Biodegradation of high-molecular weight PAHs by Rhodococcus wratislaviensis strain 9: Overexpression of amidohydrolase induced by pyrene and BaP.

A Gram-positive bacterium, Rhodococcus wratislaviensis strain 9, completely degraded 280 μM of phenanthrene, 40% of 50 μM pyrene or 28% of 40 μM benzo[a]pyrene (BaP), each supplemented in M9 medium, within 7 days. PCR screening with gene-specific primers indicated that the strain 9 harbors genes which code for 2,3-dihydroxybiphenyl 1,2-dioxygenase (bphC), 4-nitrophenol 2-monooxygenase component B (npcB) as well as oxygenase component (nphA1), 4-hydroxybenzoate 3-monooxygenase (phbH), extradiol dioxygenase (edo), and naphthalene dioxygenase (ndo), all of which are largely implicated in biodegradation of several aromatic hydrocarbons. An orthogonal design experiment revealed that BaP biodegradation was greatly enhanced by surfactants such as Tween 80, Triton X-100 and linoleic acid, suggesting that bioavailability is the major limiting factor in bacterial metabolism of BaP. Both pyrene and BaP induced the overexpression of amidohydrolase, a metallo-dependent hydrolase, possibly involved in their biodegradation by strain 9. The up-regulation of amidohydrolase gene induced by BaP, in particular, was also confirmed by semi-quantitative RT-PCR. Catechol 2,3-dioxygenase and the large subunit of ndo, but not amidohydrolase, accumulated when the strain 9 was grown on phenanthrene. To our knowledge, this is the first report on overexpression of amidohydrolase and its possible implication in bacterial degradation of high-molecular weight PAHs.