Carbenicillin

The bacterial expression vectors pCZS and pBZS (Additional file 1: Fig. S3) carrying a gene encoding methyltransferase were introduced into T7Express (New England Biolabs, Ipswich, MA, USA). The cells were inoculated in 3 mL of LB medium (1% [w/v] tryptone, 0.5% [w/v] yeast extract, and 0.5% [w/v] sodium chloride) with 100 µg/mL carbenicillin (Nacalai Tesque, Kyoto, Japan) and grown at 37 °C overnight with shaking at 200 rpm. Fifty microliters of each cell suspension was diluted in 3 mL of LB medium with carbenicillin, and the cells were cultivated at 37 °C for 3 h with shaking. For pCZS, the cell suspension was cooled in ice-cold water for 30 min, and 3 µL of 1 M isopropyl β-D-1-thiogalactopyranoside (IPTG, Nacalai Tesque) was added to induce protein expression. For pBZS, 3 µL of 30% (w/v) L-arabinose was added to induce protein expression. Cells were cultivated with shaking at 200 rpm and 16 °C overnight (pCZS) or 37 °C for 3 h (pBZS). The cells were collected via centrifugation at 2500 × g for 15 min and used for DNA purification using the DNeasy Blood & Tissue Kit (Qiagen), following the optional protocol for gram-negative bacteria provided by the manufacturer. The genomic DNA concentration was determined using the Qubit dsDNA BR Assay Kit and Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA).

The gene encoding VTopoI (GenBank: L13447.1) was synthesized by Eurofins Genomics Inc (Tokyo, Japan), with codon optimization for Escherichia coli K-12, exclusion of internal BamHI and EcoRI sites, and their inclusion at the 5′- and 3′-ends of the DNA, respectively (Supplementary Sequences). The BamHI-EcoRI fragment of the gene was subcloned into the BamHI-EcoRI site of pColdTF, pColdI, and pET28a. To produce T4 polynucleotide kinase (T4 PNK), we used polymerase chain reaction (PCR) to amplify the pseT gene of T4 phage from genomic DNA (T4 GT7 DNA, Nippon Gene, Tokyo, Japan) with BamHI and EcoRI sites at the 5′ and 3′ ends, respectively (Supplementary Sequences), and cloned it into the BamHI-EcoRI site of pColdI. These constructs were introduced into T7Express (New England Biolabs, Ipswich, MA) and cultivated in Luria–Bertani broth (LB) medium (1% (w/v) tryptone, 0.5% (w/v) yeast extract, and 0.5% (w/v) sodium chloride) with 100 µg/mL carbenicillin (for pColdTF and pColdI vector, Nacalai Tesque, Kyoto, Japan) or 50 µg/mL kanamycin (for pET28a, Fujifilm Wako Chemicals, Tokyo, Japan).

MDP1 deficient M. smegmatis (Δmdp1) was kindly provided by Dr. John L. Dahl (University of Minnesota Duluth). E. coli DH5α strain was used for all gene manipulations and was cultured in LB broth or on LB agar (both from Sigma-Aldrich, St. Louis, MO). Rosetta2 (DE3) pLyS (Novagen, Germany), BL21 Star (DE3) pLyS, and BL21Star (DE3) (invitrogen, CA), were also cultured in LB broth to obtain recombinant MDP1. M. smegmatis strain was cultured in Mueller HintonII media (BD, MD) supplemented with 0.05% (v/v) Tween 80, 50 μg/ml Hygromycin B (Hyg), and 10 μg/ml kanamycin (Km).
Hyg, Km, chloramphenicol, and isopropyl b-D-thiogalactopyranoside (IPTG) were purchased from FUJIFILM Wako Pure Chemical cooperation (Osaka, Japan). Carbenicillin was purchased from Nacalai tesque (Kyoto, Japan). Acetamide was purchased from Sigma-Aldrich (St. Louis. MO).