Sterile nitrocellulose filters

Transfer of pp3 was performed as previously described with modifications [33 (link)]. Briefly, cells from log-phase cultures of donor and recipient were washed and resuspended in LB broth to a final concentration of 1×10⁸ cfu/ml and 3×10⁸ cfu/ml, respectively. 100 μl of each suspension (representing a 1:3 ratio of donor:recipient) were mixed, placed on sterile nitrocellulose filters (0.45 μm, Millipore), and incubated at 37°C for 4 h. Cells were collected by washing the filters in 1 ml of LB medium. Transconjugants were selected on LB agar plates containing gentamicin (50 μg/ml) and tetracycline (150 μg/ml). Transfer efficiency was calculated using the total number of transconjugants divided by the total recipients in the mating mixture. To assess whether transformation is responsible for pp3 interstrain transfer, the mating mixture was supplemented with 10 mM MgCl₂, 2 μg/ml BSA and 100 μg/ml DNaseI.

Mobilization assays of pMV158, pMVS and pMVSag from S. pneumoniae 708 donor colony-forming units (CFU) harbouring pAMβ1 (as the auxiliary plasmid) and S. pneumoniae MP3008 (resistant to novobiocin) as recipients were performed as previously described [3 (link),38 (link)]. Donor and recipient cultures were grown at 37°C to 5 × 10⁸ cells ml⁻¹, mixed at a ratio of 1 (donor) to 2 (recipient), and passed through sterile nitrocellulose filters (0.22 μm pore size; Millipore, Bedford, MA). The filters were placed upside down on another filter, and placed on a plate with conjugation medium (AGCH with 10 mM MgCl₂, 0.2% albumin, 0.2% glucose, 2% agar and 5 µg ml⁻¹ DNase I). After 4 h of incubation at 37°C, the cells were recovered and transconjugants and recipients were selected by plating on AGCH supplemented with the appropriate antibiotics. Five independent experiments were performed, and mobilization efficiencies were calculated as the number of transconjugants per recipient CFU.