Genomic mini

Bacterial genomic DNA was extracted using a commercial kit (Genomic Mini, A&A Biotechnology, Gdańsk, Poland). First, 1.5 mL of log growth phase bacterial culture cultivated in brain-heart infusion medium (BHI) was applied to Eppendorf probes and centrifugated for 3 min at 13,000 RPM. The supernatant was discarded, and the sediment was suspended in 100 μL of Tris buffer. During the next stage, 10 μL of lysostaphin (1 U/μL concentration, Sigma-Aldrich) was added and the probes were incubated at 37 °C for 30 min. Then, 200 μL of LT lysing solution and 20 μL of proteinase K were subsequently added, and the probes were incubated for 20 minutes at 37 °C followed by 5 min at 70 °C. After the incubation, the probes were intensively vortexed for 20 s and centrifugated for 3 min at 13,000 RPM. The obtained supernatant was transferred to the DNA purification minicolumns and centrifugated for 1 min at 13,000 RPM. After rinsing with A1 solution twice, the minicolumns were transferred to new sterile 1.5 mL Eppendorf probes. Next, 100 uL of Tris buffer heated up to 75 °C were applied to the deposit and incubated for 5 min at room temperature, after which the minicolumns were centrifugated at 13,000 RPM. Following centrifugation, the minicolumns were discarded, and the genomic DNA obtained in the Eppendorf tube was stored at −20 °C until further analysis.

Standard DNA manipulation methods were performed as described by Sambrook and Russell (2001) . Total DNA was extracted using a kit (Genomic Mini, A&A Biotechnology) from bacterial cells harvested by centrifugation of an overnight culture carried out in LB medium. Purified DNA was used as the template for genome sequencing. Plasmid DNA was isolated analogously using a kit (Plasmid Mini, A&A Biotechnology) or by CsCl gradient method (Sambrook and Russell, 2001 ).

Genomic and plasmid DNA were isolated using Genomic Mini (A&A Biotechnology, Gdynia, Poland) and Plasmid Mini (A&A Biotechnology, Poland) kits, respectively, according to the manufacturer’s protocols.