Exogenous Plasmid Isolation

Plasmids were captured from bacteria within the biosolids through a biparental mating protocol (Smalla et al., 2000) that was modified by using multiple antibiotic disks on one large Petri dish. Recipient strains were grown overnight with antibiotic selection (Table 1, see column “Antibiotic Resistance”). About 1 ml of the culture was harvested by centrifugation at 8,000 g for 3 min. The pellets were washed two times through repeated resuspension in 1 ml of phosphate-buffered saline (PBS, pH 7.4) and centrifugation at 8,000 g for 3 min. Biosolids were suspended in sterile water at a concentration of 0.5 g ml⁻¹ and vortexed thoroughly. To each of the washed bacterial samples, 500 μl of the biosolid suspension was added. The mixtures were vortexed, centrifuged as specified above, and the pellet resuspended in 100 μl of TSB that was diluted 10-fold in sterile water (0.1 TSB). Each mixture was then pipetted individually onto sterile 0.45 μm filters placed on 3-fold diluted TSB supplemented with 15 g/L of agar and 100 μg ml⁻¹ cycloheximide and incubated overnight. Bacteria were then resuspended by vortexing the filters in 500 μl of PBS. Using a cotton swab, the cell suspensions were spread uniformly on Mueller Hinton Agar (MHA; Becton Dickinson) in 15-cm diameter Petri dishes supplemented with 100 μg ml⁻¹ cycloheximide and antibiotics to select for the recipient. Subsequently up to 10 antibiotic disks were placed on the agar (Table 1). Cycloheximide was added to limit fungal growth. After overnight incubation, colonies growing inside the zones of inhibition of each antibiotic disk were selected as potential transconjugants, i.e., recipients that have obtained a plasmid conferring resistance to the antibiotic in the respective disk. Each transconjugant was archived in 20% glycerol stocks at −70°C. Acquisition of a plasmid by the recipient was confirmed by comparing the plasmid profiles of the transconjugant with their plasmid-free counterparts on an agarose gel (0.7% w/v) electrophoresis. The plasmid DNA extraction protocol was adapted from (Sambrook and Russell, 2001) with the following modifications: Alkaline Lysis Solution I had 50 mM Tris-Cl (pH 8.0), 2 mg ml⁻¹ of lysozyme, and RNase A. The identity of the transconjugant was confirmed by amplifying the 16S rRNA genes using the universal primers 27f and 1492r (Lane, 1991) and the purified amplicons were sent to Elim Biopharm, Inc. (CA, United States) for Sanger sequencing. Details about the PCR methods can be found in the Supplementary Materials and Methods.