The TOL plasmid, originally isolated from P. putida strain mt-2, is one of the best-studied catabolic plasmids belonging to the IncP-9 group, encoding biodegradation pathways for toluenes and xylenes (Williams & Murray, 1974). The TOL plasmid can be transferred to other pseudomonads and has earlier been reported as derepressed for transfer (Benson & Shapiro, 1978; Bradley & Willams, 1982; Ramos-Gonzalez et al., 1991). During earlier studies, we had observed a stimulatory effect of TOL plasmid carriage on biofilm formation in Metformin solubility dmso P. putida
KT2440 at the air–water interface (Arango Pinedo et al., 2003). Here, we provide quantitative support for this biofilm enhancement at both the air–liquid and the liquid–solid interface and show that extracellular DNA (eDNA) may be responsible for the plasmid-stimulated interfacial growth. Pseudomonas putida KT2440 is a plasmid-free, restriction-deficient derivative of P. putida mt-2 (Bagdasarian et al., 1981). TOL is the archetypical catabolic plasmid pWWO (Williams & Murray, 1974). The TOL-free strain was chromosomally tagged with a miniTn5-Plac-gfpmut3b-kanR cassette (Normander et al., 1998), the TOL plasmid was tagged with a miniTn5-Plac-gfpmut3b-tetR cassette as per Christensen et al. (1996), and carried in a
wild-type KT2440 host. Strains were cultured in AB medium [15.1 mM (NH4)2SO4, 42.2 mM Na2HPO4, 22 mM KH2PO4, 51.3 mM NaCl, selleck chemicals llc 100 μM MgCl2, 10 μM CaCl2, 1 μM FeCl3, Clark & Maaløe, 1967] supplemented with 1 mM (flow cells) or 40 mM (static cultures) sodium citrate. Solid media were prepared by adding 20 g L−1 agar to AB medium. All cultivations were at 25 °C, unless observed otherwise. Antibiotics were added to all precultures to a final concentration of 50 μg mL−1. Static cultures were performed in replicate 500-mL Erlenmeyer flasks (70 mL medium) for bulk measurements, EPS extractions, and viscosity measurements or in 20 mL test tubes (5 mL medium) for microscopy, flow cytometry, and β-glucosidase assays. To test DNase effect, duplicate cultures were supplemented with DNaseI (Qiagen, 20 U mL−1), magnesium chloride (250 μM), and calcium Ergoloid chloride (4 μM). Pellicles were sampled with a
10-μL inoculation loop, or tweezers for very eDNA-rich pellicles, applied to a microscope slide, and stained with PI (15 μL, 10 μg mL−1) or Cytox Orange before viewing. Flow cell biofilms were established in three-channel flow cell setups, as described before (Møller et al., 1996). Flow cells were inoculated by adjusting the OD600 nm of precultures to 1.0, washing and resuspending the cells in 0.9% NaCl, and injecting 300 μL of this suspension into each channel with an insulin syringe. AB medium was continuously supplied by a peristaltic pump (Watson & Marlow 205S) to each channel at a rate of 2.7 mL h−1. After 2 and 7 days of incubation, three-dimensional image stacks of the biofilms (3 z-stacks from three replicate channels in duplicate flow cells for each strain) were recorded by confocal laser scanning microscopy (CLSM).