Electrophoretic mobility shift assay DNA fragments used for the electrophoretic mobility shift assay (EMSA) were PCR amplified using Cy5-labeled primers to perform HSP inhibitor a non-radioactive EMSA. DNA fragments used were the upstream region of acrD (246 bp), and as controls, the upstream regions of acrAB (205 bp) and tolC (291 bp). Approximately 0.16 pmol of Cy5-labeled DNA was mixed with increasing concentrations of His-tagged BaeR protein in a binding buffer reaction (50 mM

Tris–HCl, pH 7.5; 1 mM DTT; 500 mM MgCl2; 100 mM EDTA; 10 mM NaCl; 5% glycerol). To decrease unspecific binding, 500 ng competitor DNA (Salmon sperm DNA, AppliChem) was added to the reaction. Incubation was done at room temperature for 30 min. The total reaction was run on a native 4% polyacrylamide gel in 0.5x Tris-borate-EDTA (TBE)

buffer at constant 25 mA. After electrophoresis, fluorescence signals of the labeled DNA were visualized using a FLA-3000 phosphorimager (Raytest, Straubenhardt, Germany). Statistical analysis Statistical analysis was performed using R [56]. Differences between two groups were determined by a two-sided t-test with equal variances and were considered significant at P < 0.05. When necessary the standard deviation is presented in the graph when the average of several values was applied. Acknowledgments This study was supported by Jacobs University Bremen and by the MOLIFE Research Center, Selonsertib research buy Jacobs University Bremen. Electronic supplementary material Additional Flavopiridol (Alvocidib) file 1: Phylogenetic tree of AcrD. Description: The tree was calculated based on AcrD from Erwinia amylovora Ea1189 (black arrow) and its homologues from other members of the Enterobacteriaceae family, including Erwinia pyrifoliae (95% identity), E. tasmaniensis (93% identity), E. billingiae (83% identity), Pantoea agglomerans (82% identity), P. ananatis (79% identity), Enterobacter cloacae (79% identity), Salmonella enterica (79% identity), Citrobacter koseri (79%), Klebsiella pneumoniae (79% identity), Escherichia coli (78% identity) and Shigella flexneri (78% identity). The dendrogram was generated based on percentage of identity

between the sequences using the neighbor joining algorithm implemented in Jalview [25–28]. (TIFF 6 MB) Additional file 2: Sequence alignment of AcrD from Erwinia amylovora Ea1189 and Escherichia coli K-12. Description: The alignment is based on the amino acid sequences of AcrD using ClustalW for analysis and Jalview for data presentation. AcrD of Ea1189 is 79% identical and 89% similar to AcrD of E. coli K-12. Identical amino acid residues are shown in blue. Yellow bars show a quantitative measurement of conserved physico-chemical properties where the highest score shows amino acids of the same physico-chemical class [26–28]. Black bars indicate predicted transmembrane-spanning helices of AcrD from E. amylovora[29]. (TIFF 5 MB) Additional file 3: Modified view of the genomic organization of the acrD locus.