the nagR2-ORF2::lacZ transcriptional fusion in cells grown in MSB liquid media containing naphthalene crystals, salicylate, or gentisate (2.5 mM). Mean values for three independent cultures are shown with standard deviation.