Chloromycetin

The bacterial strains and plasmids used in this study are listed in Table 1. The strain A. caldus MTH-04 has been deposited in the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC 1.15711. Starkey -S⁰ liquid medium and solid Starkey-Na₂S₂O₃ plates for A. caldus growth were prepared as reported previously [34 (link)]. Chloromycetin, kanamycin, and streptomycin (Sigma, St. Louis, MO, USA) were added to a final concentration of 34, 100, and 100 μg/mL in LB medium, and 60, 100, and 100 μg/mL in both liquid and solid Starkey media, respectively. The culturing conditions were 37 °C and 200 r/min for E. coli and 40 °C and 150 r/min for A. caldus MTH-04. The cultivation method and cell measurement for A. caldus were performed according to previously reported methods [35 (link)].

The strains used in this study are listed in Additional file 1: Table S1. EC135 lacking all R-M systems and orphan MTases was used as a cloning host to construct plasmids [40 (link)]. All strains were cultured in Luria–Bertani (LB) medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) supplemented with the appropriate antibiotics (100 µg/mL ampicillin was used in E. coli and 20 µg/mL erythromycin, 10 µg/mL kanamycin, and 10 µg/mL chloromycetin were used in B. subtilis).
Kits for DNA purification/gel recovery and extracting genomic DNA, plasmid DNA, and RNA were purchased from TIANGEN Biotech (Beijing, China). DNA polymerase, restriction enzymes, and dNTPs were purchased from New England Biolabs (USA). Antibiotics, inducers, and standard chemicals, such as ampicillin, kanamycin, chloromycetin, erythromycin, IPTG, xylose, purine bases, nucleotides, and nucleosides were purchased from Sigma-Aldrich (USA). Tryptone and yeast extract were purchased from Oxoid Company (UK). Other reagents for cell culture and fermentation medium were all analytical pure, and purchased from Beijing Modern Oriental Fine Chemical Co., Ltd (China).

L. casei 393 was kindly supplied by Prof. Jos Seegers (NIZO, Ede, The Netherlands) and cultured in sterile Man, Rogosa, and Sharpe (MRS) broth at 37 °C anaerobically without shaking. To analyze the expression of Cap-LTB protein, recombinant strains were grown in basal MRS medium supplemented with 2% xylose. The antibiotic concentration used for the selection of transformants was 10 μg/mL of chloromycetin (Sigma, Ronkonkoma, NY, USA) if necessary. The plasmids pPG611.1 and pPG611.2 were kindly gifted by Prof. Jos Seegers (NIZO, Ede, The Netherlands).