Stocks of all strains were stored at −80°C in broth (BHI) (Oxoid CM225, England) containing 15% glycerol. From −80°C stocks, cultures were transferred to Blood Agar Base No. 2 (Oxoid CM271, England) supplemented with 5% horse blood and incubated for 3–4 days. One loop full of each culture was subsequently streaked onto new Blood Agar Base No. 2 plates. After 24 hours of growth, cells were harvested with 2 ml phosphate-buffered saline (PBS) (Oxoid BR0014, England). The harvested cells were adjusted to OD600 = 0.1 which has previously shown to correspond to approx. 8 log10 CFU/ml

and subsequently used as inoculum. Preparation of chemically defined broth A chemically defined medium, originally developed for N. gonorrhoeae[30], was modified in order to have an optimal broth to support growth of Campylobacter on plates. From the original medium, glucose was removed because Campylobacter is unable to ferment or oxidize hexose find more carbohydrates [31], and

different amino acids were added. The required amino acids were determined from the amino acid metabolic pathway maps listed for C. jejuni NCTC 11168 in the Kyoto Encyclopedia of Genes and Genomes (KEGG) [32] Pathway Database. If metabolic enzymes were lacking or if the amino acid biosynthetic pathway was complex, the specific amino acid was added to the modified chemically defined broth (CDB). this website Modified CDB was prepared in double-strength stock batches (see Table  1) without methionine and cysteine, which were added later. The double-strength CDB was stored at −20°C. Prior to the experiments, double-strength CDB was thawed at 4°C and diluted to single strength with MilliQ water (Table  1). Finally, the CDB was sterilized by filtration (pore heptaminol size: 0.2 μm). Table 1 Components of modified chemically defined broth (CDB) for Campylobacter jejuni Components Stock solution (mg/ml) Vol stock solution (ml) for 1 liter Final conc (mmol/l) of 1xCDB Buffer solution (10X)   100.0   K2HPO4 34.8   20.0 KH2PO4 27.2   20.0 Salt solution (20X)   50.0   NaCl 116.0   100.00 K2SO4 20.0   5.74 MgCl2,

6 H2O 8.2   2.02 NH4Cl 4.4   4.11 EDTA 0.074   0.01 Amino acid mix 1 (100X)   10.0   L-Arginine HCl 15.0   0.71 L-serine 5.0   0.48 L-leucine 9.0   0.69 L-isoleucine 3.0   0.23 L-valine 6.0   0.51 L-proline 5.0   0.43 BMN673 L-phenylalanine 5.0   0.30 L-alanine 10.0   1.12 L-histidine 5.0   0.32 L-threonine 5.0   0.42 L-lysine 5.0   0.30 L-glycine 2.5   0.33 L-trypthophan 8.0   0.39 Amino acid mix 2 (10X)   100.0   L-aspartate 5.0   3.76 L-glutamate 13.0   8.83 Individual amino acids       L-cysteine/HCl † 17.5 3.0 0.35 L-cysteine † 12.0 3.5 0.15 L-methionine 14.9 1.0 0.10 Vitamin mix (50X)   0.2   NAD 10.0   0.0030 Thiamine HCl (Vitamine B1) 10.0   0.0060 Calcium pantothenate (Vitamine B5) 10.0   0.0040 Individual components       Oxaloacetate, 2 H2O (10X) 2.5 100.0   NaHCO3 (2000×) 84.0 0.5 1.