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 (104 and 106 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

Genetic characterization of 2,6-dichlorobenzamide (BAM) degradation 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

Generic (in)stability of 2,6-dichlorobenzamide (BAM)-catanolism in Aminobacter sp. MSH1 biofilms under carbon starved 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.status: publishe

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

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-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.status: publishe

Aminobacter sp. MSH1 mineralises 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/β 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/β 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.status: publishe

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.status: publishe

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-1β 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.status: publishe