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

Abstract

Polaromonas naphthalenivorans strain CJ2 metabolizes naphthalene via the gentisate pathway and has recently been shown to carry a third copy of gentisate 1,2-dioxygenase (GDO), encoded by nagl3, within a previously uncharacterized naphthalene catabolic gene cluster. The role of this cluster (especially nagl3) in naphthalene metabolism of strain CJ2 was investigated by documenting patterns in regulation, transcription and enzyme activity. Transcriptional analysis of wild-type cells showed the third cluster to be polycistronic and that nagl3 was expressed at a relatively high level. Individual knockout mutants of all three nagl genes were constructed and their influence on both GDO activity and cell growth was evaluated. Of the three knockout strains, CJ2 Delta nagl3 showed severely diminished GDO activity and grew slowest on aromatic substrates. These observations are consistent with the hypothesis that nagl3 may prevent toxic intracellular levels of gentisate from accumulating in CJ2 cells. All three nagl genes from strain CJ2 were cloned into Escherichia coli: the nagl2 and nagl3 genes were successfully overexpressed. The subunit mass of the GDOs were similar to 36-39 kDa, and their structures were deduced to be dimeric. The Km values of Nagl2 and Nagl3 were 31 and 10 mu M, respectively, indicating that the higher affinity of Nagl3 for gentisate may protect the wild-type cells from gentisate toxicity. These results provide clues for explaining why the third gene cluster, particularly the nagl3 gene, is important in strain CJ2. The organization of genes in the third gene cluster matched that of clusters in Polaromonas sp. JS666 and Leptothrix cholodnii SP-6. While horizontal gene transfer (HGT) is one hypothesis for explaining this genetic motif, gene duplication within the ancestral lineage is equally valid. The HGT hypothesis was discounted by noting that the nagl3 allele of strain CJ2 did not share high sequence identity with its homologues in Polaromonas sp. JS666 and L. cholodnii SP-6.