, 2006; Madsen et al, 2012) Complex interspecies communities

, 2006; Madsen et al., 2012). Complex interspecies communities see more facilitate synergistic interactions between populations, affecting the function, stability and flexibility of the community (James et al., 1995; Burmølle et al., 2006). In the present work, HTG by conjugation between single populations and microbial

communities isolated from soil were investigated. The plasmid transfer frequencies and the identities of the recipients of the plasmid, when hosted by different donors, were compared. The bacterial population was analyzed based on fluorescence properties and sorted by flow cytometry (FCM) to detect and quantify the plasmid transfer to the individual isolates and the mixed community (Muller & Nebe-von-Caron, 2010). Sequencing of the 16S rRNA gene from sorted transconjugant cells was used to evaluate the host range of the plasmid when a mixed microbial community was used as recipient. Soil samples were collected from an agricultural field in Tåstrup, Denmark, in the late summer of 2009. Soil was sampled from the 5- to 10-cm layer. The soil water content upon sampling was 14.2%, and the water holding capacity (WHC) was 60%. The soil was

classified as sandy loam with pH 7.2. Leaves of baby maize seedlings were used for bacterial isolation. The seedlings were grown for 2 weeks in Tåstrup soil before harvesting. Escherichia coli CSH26::lacIq and Pseudomonas putida KT2440::lacIq1, carrying pKJK10, a conjugative, green fluorescent protein (GFP) tagged IncP1 plasmid, originally isolated from soil (Sengeløv et al., 2001; Bahl et al., RAD001 in vivo 2007) were used as donor

strains. These strains were cultured in Luria Bertani (LB) broth supplemented with kanamycin monosulfate (50 mg mL−1); 1.5% (w/v) agar was added when solid medium was needed. The recipient strains (see below) were cultured in Tryptic Soy Broth medium (TSB; 17 g peptone from casein, 3 g peptone from soymeal, 2.5 g d(+)-Glucose, 5 g NaCl, 2.5 g K2HPO4 in 1 L distilled water, pH 7.3). A 15 mg sample of a baby maize leaf was placed in 5 g Tåstrup soil adjusted to 40% WHC and incubated in triplicate at room temperature for 17 days. After 7, 12, only and 17 days, the leaves were picked up from the soil, washed with PBS (8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and KH2PO4, adjusted to 1 L distilled water and pH 7.4), placed in a microfuge tube, added 1 mL PBS and vortexed for 1 min. DNA was extracted from the cell suspension as described below. Dilutions to 10−6 were made and 100 μL were plated in triplicate onto Tryptic Soy Agar (TSA; Difco) 10% supplemented with cycloheximide (50 mg mL−1) and incubated at 25 °C for 2–5 days. Sixteen colonies from each triplicate looking phenotypically different were isolated and purified for DNA extraction.

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