YitA and YipA protein increased with an increase in yitR copy number (Figure 2, lanes 5–6). The sizes of the YitA and YipB protein produced by all the strains under environmental conditions were similar (Figure 2, lanes 2, 5, 6). No detectable YitA or YipA protein was produced by the KIM6+ΔyitR deletion mutant (data not shown). In vitro production of YitA and YipA by Y. pestis is dependent on growth temperature but not on culture Small molecule library cell line medium Y. pestis KIM6+, KIM6+ (pWKS130::yitR), and KIM6+ (pCR-XL-TOPO::yitR) were grown in BHI at 10°C, 22°C, 28°C, or 37°C overnight to determine YitA and YipA synthesis at

different growth temperatures. YitA production in parental KIM6+ was detected after growth at 10°C (Figure 3A, lane 2). Full-size YipA was not detected in KIM6+ at any temperature (Figure 3A, lanes 2, 5, 8, and 11). When plasmid pWKS130::yitR was present, YitA was seen at all temperatures, with buy PR-171 the maximum level at 10°C; the level decreased when the growth temperature was 37°C (Figure 3A, lanes 3, 6, 9, and 12). When plasmid pWKS130::yitR was present, YipA production was also greatest after growth

at 10°C (Figure 3A, lane 3) and decreased when the growth temperature was 37°C (Figure 3A, lanes 6, 9, and 12); however, very little was seen at 37°C and the larger molecular weight band was no longer present (Figure 3A, lane 12). Y. pestis KIM6+ with the high-copy number pCR-XL-TOPO::yitR had the greatest production of YitA and YipA, which also decreased when the growth temperature was 37°C (Figure 3A, lanes 4, 7, 10, and 13). For each of the strains tested, levels of YitA and YipA were comparable after growth at 22°C or 28°C (Figure 3A, lanes 5, 6, 7, 8, 9 and 10). Figure 3 Maximal synthesis of YitA and YipA during growth at low temperatures. A) KIM6+ (lanes 2, 5, 8, and 11), KIM6+ (pWKS130::yitR) (lanes 3, 6, 9, and 12) and PtdIns(3,4)P2 KIM6+ (pCR-XL-TOPO::yitR) (lanes 4, 7, 10, and 13) grown overnight

at 10°C, 22°C, 28°C or 37°C in BHI broth. YitA and YipA purified from E. coli (lane 15). B) KIM6+ (lanes 2, 5, 8, and 11), KIM6+ (pWKS130::yitR) (lanes 3, 6, 9, and 12) and KIM6+ (pCR-XL-TOPO::yitR) (lanes 4, 7, 10, and 13) grown overnight at 22°C or 37°C in either RPMI 1640 (RPMI) or whole sheep blood (Blood). YitA and YipA purified from E. coli (lanes 15 and 16). Panels show Western blots probed with anti-YitA, anti-YipA, or anti-Ail (sample loading control) antiserum. YitA and YipA production following growth in both blood and RPMI 1640 was equivalent to production following growth in BHI. YitA and YipA were produced to the greatest extent after growth at 22°C in RPMI 1640 and blood (Figure 3B, lanes 2–7) and levels dramatically decreased following growth at 37°C (Figure 3B, lanes 8–12). As with growth in BHI, Y.