Figure 1. Trans-complementation of an rrp2 point mutant using a shuttle plasmid.

(A) Construction of an IPTG-inducible rrp2 expression shuttle plasmid. To create pRrp2, rrp2 was amplified from B. burgdorferi and cloned into pJSB275. The plasmid pRrp2 was then introduced into strain OY01 (rrp2[G239C]), yielding OY160. SDS-PAGE (B) and immunoblot (C) were performed to analyze gene expression in OY160. Bacteria were grown at 37°C in BSK-II medium with various concentrations of IPTG. When bacterial growth reached ∼10⁸ cells per ml, spirochetes were harvested. Approximately 4×10⁷ spirochetes were loaded onto each lane of a 12.5% SDS-PAGE gel. In (B), approximate molecular masses are indicated at the left in kDa; concentrations of IPTG are indicated above the image; and the arrow indicates OspC. Specific antibodies, denoted as α- used in the immunoblot (C), are indicated on the left.