FIG 1.

Expression of TraK and TraB-FLAG in N. gonorrhoeae. (A) Western blot with the 2E5 TraK monoclonal antibody using lysates from gonococcal strains MR535 (ΔtraK), MS11 (WT), and MR537 (traK expressed from an IPTG-inducible promoter in the WT background [traK⁺]). This Western blot using the 2E5 anti-TraK antibody is representative of those obtained with the 3H4 and 3B8 anti-TraK monoclonal antibodies. The black triangle indicates TraK. Approximately 16.5 μg of protein from cell lysates was subjected to SDS-PAGE. (B) Detection of traK and rpoB mRNA by two-step RT-PCR. Total RNA was isolated from gonococcal strains ND500 (ΔGGI), MS11, and MR537. Reverse transcription reactions were carried out in the presence (+RT) or absence (−RT) of reverse transcriptase and amplified with primers to detect traK and rpoB. L, nucleotide ladder in base pairs. (C) Western blots using the anti-FLAG antibody (Sigma) to detect TraB-FLAG in gonococcal strains MS11 (WT), MR646 (traB-FLAG expressed from the native locus in the GGI [traB-FLAG]), and MR565 (traB-FLAG expressed from an IPTG-inducible promoter in the WT background, [traB-FLAG⁺]). Full-length TraB-FLAG and a smaller FLAG-reactive band are indicated. Approximately 15 μg of protein from cell lysates was subjected to SDS-PAGE.