SpR. This study NVH-1311 NVH-1307 with pHT315_MW3gerA. SpR and EmR. This study ATCC 14579 Bacillus cereus type strain [72, 73] B252 Bacillus subtilis

isolated from tap water [71] Plasmids     pMAD E. coli/B. licheniformis shuttle plasmid. ApR, EmR, ori Bacillus ts and pclpB-bgaB selleck chemicals llc [75] pMAD_SpR pMAD-derivate supplemented with a SpR cassette in the SalI site. ApR, EmR, SpR, ori Bacillus ts and pclpB-bgaB [76] pMAD_SpRΔgerAA pMAD_SpR-derivate allowing substitution of parts of gerAA in MW3 with a SpR cassette. ApR, EmR, SpR, ori Bacillus ts and pclpB-bgaB This study pHT315 E. coli/B. licheniformis shuttle plasmid. ApR and EmR [52] pHT315_MW3gerA pHT315-derivate containing gerA fragment b amplified from MW3 DNA template. ApR and EmR This study a ApR; resistance to ampicillin, EmR; resistance to erythromycin, SpR; resistance to spectinomycin, ori Bacillus ts; temperature-sensitive Bacillus origin of replication, pclpB-bgaB; constitutively expressed termostable β-galactosidase Citarinostat (allowing blue/white screening of transformants on X-Gal plates). b gerA fragment contains a sequence

151 bp upstream of gerAA, gerAA, gerAB, gerAC and 177 bp downstream of gerAC. Preparation and transformation of B. licheniformis electrocompetent cells Electrocompetent B. licheniformis was prepared and transformed by a modified version of the protocol described by Mahillion et al.[74] as follows. A preculture in Brain Heart Infusion broth (BHI) (Oxoid, Cambridge, United Kingdom) was grown overnight at 37 °C, and 1 ml was used to inoculate 200 ml pre-warmed BHI in a 1 l Erlenmeyer. The culture was incubated 4 to 5 h at 37 °C and 150 rpm (HT-Infors AG CH-4103, Bottmingen, Switzerland) until A600 of 0.9-1.0 was reached (Shimadzu UV-VIS 160A, Shimadzu Europa GMBH). Cells were pelleted and washed twice with 200 ml RT autoclaved MilliQ water (MQ) by 15 min centrifugations at 3.300 and 10.400 × g. The pellet was resuspended in a 10 ml filter sterilised solution of freshly prepared polyethylene glycol (PEG) 6000 (Merck, Darmstadt, Germany), made by dissolving 40 g PEG6000 in 100 ml MQ. Following 15 min centrifugation at 4.080 × g,

cells were resuspended Montelukast Sodium in 0.5-1 ml of the PEG6000/MQ solution, aliquoted (100 µl) and stored at -80 °C. Transformation was conducted by adding 2 µl plasmid to 100 µl electro competent cells thawed on ice. Following ~1 min incubation on ice, electroporation was performed at 1.4 to 2.5 kV (Eppendorf Eporator, Eppendorf AG, Hamburg, Germany or MicroPulser™, Bio-Rad, Hercules, CA), using 0.2 cm gap width electroporation cuvettes (selleck Bio-Rad Laboratories, Hercules, CA). Before plating on selective LB-agar plates, cells were recovered in LB or S. O. C. medium (Invitrogen) at 37 °C, 150 rpm, for 4 to 5 h. Construction of B. licheniformis MW3ΔgerAA::spc The shuttle vector used for construction of a spectinomycin resistant (SpR) insertion deletion in the gerAA was pMAD_SpR.