Methods Bacterial strains and routine culture conditions Campylobacter jejuni strains derived from the parent 81–176 [30, 31] (Table 1) were routinely maintained with minimal passage on blood agar plates (Remel; Lenexa, KS) at 37°C in sealed culture boxes (Mitsubishi Gas AZD2281 Chemical [MGC], New York, NY) containing a microaerobic atmosphere generated by Pack-Micro Aero (MGC). Liquid cultures of C. jejuni were grown in Brucella broth or Mueller-Hinton (MH) broth and cultured in microaerobic environments. When appropriate, strains were cultured in the presence of chloramphenicol (30 μg/ml) or streptomycin (30 μg/ml) to select for antibiotic resistance markers. Table 1 Strains used in this

study Strain Reference or source C. jejuni 81–176 [30] C. jejuni 81–176cj0596 This study C. jejuni 81–176cj0596 + This study C. jejuni NCTC11168 [22] C. jejuni 81116 [43] C. jejuni HB95-29

[44] C. jejuni INP44 [44] C. jejuni INP59 [44] C. coli D3088 [44] C. jejuni RM1221 TIGR CMR [62] C. jejuni subsp. doylei 269.97 TIGR CMR [62] C. jejuni subsp. jejuni 260.94 TIGR CMR [62] C. jejuni subsp. jejuni 84-25 TIGR CMR [62] C. jejuni subsp. jejuni CF93-6 TIGR CMR [62] C. jejuni subsp. jejuni CG8486 [45] C. jejuni subsp. jejuni HB93-13 TIGR CMR [62] C. coli RM2228 TIGR CMR [62] C. concisus 13826 TIGR CMR [62] C. curvus 525.92 TIGR CMR [62] C. fetus subsp. fetus see more 82–40 TIGR CMR [62] C. hominis Methane monooxygenase ATCC BAA-381 TIGR CMR [62] C. lari RM2100 TIGR CMR [62] C. upsaliensis RM3195 TIGR CMR [62] E. coli BL21(DE3)pLysS [32] H. pylori 84–183 [50] Escherichia coli JM109 was used as the host strain for cloning experiments and E. coli

BL21(DE3)pLysS [32] was used as the host strain for expression of the his-tagged Cj0596 protein. E. coli strains were cultured in Luria-Bertani (LB) broth or agar [33], supplemented with the following antibiotics as appropriate for selection of plasmids: ampicillin, 50 μg/ml; chloramphenicol, 30 μg/ml; streptomycin, 30 μg/ml. Proteome analysis of C. jejuni strains Proteomics experiments were performed on C. jejuni cells grown at 37°C and 42°C as described [34]. Briefly, cells were grown overnight at 37°C in Brucella broth, then diluted the following morning into two aliquots of fresh Brucella broth (OD600 = 0.1), which were grown at 37°C and 42°C to mid-log phase (OD600 = 0.1). Chloramphenicol (187 μg/ml) was added to stop protein synthesis [35], and the cells were harvested for proteome analysis as described [34]. Proteomics experiments were performed using Differential In-Gel Electrophoresis (DIGE) technology from GE Biosystems (Piscataway, NJ), Whole-cell protein lysates from the 37°C- and 42°C-grown C. jejuni (25 μg each) were labelled individually with Cy3 and Cy5 dyes according to the protocol supplied by the manufacturer (GE Biosystems), then mixed in equal mass and separated using two-dimensional (2D) SDS-PAGE.