Based on analysis of 43 colonies resistant to both spectinomycin and kanamycin, Selleckchem Vismodegib similar results were obtained using strain serotype M1 strain as the recipient strain (MGAS2221ΔcovRS, resistant to kanamycin). Figure 2 RD2 encodes homologues of conjugative transfer genes present in the ICE St1 and ICE St3 elements of S. thermophilus. Figure 3 Detection of RD2 transfer from donor strain MGAS6180 ( emm28 ) to recipient strain MGAS10750 ( emm4 ). Amplicons 1-12 generated by PCR tiling across the RD2 element. A. transconjugant; * denote amplicons encompassing deleted M28_Spy1325-1326 region that is replaced by spectinomycin resistance cassette; B. control with chromosomal DNA isolated from strain MGAS6180. M -

1 kb ladder (Invitrogen) RD2 is present in multiple, likely

extrachromosomal, copies in GAS Many gene transfer processes, including conjugation, require circular form of the transferred molecule or that more than one copy of the element exists during at least one point in the transfer cycle [20–22]. Therefore, we tested the hypothesis that multiple copies of the RD2 are present in the bacterial cell. PCR primers were used that allow detection of a circular form of RD2, and permit assessment of the orientation of chromosomal integration of multiple copies of RD2 (Figure 4A). Primers #1 and #4 recognize chromosomal sequences, whereas primers MAPK Inhibitor Library molecular weight #2 and #3 recognize RD2 element sequences. Depending on the direction and/or arrangement of multiple copies of RD2 (i.e., head-to-head, tail-to-tail, head-to-tail), the different primer combinations would yield distinct amplicons. Based on the

genome sequence of strain MGAS6180 [1] primer pairs #1-#2 and #3-#4 would Progesterone amplify the junction region between the chromosome and RD2 on the left and right flank, respectively (positive control reactions). Using total DNA isolated from an overnight culture of MGAS6180 as template, PCR analysis yielded products amplified with primers #1-#2 and #3-#4, as expected. However, we also observed that primers #2 and #3 amplified a product, a result suggesting the presence of either multiple integrated copies of RD2 or a circular form of RD2 (Figure 4B). Next, we analyzed nine other GAS strains of multiple M protein serotypes using primers #2-#3 to determine if this was a general phenomenon. Regardless of emm type, all RD2-positive strains yielded an amplicon with the primer #2-#3 combination whereas RD2-negative organism did not (Figure 4C). Further, DNA sequence analysis revealed that all PCR amplicons generated with primers #2-#3 contained the sequence CGGTGGTGGCA, corresponding to a junction between the left and right flanking regions of RD2 (Figure 4). Figure 4 PCR screen detects multiple or circular copy of RD2. A. Primer combinations used for detection of seven potential arrangements of RD2. Thick black arrows represent RD2 element; thin gray line represents the chromosome.