Cloning and characterization of a 4-nitrophenol hydroxylase gene cluster from Rhodococcus sp. PN1.

A 4-nitrophenol (4-NP)-degrading bacterium was isolated from activated sludge and identified as a Rhodococcus sp. This bacterium, designated as strain PN1, could utilize 4-NP as a sole carbon, nitrogen and energy source. Degradation tests of 4-NP using cell suspensions of strain PN1 revealed that the degradation was induced by 4-NP and that 4-nitrocatechol (4-NC) was one of the metabolites. A gene library was constructed from the total DNA of strain PN1 and introduced into Rhodococcus rhodochrous ATCC 12674. Two recombinant strains showed 4-NP hydroxylase activity, and a 9.1-kb DNA fragment encoding the activity was isolated from one of the strains. In addition, a 2.4-kb smaller fragment expressing the activity was subcloned from the 9.1-kb fragment and sequenced. The sequence analysis showed that the fragment encodes a two-component 4-NP hydroxylase, the predicted amino acid sequence of which exhibits significant similarity to those of phenol hydroxylases and 4-hydroxyphenylacetate 3-hydroxylases belonging to the two-component flavin diffusible monooxygenase (TC-FDM) family proposed by Galán et al. (J. Bacteriol., 182, 627-636, 2000).

Plasmid-encoded genes specifying aniline oxidation from Acinetobacter sp. strain YAA.

Acinetobacter sp. strain YAA is able to use aniline and o-toluidine as the sole carbon and energy source. This strain has several different plasmids and acridine orange curing suggested that aniline utilization in strain YAA was plasmid-encoded. The gene cluster involved in aniline oxidation was cloned in Escherichia coli JM109 from the total plasmid DNA of strain YAA. A recombinant E. coli containing an 18.5 kb insert fragment showed yellow colouration on aniline-containing plates, indicating the formation of 2-hydroxymuconic semialdehyde from aniline. In addition, subcloning of a 9.0 kb SalI fragment from the insert in E. coli resulted in the accumulation of catechol. Southern hybridization studies indicated that the aniline oxygenase gene (atdA) was present on one of the plasmids, pYA1. These results suggest that in strain YAA aniline is degraded via catechol through a pathway involving meta-cleavage of the benzene-ring by plasmid-encoded genes including atdA.