diphtheriae protein DIP1281 was, as its homologs Ce1659, Cg1735, and JK0967 in Corynebacterium efficiens, Corynebacterium glutamicum, and Corynebacterium jeikeium, previously annotated as hypothetical invasion-associated protein. Generation and analyses Smad inhibitor of mutant strains indicate that DIP1281 is predominantly involved in the organization of the outer surface protein layer of C. diphtheriae rather than in the

separation of the peptidoglycan cell wall of dividing bacteria. The adhesion- and invasion-negative phenotype of corresponding mutant strains is an effect of rearrangements of the outer surface of bacteria. Specific interaction partners for DIP1281 and its homologs in other corynebacteria are unknown and might be the focus of further studies to unravel the specific functions and targets of these proteins on a molecular level. Methods Bacterial strains and growth Strains used in this study are listed in check details Table 2. Escherichia coli DH5αMCR was grown in Luria Bertani (LB) medium at 37°C, C. diphtheriae in Heart Infusion (HI) broth at 37°C. If appropriate, kanamycin was added (30 μg/ml for E. coli; 50 μg/ml for C. diphtheriae). Table 2 Bacterial strains and eukaryotic cells used in this study. Strains Description Reference C. diphtheriae     DSM44123 non-toxigenic isolate, type strain DSMZ (Braunschweig) ISS3319 C. diphtheriae var. mitis, non-toxigenic isolate [9] ISS4060

C. diphtheriae var. gravis, non-toxigenic isolate [9] Lilo1 ISS3319 DIP1281::JNK-IN-8 purchase pK18mob’DIP1281” This study Lilo2 ISS4060 DIP1281::pK18mob’DIP1281” This study E. coli     DH5αMCR endA1 supE44 thi-1 λ- recA1 gyrA96 relA1 deoR Δ(lacZYA-argF) U196 φ80ΔlacZ ΔM15 mcrA Δ(mmr hsdRMSmcrBC) [28] Cell lines     Detroit562 human hypopharyngeal carcinoma cells [29] Preparation of C. diphtheriae protein extracts To prepare surface proteins, bacteria

were grown in 20 ml HI broth (with kanamycin added for the mutant strains) for approximately six hours and used to inoculate 250 BCKDHA ml HI broth for overnight growth. Bacteria were harvested by centrifugation at 5,000 × g for 20 min, washed twice with pre-cooled (4°C) 50 mM Tris-HCl buffer (pH 7.2), resuspended in 50 mM Tris-HCl (pH 7.2) containing 2% 3-[(3-choamidopropyl)-dimethylammonio] propanesulfonate (CHAPS) and incubated on ice overnight, followed by centrifugation at 3,500 × g and 4°C for 30 min to separate the cell surface proteins. After filtration of the protein solution using 0.45 μm pore-size filters (SARSTEDT, Nümbrecht, Germany), further preparation of the surface proteins by phenolic acid extraction and methanol precipitation followed a protocol described by Watt and co-workers [23]. The precipitated proteins were harvested by centrifugation at 3,500 × g and 4°C for 30 min. The pellet was washed twice with 3 ml of 70% ethanol (-20°C) and once with 3 ml of acetone (-20°C). Finally, the protein pellet was dried on ice and solubilised in 450 μl of dehydration buffer (8 M urea, 20 mM DTT, 2% CHAPS).