Characterization of a para-nitrophenol catabolic cluster in Pseudomonas sp. strain NyZ402 and construction of an engineered strain capable of simultaneously mineralizing both para- and ortho-nitrophenols.

Pseudomonas sp. strain NyZ402 was isolated for its ability to grow on para-nitrophenol (PNP) as a sole source of carbon, nitrogen, and energy, and was shown to degrade PNP via an oxidization pathway. This strain was also capable of growing on hydroquinone or catechol. A 15, 818 bp DNA fragment extending from a 800-bp DNA fragment of hydroxyquinol 1,2-dioxygenase gene (pnpG) was obtained by genome walking. Sequence analysis indicated that the PNP catabolic gene cluster (pnpABCDEFG) in this fragment shared significant similarities with a recently reported gene cluster responsible for PNP degradation from Pseudomonas sp. strain WBC-3. PnpA is PNP 4-monooxygenase converting PNP to hydroquinone via benzoquinone in the presence of NADPH, and genetic analysis indicated that pnpA plays a key role in PNP degradation. pnpA1 present in the upstream of the cluster (absent in the cluster from strain WBC-3) encodes a protein sharing as high as 55% identity with PnpA, but was not involved in PNP degradation by either in vitro or in vivo analyses. Furthermore, an engineered strain capable of growing on PNP and ortho-nitrophenol (ONP) was constructed by introducing onpAB (encoding ONP monooxygenase and ortho-benzoquinone reductase which catalyzed the transformation of ONP to catechol) from Alcaligenes sp. strain NyZ215 into strain NyZ402.