p-Nitrophenol degradation via 4-nitrocatechol in Burkholderia sp. SJ98 and cloning of some of the lower pathway genes.

Microbial degradation studies have pointed toward the occurrence of two distinct PNP catabolic pathways in Gram positive and Gram negative bacteria. The former involves 4-nitrocatechol (4-NC), 1,2,4-benzenetriol (BT), and maleylacetate (MA) as major degradation intermediates, whereas the later proceeds via formation of 1,4-benzoquinone (BQ) and hydroquinone (HQ). In the present study we identified a Gram negative organism viz. Burkholderia sp. strain SJ98 that degrades PNP via 4NC, BT, and MA. A 6.89 Kb genomic DNA fragment of strain SJ98 that encompasses seven putatively identified ORFs (orfA, pnpD, pnpC, orfB, orfC, orfD, and orfE) was cloned. PnpC is benzenetriol dioxygenase belonging to the intradiol dioxygenase superfamily, whereas PnpD is identified as maleylacetate reductase, a member of the Fe-ADH superfamily showing NADH dependent reductase activity. The in vitro activity assays carried out with purified pnpC and pnpD (btd and mar) gene products transformed BT to MA and MA to beta-ketoadipate, respectively. The cloning, sequencing, and characterization of these genes along with the functional PNP degradation studies ascertained the involvement of 4-NC, BT, and MA as degradation intermediates of PNP pathway in this strain. This is one of the first conclusive reports for 4-NC and BT mediated degradation of PNP in a Gram negative organism.