The Locus Coding for the 3-Nitrobenzoate Dioxygenase of Comamonas sp. Strain JS46 Is Flanked by IS 1071 Elements and Is Subject to Deletion and Inversion Events

Abstract

In Comamonas sp. strain JS46, 3-nitrobenzoate (3Nba) is initially oxidized at the 3,4 position by a dioxygenase, which results in release of nitrite and production of protocatechuate. The locus coding for the 3Nba dioxygenase (designated mnb, for m-nitrobenzoate) was mobilized from strain JS46 using a plasmid capture method, cloned, and sequenced. The 3Nba dioxygenase (MnbA) is a member of the phthalate family of aromatic oxygenases. An open reading frame designated mnbB that codes for an NAD(P)H-dependent class IA aromatic oxidoreductase is downstream of mnbA. MnbB is tentatively identified as the oxidoreductase that transfers reducing equivalents to MnbA in strain JS46. The mnb locus is flanked by IS1071 elements. The upstream element is interrupted by a novel insertion sequence designated ISCsp1, and the transposase genes of the flanking insertion elements are transcribed in the direction opposite the direction of mnbA transcription. Spontaneous deletion of mnb occurs because of homologous recombination between the directly repeated flanking IS1071 elements. In addition, in ϳ0.007 to 0.2% of any population of JS46 cells growing on 3Nba, alternative orientations of mnb relative to the flanking IS1071 elements are detected. These alternative forms are the result of inversions of mnb and the flanking IS1071 elements. Inversions appear to occur because of homologous recombination between the inverted repeats that flank the IS1071 elements.Nitroaromatic compounds are used extensively in industry (e.g., for the synthesis of pesticides, dyes, and explosives) and are the by-products of natural processes (e.g., nitration during incomplete combustion of organic matter). Many nitroaromatic compounds are widely dispersed pollutants, and because of their toxicity and/or carcinogenicity, their presence in the environment is a cause for concern. Biodegradation of the simpler nitroaromatic compounds is a well-recognized phenomenon, and this natural process is currently being exploited for bioremediation of contaminated sites (34).Bacterial degradation pathways for aerobic catabolism of nitrobenzoic acids have been described for all three isomers. With 2-nitrobenzoic acid (2Nba) and 4-nitrobenzoic acid (4Nba), the NO 2 group initially undergoes reduction prior to final ring cleavage (6, 13-16, 27, 40). In contrast, with 3-nitrobenzoic acid (3Nba), O 2 is added directly at the 3,4 position of the aromatic ring by a dioxygenase. The NO 2 moiety is spontaneously released to produce protocatechuate (Pca) (20). Genes for the initial metabolism of 4Nba have been described (16, 40), but no genes for metabolism of 2Nba and 3Nba have been described.Insertion elements have been implicated in the development and horizontal dissemination of various genetic elements in microorganisms. Various biodegradative genes are part of composite transposons, and IS1071 is an insertion sequence that flanks a range of catabolic operons, such as the operons for metabolism of various aromatic substrates and halogenated alkanes (25,37). In this study, we...

Nitroaromatic Compounds, from Synthesis to Biodegradation

“…Through the use of 18 O 2 incorporation experiments, the oxidation of 3-nitrobenzoate to protocatechuate was shown to be catalyzed by a dioxygenase in both JS46 and JS51 (126). The genes encoding 3-nitrobenzoate dioxygenase in JS46 were later localized to a region of the chromosome that is flanked on both sides by IS1071 elements, which may explain why the degradation phenotype of this strain is unstable (151).…”

The TetR Family of Regulators

“…TFRs have been implicated in both central pathways for carbon metabolism as well as peripheral pathways for the catabolism of specific carbon sources, including the degradation of pollutants and other waste products (e.g., DhaR and MnbR [143,144]). AcnR in Corynebacterium glutamicum controls the expression of the aconitase gene, acn.…”

Degradation of chloroaromatics by Pseudomonas putida GJ31: assembled route for chlorobenzene degradation encoded by clusters on plasmid pKW1 and the chromosome

“…2), the predicted encoded peptide being identical with the Nterminal portion of TnpA of IS1071 of Pseudomonas sp. strain H and Comamonas testosteroni strain BR60 (Providenti et al, 2006;Sota et al, 2006). At 159 bp downstream of the tnpA3 start codon, an inverted repeat of 38 nt (IR2 'left'), identical to that of transposon Tn5501 (28 % identical nucleotides to IS1 'left'), disrupts the tnpA3 gene.…”