1887

Abstract

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 within a previously uncharacterized naphthalene catabolic gene cluster. The role of this cluster (especially ) 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 was expressed at a relatively high level. Individual knockout mutants of all three genes were constructed and their influence on both GDO activity and cell growth was evaluated. Of the three knockout strains, CJ2Δ showed severely diminished GDO activity and grew slowest on aromatic substrates. These observations are consistent with the hypothesis that may prevent toxic intracellular levels of gentisate from accumulating in CJ2 cells. All three genes from strain CJ2 were cloned into : the and genes were successfully overexpressed. The subunit mass of the GDOs were ~36–39 kDa, and their structures were deduced to be dimeric. The values of NagI2 and NagI3 were 31 and 10 µM, respectively, indicating that the higher affinity of NagI3 for gentisate may protect the wild-type cells from gentisate toxicity. These results provide clues for explaining why the third gene cluster, particularly the gene, is important in strain CJ2. The organization of genes in the third gene cluster matched that of clusters in sp. JS666 and 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 allele of strain CJ2 did not share high sequence identity with its homologues in sp. JS666 and SP-6.

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2011-10-01
2020-01-23
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