Panels show Western blots probed with A) anti-YitA, B) anti-YipA, or C) anti- β-lactamase antiserum. Anti-YipA serum detected YipA-β-lactamase as two prominent bands. The

YipA-β-lactamase lower band at ~73 kDa (Figure 5B, lane 4) was the same size as the lower band seen with wild-type YipA (Figure 5B, lane 2). The upper band of YipA-β-lactamase was detected at ~135 kDa (Figure 5B, lane 4), whereas the upper band of wild-type YipA was detected at ~106 kDa (Figure 5B, lane 2). Anti-β-lactamase antibody detected the upper ~135 kDa band corresponding to full-length YipA-β-lactamase (Figure 5C, lane 4). However, the lower ~73 kDa band was not detected by anti-β-lactamase antibody (Figure 5C, lane 4); although a see more distinct band at ~62 kDa was detected by anti-β-lactamase antibody (Figure 5C, lane 4). This indicates that the YipA molecular weight band detected by anti-YipA at ~73 kDa (Figure 5B, lane 4) represents the N-terminus of YipA, whereas the smaller molecular weight band detected by anti-β-lactamase antibody (~62 kDa) represents the C-terminal region of YipA fused to β-lactamase (Figure 5C, lane 4). YitA and YipA are MAPK Inhibitor Library localized in the outer membrane of Y. pestis To determine where YitA and YipA are localized within Y. pestis, cytoplasmic, periplasmic, inner membrane and outer membrane fractions were collected from KIM6+ YitA-β-lactamase

(pCR-XL-TOPO::yitR) and KIM6+ YipA-β-lactamase (pCR-XL-TOPO::yitR) grown in BHI overnight at 22°C. YitA-β-lactamase was detected by anti-YitA (Figure 6a, top panel) and anti-β-lactamase (Figure 6A, bottom panel) antibodies predominately in the outer membrane fraction (Figure 6A, lane 6) and HDAC inhibition to a lesser extent in the periplasm (Figure 6A, lane 4). Wild-type YitA was detected in the cytoplasmic, periplasmic, inner membrane and outer membrane fractions of KIM6+ YipA-β-lactamase (Figure 6A, lanes 8–11). 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 Progesterone 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.