0 mL saline, diluted 105-fold, learn more spread on MRS agar plates, and incubated overnight at 37 °C. The cells were then overlaid with 3 mL soft (0.75%) agar medium inoculated with 30 μL culture of the indicator strain (at c. l06 CFU mL−1). The plates

were then incubated again overnight at 37 °C and colonies with no clear zone surrounding them were randomly selected, isolated, purified, and tested for the presence of plasmids. Total DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega, Madison) and plasmid DNA using the ‘Qiagen plasmid kit’ (Diagen, Dusseldorf, Germany) according to the manufacturers’ instructions (the latter preparation is henceforth called ‘crude plasmid DNA’). When specified, a single plasmid band was excised and cleaned according to instructions of the Silica Bead DNA gel extraction kit (Fermentas, Dusseldorf, Germany) and used for further study (such preparations are henceforth called ‘gel-purified

plasmid DNA’). First, a digoxigenin (DIG)-labelled probe corresponding to part of the SppA gene of strain wt was generated using the PCR DIG Probe Synthesis kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Total wt DNA was used as a template and the primers were SakPfw (5′-GAA (T/A)T(A/G)(C/A)(C/A)A NCA ATT A(C/T)(A/C) GGT GG-3′) and SakPrev (5′-GGC CCA GTT TGC AGC TGC AT-3′), based on the SppA sequence deposited in the GenBank database. Southern blotting was then performed on restriction fragments of gel-purified plasmid from wt (the products find more of separate HindIII, CfoI, and EcoRI digestions were run in parallel).

Restriction mixtures were electrophoresed through a 0.8% agarose gel, along with the 500-bp marker (Biorad) and the Big Dye Marker (Roche, Penzberg, many Germany). The resulting bands were blotted onto a Hybond N+nylon membrane and allowed to hybridize with the DIG-labelled probe for 20 h at 65 °C. Chemiluminescence detection was performed using the DIG-DNA kit (Boehringer, Mannheim, Germany) according to the manufacturer’s instructions. The plasmid location of the SppA gene was confirmed by subjecting gel-purified plasmid DNA to PCR with Taq DNA polymerase (Applied Biosystems, Milan, Italy). The reaction mixture (final volume: 25 μL, placed in a 0.2-mL Eppendorf tube) contained 10 × Taq Buffer, 1.5 mM MgCl2, 0.2 mM dNTP, each primer at 0.5 μM, 5 U μL−1 Taq DNA polymerase (Applied Biosystems), and 25 μg mL−1 DNA in sterile milli-Q water. Amplification was carried out in a Mastercycler Personal thermocycler (Eppendorf, Pecq, France). The heating/cooling program was as follows: a first cycle at 94 °C for 2 min, 55 °C for 1 min, 72 °C for 1 min, followed by 32 cycles of 94 °C for 1 min, 50 °C for 45 s, and 72 °C for 1 min. The target amplicon was detected with the DIG-labelled probe. To electroporate LMG with the wt-derived plasmid, the method described by Kim et al.