(A) Diagram of the rpoS locus (bb0771) and adjacent ORFs. The binding sites of six primers, i.e. P1F to P3F and P1R to P3R, are also indicated. (B) Diagram of the disrupted rpoS locus, showing a major portion of rpoS gene is replaced with the aacC1 cassette (grey arrow). The small arrow points a residual portion of rpoS. The binding sites of primers P4F, P5F, P5R and P4R are also indicated. (C&D) PCR analysis of rpoS mutant. The 13A spirochetes, the disruption plasmid pSKO, and ΔrpoS were used as DNA sources and subjected to PCR amplification using the primers P3F and P3R producing an amplicon of 111 bps (C), and the primers P5F and P5R generating an amplicon of 517 bps (D).