To ensure high fidelity, PCR amplification was performed with Phusion Hot Start II High-Fidelity DNA Polymerase kit (Finnzymes).
Akt inhibitor The amplification protocol was as follows; initial denaturation for 30s at 98°C, 30 cycles of 10 s at 98 °C, 30 s at 58 °C and 2 min at 72 °C, and final extension at 72 °C for 10 min. The amplified fragments were cloned into the SalI/XbaI restriction site of pHT315, giving the see more complementation plasmid pHT315_MW3gerA. The purified plasmid was controlled by sequencing using primers hybridizing to pHT315 and internal gerA. The verified plasmid was introduced into the disruption mutant (NVH-1307) by electroporation as described earlier, giving the strain B. licheniformis MW3ΔgerAA::spcpHT315_MW3gerA (NVH-1311). The strain was used in sporulation and germination assays. Sporulation Sporulation was performed by a modified version of the sporulation protocol and medium described by van der Voort [42] as outlined below. Bacteria were pre-cultivated for 5 to 6 h in 50 ml LB-Broth with agitation (225 rpm) at MK-0457 50 °C. Pre-culture of NVH-1307 was supplemented with 250 µg/ml spectinomycin, while the culture
of NVH-1311 was supplemented with 250 µg/ml spectinomycin and 1 µg/ml erythromycin. Twenty µl of pre-culture was added to 100 ml sporulation medium, containing 8 g of nutrient broth (Difco, Becton, Dickinson and Company, NJ, USA) per liter, 1 μM FeSO4·7H2O (Merck KGaA, Darmstadt, Germany), 2.5 μM CuCl2·2H2O (Sigma-Aldrich, Steinheim, Germany), 12.5 μM ZnCl2 (Sigma-Aldrich, Steinheim, Germany), 66 μM MnSO4·4H2O DCLK1 (BDH Prolabo, VWR International AS, Oslo, Norway), 1 mM MgCl2·6H2O (J. T. Baker Chemicals B. V., Deventer, Holland), 5 mM (NH4)2SO4 (Merck KGaA, Darmstadt, Germany), 2.5 µM Na2MoO4·2H2O (Riedel-de Häen, Sigma-Aldrich, Seelze, Germany), 2.5 µM CoCl2·6H2O (Sigma-Aldrich, Steinheim, Germany) and 1 mM Ca(NO3)2·4H2O (Merck KGaA, Darmstadt, Germany). Filter sterilised Ca(NO3)2·4H2O, MnSO4·4H2O and FeSO4·7H2O were added to the medium after it had been autoclaved. pH was adjusted to 7.6
before autoclaving, and the pH of the final sporulation medium was 7.2. Sporulation medium of NVH-1311 was supplemented with 1 µg/ml erythromycin. The cultures were incubated with agitation (225 rpm) at 50 °C for 1 to 2 days for B. licheniformis strains MW3, NVH-1307 and NVH-1311, or for 2 days at 30 °C for B. subtilis B252 and B. cereus ATCC 14579 until ≥90% phase bright spores as judged by phase contrast microscopy. Spores were harvested by centrifugation for 10 min at 6000 × g at 4 °C, and resuspended in 10 ml cold autoclaved MQ. Washing of spores was done by centrifugation and resuspension in MQ a total of ten times. The resulting spore crops, < 10% germinated spores, were stored refrigerated in MQ. When used in the following germination studies, spore crops were between 2 and 7 months old.