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.

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.

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.

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.

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 a sole source of carbon, nitrogen and energy and is considered a prime biocatalyst for groundwater bioremediation in DWTPs. Its use in bioremediation requires knowledge on its BAM-catabolic pathway which is currently restricted to the amidase BbdA converting BAM into 2,6-dichlorobenzoic acid (2,6-DCBA) and the mono-oxygenase BbdD transforming 2,6-DCBA in 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 catalyses a second hydroxylation forming 2,6-dichloro-3,5-dihydroxybenzoic acid. Subsequently, glutathione-dependent dehalogenases (BbdI and BbdE) catalyse the thiolytic removal of a first chlorine. The remaining chlorine is then removed hydrolytically by a dehalogenase of the / 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 / 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.