The availability of both disruption and complementation mutants will facilitate further research on the function of the GerA receptor of B. licheniformis MW3, as well as its potential involvement in germination triggered by alternative nutrients and cooperation with other germinant receptors. Further bioinformatic selleck screening library and phenotypic investigations are in progress in our laboratory and might eventually

provide insight relevant for improved spore decimation techniques by the use of induced germination. Methods Bacterial strains and DNA extraction The strains used in this study were B. licheniformis MW3 [50], B. subtilis B252 [71] and the B. cereus type-strain ATCC 14579 [72, 73] (Table 1). B. licheniformis MW3 is a mutant created from B. licheniformis DSM13 (isogenic to ATCC 14580) with targeted deletions of the hsdR loci of two type I restriction modification systems making the strain readily transformable. B. licheniformis MW3 was used as host for Seliciclib creating disruption and complementation mutants of the gerA locus. When not stated otherwise, bacteria were cultured at 37 °C on LB agar or broth containing appropriate selective antibiotics (Table 1). Genomic DNA for PCR amplifications and sequencing was extracted from B. licheniformis MW3 and B. licheniformis NVH-1307 by a method slightly modified from [71], as follows. An overnight culture was transferred to fresh growth medium and grown at 37 °C, 225

rpm (HT-Infors AG CH-4103, Bottmingen, Switzerland), to turbidity (4-5 h). Cells from 1 find more ml culture was harvested by centrifugation (3 min at 16.100 × g), and the pellet was frozen at -20 °C. Thawed pellet was resuspended in 495 µl SET buffer (75 mM NaCl, 25 mM EDTA, 20 mM Tris, pH 7.5) and 50 µl 10 mg/ml lysozyme before incubation at 37 °C for 1 h. Further, 50 µl 10% sodium dodecyl sulfate and 5 µl 25 mg/ml proteinase K was added, and the sample was incubated at 50 °C for 2 h. At room temperature (RT), the sample was mixed with 200 µl 5 M NaCl and

700 µl of chloroform-isoamyl alcohol (24:1), and incubated with frequent inversions for 30 min. The aqueous Niclosamide phase was separated by centrifugation (20-30 min at 16.100-20.800 × g), transferred to a fresh tube, and DNA was precipitated by addition of an equal volume of isopropanol followed by centrifugation (20 min at 16.100-20.800 × g). The precipitate was washed with 70% ethanol and centrifuged (15 min at 16.100-16.500 × g), and the supernatant was removed before the precipitate was left to air dry. DNA was resuspended in 100 µl 10 mM Tris HCl buffer (pH 8.5). Plasmid DNA was purified according to the manual provided with the Plasmid Mini/Midi kits (QIAGEN®). Table 1 Strains and plasmids used in this study strain or plasmid description, phenotype or genotype relevant for this study a reference Strains     Escherichia coli TOP10 One Shot® TOP10 electro/chemically competent E. coli for cloning Invitrogen MW3 Bacillus licheniformis DSM13 (ΔhsdR1, ΔhsdR2) [50] NVH-1307 B.