38 0.29 0.76 0.38 0.0468 0.38 a) spot number as denoted

in Figure 4; b) protein accession number and locus tag as listed in Y. pestis KIM genome database (NCBI); c) gene name and protein description from the KIM database or a Epoxomicin conserved E. coli K12 ortholog http://​www.​ecocyc.​org, if >65 pct. sequence identity; BLZ945 d) subcellular localization based on PSORTb data: CY, cytoplasm; ML: multiple localizations; PP, periplasm; U: unknown; e) proven or putative regulation by Fur or a Fur-dependent small RNA (e.g. RyhB); f) highest Mascot score for a protein from LC-MS/MS or MALDI data; g) Vs (-Fe): average spot volume (n ≥ 3) in 2D gels for iron-depleted growth conditions at 26°C as shown in Figure 4; h) Vs (+Fe): average spot volume (n ≥ 3) in 2D gels for iron-supplemented growth conditions at 26°C; i) spot volume ratio (-Fe/+Fe) at 26°C, N.D.: not determined; -: no spot detected; j) two-tailed t-test p-value for spot abundance change at 26°C, 0.000 stands for < 0.001; k) average spot volume ratio (-Fe/+Fe) at 37°C; additional data for the statistical spot analysis at 37°C are part of Additional Table 1. Y. pestis iron acquisition systems Proteomic profiling of characterized Y. pestis iron/siderophore and heme transporters (Ybt, Yfe, Yfu, Yiu AC220 clinical trial and Hmu) was in good agreement with negative regulation of the respective operons by Fur and iron [15, 16, 20, 49, 50]. The subscript number following a protein name represents the

spot number displayed in Figures 1, 2, 3 and 4, and is also denoted in the left-most column of Tables 1, 2 and 3. Periplasmic binding proteins of four of the ABC transporters (YfeA#68, YfuA#65, YiuA#82 and HmuT#56; Figures 1 and 2) were increased in abundance in iron-starved cells. The integral IM proteins YbtP and YbtQ

were identified from streaky 2D spots of the usb-MBR fraction of iron-depleted cells, but could not be differentially quantitated. Two RVX-208 of these five transporters have an OM receptor responsible for iron/yersiniabactin or heme uptake (Psn#102 and HmuR#95, respectively; Figure 3), both of which were increased in iron-starved cells. Y0850#96 (Figure 3) is hypothesized to be a TonB-dependent OM receptor with Fe3+/siderophore uptake activity. This protein was also more abundant in iron-depleted cells. Detection in the usb-MBR fraction, its Mr of ca. 75-85 kDa and the presence of a highly conserved Fur-box upstream of the gene’s transcriptional start site (AATGATAATTGATATCATT, -100 to -82) with a position weight matrix score of 13.2 using the patser-matrix tool [51] further supported the assignment as a Fur-regulated TonB-dependent OM receptor. Fur#18 was also detected in the cytoplasm, but not altered in abundance (Figure 4). Figure 1 Protein display in 2D gels of Y. pestis KIM6+ periplasmic fractions in the pI range 4-7 (-Fe vs. +Fe conditions). Proteins were derived from cell growth in the presence of 10 μM FeCl3 at 26°C (top) or the absence of FeCl3 at 26°C (bottom).