RFLP Analysis of the BIR, blpC Sequencing and Detection of the blpA 4 bp Repeat

The DNA fragments of BIR were PCR amplified using primers 1 and 2, which bind to the highly conserved regions within blpA and blpY genes, respectively. PCR products were purified and the product was digested with restriction enzyme AseI. The size pattern of each isolate was compared to the predicted patterns from the known distinct BIR regions from the sequence database (Table S2). Isolates with identical patterns were assigned to the same group. We confirmed the absence of a blpA repeat or deletion in all strains with pheromone signaling as follows: a 761 bp region of blpA was amplified with primers 3 and 4. The product was digested with enzyme Cac8I, which will only cleave a product containing the common frame shifting 4 bp repeat (AAGC) in the blpA gene. The DNA fragment containing a frame shift repeat would separate into two pieces of fragments while the wild type sequence would remain intact as a single piece fragment.

Construction of deletion mutant strains can be found in the Supplemental Material.