5% sucrose and Vorinostat mw incubated at 30°C for four days. pDK001-cured strains were finally streaked on MM9-succinate gentamicin. Phage ΦM12 was used for transductions following the usual procedure [56], except that TY media was used instead of LBmc media to prepare and dilute lysates. High yield of transductants required the use of Bacto™-Agar, -Tryptone, and -Yeast extract (BD). Diluted lysate (0.5 ml) was mixed with equal volume of cell suspension and incubated at room temperature for 30 minutes. Cells were then recovered by centrifugation in a microcentrifuge for 10 minutes and washed twice with 2 ml of saline. Final resuspension

was done with find more 400 μl saline and then spread on two agar plates. Plates were incubated at 30°C for four days. Growth in liquid media Inocula were prepared by resuspending AG-881 purchase bacterial biomass from MM9-succinate-agar plates into a saline solution (0.85% NaCl) to obtain an optical density (OD600) of 0.8. Test tubes containing 5-ml liquid media made of MM9-succinate with/without 0.1% proline and/or 0.1% uracil where inoculated with the inoculum at a 10% concentration. Test tubes were incubated at 30°C with constant

shaking. Growth was monitored by reading the absorbance at 600 nm. Growth rate constants (μ) were calculated based on absorbance values during the exponential growth phase and using the formula: μ = ( (log10 N – log10 N0) 2.303) / (t – t0). Results represent the average of duplicates and the standard deviation was calculated as the error. β-Glucuronidase assay To measure transcription from reporter gene fusion strains, the β-glucuronidase assay described in Cowie et al. [20] was adapted. Strains were grown in MM9-succinate plus 0.1% proline, 0.1% uracil, and gentamicin until OD600 of 0.2 – 0.8. These cells were then used directly for the assay in microplates as described previously [20]. Assays were

done in triplicate and standard deviation calculated. Acknowledgements This research was supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery BCKDHA Grant to T.C.C. L.B. received a scholarship from “Fonds québécois de la recherche sur la nature et les technologies” (FQRNT). We thank Professor Bi-Cheng Wang and Dr. Hao Xu at University of Georgia (USA) for provision of the purified ChvI protein and Professor Turlough M. Finan from McMaster University (Canada) who made the fusion library available to us. We are grateful to Jennifer Moore and Jacquelyn Fleming for technical assistance, Dr. Jiujun Cheng for critically reading the manuscript, and Kathy Lam and John Heil for assistance with data analysis. Electronic supplementary material Additional file 1: Gel image of PD.EMSA to compare DNA shifts on 6-cm versus 14-cm 5% nondenaturing polyacrylamide gel and using SB buffer. Prior to the electrophoresis, the Bsp143I restricted pTC198 plasmid was incubated or not with the HisTag-ChvI protein. (PNG 224 KB) Additional file 2: Gel image of PD.