Proteomic analyses of mviN knock-down strains

Strains GV341 and GV342 were grown in TY broth with thiamphenicol (15 μg/mL) overnight, then diluted 1:50 in TY and thiamphenicol with or without anhydrotetracycline (500ng/mL). Samples were collected at mid-log and processed for iTRAQ proteomic analysis as described previously [40,41]. Briefly, a total of 100 μg of protein in 20 μl of 7M urea, 2M thiourea, and 4% CHAPS (Sigma-Aldrich, St. Louis, MO) was denatured in 2% SDS, reduced in 100mM tris-(2-carboxyethyl) phosphine, and alkylated in 84mM iodoacetamide. The urea concentration of the samples was diluted to 2M using 100mM TEAB prior to digestion with trypsin at a ratio of 1:10. iTRAQ reagent labeling was performed according to manufacturers’ instructions (AB SCIEX, Framingham, MA). Strong cationic exchange (SCX) fractionation was performed using Waters 600E HPLC system, fractions collected, resuspended in acetonitrile/ trifluoroacetic acid, injected onto a Merck Chromolith CapRod column, and eluates automatically spotted on a stainless steel MALDI target plate for spot analysis (ABSciexTripleTOF 5600+ mass spectrophotometer; AB SCIEX, Framingham, MA). Protein identification and quantitation was performed using the Protein Pilot 3.0 software against the C. difficile strain 630 protein database, differentially abundant proteins were identified via multiple stringent statistical tests as described previously [40,41], and in the Statistics section below.