Dna mini preparation kit

For the determination of the chromosomal integration sites (attB) of P282, genomic DNA (gDNA) of various lysogens was isolated using the DNA mini preparation kit (Qiagen, Hilden, Germany). DNAs were digested with the restriction endonucleases Eco32I, HindIII, and XbaI according to the manufacturer’s recommendations (Thermo, St. Leon Roth, Germany). Following digestion, restriction fragments were treated with T4 ligase (Thermo) and used as template for PCR. Outward primers deduced from the 5′- and 3′-coding region of the P282 int gene were used under the following conditions: Initial PCR activation and template denaturation at 96 °C for 120 s followed by 35 cycles including denaturation phase at 96 °C for 15 s, annealing at 55 °C for 5 min and elongation for 210 s at 72 °C. In addition, a final elongation step at 72 °C for 1 min was included, before the amplicons were purified (QIAquick PCR purification kit, Qiagen) and sequenced according to Sanger (Eurofins Genomics,). The obtained nucleotide sequences of the amplicons were compared with whole genome sequences of S. aureus strains of the GenBank database (National Center for Biotechnology Information).

To determine the chromosomal integration site of the phage, gDNA of B. inopinata BO1 and B. abortus S19lysBiPBO1 was isolated using the DNA mini preparation kit (Qiagen, Hilden, Germany). The DNAs were digested with several restriction endonucleases (Bsp143I, DpnI, DraI, Eco32I, HindIII) according to the manufacturer's recommendations (Fermentas, St. Leon Roth, Germany). Restriction fragments of each digest were treated with T4 ligase (Fermentas) and used as template in a PCR reaction applying outward facing primers deduced from the coding region of the BiPBO1 integrase gene (Table S5). PCR products were purified (QIAquick PCR purification kit, Qiagen) and sequenced. The obtained nucleotide sequences of the amplicons were compared with whole genome sequences of the respective strains. To study the BiPB01 integration site in other Brucella strains, a Multiplex-PCR was developed. Primers deduced from the Brucella chromosome left and right of the BiPB01 integration site provided information on the presence of foreign DNA at this position. Combinations of these primers with primers deduced from the BiPB01 genome were used to detect BiPB01-related prophage DNA. All primers are listed in Supplementary Material Table S5.