The regions in S. agalactiae genomes homologous to genes M28_Spy1303- M28_Spy1325 are located in majority of analyzed GBS strains within single chromosomal location, while genes M28_Spy1326-M28_Spy1337

are located in other chromosomal location or locations (Figure 1 and Additional File 6, Table S3). Figure 1 Comparison of the organization of RD2 ORFs in diverse GAS and GBS strains. Slanted red lines indicate discontinuity between fragments homologous to RD2 element present in MGAS6180. RD2 open reading frames are marked as white rectangles, open reading frames of GBS are color coded according to their homology to RD2 on the protein level. Numbers within each rectangle represent ORF designation ISRIB clinical trial number in particular GBS strain

RD2 encodes a putative conjugation module Based on DNA sequence analysis, RD2 does not appear to encode genes involved in replication as a circular plasmid. GAS is not considered to be naturally TPX-0005 mouse transformable under standard laboratory growth conditions, suggesting that other mechanisms must be used to transfer RD2-related genes between cells. DNA sequence analysis identified a putative transfer module encoded by RD2 with similarity to the ICESt1 and ICESt3 conjugation modules present in Streptococcus thermophilus (Figure 2) [18]. Thus, we hypothesized that RD2 uses a conjugation-like mechanism to transfer from donor to recipient strains. To test this hypothesis, we

performed filter mating using donor strain MGAS6180Δ1325-1326spcR, which contains a spectinomycin resistance cassette integrated into the chromosomal copy of RD2. The recipient strain used was strain MGAS10750, a type emm4 organism that is naturally OSI-744 supplier resistant to erythromycin. After filter mating of strain MGAS6180Δ1325-1326spcR and RANTES strain MGAS10750 for 3 h, 6 h, and 16 h, we obtained 1, 3, and 202 colonies, respectively (transfer frequency ~10-6 of transconjugants per donor cell), which were resistant to both antibiotics (spectinomycin 150 μg/ml and erythromycin 1 μg/ml). Eight putative transconjugant colonies were tested for the presence of the RD2 element and characterized for the emm gene sequence. In group A Streptococcus, emm gene is highly polymorphic in sequence and encodes for major surface protein M that is responsible for GAS serotype. Amplification of hyper-variable region of emm gene with primers CDCemm1 and CDCemm2 yields products that differ in size depending on the M serotype [19]. RD2 positive transconjugants were first screened based on the emm amplicon size (data not shown), and the amplified product was sequenced to confirm that transconjugants belong to M4 serotype, the same as the recipient. Successful transfer of the RD2 element from the emm28 strain to the emm4 strain was confirmed by PCR tiling across the entire RD2 element (Figure 3).