Arabinose

Growth assays were performed as previously described^{31 (link)}. Briefly, the cobalamin-deficient E. coli ΔFEC strain was transformed with various expression vectors to express BacA_Mt, ECF-CbrT or ECF-FolT2. Single colonies were then innoculated into M9 medium supplemented with 0.00001% (w/v) arabinose (Sigma-Aldrich) and 50 µg ml⁻¹ methionine (Sigma-Aldrich). Cells were grown at 37 °C for 24 h and were then diluted with a ratio of 1:250 into 200 µl fresh M9 medium supplemented with 0.00001% (w/w) arabinose and either 1 nM cyano-cobalamin or 50 µg ml⁻¹ methionine (Sigma-Aldrich) in 96-well plates (Greiner). Plates were sealed with sterile and gas-permeable foil (Breathe-Easy, Diversified Biotech) and incubated at 37 °C for 24 h. The OD₆₀₀ was measured every 10 min on a SpectraMax ABS plus Microplate reader (Molecular Devices). The measurements were performed as biological triplicates each containing technical triplicates and collected using the instruments’ SoftMax Pro version 7.1.2 (Molecular Devices).

In this study, we used Escherichia coli K12 MG1655 bearing a ColE1-like (p15A) plasmid, pBGT, encoding for the β-lactamase resistance gene bla_TEM-1 that confers resistance to ampicillin under a constitutive promoter, an GFPmut2 gene under an arabinose inducible promoter, and the araC repressor. Mean PCN = 19.12, s.d. = 1.53^{36 (link)}. As a control, a strain E. coli K12 MG1655 was used, carrying the construct araC − pBAD − gfp2 − bla_TEM-1 inserted into the chromosome at the λ-phage integration site (attB). Strains bearing plasmid variants G54U and G55U contained a point mutation in the origin of replication: G to U changes at positions 54 and 55 of the RNAI placed in the loop of the central hairpin and affect the RNAI-RNAII kissing complex that controls plasmid replication and PCN. All experiments were conducted in Lysogeny Broth- Lenox (LB) (Sigma-L3022) supplemented with arabinose (0.5% w/v) and appropriate ampicillin concentrations were supplemented as indicated in each experiment. arabinose stocks solutions were prepared at 20% w/v by diluting 2 g of arabinose (Sigma-A91906) in 10 ml DD water sterilized by 0.22 μm filtration. AMP stock solutions (100 mg/ml) were prepared by diluting ampicillin (Sigma-A0166) directly in 0.5% w/v arabinose LB.

The wolfberry pollen was collected from the Ningxia Academy of Agriculture and Forestry Sciences (Yinchuan, Ningxia, China). Cellulose DEAE-52, Sephadex G-100 columns and monosaccharide standards (mannose, glucuronic acid, galacturonic acid, xylose, galactose, arabinose, and trehalose) were purchased from Sigma Chemical Co., Ltd. (St. Louis, MO, USA). HPLC-grade acetonitrile was purchased from TEDIA Co., Inc. (Fairfield, IA, USA). 1-phenyl-3-methyl-5-pyrazolone (PMP) high-glucose DMEM medium and fetal bovine serum were purchased from Gibco (Carlsbad, CA, USA). Penicillin-streptomycin double antibody and 0.25% trypsin were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from KeyGEN BioTech Co., Ltd. Nanjing, China). In situ cell death detection kit and POD were purchased from Roche Diagnostics (Indianapolis, IN, USA) and DAPI was obtained from ZSGB-BIO Biological Co., Ltd. (Beijing, China). Annexin V-FITC cell apoptosis detection kit was purchased from Best Bio Biological Co., Ltd. (Shanghai, China).