Mobilization assays were performed as previously described (Lorenzo-Diaz and Espinosa, 2009a,b) , with minor modifications. Briefly, cultures of donor and recipient cells were grown overnight at 37°C with shaking in 5 ml of THY containing the appropriate antibiotics. Cells were centrifuged and washed to remove the antibiotics, and then diluted 1:10 in pre-warmed fresh THY medium to ensure that cultures were in exponential phase during the matings. Two hundred microlitres of donor culture and 1 ml of recipient culture (1:5 ratio) were mixed in a 1.5 ml Eppendorf tube and spun for 2 min in a microfuge at 14 000 r.p.m. to pellet the cells. The pellet was resuspended in 50 ml pre-warmed THY supplemented with 10 mM MgCl2, 2 mg ml -1 BSA and 100 U DNase I. The cell mixtures were placed on sterile nitrocellulose filters (0.45 mm, Millipore) and incubated at 37°C for 6 h. Cells were removed by washing the filters in 1 ml of THY medium. Transconjugants were selected on THY medium with spectinomycin and chloramphenicol. Conjugative transfer frequencies were calculated as the number of transconjugant cells per donor. To confirm the occurrence of transfer, 10 transconjugants from each mating were randomly selected for PCR analysis with specific primers.