Figure 6.

YitA and YipA are localized to the outer membrane fraction of Y. pestis and YitA is detectable on the surface of the bacteria.A) Y. pestis KIM6+ (pCR-XL-TOPO::yitR) YitA-β-lactamase (Lanes 2–6) or YipA-β-lactamase (Lanes 7–11) grown overnight at 22°C in BHI were lysed and separated into cytoplasmic (C), periplasmic (P), cytosolic inner membrane (I), and outer membrane (O) fractions and analyzed by Western blot. Whole cell lysates (W) are provided as a control for both strains. Panels show Western blots probed with antisera to YitA, YipA, and β-lactamase, or Ail (a known Y. pestis outer membrane protein). B) Evidence of surface exposed YitA on Y. pestis. The top panel includes images of Y. pestis KIM6+ (pCR-XL-TOPO::yitR) (pAcGFP1, fluoresces green) grown overnight at 22°C in BHI. YitA was detected by incubating fixed bacteria with anti-YitA serum and staining with Alexa Fluor 568 goat anti-rabbit IgG (fluoresces red). Fluorescence was imaged under green (FITC) and red (TRITC) filters, artificially colored, and merged. Images of KIM6+ΔyitA-yipB (pCR-XL-TOPO::yitR) (pAcGFP1) prepared and imaged by the same method are shown in the bottom row as a background control