The MIC was defined as the lowest concentration of metal that allowed no bacterial growth. For each metal and bacterial strain, at least three independent experiments were carried out. β-galactosidase assay Enzyme activities were measured from bacteria grown overnight in LB or in LB Stattic price supplemented with different metal salts (concentrations are specified in Results). β-galactosidase activity was assayed according to a previously described protocol [68]. Western blotting Cell lysates were prepared from bacteria grown overnight in LB or in LB supplemented with either 0.6 mM ZnSO4
or 0.15 mM FeSO4. Equal amounts of total protein (3 μg) were separated by Tricine-SDS-PA gel electrophoresis, followed by protein transfer to a nitrocellulose membrane. For Western blotting, the membranes were probed with ColR-specific polyclonal antibodies, followed by treatment SHP099 nmr with alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G. The blots
were developed using bromochloroindolyl phosphate/nitro blue tetrazolium (BCIP/NBT). Acknowledgments We are grateful to Hedvig Tamman, Andres Ainelo and Hanna Hõrak for critically reading the manuscript. We thank Külliki Holtsmann for assistance in the cloning and Peeter Hõrak and Riho Teras for advice in the statistical analysis. This work was supported by the grant 7829 from the Estonian Science Foundation, by Targeted Financing Project TLOMR0031 and by Institutional Abemaciclib mw Research grant IUT20-19.
Electronic supplementary material Additional file 1: Table S1: Bacterial strains and plasmids. (DOCX 56 KB) Additional file 2: Table S2: The oligonucleotides. (DOCX 22 KB) Additional file 3: Table S3: The oligonucleotide pairs used in two sequential PCRs for site-directed mutagenesis of colS. (DOCX 18 KB) References 1. Andreini C, Bertini I, Cavallaro G, Holliday GL, Thornton JM: Metal ions in biological catalysis: from enzyme databases to general principles. J Biol Inorg Chem 2008,13(8):1205–1218.CrossRef 2. Touati D: Iron and oxidative stress in bacteria. Arch Biochem Biophys 2000,373(1):1–6.PubMedCrossRef 3. Imlay JA: Iron-sulphur next clusters and the problem with oxygen. Mol Microbiol 2006,59(4):1073–1082.PubMedCrossRef 4. McDevitt CA, Ogunniyi AD, Valkov E, Lawrence MC, Kobe B, McEwan AG, Paton JC: A molecular mechanism for bacterial susceptibility to zinc. PLoS Pathog 2011,7(11):e1002357.PubMedCentralPubMedCrossRef 5. Outten CE, O’Halloran TV: Femtomolar sensitivity of metalloregulatory proteins controlling zinc homeostasis. Science 2001,292(5526):2488–2492.PubMedCrossRef 6. Changela A, Chen K, Xue Y, Holschen J, Outten CE, O’Halloran TV, Mondragon A: Molecular basis of metal-ion selectivity and zeptomolar sensitivity by CueR. Science 2003,301(5638):1383–1387.PubMedCrossRef 7. Nies DH: Efflux-mediated heavy metal resistance in prokaryotes. FEMS Microbiol Rev 2003,27(2–3):313–339.PubMedCrossRef 8.
Related posts:
- Funding This work was supported by the National Institutes of Hea
- Metal complexes, as models with known structures, have been essen
- MtrB homologs with an N-terminal CXXC motif may thus represent a
- The mesophase of metal alkanoates can be used as a nanoreactor fo
- Depending on the environment, the metal release from stainless st