Bacillus spp. Cultivation and Lysis

Bacillus cereus (ATCC 14579) and Bacillus thuringiensis (strain 407) were obtained from Nalini Ramarao (INRA, La Minière, Guyancourt, France). The B. cereus ATCC 14579 deletion mutant lacking the plcR regulon (BcΔplcR) was a kind gift from Michel Gohar (INRA, Génétique Microbienne et Environnement Jouy-en-Josas, France). Bacillus subtilis (DSM 402), Bacillus weihenstephanensis and Bacillus megaterium were included as further controls. All bacteria were grown in brain heart infusion (BHI) medium (Sigma Aldrich) at 37°C overnight shaking at 200 rpm. Overnight cultures were diluted 1:20 in fresh BHI medium and bacteria and their corresponding supernatants from the liquid cultures were harvested after indicated time periods by centrifugation at 3000 x g for 10 min at 4°C. Supernatants were sterile-filtered (0.22 nm filter, Greiner). Bacterial pellets were resuspended in lysis buffer (20 mM Tris pH 7.5, 100 mM NaCl, 1% Triton X-100, 0.5% DOC, 0.1% SDS, 0.5% NP-40) and sonicated 3 x for 30 sec with 50% power [35 (link)]. Bacterial lysates were cleared from debris by centrifugation at 16000 x g for 10 min at 4°C and protein amounts were determined by Bradford protein assay (Carl Roth).