Restriction enzyme

All chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA), and restriction enzymes were obtained from Takara Biotechnology Co., Ltd. (Dalian, China). All primers used in this study were synthesized by Beijing Genome Institute (BGI) and are listed in Table 1. Norepinephrine and epinephrine were purchased from Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China) and Aike Reagent Co., Ltd. (Chengdu, China), respectively. Antibodies for β-actin (#4970), phosphorylated CREB (#9198) and phosphorylated ERK1/2 (#9101) were purchased from Cell Signaling Technology (CST, Bevely, MA, USA). The pharmacological agents including H89, U73122, PD98059 and 2-aminoethoxydiphenyl borate (2-APB) were purchased from Calbiochem (Merck KGaA, Darmstadt, Germany). The EdU Cell Proliferation Kit was purchased from RiboBio Co., Ltd. (Guangzhou, China).

Blend Taq^® and Taq DNA polymerase were purchased from TOYOBO (Osaka, Japan) and New England Biolabs (Ipswich, MA, USA), respectively. T4 DNA Ligase was purchased from Takara Bio (Kusatsu, Japan). Restriction enzymes were purchased from Takara Bio and TOYOBO. Other general reagents were purchased from Wako Pure Chemical Industries (Osaka, Japan) and Nacalai Tesque (Kyoto, Japan).

Combined with lysozyme, genomic DNA and/or plasmids were extracted from B. subtilis and L. lactis strains using a rapid bacterial genomic DNA isolation kit (Sangon Biotech, Shanghai, China) and a rapid mini plasmid kit (Tiangen Biotech, Beijing, China), respectively. To study the effect of codon bias on the expression of Subtilisin QK-2 in L. lactis, codon optimization was performed using the JCat program [33 (link)] according to the protein sequence of Subtilisin QK-2 (GenBank: AJ579472.2). The codon-optimized gene of Subtilisin QK-2 (qk′, GenBank accession number KT725198) was synthesized by TsingKe Biological Technology (Wuhan, China) and was ligated into the pUC19 plasmid, resulting in pUC19-qk′. On the basis of the qk′, qkpro′ (GenBank accession number KT991841) containing the codon-optimized propeptide sequence only was obtained by overlap extension (SOE)-PCR. KOD Dash DNA polymerase (TOYOBO) was used to maintain the veracity of the PCR process. Restriction enzymes and T4 DNA ligase were purchased from TaKaRa and were used according to the manufacturer’s instructions. General molecular biological procedures were performed according to [34 ].

Lysobacter strains and the derived mutants were grown in 40% strength TSB medium. Davis minimal medium without methionine was used for the vitamin B₁₂ utilization assay (35 (link)). The concentration of indole in all experiments was 0.5 mM. The supplemental concentration of LeDSF (13-methyltetradecanoic acid) in the experiments was 5 μM. E. coli strains DH5α and S17-1 were used for DNA manipulation and conjugation assays, respectively. Additional bacterial strains and plasmids used in this study are described in Table S1 in the supplemental material. Extraction of plasmids and DNA fragments was performed following the instructions included with the kits purchased from Omega (plasmid mini kit I and gel extraction kit, Omega USA). All molecular manipulations were carried out according to methods described previously (30 (link), 36 (link)). Restriction enzymes and molecular biology reagents were purchased from TaKaRa (TaKaRa Bio Group, Japan). PCR primers were synthesized by Tsingke Biological Technology Company.