Search

Aminobacter sp. MSH1 uses the groundwater micropollutant 2,6-dichlorobenzamide (BAM) as sole source of carbon and energy. In the first step, MSH1 converts BAM to 2,6-dichlorobenzoic acid (2,6-DCBA) by means of the BbdA amidase encoded on the IncP-1β plasmid pBAM1. Information about the genes and degradation steps involved in 2,6-DCBA metabolism in MSH1 or any other organism is currently lacking. Here, we show that the genes for 2,6-DCBA degradation in strain MSH1 reside on a second catabolic plasmid in MSH1, designated as pBAM2. The complete sequence of pBAM2 was determined revealing that it is a 53.9 kb repABC family plasmid. The 2,6-DCBA catabolic genes on pBAM2 are organized in two main clusters bordered by IS elements and integrase genes and encode putative functions like Rieske mono-/dioxygenase, meta-cleavage dioxygenase, and reductive dehalogenases. The putative mono-oxygenase encoded by the bbdD gene was shown to convert 2,6-DCBA to 3-hydroxy-2,6-dichlorobenzoate (3-OH-2,6-DCBA). 3-OH-DCBA was degraded by wild-type MSH1 and not by a pBAM2-free MSH1 variant indicating that it is a likely intermediate in the pBAM2-encoded DCBA catabolic pathway. Based on the activity of BbdD and the putative functions of the other catabolic genes on pBAM2, a metabolic pathway for BAM/2,6-DCBA in strain MSH1 was suggested. [ABSTRACT FROM AUTHOR]

2,6-dichlorobenzamide (BAM) is a recalcitrant groundwater micropollutant that poses a major problem for drinking water production in European countries. Aminobacter sp. MSH1 and related strains have the unique ability to mineralize BAM at micropollutant concentrations but no information exists on the genetics of BAM biodegradation. An amidase--BbdA--converting BAM to 2,6-dichlorobenzoic acid (DCBA) was purified from Aminobacter sp. MSH1. Heterologous expression of the corresponding bbdA gene and its absence in MSH1 mutants defective in BAM degradation, confirmed its BAM degrading function. BbdA shows low amino acid sequence identity with reported amidases and is encoded by an IncP1-ß plasmid (pBAM1, 40.6 kb) that lacks several genes for conjugation. BbdA has a remarkably low KM for BAM (0.71 µM) and also shows activity against benzamide and ortho-chlorobenzamide (OBAM). Differential proteomics and transcriptional reporter analysis suggest the constitutive expression of bbdA in MSH1. Also in other BAM mineralizing Aminobacter sp. strains, bbdA and pBAM1 appear to be involved in BAM degradation. BbdA's high affinity for BAM and its constitutive expression are of interest for using strain MSH1 in treatment of groundwater containing micropollutant concentrations of BAM for drinking water production. [ABSTRACT FROM AUTHOR]