R2a agar

Water samples were gathered from 49 sites (Figure 1) on non-rainy days and bacteria were isolated using cultivation-based methods. Similar to eDNA isolation, first, water samples were filtered through a pre-filter (Y100A047A, Advantec^®, diameter: 47 mm, pore size: 10 mm) and 0.1 mL of each filtrate was inoculated onto R2A agar (Becton Dickinson) and BBL™ Standard Methods Agar (Tryptone Glucose Yeast Agar) (Becton Dickinson) plates for bacterial isolation. The plates were incubated at 25°C for 2–4 days. Each colony grown on the plate was transferred aseptically into nutrient broth (Becton Dickinson) medium. The pure liquid culture was incubated at 37°C overnight and used for antimicrobial susceptibility testing.

The mycelia of 7-day-old CBS 648.67 and CFCC 80795 grown in liquid-submerged cultures were filtered and cryo-ground (0–4°C) using RETSCH’s Mixer Mill MM 400. After grinding, products were mixed with 0.25 M sucrose solution and centrifuged at different rotational speeds to obtain isolated bacteria. Supernatants were collected and filtered through cellulose nitrate (Cellulose Nitrate [Mixed Cellulose Ester] Membrane Filters, 5‍ ‍μm, Sartorius) to obtain suspensions containing endobacteria. They were then centrifuged at 13,000×g at 4°C for 20‍ ‍min. The supernatants were gently removed to avoid losing endobacteria, which were resuspended in 0.25 M sucrose solution. Suspensions were plated onto agar plates and incubated at 25°C.
Each bacterial suspension was spread onto two media (200‍ ‍μL of suspensions per plate): LB agar (L^–1: 10‍ ‍g tryptone [Becton, Dickinson and Co.], 10‍ ‍g sodium chloride [Sinopharm Chemical Reagent], 5‍ ‍g yeast extract [Becton, Dickinson and Co.], and 1.5% [w/v] agar [Sinopharm Chemical Reagent]) and R2A agar (Becton, Dickinson and Co.), and then incubated at 25°C for 2‍ ‍weeks. R2A agar is suitable for Pelomonas growth according to Gomila et al. (2007) (link), and LB agar is commonly used as a bacterial medium. Therefore, these two media were selected for the cultivation of endobacteria. Ten plates were used to observe the growth of endobacteria.

Ginsenoside standards that are over 98% purity, such as Rb₁, Rb₂, Rc, Rd, Rg₃(S), Rh₂(S), F₂, compound K (C-K), protopanaxadiol (PPD), Rg₁, Re, Rg₂(S), Rh₁(S), and protopanaxatriol (PPT), were purchased from (Zelang Medical Technology Co., Ltd., Nanjing, China). HPLC grade acetonitrile was obtained from Merck (Darmstadt, Germany). GypXVII, GypLXXV, and ginsenosides compound O (C-O), compound Y (C-Y), compound Mc₁ (C-Mc₁), and compound Mc (C-Mc), were prepared as described by Cui et al. [23 (link)]. 5-bromo-4-chloro-3-indolyl β-d-glucopyranoside (X-Glc) was purchased from Wako Co. Ltd. (Tokyo, Japan). The other chemicals used were a minimum of analytical reagent grade. Microbacterium sp. Gsoil 167, was isolated from a soil sample of a ginseng field, Pocheon Province, South Korea, and cultivated on R2A agar (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) under aerobic conditions at 30 °C and used for the gene cloning experiment. Escherichia coli BL21 (DE3), and pGEX 4T-1 plasmid (GE Healthcare, Chicago, IL, USA) were used as host and expression vector sources, respectively. The E. coli cells harboring the BglG167b-containing recombinant plasmid for protein expression was cultivated in a Luria-Bertani (LB) medium supplemented with ampicillin (100 mg/L).

Bacterial strains were isolated from a xenic KW culture, according to a procedure described previously with some modifications (55 (link)). Briefly, the xenic KW culture was mechanically homogenized, serially diluted, and spread on R2A agar (BD, USA). After 3 d of incubation at 25°C, 16S rRNA genes from 50 randomly selected colonies were amplified and double-digested with a mixture of HaeIII and HhaI. Representative PCR products of different fragment patterns were sequenced, and their sequences were compared with those of all validated type strains using the Nucleotide Similarity Search program in the EzBioCloud (56 (link)). To evaluate the effects of the bacterial isolates on the growth of NIES-298 culture, bacterial isolates were cultured in R2A broth, washed using BG-11 broth, and inoculated into fresh NIES-298 cultures to a final concentration of 10⁷ bacterial cells/mL.
The inactivation test for the bleaching property of bleached culture by strain MAE1-K, bleached NIES-298 culture obtained from the end of the death phase was filtered using a 0.45 μm filter (Millipore, USA). Strain MAE1-K was cultured in R2A broth, washed with BG-11 broth, and inoculated into the filtered bleached culture (approximately 10⁶ cells/mL). After 24 h of aerobic incubation at 25°C, bleached culture treated with strain MAE1-K was filtered again using a 0.45 μm filter and used for the growth tests.

Theginsenoside Re (purity: 87.6%) from the root of Panax quinquefolius from FusongBiotech Co. Ltd. (China) was used as the substrate in the currentinvestigation. Ginsenosides standards which are over 98% purity such as Rb₁, Rb₂, Rc, Rd, Rg₂(S), Rh₂(S), F₂, compound K (C-K), protopanaxadiol (PPD), Rg₁, Re, Rg₂(S), Rh₁(S), and protopanaxatriol (PPT) were purchased from Nanjing Zelang Medical Technology Co. Ltd. (China). Methanol and acetonitrile with HPLC grade were obtained from Merck (Darmstadt, Germany). The other chemicals used in this study were a minimum of analytical reagent grade, and the sources are noted individually in the Methods section. Pseudonocardia sp. Gsoil 1536, which has ginsenoside-hydrolyzing activity, was isolated from the soil sample in ginseng filed, Pocheon Province, South Korea, and cultivated on R2A agar (BD, USA) under aerobic conditions at 30°C and used for the gene cloning experiment. Escherichia coli BL21 (DE3) and pGEX 4T-1 plasmid (GE Healthcare, USA) were used as host, and expression vectorsources, respectively. The recombinant E. coli for protein expression was cultivated in a Luria-Bertani (LB) medium supplemented with ampicillin (100 mg/l).