Tetracycline

5-FOA (Tokyo Chemical Industry, Tokyo, Japan), uracil and chloramphenicol (FUJIFILM, Tokyo, Japan), ampicillin (Sigma-Aldrich, St. Louis, MO, USA), tetracycline, isopropyl β-D-1-thiogalactopyranoside (IPTG), and a protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan) were used for mutant selection, gene expression, and protein analysis. DNA polymerase for polymerase chain reaction (PCR) and In-Fusion HD Cloning Kit were purchased from TaKaRa Bio Inc. (Shiga, Japan). DNA purification was performed using Spin Miniprep Kit for plasmid DNAs and Gentra Puregene Yeast/Bact. Kit for genomic DNAs (QIAGEN, Hilden, Germany). Guide-it Cas9 polyclonal antibody and goat anti-rabbit IgG horseradish peroxidase (HRP)-linked antibody were purchased from TaKaRa Bio Inc. and Agilent (P0448, Agilent, Santa Clara, CA, USA), respectively. Enhanced chemiluminescence (ECL) prime, 2-D Clean-Up Kit, and Immobiline DryStrips were purchased from Cytiva (Tokyo, Japan).

A total of 43 OS-MRSA isolates from various clinical samples were collected from routine clinical laboratories in hospitals across Japan and Taiwan between 1998 and 2015 (Table S1^{63 (link)–65 (link)}). Mueller–Hinton broth (MHB; Becton Dickinson Co., Ltd., Sparks, MD, USA) and tryptic soy broth (TSB; Becton Dickinson) were used to culture S. aureus, whereas Escherichia coli was grown in Luria–Bertani (LB; Becton Dickinson) medium. In some experiments, antibiotics were added to the medium at the following concentrations: ampicillin (Nacalai Tesque, Inc., Kyoto, Japan) at 100 µg/mL for E. coli, tetracycline (Nacalai Tesque) at 5 µg/mL for S. aureus, and chloramphenicol (Nacalai Tesque) at 10 µg/mL for S. aureus and E. coli. For preservation, bacterial cells were cultivated on tryptic soy agar (TSA; Becton Dickinson) and incubated at 37 °C upon receipt. A single colony was then selected and grown overnight in TSB at 37 °C. The overnight culture was aliquoted and stored at − 80 °C in 50% glycerol (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) until use.

Reticuline-producing E. coli harboring pCOLADuet-1 or pCOLADuet-1_AtDTX1 were cultured overnight in liquid LB containing 80 mg/L ampicillin (Sigma-Aldrich, St. Louis, MO, USA), 2 mg/L tetracycline (Nacalai Tesque), 100 mg/L spectinomycin (Nacalai Tesque), 30 mg/L chloramphenicol (Nacalai Tesque), and 50 mg/L kanamycin at 30 °C with shaking at 200 rpm. The overnight grown cultures were treated as described in section 2.5. Growth was measured by OD₆₀₀.