Bacillus subtilis Cultivation and Genetic Manipulation

B. subtilis strains derived from CU1065 (WT) were grown on lysogeny broth (LB) medium (10 g l⁻¹ casein digest peptone, 5 g l⁻¹ yeast extract, 5 g l⁻¹ NaCl) at 37°C with vigorous shaking. The strains used in this study are listed in Table S1, available in the online version of this article. Ferrous iron (FeSO₄·7H₂O) was titrated into the media as indicated from a freshly made 100 mM iron stock solution dissolved in 1 N HCl unless otherwise indicated. SPβ phage are derivatives of SPβc2Δ2 and were constructed by integration of a promoter region–cat–lacZ operon fusion constructed in pJPM122 into strain ZB307A as described previously [38 (link)]. Ampicillin (amp; 100 μg ml⁻¹) was used to select E. coli transformants. Erythromycin (ery; 1 μg ml⁻¹) and lincomycin [linc; 25 μg ml⁻¹; for testing macrolide–lincosamide–streptogramin B (MLS) resistance], spectinomycin (spec; 100 μg ml⁻¹), kanamycin (kan; 10 μg ml⁻¹) and neomycin (neo; 10 μg ml⁻¹) were used for the selection of various B. subtilis strains.