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 and additional 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 (nagRAaGHAbAcAdBFCQEDJIORF1tnpA) 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.The degradation of naphthalene has been studied extensively in two Pseudomonas species that carry the archetypal catabolic plasmids, NAH7 (in P. putida G7) and pDTG1 (in P. putida NCIB9816-4) (6,11,41). In both strains, the nah dissimilatory genes are organized into two operons: one coding for the enzymes involved in the conversion of naphthalene to salicylate (naphthalene degradation upper pathway) and another coding for the conversion of salicylate to pyruvate and acetyl coenzyme A via meta-cleavage (naphthalene degradation lower pathway) (2,42,42,54). In contrast to the nah naphthalene catabolic pathway in Pseudomonas species, the nag genes of Ralstonia sp. strain U2 encode the alternative gentisate pathway, which converts naphthalene to fumarate and pyruvate via salicylate and gentisate (14). The nag genes are organized in a single operon (56,57). Despite the contrasts in structural genes and their arrangements, the nah and nag systems exhibit strikingly similar regulation. Transcription of nag and both nah operons is controlled by a single Lys-R-type regulatory protein (NagR and NahR, respe...

Naphthalene metabolism and growth inhibition by naphthalene in Polaromonas naphthalenivorans strain CJ2

“…The naphthalene catabolic genes in strain CJ2 are homologous to the nag operon of Ralstonia sp. strain U2, but the genes are arranged in two separate clusters, each with its own regulatory protein (Jeon et al, 2006). In the present investigation, we show that strain CJ2 metabolizes naphthalene via the gentisate pathway using respirometry, GC-MS and cell-free enzyme assays.…”

“…Sequencing of the naphthalene catabolic genes has revealed a novel arrangement of structural and regulatory genes in strain CJ2 (Jeon et al, 2006). In this study, we gained insight into the physiology of naphthalene metabolism in strain CJ2 by comparing it to two archetypal naphthalene-degrading bacteria.…”

“…strain U2 (Zhou et al, 2002) were included in this study for comparison. Strain CJN110 is a mutant of strain CJ2 in which the regulatory gene nagR has been disrupted by a suicide vector carrying a kanamycin resistance gene (Jeon et al, 2006). Growth assays.…”

“…To determine if naphthalene is directly toxic to strain CJ2, we measured the growth of CJN110, a NagR1 regulatory mutant of CJ2 that is unable to grow on naphthalene (Jeon et al, 2006), in MSB supplemented with 6 mM glucose in the presence of naphthalene at concentrations that were inhibitory or non-inhibitory to the wild-type strain. Partially inhibited growth occurred on glucose in the presence of 23 mM naphthalene.…”

Metabolism of 2-hydroxy-1-naphthoic acid and naphthalene via gentisic acid by distinctly different sets of enzymes in Burkholderia sp. strain BC1

“…Whereas the specific activities of both salicylaldehyde dehydrogenase and gentisate dioxygenase were comparable in the two extracts (data not shown), a much higher catechol 1,2-dioxygenase activity (0.325 U mg 21 ) was found in the salicylic acid-grown enzyme preparation than in the naphthalene-grown preparation (0.071 U mg 21 ). These data suggest that during naphthalene metabolism, salicylic acid is present as a transient intermediate, which is metabolized primarily via the gentisate pathway with the possible involvement of the nag operon (Jeon et al, 2006;Zhou et al, 2001). On the other hand, when cells are grown in the presence of a higher concentration (1.0 g l 21 ) of salicylic acid, apart from the usual induction of the nag operon, both salicylate 1-hydroxylase and catechol ortho cleavage dioxygenase are induced to a much higher level.…”