General
Electron ionization mass spectrometry (EI-MS), field desorption mass spectrometry (FD-MS), and field desorption high-resolution mass spectrometry (FD-HR-MS) were conducted using MS spectrometers JMS-T100GCV and JMS-SX-102 (JEOL, Tokyo, Japan). 1H-/13C-NMR spectra were recorded using a JEOL JNM-EX270 (JEOL, Tokyo, Japan) at 270/67.5 MHz and a Bruker AMX-500 (Bruker, Billerica, MA, USA) at 500/125 MHz.
Diacetonamine hydrogen oxalate (1a) was purchased from Sigma-Aldrich (St. Louis, MO, USA), and diacetonamine hydrochloride (1b) and N-acetyldiacetonamine (2) were prepared from 1a. As another chemical derivatizable from 1, diacetone acrylamide (3) was purchased from Wako (Osaka, Japan), while a β-amino acid, 3-amino-3-metylbutyric acid (4), used as a negative control was purchased from Combi-Blocks (San Diego, CA, USA). Their chemical structures are shown in Fig. 2.
Bacterial strains and culture conditions
For the sporulation assay, we used B. amyloliquefaciens AHU 2170 isolated from the rhizosphere of Chinese cabbage (Brassica rapa var. pekinensis) and stocked in our laboratory (Sugita 2008) and B. megaterium NBRC 15308 purchased from NITE Biological Resource Center, Kisarazu, Japan. Both strains of bacilli were pre-cultured at 30 °C for 24 h on a nutrient-broth (NB) agar plate before use. The B. amyloliquefaciens wild-type strain easily formed spore-based pellicle in NB liquid medium, whereas B. megaterium was a rarely sporulating Bacillus species (Strnadová et al. 1991; Larsen et al. 2014).
Bioassay for sporulation
After shaking culture of B. amyloliquefaciens or B. megaterium in NB medium (40 mL) at 30 °C for 48 h in the dark as reciprocation of 100 rpm, the samples were divided into four portions (10 mL each in 15-mL Falcon tube) and centrifuged at 3500g at 4 °C for 15 min. The resulting bacterial cell pellet was re-suspended in 10 mL MSG (minimal salts mixture +0.5% glutamic acid) medium (Freese et al. 1979) containing a test substance equivalent to that originally contained in 10 g soybean curd residue, or maximum concentration at 400 µM. When 400 µM of a chemical solution should be prepared, a 400 mM solution of the compound dissolved in dimethyl sulfoxide (DMSO) was first passed through a sterile Millex®-GV membrane filter (0.22 µm polyvinylidene fluoride, Merck Millipore Ltd., Billerica, MA), and a 10 µL portion of the solution was added to 10 mL MSG medium previously autoclaved. The 10-mL cell suspension was linear-shaken in a 30-mL Erlenmeyer flask at 80 rpm at 30 °C for 48 h for B. amyloliquefaciens. B. megaterium required 72–120 h for sporulation.
A LIVE/DEAD BacLight Bacterial Viability Kit (L13152) (Laflamme et al. 2004) was used to evaluate bacterial sporulation induction. The bacterial cells exposed to a test substance or fraction for the incubation time were collected by brief centrifugation, and the resulting cell pellet was washed with 1 mL saline twice by pipetting and then suspended in 1 mL or 100 µL Milli-Q water. The cell suspension was then mixed with 10 µL of LIVE/DEAD BacLight Bacterial Viability Kit solution (SYTO 9: propidium iodide, 9:1) and incubated for 15 min at room temperature in the dark, and then centrifuged at 10,000g for 1 min to remove the supernatant. The resulting cell pellet was re-suspended in 100 or 20 µL Milli-Q water, and a 3-µL portion of the resulting cell suspension was dropped on a glass slide (S1111, Matsunami Glass, Kishiwada, Japan) and pressed tightly with a cover glass (Matsunami Glass). The prepared slide was observed using a fluorescent microscope BioRevo (Keyens, Osaka, Japan) under 1500× magnification with an oil immersion lens.
Spores of B. amyloliquefaciens are barrel-like short-rods (physical size of approximately 0.8 µm × 1.2 µm) distinguishable from vegetative cells (typical rods, approximately 0.7 × 2.5 µm). Using a GFP-BP filter (Ex 470/40, Em 535/50, dichroic mirror 495), live cells and spores were stained as green with SYTO 9, whereas propidium iodide with a TRITC filter (Ex 540/25, Em 605/70, dichroic mirror 565) stained dead cells red. Competent cells (Yang et al. 2015) were observed as orange-colored rods. The spore cells stained with SYTO 9 were observed as green along the border with the unstained central part. Sporulation frequency (numbers of spore/numbers of spore plus vegetative cells) was calculated.
Vegetative cells of B. megaterium are relatively large (1.0 × 15 µm or more), while sporulated cells were more spherical endospores (approximately 0.8 µm × 1.0 µm) than those of B. amlyloliquefaciens and easily distinguishable from vegetative cells. Immature spores of B. megaterium were stained by SYTO 9, while mature spores were not.
Heat-tolerance assay for evaluating sporulation frequency
A 100-µL portion of spore suspension diluted 101–105-fold with Milli-Q water in a polymerase chain reaction (PCR) tube was heated at 95 °C for 10 min in a PCR thermocycler. After cooling to room temperature, the suspension was well mixed by pipetting and spread over a NB agar plate using a disposable bacteria spreader. After 24-h incubation at 30 °C in the dark, colonies were counted. As a blank, all bacterial cell suspensions were spread on NB agar plates without heating treatment.
Extraction of sporulation-inducing factor (SIF) from soybean curd residue
A 10-kg fresh weight soybean curd residue (1.85 kg from Miyoshiya Tofu, Bibai, Japan, 2.45 kg, Mameta, Sapporo, Japan, 3.0 kg, Marukawa Foods, Sapporo, Japan, and 2.7 kg, At the Bran Made of Koyama, Sapporo, Japan) was collected from grocery shops and mixed for extraction with methanol (MeOH). The soybean curd residues were soaked in 10 L of MeOH, and the methanolic extract was collected by decantation and squeezing the remaining residues in a cotton cloth. The squeezed residues were re-extracted with another 6 L of MeOH. The methanolic extracts further collected and combined were filtered in vacuo and concentrated under a low pressure to 500 mL of an aqueous solution. This aqueous solution was partitioned with hexane (500 mL × 3, 0.7 g), ethyl acetate (EtOAc) (500 mL × 3, 2.0 g), and normal butanol (n-BuOH) (500 mL × 3, 9.6 g). The resulting aqueous layer was the only fraction active as SIF.
Column chromatography in Sephadex G-25 and Sephadex LH-20
A half volume of the aqueous layer concentrated to 50 mL (over 25 g) was subjected to gel filtration using a 1.5 L Sephadex G-25 (200 g, GE Healthcare Little Chalfont, UK) column (400 mL per fraction, more than 8 fractions). Sporulation-inducing activities were observed in the first three fractions, from Fr-G-1 to Fr-G-3. The remaining half portions of the extracts were also subjected to gel filtration at the same scale as the Sephadex G-25 column, and all the active fractions obtained were combined (Fr-G-1–3, 21.76 g).
Fraction Fr-G-1–3 was further fractionated by column chromatography using a 450-mL Sephadex LH-20 (Amersham Pharmacia Biotech AB, Uppsala, Sweden) column by eluting with 5% MeOH (300 mL per fraction, total of 12 fractions), and Fr-LH-2 and LH-3 (Fr-LH-2–3, 16.55 g) showed active sporulation induction toward B. amyloliquefaciens.
Cation- and anion-exchange chromatography
For cation-exchange chromatography, Fr-LH-2–3 containing active components were applied to a 600 mL-bed volume of a CM-cellulose (cation exchange carrier, Wako) open column, previously conditioned with 0.2 M aq. HCl followed by washing with Milli-Q water. The active component was eluted with Milli-Q water in the second fraction (600 mL, Fr-CMC-2) obtained as 4.08 g of syrup.
Anion-exchange column chromatography using DEAE-cellulose (Wako, 600 mL-bed volume) was subsequently conducted for Fr-CMC-2. The DEAE-cellulose column, previously conditioned with 0.2 M aq. NaOH solution followed by washing with Milli-Q water, retained the active component in the column, despite elution with Milli-Q water (1200 mL). Therefore, the column was continuously eluted with 600 mL of 0.2 M Na2CO3 solution. As the concentrated solutes (Fr-DEAE-2, 9.71 g containing excessive Na2CO3 salts) exhibited sporulation induction, all solutes were subjected to a second gel filtration step in a 400-mL Sephadex G-25 column for elution with Milli-Q water (100 mL for each fraction). The third fraction (Fr-DS-3, 1.02 g) showed sporulation-inducing activity.
Normal phase silica gel TLC
Fraction Fr-DS-3 was analyzed by normal phase silica gel thin-layer chromatography (TLC) (Merck Kieselgel 60F254 Art. 1.05715, Merck, Kenilworth, NJ, USA) developed in n-BuOH:water (H2O):acetic acid (AcOH) 4:1:1. The main substance in the fraction gave a dark brown-colored spot at R
f
0.4 by spraying with vanillin-sulfuric acid reagent. The main compound was obtained by preparative TLC (10 × 10 cm) in n-BuOH:H2O:AcOH 4:1:1 for a small portion of Fr-DS-3. However, the main compound obtained and eluted from silica gel with 50% MeOH showed no sporulation induction. The chemical substance recovered from the plate lower zones of the main band also eluted with 50% MeOH and showed clear sporulation activity.
This fraction exhibited a characteristic unique pinkish color by spraying with p-anisaldehyde-sulfuric acid reagent followed by heating, but this compound showed a severe tailing in the acidic solvent. Thus, we replaced AcOH with 25% aq. NH4OH solution, and the substance gave a fine spot on normal phase silica gel TLC in n-BuOH:25% aq. NH4OH:acetone 4:5:2.
Silica gel column chromatography with n-BuOH:25% aq. NH4OH:acetone
The active component in Fr-DS-3 (1.00 g) was further purified by silica gel column chromatography (174 g, Wakogel C60, Wako) and eluted with n-BuOH: 25% aq. NH4OH: acetone (4:5:2). Silica gel column was first conditioned with Milli-Q water followed by the eluting solution, and then Fr-DS-3 was loaded to obtain fractions (25 mL each). Each fraction was immediately concentrated in vacuo. The target substance responsive to p-anisaldehyde-sulfuric acid reagent was detected in Fr-15–Fr-22 as a single spot on TLC. All fractions (SIF-Fr-15-22) together (450 mg of a colorless solid) exhibited significant sporulation-inducing activity at a concentration equivalent to 1 g soybean curd residue per mL medium (45 µg SIF-Fr-15-22 per mL).
FD-MS (m/z, %) of SIF-Fr-15–22 showed several parent ion peaks, such as 407.0 (42), 385.0 (20), 308.1 (21), 215.0 (25), 193.0 (100), and 116.1 (81). We also tested several coloring reagents for the detection of active spots on TLC, including p-anisaldehyde-sulfuric acid reagent and ninhydrin. The target compound showed a pale pink color on spraying with p-anisaldehyde-sulfuric acid reagent followed by strong heating, but no response to ninhydrin.
Acetylation of SIF-fraction
Although the active component was highly concentrated in SIF-Fr-15–22, the active fraction contained a relatively high amount of silica gel and contaminants. To remove inactive contaminants, a portion of the dried active fraction was subjected to an acetylation reaction. A portion of SIF-Fr-15–22 (29.5 mg) re-dissolved in 2 mL of acetic anhydride and pyridine (1:1) was incubated at 70 °C for 1 h. One of the main products, 2, was detected at an R
f
0.15 on silica gel TLC (hexane:EtOAc 1:1) as a purple-colored spot by spraying with p-anisaldehyde-sulfuric acid reagent. This product was purified by preparative TLC developed twice in hexane:EtOAc 1:1 to yield 4.0 mg of colorless syrup. EI-MS (m/z, %): 157 ([M]+, 11), 142 ([M–CH3]+, 9.4), 114 ([M–COCH2 + H]+, 18), 100 ([M–CH3-COCH2 + H]+, 94), 98 (42), 83 (57), 72 (24), 60 (22), 58 (100), and 43 (85). 1H-NMR (270 MHz, δH, in CDCl3): 5.8 (1H, br., NH), 2.94 (2H, s, CH2), 2.12 (3H, s, CH3), 1.90 (3H, s, Ac), and 1.39 (6H, s, 2 × CH3). 13C-NMR and distorsionless enhancement by polarization transfer (DEPT) (67 MHz, δH, in CDCl3): δC 208.18 (CO), 170.11 (COCH3), 52.15 (C), 50.93 (CH2), 31.77 (CH3), 27.52 (CH3 × 2), and 24.36 (COCH3).
Preparation of few diacetonamine derivatives from diacetonamine hydrogen oxalate
Commercially available diacetonamine hydrogen oxalate (1a, Sigma-Aldrich) was subjected to an anion exchange reaction. Compound 1a (1.23 g, 6 mmol) dissolved in 10 mL Milli-Q water was passed through a conditioned DEAE cellulose column (200 mL bed volume). An alkaline fraction eluted first was collected and neutralized using 1 M aq. HCl solution and then concentrated to yield 0.82 g of diacetonamine hydrochloride (1b) as a colorless syrup (90% yield).
For the acetylation reaction, 1a (196.4 mg) dissolved in a 1:1 mixture of Ac2O and pyridine (4 mL) was incubated at 70 °C for 1 h. A major product, N-acetyldiacetonamine (2), was obtained by preparative TLC (134.2 mg, 89% yield) as a pinkish purple-colored spot upon spraying with p-anisaldehyde-sulfuric acid spray reagent followed by heating.
Assay for diacetonamine and its amide derivatives
Diacetonamine hydrochloride (1b) was preliminarily tested at concentrations of 4, 40, and 400 µM for sporulation-inducing activities toward B. amyloliquefaciens and B. megaterium. All the test compounds, including 1b, N-acetyldiacetonamine (2), and diacetone acrylamide (3), were dissolved in DMSO to be 400 mM, and diluted with MSG medium into final concentrations of 4, 40, and 400 µM. The MSG medium for the sporulation assay always contained 0.1% DMSO.