Initial reactions in the biodegradation of 1-chloro-4-nitrobenzene by a newly isolated bacterium, strain LW1

Abstract

Bacterial strain LW1, which belongs to the family Comamonadaceae, utilizes 1-chloro-4-nitrobenzene (1C4NB) as a sole source of carbon, nitrogen, and energy. Suspensions of 1C4NB-grown cells removed 1C4NB from culture fluids, and there was a concomitant release of ammonia and chloride. Under anaerobic conditions LW1 transformed 1C4NB into a product which was identified as 2-amino-5-chlorophenol by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. This transformation indicated that there was partial reduction of the nitro group to the hydroxylamino substituent, followed by Bamberger rearrangement. In the presence of oxygen but in the absence of NAD, fast transformation of 2-amino-5-chlorophenol into a transiently stable yellow product was observed with resting cells and cell extracts. This compound exhibited an absorption maximum at 395 nm and was further converted to a dead-end product with maxima at 226 and 272 nm. The compound formed was subsequently identified by 1H and 13C NMR spectroscopy and mass spectrometry as 5-chloropicolinic acid. In contrast, when NAD was added in the presence of oxygen, only minor amounts of 5-chloropicolinic acid were formed, and a new product, which exhibited an absorption maximum at 306 nm, accumulated.

FIG. 1

Putative initial reactions with 1C4NB based on studies of nitrobenzene degradation via catechol (40) or 2-aminophenol (30), nitrobenzene transformation into aniline (34), reductive dehalogenation (13), and dehalogenation due to hydrolytic (25) or dioxygenolytic (32) activity.

FIG. 2

Growth of bacterial strain LW1 with 1C4NB as the sole carbon source. The initial concentration of the dissolved carbon source in the culture fluid was 1.2 mM. The culture was inoculated (10%, vol/vol) with a preculture grown with 1C4NB and harvested during the exponential phase. Substrate depletion, formation of chloride, and TOC values were determined as described in Materials and Methods. Growth was monitored by monitoring the increase in OD₅₄₆.

FIG. 3

Time course for conversion of 1C4NB by resting cells of strain LW1 grown with 1C4NB under aerobic conditions. Cell suspensions at an OD₅₄₆ of 6.0 were resuspended in 50 mM sodium phosphate buffer (pH 7.2), and substrate was added to a final concentration of 1.36 mM. Substrate depletion was determined by HPLC.

FIG. 4

Time course for conversion of 1C4NB by resting cells of strain LW1 grown with 1C4NB under anaerobic conditions. Cell suspensions at an OD₅₄₆ of 5.0 were resuspended in 50 mM sodium phosphate buffer (pH 7.2). Substrate was added to a final concentration of 1.2 mM. Substrate depletion and product formation were determined by HPLC.

FIG. 5

Spectral changes due to 2-amino-5-chlorophenol 1,6-dioxygenase activity in extracts of cells pregrown with 1C4NB. (A) Reaction mixture containing 2-amino-5-chlorophenol (100 μM) and LW1 cell extract (0.1 mg of protein) in 1 ml of 50 mM sodium phosphate buffer (pH 7.2). (B) Reaction mixture also containing NAD (100 μM). Overlay spectra were recorded for 6 min from 220 to 450 nm as indicated.

FIG. 6

Proposed pathway for catabolism of 1C4NB by bacterial strain LW1. The ring cleavage product 2-amino-5-chloromuconic semialdehyde can be subject to dehydrogenation (probably producing 2-amino-5-chloromuconic acid) or intramolecular condensation to form the dead-end product 5-chloropicolinic acid as previously reported for 2-aminophenol transformation by P. pseudoalcaligenes JS45 (20) or by Pseudomonas sp. strain AP-3 (48).