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

This study was designed to characterize naphthalene metabolism in Polaromonas naphthalenivorans CJ2. Comparisons were completed using two archetypal naphthalene-degrading 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 mu M (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.