As selective antibiotics for the presence of pMAD_SpR or its derivative constructs, 100 µg/ml ampicillin and 100 µg/ml spectinomycin was used for E. coli TOP10 growth,

and 3 µg/ml erythromycin and 250-300 µg/ml spectinomycin for B. licheniformis growth. This vector carries a constitutively expressed β-galactosidase gene, allowing blue-white screening on plates spread with X-Gal (40 µl 40 mg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, VWR, BDH Prolabo). This screening was, however, not always #CRT0066101 cell line randurls[1|1|,|CHEM1|]# unambiguous following long incubations of plates with B. licheniformis MW3 transformants, probably due to the natural precence of β-galactosidase in B. licheniformis DSM 13 [77]. To construct the gene replacement vector, primers (Table 2) were designed to amplify two DNA fragments, one homologous to upstream (709 bp) and one to downstream (696 bp) regions of the deletion target (567 bp) in the gerAA. Platinum Taq DNA Polymerase High Fidelity kit (Invitrogen) was used for PCR amplification

with the following amplification procedure: initial denaturation for 2 min at 94°C, 30 cycles of 30 s at 94 °C, 30 s at 50 °C and 1 min at 68 °C, and final extension at 68 °C for 10 min. Primers of the upstream and downstream amplicons Selleck H 89 contained restriction sites BamHI and EcoRI respectively (Table 2), allowing a two_step ligation into the corresponding restriction sites on either side of the (SpR)-cassette in pMAD_SpR. The Succinyl-CoA resulting gene replacement plasmid, pMAD_SpRΔgerAA, was controlled for correct orientation of the upstream and downstream fragments by PCR. pMAD_SpRΔgerAA was introduced into B. licheniformis MW3 by electroporation, and allelic exchange

of internal parts of gerAA (567 bp) with the (SpR)-cassette of pMAD_SpRΔgerAA was allowed by double crossover. The protocol was performed as described by Arnaud et al.[75], except using growth temperatures of 37 °C following initial transformation, an incubation temperature of 45 °C and spectinomycin present during plasmid curing, and an incubation temperature of 37 °C when screening for the double crossover phenotype (spectinomycin resistant and erythromycin sensitive colonies). Chromosomal DNA was purified from a candidate colony and used in PCR amplifications (as described above) with primers hybridizing outside the cloned DNA fragment and inside the spectinomycin cassette (Table 2) to verify the deletion and insertion by sequencing. The disruption mutant was named B. licheniformis MW3ΔgerAA::spc (NVH-1307) and used in the following complementation, sporulation and germination assays.