Recombinant DNA techniques and oligonucleotides.

The techniques of standard molecular cloning were performed as described previously (60). The GenElute genomic DNA kit (Sigma-Aldrich, St. Louis, MO) was used to isolate genomic DNA of L. lactis. The NucleoSpin Plasmid EasyPure kit (Bioke, Leiden, the Netherlands) and the NucleoSpin gel and PCR clean-up kit (Bioke, Leiden, the Netherlands) were employed to extract plasmids and purify PCR products following the manufacturer’s instructions, respectively. PCRs were conducted with PrimeStar Max DNA polymerase (TaKaRa Bio Europe SAS, Saint-Germain-en-Laye, France) referring to the manufacturer’s protocol. The obtained PCR products were mixed and treated with the Gibson Assembly master mix (Bioke, Leiden, the Netherlands), yielding 20-nucleotide overhangs annealing to complementary overhangs. The mixtures were applied to transform E. coli DH5α directly to generate plasmids. Oligonucleotides used in this work were purchased from Biolegio BV (Nijmegen, the Netherlands) and are given in Table S4. The transformation of E. coli strains was performed following the standard procedures (60). B. subtilis 168 and WB800 were transformed based on natural competence (61). In miniBacillus PG10, competence genes (comK and comS) controlled by the mannitol-inducible promoter (P_mtlA) were induced by adding 0.5% (wt/vol) mannitol (62). All nucleotide sequencing was performed at Macrogen Europe (Amsterdam, the Netherlands). The detailed procedure of all the plasmid constructions is described in Text S1.

Oligonucleotides used in this study. Download Table S4, DOCX file, 0.01 MB.