Genetische karakterisering van 2,6-dichlorobenzamide (BAM) degradatie in Aminobacter sp. MSH1

2,6-dichlorobenzamide (BAM) is a pesticide transformation product and a frequently detected groundwater micropollutant. Concentrations often exceed the EU drinking water threshold limit of 0.1 μg/L causing the costly closure of groundwater abstraction wells and the installation of expensive activated carbon filters in drinking water production plants. A promising alternative to treat BAM contaminated groundwater is the bioaugmentation of water treatment units (such as sand filters) with Aminobacter sp. MSH1. This strain has the rare ability to use BAM as sole source of carbon, nitrogen and energy resulting in its mineralization. The BAM metabolic pathway used by strain MSH1 and the genetics involved are however largely unknown and need to be characterized to evaluate the production of potentially toxic intermediates, define potential pathway bottlenecks, and develop monitoring tools. This work aims at the genetic characterization of BAM metabolism in Aminobacter sp. MSH1. A first part of this work focused on the identification and characterization of the initial catabolic step in BAM degradation, i.e., the conversion of BAM to 2,6-dichlorobenzoic acid (2,6-DCBA). Protein fractionation followed by mass spectrometry identified a protein, BbdA, that showed amidase activity converting BAM to 2,6-DCBA in crude cell extracts of Aminobacter sp. MSH1. The corresponding bbdA gene was identified in the draft genome sequence of strain MSH1 and the heterologous expression of BbdA in E. coli confirmed its activity of converting BAM to 2,6-DCBA. The bbdA gene was absent in spontaneous mutants of MSH1 that were defective in BAM degradation suggesting that there is no functional redundancy in strain MSH1 to degrade BAM. BbdA shows low amino acid sequence identity to other amidases and is predicted to contain a N-terminal domain involved in dimerization and forming a narrow substrate binding tunnel. bbdA is carried by an unusual IncP-1β plasmid, pBAM1 (40.6 kb), that lacks several genes for conjugation (trbE to trbP) conserved in other IncP-1 plasmids. Homologs of bbdA and plasmid pBAM1 were also present in other BAM mineralizing Aminobacter strains. BbdA displayed a broad substrate range (including benzamide and ortho-chlorobenzamide (OBAM)), a high optimal temperature (Topt = 62.5 °C) and a high affinity for BAM (KM = 0.71 μM). Moreover, differential proteomics and transcriptional reporter analysis suggested the constitutive expression of BbdA. Both the constitutive expression of BbdA and the high affinity of BbdA for BAM are of interest for strain MSH1 to degrade BAM at micropollutant concentrations. The second part of this work covers the identification of gene functions involved in the downstream pathway of BAM metabolism, i.e., the further degradation of 2,6-DCBA. 2,6-DCBA degradation was found to be encoded by a second plasmid of 57.8 kb, pBAM2, that was identified as a repABC type plasmid. Due to the presence of several perfect repeats, the complete sequence of pBAM2 could not be determined but several genes with putative catabolic gene functions for chloro-aromatic catabolism (such as a dioxygenase, mono-oxygenase and dehalogenase) were identified. These catabolic genes are encoded by two gene clusters, i.e., bbdB1B2B3CDE and bbdFGHIJK. Expression of bbdD (encoding a putative mono-oxygenase) in a MSH1 mutant that lacked the ability to metabolize 2,6-DCBA resulted in the conversion of 2,6-DCBA to 3-hydroxy-2,6-dichlorobenzoic acid (3-OH-2,6-DCBA) indicating that further degradation of 2,6-DCBA starts with conversion to 3-OH-2,6-DCBA. Based on this reaction and the annotated gene functions of the catabolic genes found on pBAM2, a putative metabolic pathway of BAM degradation beyond 2,6-DCBA was proposed. The third part aimed at acquiring insight into the regulation of 2,6-DCBA metabolism in Aminobacter sp. MSH1. The transcriptional activity of several possible promoters combined with adjacent regulatory genes on pBAM2 was evaluated by transcriptional fusions with a promoterless gfpmut3.1 gene in wild type MSH1 and spontaneous mutants lacking pBAM1 and/or pBAM2. The results revealed that P1, located in front of gene cluster bbdB1B2B3CDE, is constitutively transcribed. In contrast, P4, located in front of bbdFGHIJK, shows background transcriptional activity and is upregulated by the upstream and minus strand encoded repressor BbdR2 in the presence of an unknown 2,6-DCBA degradation intermediate. Increased transcription from the P4 region was only observed at concentrations of 1 mg/L BAM/2,6-DCBA or higher. However, the leaky transcription from the P4 region at lower concentrations ensures that no induction is needed for low level expression and thus that no threshold concentration for BAM degradation at micropollutant concentrations is created by lack of induction. Differential proteomics showed that up to 6.5 % of the proteome of MSH1 is significantly altered upon growth on BAM compared to growth on glucose. Differences in protein expression mainly related to environmental information processing (transporters) and metabolic processes, including carbohydrate and xenobiotic metabolic proteins. Furthermore, several stress associated proteins showed an increased abundance upon growth on BAM compared to growth on glucose indicating that MSH1 experiences a stress situation when grown on BAM. We conclude that important steps were taken to unravel the genetics of BAM degradation in Aminobacter sp. MSH1. Key catabolic steps were identified and first knowledge was acquired on the regulation of the bbd genes. Together they provided insight into the BAM metabolic pathway, the possible formation of BAM degradation bottlenecks in MSH1, the evolutionary path of BAM degradation in Aminobacter spp. and defined targets for genetic improvement and molecular tools for monitoring purposes.status: publishe