The φX216 scrnA and scrnB probes are specific to φX216/φ52237 and amplify DNA fragments from φX216 gene #46 and from the intergenic region between φX216 genes #30 and #31, respectively. The GI2 (Genomic island 2) probe amplifies the junction between the bacterial and prophage genomes at tRNA-Phe, predicted to serve as the attB site for Burkholderia
subgroup A phages [8, 9]. We found that P2-like prophages are very common in B. pseudomallei strains (Table 1). Indeed, PCR analysis revealed that 30 out of 72 B. pseudomallei strains tested allowed amplification of DNA fragments indicative of the presence of a P2-like prophage (see Figure 3 for representative examples). Of those 30, 25 tested positive for subgroup A prophages. Six of Small molecule library cost those, including E0237, selleck inhibitor produced PCR results indicative of a close relationship with φ52237/φX216. B. pseudomallei 1710b, K96243, S13 and 1026b each produced PCR results that match sequence-based predictions for the presence of prophages [7, 8, 15]. Whereas strain 1710b is Selleck ��-Nicotinamide negative for a P2-like prophage, K96243 and S13 are both positive for subgroup A prophages (Table 1). Furthermore,
1026b is predicted to carry a φ52237-like prophage that is split into two fragments located in different regions of chromosome I (GenBank:CP002833.1, Locus # BP1026B_I0126- I0172 and BP1026B_ I3339-I3345). It is important to note that a positive hit for a subgroup A prophage does not exclude the possibility
of a strain possessing multiple subgroup A prophages or more distantly related P2-like prophages. For instance, B. pseudomallei K96243 encodes both the φK96243 subgroup A prophage in genomic island 2, as well as the predicted subgroup B prophage GI15 on chromosome II, but the subgroup A PCR results hide the presence of the subgroup B GI15 prophage due to the fact that the GI15 probe amplicons are identical in size to those from the φK96243 prophage. The PCR probe results also do not indicate whether the candidate prophages can release viable phage progeny or are defective, as observed with the 1026b split φ52237-like prophage. The 30 strains that Avelestat (AZD9668) produced positive hits for P2-like prophages were additionally screened with the GI2 PCR probe. Strain 1710b was used as a P2-like-minus negative control. The 25 subgroup A candidate strains all produced positive PCR results for prophage integration into the 3’ end of the tRNA-Phe gene resulting in the formation of genomic island 2. The five candidates that failed to produce a positive GI2 PCR result were categorized as P2-like only. While our results do not definitively identify these five P2-like candidates as subgroup B members, subgroup B phages are predicted to use a different attB site and integration mechanism . Table 1 B. pseudomallei P2-like prophage distribution screen P2-like prophage PCR probe results Multiplex B.