Identification of the Amidase BbdA That Initiates Biodegradation of the Groundwater Micropollutant 2,6-dichlorobenzamide (BAM) in Aminobacter sp. MSH1

Abstract

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-β 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.

Genetic (In)stability of 2,6-Dichlorobenzamide Catabolism in Aminobacter sp. Strain MSH1 Biofilms under Carbon Starvation Conditions

“…The bbdD/16S rRNA gene ratio, however, clearly changed. In biofilms originating from the DCBA ϩ inoculum, the bbdD/16S rRNA gene ratios decreased significantly (by factors of 7,10,8,12, and 2 when feeds containing 0, 1, 10, 100, and 1,000 g/liter BAM, respectively, were used). Similarly, biofilms originating from the DCBA Ϫ inoculum became depleted of bbdD when they were grown on 0, 1, …”

“…Whether this is due to loss of the entire plasmid is currently unclear, but PCR analysis of Dcba Ϫ mutants of MSH1 targeting the pBAM2 backbone genes indicates that this is the case (T'Syen, unpublished). With regard to the loss of the BbdA ϩ phenotype, we have previously shown that in a BbdA Ϫ mutant, the strain contains a version of pBAM1 which lacks the whole accessory gene region, indicating that the whole pBAM1 plasmid is not necessarily lost (12). Further increases in the incidence of the Dcba Ϫ variant during the growth of MSH1-GFP can be either due to the above-mentioned genetic instability during cell division or due to the growth rate advantage of Dcba Ϫ cells over Dcba ϩ cells.…”

“…MSH1 16S rRNA gene and the catabolic genes bbdA, bbdD, and bbdF. bbdA encodes the conversion of BAM into 2,6-DCBA (12). Both bbdD and bbdF are involved in the degradation of 2,6-DCBA.…”

“…The genes that encode BAM metabolism and hence BAM mineralization in MSH1 are carried by two plasmids, i.e., the IncP1 plasmid pBAM1, which contains the bbdA gene responsible for conversion of BAM to 2,6-dichlorobenzoic acid (2,6-DCBA) (BbdA ϩ phenotype) (12), and the repABC family plasmid pBAM2, which carries two gene clusters responsible for degradation of 2,6-DCBA to Krebs cycle intermediates (Dcba ϩ phenotype), i.e., bbdR1B1B2B3CDE and bbdFGHIJK (J. T'Syen, unpublished results). Mutants that lack one of the two or both genotypes and hence are defective in BAM mineralization were isolated when MSH1 was grown on nonselective medium, indicating that the strain is genetically unstable with regard to BAM mineralization and catabolism (12).…”

Mineralization of the herbicide swep by a two-strain consortium and characterization of a new amidase for hydrolyzing swep

“…LibA and TccA2 were identified from Variovorax [16] and Diaphorobacter [18] and are responsible for the initial degradation of linuron. BbdA was identified from Aminobacter and initiates the degradation of 2,6-dichlorobenzamide [36]. TccA was identified from Ochrobactrum and is involved in the hydrolysis of triclocarban [28].…”