The complete genome of 2,6-dichlorobenzamide (BAM) degrader Aminobacter sp. MSH1 suggests a polyploid chromosome, phylogenetic reassignment, and functions of plasmids.

Aminobacter sp. MSH1 (CIP 110285) can use the pesticide dichlobenil and its recalcitrant transformation product, 2,6-dichlorobenzamide (BAM), as sole source of carbon, nitrogen, and energy. The concentration of BAM in groundwater often exceeds the threshold limit for drinking water, requiring additional treatment in drinking water treatment plants or closure of the affected abstraction wells. Biological treatment with MSH1 is considered a potential sustainable alternative to remediate BAM-contamination in drinking water production. We present the complete genome of MSH1, which was determined independently in two institutes at Aarhus University and KU Leuven. Divergences were observed between the two genomes, i.e. one of them lacked four plasmids compared to the other. Besides the circular chromosome and the two previously described plasmids involved in BAM catabolism, pBAM1 and pBAM2, the genome of MSH1 contained two megaplasmids and three smaller plasmids. The MSH1 substrain from KU Leuven showed a reduced genome lacking a megaplasmid and three smaller plasmids and was designated substrain MK1, whereas the Aarhus variant with all plasmids was designated substrain DK1. A plasmid stability experiment indicate that substrain DK1 may have a polyploid chromosome when growing in R2B medium with more chromosomes than plasmids per cell. Finally, strain MSH1 is reassigned as Aminobacter niigataensis MSH1.

Aminobacter sp. MSH1 Mineralizes the Groundwater Micropollutant 2,6-Dichlorobenzamide through a Unique Chlorobenzoate Catabolic Pathway.

2,6-Dichlorobenzamide (BAM) is a major groundwater micropollutant posing problems for drinking water treatment plants (DWTPs) that depend on groundwater intake. Aminobacter sp. MSH1 uses BAM as the sole source of carbon, nitrogen, and energy and is considered a prime biocatalyst for groundwater bioremediation in DWTPs. Its use in bioremediation requires knowledge of its BAM-catabolic pathway, which is currently restricted to the amidase BbdA converting BAM into 2,6-dichlorobenzoic acid (2,6-DCBA) and the monooxygenase BbdD transforming 2,6-DCBA into 2,6-dichloro-3-hydroxybenzoic acid. Here, we show that the 2,6-DCBA catabolic pathway is unique and differs substantially from catabolism of other chlorobenzoates. BbdD catalyzes a second hydroxylation, forming 2,6-dichloro-3,5-dihydroxybenzoic acid. Subsequently, glutathione-dependent dehalogenases (BbdI and BbdE) catalyze the thiolytic removal of the first chlorine. The remaining chlorine is then removed hydrolytically by a dehalogenase of the α/ß hydrolase superfamily (BbdC). BbdC is the first enzyme in that superfamily associated with dehalogenation of chlorinated aromatics and appears to represent a new subtype within the α/ß hydrolase dehalogenases. The activity of BbdC yields a unique trihydroxylated aromatic intermediate for ring cleavage that is performed by an extradiol dioxygenase (BbdF) producing 2,4,6-trioxoheptanedioic acid, which is likely converted to Krebs cycle intermediates by BbdG.

Catabolism of the groundwater micropollutant 2,6-dichlorobenzamide beyond 2,6-dichlorobenzoate is plasmid encoded in Aminobacter sp. MSH1.

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-1ß 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.

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

Aminobacter sp. strain MSH1 grows on and mineralizes the groundwater micropollutant 2,6-dichlorobenzamide (BAM) and is of interest for BAM removal in drinking water treatment plants (DWTPs). The BAM-catabolic genes in MSH1 are located on plasmid pBAM1, carrying bbdA, which encodes the conversion of BAM to 2,6-dichlorobenzoic acid (2,6-DCBA) (BbdA+ phenotype), and plasmid pBAM2, carrying gene clusters encoding the conversion of 2,6-DCBA to tricarboxylic acid (TCA) cycle intermediates (Dcba+ phenotype). There are indications that MSH1 easily loses its BAM-catabolic phenotype. We obtained evidence that MSH1 rapidly develops a population that lacks the ability to mineralize BAM when grown on nonselective (R2B medium) and semiselective (R2B medium with BAM) media. Lack of mineralization was explained by loss of the Dcba+ phenotype and corresponding genes. The ecological significance of this instability for the use of MSH1 for BAM removal in the oligotrophic environment of DWTPs was explored in lab and pilot systems. A higher incidence of BbdA+ Dcba- MSH1 cells was also observed when MSH1 was grown as a biofilm in flow chambers under C and N starvation conditions due to growth on nonselective residual assimilable organic carbon. Similar observations were made in experiments with a pilot sand filter reactor bioaugmented with MSH1. BAM conversion to 2,6-DCBA was not affected by loss of the DCBA-catabolic genes. Our results show that MSH1 is prone to BAM-catabolic instability under the conditions occurring in a DWTP. While conversion of BAM to 2,6-DCBA remains unaffected, BAM mineralization activity is at risk, and monitoring of metabolites is warranted.IMPORTANCE Bioaugmentation of dedicated biofiltration units with bacterial strains that grow on and mineralize micropollutants was suggested as an alternative for treating micropollutant-contaminated water in drinking water treatment plants (DWTPs). Organic-pollutant-catabolic genes in bacteria are often easily lost, especially under nonselective conditions, which affects the bioaugmentation success. In this study, we provide evidence that Aminobacter sp. strain MSH1, which uses the common groundwater micropollutant 2,6-dichlorobenzamide (BAM) as a C source, shows a high frequency of loss of its BAM-mineralizing phenotype due to the loss of genes that convert 2,6-DCBA to Krebs cycle intermediates when nonselective conditions occur. Moreover, we show that catabolic-gene loss also occurs in the oligotrophic environment of DWTPs, where growth of MSH1 depends mainly on the high fluxes of low concentrations of assimilable organic carbon, and hence show the ecological relevance of catabolic instability for using strain MSH1 for BAM removal in DWTPs.

Biocarriers Improve Bioaugmentation Efficiency of a Rapid Sand Filter for the Treatment of 2,6-Dichlorobenzamide-Contaminated Drinking Water.

Aminobacter sp. MSH1 immobilized in an alginate matrix in porous stones was tested in a pilot system as an alternative inoculation strategy to the use of free suspended cells for biological removal of micropollutant concentrations of 2,6-dichlorobenzamide (BAM) in drinking water treatment plants (DWTPs). BAM removal rates and MSH1 cell numbers were recorded during operation and assessed with specific BAM degradation rates obtained in lab conditions using either freshly grown cells or starved cells to explain reactor performance. Both reactors inoculated with either suspended or immobilized cells showed immediate BAM removal under the threshold of 0.1 µg/L, but the duration of sufficient BAM removal was 2-fold (44 days) longer for immobilized cells. The longer sufficient BAM removal in case of immobilized cells compared to suspended cells was mainly explained by a lower initial loss of MSH1 cells at operational start due to volume replacement and shear. Overall loss of activity in the reactors though was due to starvation, and final removal rates did not differ between reactors inoculated with immobilized and suspended cells. Management of assimilable organic carbon, in addition to cell immobilization, appears crucial for guaranteeing long-term BAM degradation activity of MSH1 in DWTP units.

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

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.

The T7-related Pseudomonas putida phage φ15 displays virion-associated biofilm degradation properties.

Formation of a protected biofilm environment is recognized as one of the major causes of the increasing antibiotic resistance development and emphasizes the need to develop alternative antibacterial strategies, like phage therapy. This study investigates the in vitro degradation of single-species Pseudomonas putida biofilms, PpG1 and RD5PR2, by the novel phage ϕ15, a 'T7-like virus' with a virion-associated exopolysaccharide (EPS) depolymerase. Phage ϕ15 forms plaques surrounded by growing opaque halo zones, indicative for EPS degradation, on seven out of 53 P. putida strains. The absence of haloes on infection resistant strains suggests that the EPS probably act as a primary bacterial receptor for phage infection. Independent of bacterial strain or biofilm age, a time and dose dependent response of ϕ15-mediated biofilm degradation was observed with generally a maximum biofilm degradation 8 h after addition of the higher phage doses (10(4) and 10(6) pfu) and resistance development after 24 h. Biofilm age, an in vivo very variable parameter, reduced markedly phage-mediated degradation of PpG1 biofilms, while degradation of RD5PR2 biofilms and ϕ15 amplification were unaffected. Killing of the planktonic culture occurred in parallel with but was always more pronounced than biofilm degradation, accentuating the need for evaluating phages for therapeutic purposes in biofilm conditions. EPS degrading activity of recombinantly expressed viral tail spike was confirmed by capsule staining. These data suggests that the addition of high initial titers of specifically selected phages with a proper EPS depolymerase are crucial criteria in the development of phage therapy.