Construction of plasmids for gene fragments heterologous expression

To verify that Cd²⁺ resistance is conferred by genes, recombinant pUC19 and pUBC19 plasmids were constructed. The 3444-bp Sac I-Xba I DNA fragment amplified from B. vietamensis 151–6 genome with the primers 4111–4112-4113-F/4111–4112-4113-R (Additional file 2: Table S2), containing copper-translocating P-type ATPase gene (orf4111), cadmium efflux system accessory gene cadC (orf4112), cadmium resistance gene cadD (orf4113) and 217-bp upstream of orf4113, was cloned into E. coli pUC19 vector and E. coli-B. subtilis shuttle vector pUBC19, respectively. The gene fragments 4087–4088, 4093–4094-4095, 4102–4103 and 4108–4109 amplified from B. vietamensis 151–6 genome with corresponding primers (Additional file 2: Table S2) were also cloned into pUC19 and pUBC19. For B. marisflavi 151–25, the gene fragments 4774–4775, 4776–4777, 4779–4780, 4781–4782 and 4802–4803 amplified from plasmid p25 of B. marisflavi 151–25 with the corresponding primers (Additional file 2: Table S2) were cloned into pUC19 and pUBC19. The ligation mixture was transformed into E. coli TOP10, and the correct plasmids were identified through colony PCR with corresponding sequencing primers (Additional file 2: Table S2). After confirmation via sequencing, recombinant pUBC19 plasmids were extracted from E. coli TOP10 and transformed into B. subtilis, as described previously [46]. Transformants were harvested by screening the clones on LB agar containing 10 μg/mL kanamycin, and the correct plasmids were verified through colony PCR with the pUBC19 sequencing primers CX-F/CX-R (Additional file 2: Table S2).