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 nagI3, within a previously uncharacterized naphthalene catabolic gene cluster. The role of this cluster (especially nagI3) 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 nagI3 was expressed at a relatively high level. Individual knockout mutants of all three nagI genes were constructed and their influence on both GDO activity and cell growth was evaluated. Of the three knockout strains, CJ2DnagI3 showed severely diminished GDO activity and grew slowest on aromatic substrates. These observations are consistent with the hypothesis that nagI3 may prevent toxic intracellular levels of gentisate from accumulating in CJ2 cells. All three nagI genes from strain CJ2 were cloned into Escherichia coli: the nagI2 and nagI3 genes were successfully overexpressed. The subunit mass of the GDOs were~36-39 kDa, and their structures were deduced to be dimeric. The K m values of NagI2 and NagI3 were 31 and 10 mM, 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 nagI3 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 nagI3 allele of strain CJ2 did not share high sequence identity with its homologues in Polaromonas sp. JS666 and L. cholodnii SP-6.

Methane emission and dynamics of methanotrophic and methanogenic communities in a flooded rice field ecosystem

“…For the analysis of pmoA and mcrA gene expressions, quantitative reverse transcriptase real‐time PCR (qRT‐PCR) was performed as described previously with some modifications (Lee et al ., ). Briefly, to avoid mRNA degradation, 10 volumes of RNAlater‐ICE (Ambion, Austin, TX) were added to 2 g of the soil samples and the samples were stored overnight at −20 °C.…”

Function of different amino acid residues in the reaction mechanism of gentisate 1,2-dioxygenases deduced from the analysis of mutants of the salicylate 1,2-dioxygenase from Pseudaminobacter salicylatoxidans

“…Polaro_naph_1: Polaromonas naphthalenivorans CJ2-AAZ93402 -(nagI″) Jeon et al [47]. Polaro_naph_2: Polaromonas naphthalenivorans CJ2-AAZ93397 (nagI´) -Jeon et al [47]. Polaro_naph_3: Polaromonas naphthalenivorans CJ2-ABM37760-(nagI3) -Lee et al [48].…”

“…U2-AAD12619 -Zhou et al [46]. Polaro_naph_1: Polaromonas naphthalenivorans CJ2-AAZ93402 -(nagI″) Jeon et al [47]. Polaro_naph_2: Polaromonas naphthalenivorans CJ2-AAZ93397 (nagI´) -Jeon et al [47].…”

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

“…Moreover, realtime analysis suggests the presence of multiple gentisate dioxygenases in strain BC1 apart from NagI, similar to that observed in Polaromonas sp. CJ2 (Lee et al, 2011). Further characterization of the catabolic operons will shed light on the regulatory mechanisms involved in the metabolism of 2H1NA and naphthalene/salicylic acid in this strain.…”