title: Genetic (In)stability of 2,6-Dichlorobenzamide Catabolism in Aminobacter sp. Strain MSH1 Biofilms under Carbon Starvation Conditions.
abstract: Aminobactersp. 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, carryingbbdA, 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.IMPORTANCEBioaugmentation 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 thatAminobactersp. 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.
authors.affiliation: Division of Soil and Water Management, Department of Earth and Environmental Sciences, Faculty of Bioscience Engineering, KU Leuven, Heverlee, Belgium Benjamin.horemans@kuleuven.be.
authors.forename: Benjamin
authors.identifier: http://orcid.org/0000-0001-6980-3715
authors.initials: B
authors.lastname: Horemans
authors.affiliation: Division of Soil and Water Management, Department of Earth and Environmental Sciences, Faculty of Bioscience Engineering, KU Leuven, Heverlee, Belgium.
authors.forename: Bart
authors.initials: B
authors.lastname: Raes
authors.affiliation: Division of Soil and Water Management, Department of Earth and Environmental Sciences, Faculty of Bioscience Engineering, KU Leuven, Heverlee, Belgium.
authors.forename: Hannelore
authors.initials: H
authors.lastname: Brocatus
authors.affiliation: Division of Soil and Water Management, Department of Earth and Environmental Sciences, Faculty of Bioscience Engineering, KU Leuven, Heverlee, Belgium.
authors.forename: Jeroen
authors.initials: J
authors.lastname: T'Syen
authors.affiliation: Faculty of Veterinary Medicine, Department of Veterinary Public Health and Food Safety, Laboratory of Chemical Analysis, UGent, Merelbeke, Belgium.
authors.forename: Caroline
authors.initials: C
authors.lastname: Rombouts
authors.affiliation: Faculty of Veterinary Medicine, Department of Veterinary Public Health and Food Safety, Laboratory of Chemical Analysis, UGent, Merelbeke, Belgium.
authors.forename: Lynn
authors.initials: L
authors.lastname: Vanhaecke
authors.affiliation: Molecular Imaging and Photonics, KU Leuven, Leuven, Belgium.
authors.forename: Johan
authors.initials: J
authors.lastname: Hofkens
authors.affiliation: Division of Soil and Water Management, Department of Earth and Environmental Sciences, Faculty of Bioscience Engineering, KU Leuven, Heverlee, Belgium.
authors.forename: Dirk
authors.initials: D
authors.lastname: Springael
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001549:Benzamides / Q000378:metabolism*; D018441:Biofilms; D002244:Carbon / Q000378:metabolism; D042822:Genomic Instability; D059127:Phyllobacteriaceae / Q000235:genetics* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D001549:Benzamides; D002244:Carbon; C057454:2,6-dichlorobenzamide
keywords: 2,6-dichlorobenzamide; Aminobacter sp. strain MSH1; catabolic instability; micropollutant; starvation conditions