Antibiotics were used in the following concentrations: Ampicillin, 100 μg/ml; kanamycin, 50 μg/ml and chloramphenicol, 10 μg/ml. Plasmids used in DNA manipulations are listed in the Additional file 4: Table S4. Restriction enzymes and Dream-Taq polymerase (Fermentas, Thermo Scientific, Denmark) were used with the supplied buffers and according to the instruction of the manufacturer. Plasmids and PCR fragments were purified using the GeneJET Plasmid Miniprep (Thermo Scientific) – and illustra GFX Copanlisib price PCR DNA

and Gel Band Purification (GE Healthcare) kits. Deletion mutant strains of S. Typhimurium 4/74 were constructed by lambda red recombination using a published protocol [71]. The pKD3 plasmid was used as the template to prevent polar effects and the primers used for generation of PCR products for the mutagenesis

of the Vistusertib selected genes are listed in the Additional file 5: Table S5. The constructs were verified by PCR utilizing primers flanking the this website insertion sites, listed in in the Additional file 5: Table S5, checking for correct fragment size. Furthermore, the PCR products from the verification were sequenced (Macrogen) with the flanking primers to ensure correct constructs. Transduction into a clean wild-type background was performed with the P22 HT105/1 int-201 phage as previously described [72] and lysogen-free colonies were obtained by streaking on green-plates followed by sensitivity testing of the colonies with the P22-H5 phage [73]. The pCP20 plasmid was used for removal of the inserted resistance gene by utilizing for FLP-FRT mediated excision [71]. The non-selective growth was performed at 37°C. Correct removal of the resistance gene was confirmed by sequencing. After removal of the resistance

gene, double mutants were constructed by transduction with the previously made P22 HT105/1 int-201 phage lysates, again ensuring lysogen-free colonies. Growth and stress adaptation investigations in mutants To compare the growth ability of mutant strain to wild type strain, overnight cultures of were inoculated in LB. The strains were incubated at 37°C with shaking to balanced growth after 8–10 generations, including dilution of the cultures midway. Etomidate At OD600 = 0.4 serial dilutions were prepared and 10 μl of the 10-3 to 10-6 dilutions were spotted on solid media of varying composition according the methods described elsewhere [74]: 1) Standard LB plates were incubated at different temperatures; 15°C, 37°C and 44°C; 2) growth at different NaCl concentrations were examined by spotting onto plates supplemented to contain an additional 2% or 4% NaCl; 3) growth at different pH values was investigated by plating onto plates where the pH values were: 5, 9, 10 and 11. These plates were prepared by mixing filter sterilized liquid LB medium at high or low pH with normal autoclaved LB media at predetermined ratios. The autoclaved LB media was supplemented with agar to obtain a final concentration of 1.5%.