Figure 2.

Generation of B. turicatae btpA mutants. (A) Organization of btpA::PflgB-aacC1 mutant. A segment of btpA in wild-type B. turicatae (WT) was replaced with the aacC1 gentamicin resistance marker expressed from the B. turicatae flgB promoter (btpA::PflgB-aacC1) via allelic exchange. (B) Organization of btpA::aacC1 mutant. A segment of btpA in wild-type B. turicatae (WT) was replaced with a promoterless aacC1 gentamicin resistance marker via allelic exchange. (C) Genotypic confirmation of btpA mutants. PCRs were conducted to amplify a region flanking the insertion site of the aacC1 marker in btpA (P1+P2; WT, 1,649 bp; btpA::PflgB-aacC1, 1,319 bp; or btpA::aacC1, 745 bp) or internal segments of btpA (P3+P4; 305 bp), aacC1 (689 bp), and flaB (519 bp). PCRs were conducted using genomic DNA (gDNA) from wild-type B. turicatae (WT), btpA::PflgB-aacC1, and btpA::aacC1, as well as the plasmids used to generate the mutants. PCRs were also conducted with no DNA as a purity control (H₂O). MW denotes the DNA standard, and numbers to the left indicate molecular weights in kb. (D) Transcriptional confirmation of btpA mutants. RNA was isolated from wild-type B. turicatae (WT), btpA::PflgB-aacC1, and btpA::aacC1 culture and converted to cDNA (+). Mock reactions without reverse transcriptase were performed as a control (–). Wild-type B. turicatae genomic DNA (gDNA) was included as a positive control for each reaction. PCRs were conducted with cDNA to amplify internal regions of bt0790 (450 bp), btpA (305 bp), bt0791 (505 bp), and flaB (519 bp). MW denotes the DNA standard, and numbers to the left indicate molecular weights in kb.