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

Abstract

This study was designed to characterize naphthalene metabolism in Polaromonas naphthalenivorans CJ2. Comparisons were completed using two archetypal naphthalenedegrading bacteria: Pseudomonas putida NCIB 9816-4 and Ralstonia sp. strain U2, representative of the catechol and gentisate pathways, respectively. Strain CJ2 carries naphthalene catabolic genes that are homologous to those in Ralstonia sp. strain U2. Here we show that strain CJ2 metabolizes naphthalene via gentisate using respirometry, metabolite detection by GC-MS and cell-free enzyme assays. Unlike P. putida NCIB 9816-4 or Ralstonia sp. strain U2, strain CJ2 did not grow in minimal medium saturated with naphthalene. Growth assays revealed that strain CJ2 is inhibited by naphthalene concentrations of 78 mM (10 p.p.m.) and higher, and the inhibition of growth is accompanied by the accumulation of orange-coloured, putative naphthalene metabolites in the culture medium. Loss of cell viability coincided with the appearance of the coloured metabolites, and analysis by HPLC suggested that the accumulated metabolites were 1,2-naphthoquinone and its unstable auto-oxidation products. The naphthoquinone breakdown products accumulated in inhibited, but not uninhibited, cultures of strain CJ2. Furthermore, naphthalene itself was shown to directly inhibit growth of a regulatory mutant of strain CJ2 that is unable to metabolize naphthalene. These results suggest that, despite being able to use naphthalene as a carbon and energy source, strain CJ2 must balance naphthalene utilization against two types of toxicity.

Gentisate 1,2-dioxygenase, in the third naphthalene catabolic gene cluster of Polaromonas naphthalenivorans CJ2, has a role in naphthalene degradation

“…The second catabolic gene cluster is also essential for the degradation of 3-hydroxybenzoate (Park et al, 2007a). These findings have been confirmed biochemically, and other physiological traits of strain CJ2, particularly the accumulation of toxic metabolites during naphthalene metabolism, have been elucidated (Park et al, 2007b;Pumphrey & Madsen, 2007). The genome of P. naphthalenivorans CJ2 contains a third copy of the GDO gene (Yagi et al, 2009) separated from nagI1 by approximately 16.5 kb on the chromosome; however, the physiological role and genetic regulation of this third GDO catabolic gene in naphthalene catabolism by strain CJ2 have not been investigated to our knowledge.…”

“…Some metabolites of aromatic compounds, such as catechols and quinones, can be more toxic than the parent compounds due to an increase in solubility, production of reactive oxygen species or adduct formation with DNA and proteins (Penning et al, 1999;Schweigert et al, 2001). In a prior study of P. naphthalenivorans CJ2, Pumphrey & Madsen (2007) documented both direct inhibition of growth by naphthalene at high concentrations and the accumulation of toxic oxidation products derived from 1,2-naphthoquinone, which resulted in a complete loss of cell viability. Here we discovered a severe growth defect (Table 3) and nearly complete loss of GDO activity in a NagI3 deletion mutant.…”

Field-Based Stable Isotope Probing Reveals the Identities of Benzoic Acid-Metabolizing Microorganisms and Their In Situ Growth in Agricultural Soil

“…Researchers can use phylogenetic analysis of labeled sequences, along with chemical knowledge of both the labeled compound and the experimental environment, to gain further insight into the physiology of the metabolically active population. Furthermore, the successful isolation of a strain representative of an active population identified by SIP allows for more detailed genetic and physiological investigation (23,24,25,30,39).…”

Enhanced Tolerance to Naphthalene and Enhanced Rhizoremediation Performance for Pseudomonas putida KT2440 via the NAH7 Catabolic Plasmid

“…The tolerance of KT2440 for naphthalene was somewhat surprising considering that even microorganisms able to degrade naphthalene are more sensitive to it, like Polaromonas naphthalenivorans, whose growth was completely inhibited in medium containing only 78 M naphthalene, whereas for a nondegrading variant of this bacterium, the naphthalene tolerance limit dropped to 23 M (41). The ability to degrade naphthalene was previously considered an essential mechanism for conferring naphthalene tolerance (37,41); however, Kang and colleagues (28) reported a direct correlation between antioxidant enzyme levels and naphthalene biodegradation in the Pseudomonas sp. strain As1.…”

“…Naphthalene is toxic for eukaryotic and prokaryotic cells (26,40,48) and even for naphthalene-metabolizing microorganisms (2,23,37,39,41). However, a number of bacteria belonging to different genera have been shown to use naphthalene as a source of carbon and energy (45).…”