Thiamine hydrochloride

Bacillus subtilis NCIB 3610 amyE::P_tapA-Ypet strain (ES2107) was used for all experiments. The strain was cultured on lysogeny broth (LB)-agar plates prepared from Miller LB (Acros Organics, Fair Lawn, NJ) and 1.5% agar powder (Molecular Genetics, Fisher Scientific, Waltham, MA). LB agar plates were streaked with B. subtilis ES2107 from frozen stocks and incubated at 30°C for 15 h.
B. subtilis biofilms were grown on MSgg, a B. subtilis biofilm-promoting medium (5 mM potassium phosphate [pH 7]) (Fisher Scientific, Waltham, MA), 100 mM MOPS (morpholinepropanesulfonic acid [pH 7]) (Sigma-Aldrich, St. Louis, MO), 2 mM MgCl₂ (Fisher Scientific, Waltham, MA), 700 μM CaCl₂ (Alfa Aesar, Haverhill, MA), 50 μM MnCl₂ (Fisher Scientific, Waltham, MA), 50 μM FeCl₃ (Sigma-Aldrich, St. Louis, MO), 1 μM ZnCl₂ (Sigma-Aldrich, St. Louis, MO), 2 μM thiamine hydrochloride (Fisher Scientific, Waltham, MA), 0.5% glycerol (Fisher Scientific, Waltham, MA), and 0.5% glutamate (Sigma-Aldrich, St. Louis, MO). For MSgg agar plates, 1.5% agar (BD) was added to the medium, and approximately 5-ml plates were poured.

E. coli K-12 MG1655 (ATCC #47076) was used for this study. It was chosen because of its sensitivity to metals and its lack of metal or antibiotic resistance. E. coli K-12 MG1655 does not contain any plasmids and its chromosome has 4,641,652 nucleotides (GenBank:NC_000913.3). All growth experiments in this study were carried out using Davis Minimal Broth (DMB, Difco^TM, Sparks, MD, USA) supplemented with 1 g per liter dextrose as a sole carbon source, with Thiamine Hydrochloride 0.1% (Thiamine Hydrochloride, Fisher Scientific, Fair Lawn, NJ, USA).