Microbial metabolism of nitroaromatic compounds and molecular analysis of genes for catabolism

Abstract

Pseudomonas putida HS12 isolated from soil was able to use nitrobenzene (NB) as a sole source of carbon, nitrogen, and energy and was found to possess a partial reductive pathway for the degradation of NB. From LC/MS and 1H-NMR spectroscopic analyses, NB-grown cells of P. putida HS12 were found to convert 3- and 4-chloronitrobenzenes (3- and 4-CNBs) to the corresponding 5- and 4-chloro-2-hydroxyacetanilides, respectively, by partial reduction and subsequent acetylation. For complete mineralization of CNBs, Rhodococcus sp. HS51, which degrades 4- and 5-chloro-2-hydroxyacetanilides, was isolated and combined with P. putida HS12 to give a co-culture. This co-culture was confirmed to mineralize 3- and 4-CNBs in the presence of an additional carbon source. The degradation pathways for NB, 3-CNB, and 4-CNB by the two isolated strains were also proposed. Pseudomonas putida HS12 was found to carry two plasmids, pNB1 and pNB2. The activity assay experiments of wild-type HS12(pNB1 and pNB2), a spontaneous mutant HS121(pNB2), and a cured derivative HS124(pNB1), demonstrated that the catabolic genes coding for the nitrobenzene-degrading enzymes, designated nbz, are located on the two plasmids, pNB1 and pNB1. The genes nbzA, nbzC, nbzD, and nbzE, encoding nitrobenzene nitroreductase, 2-aminophenol 1,6-dioxygenase, 2-aminomuconic 6-semialdehyde dehydrogenase, and 2-aminomuconate deaminase, respectively, are located on pNB1 (59.1-kb). Meanwhile, the nbzB gene encoding hydroxylaminobenzene mutase, a second step enzyme in the nitrobenzene catabolic pathway, was found in pNB2 (43.8-kb). Physical mapping, cloning, and functional analysis of the two plasmids and their subclones in E. coli strains revealed in more detail the genetic organization of the catabolic plasmids pNB1 and pNB2. The genes nbzA and nbzB are located on the 1.1-kb SmaI-SnaBI fragment of pNB1 and the 1.0-kb SspI-SphI fragment of pNB1, respectively, and their expressions were not tightly regulated. On the other h...