Metabolism of 2-chloro-4-nitroaniline via novel aerobic degradation pathway by Rhodococcus sp. strain MB-P1

Abstract

2-chloro-4-nitroaniline (2-C-4-NA) is used as an intermediate in the manufacture of dyes, pharmaceuticals, corrosion inhibitor and also used in the synthesis of niclosamide, a molluscicide. It is marked as a black-listed substance due to its poor biodegradability. We report biodegradation of 2-C-4-NA and its pathway characterization by Rhodococcus sp. strain MB-P1 under aerobic conditions. The strain MB-P1 utilizes 2-C-4-NA as the sole carbon, nitrogen, and energy source. In the growth medium, the degradation of 2-C-4-NA occurs with the release of nitrite ions, chloride ions, and ammonia. During the resting cell studies, the 2-C-4-NA-induced cells of strain MB-P1 transformed 2-C-4-NA stoichiometrically to 4-amino-3-chlorophenol (4-A-3-CP), which subsequently gets transformed to 6-chlorohydroxyquinol (6-CHQ) metabolite. Enzyme assays by cell-free lysates prepared from 2-C-4-NA-induced MB-P1 cells, demonstrated that the first enzyme in the 2-C-4-NA degradation pathway is a flavin-dependent monooxygenase that catalyzes the stoichiometric removal of nitro group and production of 4-A-3-CP. Oxygen uptake studies on 4-A-3-CP and related anilines by 2-C-4-NA-induced MB-P1 cells demonstrated the involvement of aniline dioxygenase in the second step of 2-C-4-NA degradation. This is the first report showing 2-C-4-NA degradation and elucidation of corresponding metabolic pathway by an aerobic bacterium.

Figure 1. Growth characterization of Rhodococcus sp. strain MB-P1 on 2-C-4-NA as the sole carbon, nitrogen, and energy source.

(•), 2-C-4-NA; (▴), NO₂ ⁻; (Δ), NH₄ ⁻; (○), Cl⁻; (▪), total protein. Values are presented as arithmetic means of data obtained from experiments carried out in triplicates; error bars represent standard deviation.

Figure 2. Degradation kinetics of 2-C-4-NA and 4-A-3-CP during resting cell studies performed by 2-C-4-NA-induced cells of strain MB-P1.

(A) 2-C-4-NA degradation kinetics. (•), 2-C-4-NA; (▪), 4-A-3-CP; (Δ), 6-CHQ; (○), 2-C-4-NA in abiotic control. (B) 4-A-3-CP degradation kinetics. (•), 4-A-3-CP; (Δ), 6-CHQ. Values are presented as arithmetic means of data obtained from experiments carried out in triplicates; error bars represent standard deviation.

Figure 3. Representative HPLC and GC-MS chromtograms along with mass spectra of metabolites identified during the degradation of 2-C-4-NA by resting cells of Rhodococcus sp. strain MB-P1.

(A) HPLC chromatograms of metabolites identified at different time intervals. Peak at Rt value of 8.5 min corresponds to 2-C-4-NA; Peak at Rt value of 4.86 min corresponds to 4-A-3-CP; Peak at Rt value of 3.28 min corresponds to 6-CHQ. (B) GC-MS chromatogram of the authentic standards and the identified metabolites. Peak at Rt value of 6.41 min corresponds to 2-C-4-NA; Peak at Rt value of 7.62 min corresponds to 4-A-3-CP; Peak at Rt value of 9.52 min corresponds to 6-CHQ. (C) Mass spectra of metabolites (Left side; 4-A-3-CP and right side; 6-CHQ) of 2-C-4-NA as analyzed by GC-MS.

Figure 4. Ring cleavage inhibition studies using 2, 2-dipyridyl.

(A) Treated with 2, 2-dipyridyl. (B) Untreated with 2, 2-dipyridyl. 2-C-4-NA (•); 4-A-3-CP (▴); and 6-CHQ (○).Values are presented as arithmetic means of data obtained from experiments carried out in triplicates; error bars represent standard deviation.

Figure 5. Proposed metabolic pathway for aerobic degradation of 2-C-4-NA by Rhodococcus sp. strain MB-P1.

According to the results obtained presented here, 4-A-3-CP and 6-CHQ are identified as major intermediates during degradation of 2-C-4-NA.