licheniformis ATCC14580/DSM 13 (YP_080584.1; YP_080585.1; YP_080586.1) [25] and B. subtilis subsp. subtilis str. 168 (NP_391185.2; NP_391186.1; NP_391187.1) [23, 63]. Construction of B. licheniformis MW3∆gerA complementation mutants The entire gerA operons including

the putative sigG promoter from B. licheniformis strain NVH1032, NVH800 and NVH1112 were cloned into the pHT315 [47] shuttle vector and introduced into the gerAA deletion mutant strain MW3∆gerAA by electroporation as described previously [28]. Briefly, PCR, with primers (Table  2) containing SalI and XbaI restriction sites, was used to amplify the gerA operon including 151 bp upstream of the gerAA start codon and 177 bp downstream of the gerAC STOP codon. The amplified fragments were cloned into the SalI/XbaI restriction site of pHT315, giving the complementation AZD4547 datasheet plasmids.

For details regarding primers, PCR conditions, DNA isolation and electroporation see Løvdal et al. 2012 [28]. The strains created in this study were designated as follows: B. licheniformis NVH1309 (MW3∆gerAA _NVH1032gerA); NVH1321 (MW3∆gerAA _NVH1112gerA) and NVH1322 (MW3∆gerAA _NVH800gerA). Correct construction of the complementation plasmids was confirmed by sequencing and the complementation mutants were verified by PCR analysis. Sequence editing and alignments were buy 4SC-202 performed as already described in the Data analysis section. Bacterial growth 3-Methyladenine mouse and sporulation Sporulation was performed according to Løvdal et al. 2012 [28], with minor modifications. Bacteria were pre-cultured

overnight in LB-broth with agitation (230 rpm) at 37°C. Complementation mutants were grown in presence of 1 μg mL-1 erythromycin. 10 μL of preculture was transferred to 50 mL of the non-defined, rich sporulation medium [28] in 500 mL EM flasks. Incubation was performed with agitation (230 rpm) at 37°C for 3–7 days until ≥ 80% phase bright spores as judged by phase contrast microscopy. Seven of the strains (M55, ATCC9945A, NVH622, 749, M46, NVH1079 and LMG6934) did not sporulate adequately and were excluded from further analysis. Spores were harvested by centrifugation Amino acid for 10 min at 3900 × g (Eppendorf) at 4°C and resuspended in 10 mL ice-cold autoclaved Milli-Q water. The spores were centrifuged at 10000 × g through a 50% (w/v) Nycodenz (Axis-Shield) gradient in order to remove cell debris and vegetative cells. The spores were washed three times in ice-cold autoclaved Milli-Q water before storage (1–3 months) in the dark at 4°C. The final spore suspensions were 98% free of vegetative cells, not fully sporulated cells, cell debris and germinated cells as judged by phase contrast microscopy. Quantitative RT-PCR Quantitative RT-PCR experiments were performed on mRNA isolated from B. licheniformis cultures harvested after ~ 50% sporulation judged by phase contrast microscopy.