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

Abstract

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 ϩ phenotype), and plasmid pBAM2, carrying gene clusters encoding the conversion of 2,6-DCBA to tricarboxylic acid (TCA) cycle intermediates (Dcba ϩ 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 ϩ 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 ϩ Dcba Ϫ 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.

PromA Plasmids Are Instrumental in the Dissemination of Linuron Catabolic Genes Between Different Genera

“…The mobile elements associated with these catabolic clusters seem to be unstable, and are lost from most of the PromA plasmid copies in both populations. The loss of catabolic clusters from plasmids, both in the presence and absence of other C sources than the xenobiotic compound, was shown in various studies (Takahashi et al, 2009;Changey et al, 2011;Horemans et al, 2017). In case of plasmid pADP1 from Pseudomonas huttiensis, it was observed that the loss of a 47 kbp upstream atrazine catabolic region via an ISPps1-mediated homologous recombination event led to an overall fitness increase in the host when growing on the atrazine downstream degradation product cyanuric acid.…”

Isolation, identification and selection of bacteria with the proof-of-concept for bioaugmentation of whitewater from woodfree paper mills

“…A, Section A.1). Stable catabolism of the four selected isolates, probably developed under nutrient-poor conditions during isolation (Horemans et al, 2017b), was confirmed with an 88% decrease in COD even on day 21 of the pilot experiment. The coexistence tests that gave the best combination of 4 bacteria (Suppl.…”

“…In preparing the consortium, we pursued specific aims (Fig. 1): (i) Isolation of individual strains capable of degrading a range of papermaking chemicals; to isolate bacteria with stable catabolism at low nutrient concentrations, we performed bacterial isolation under nutrient-poor conditions (Horemans et al, 2017b), (ii) construction of the most suitable inoculum; we prepared different combinations of active bacteria in synthetic and real whitewater to select the combination with the broadest repertoire of carbon source use, and (iii) upscaling the use of the prepared inoculum by applying an immobilisation strategy.…”

Biotransformation of insecticide flonicamid by Aminobacter sp. CGMCC 1.17253 via nitrile hydratase catalysed hydration pathway

“…MSH1 can mineralize 2,6‐dichlorobenzamide, a major organic pollutant in groundwater, through the metabolic pathway of chlorobenzoic acid (Horemans et al . 2017). TFNG‐AM is the product of flonicamid transformed through hydration pathway, A. faecalis CGMCC 17553 could transform flonicamid into TFNG‐AM through hydration pathway mediated by nitrilase NitA, (Yang et al .…”