7 azaindole

C. acnes KCCM 41747 (ATCC 6919) (isolated from human facial acne), methicillin-sensitive S. aureus ATCC 6538, and fluconazole-resistant C. albicans strain DAY185 were used in this study. The C. acnes strain was cultured on Reinforced Clostridium Media (RCM)-agar plates for colony preparation and in liquid RCM at 37°C under anaerobic conditions (BD GasPak EZ Gas Generating Anaerobic Pouch Systems; Fisher Scientific, Pittsburgh, PA, USA) for all other experiments. The S. aureus strain was cultured in LB medium at 37°C, and the C. albicans strain was cultured in Potato Dextrose Broth (PDB) medium at 37°C.
Twenty indoles, namely, 7-azaindole, 7-benzyloxyindole, 3,3′-diindolylmethane (DIM), 7-formylindole, 7-hydroxyindole, indole, indole-3-acetamide, indole-3-acetic acid, indole-3-acetonitrile, indole-3-butyric acid, indole-3-carbinol, indole-3-carboxyaldehyde, indole-7-carboxylic acid, indole-3-propionic acid, isatin, 7-methoxyindole, 7-methylindole, methyl indole-7-carboxylate, 7-nitroindole, and 2-oxindole were purchased from Sigma-Aldrich (St. Louis, MO, USA), Wako Chemicals Inc. (Richmond, VA, USA) or Combi-Blocks, Inc. (San Diego, CA, USA). Dimethyl sulfoxide (DMSO) was used to dissolve all indoles, and 0.1% (vol/vol) DMSO was used as the negative control; at this concentration, DMSO did not affect bacterial growth or biofilm formation.

Screening of small molecules changing circadian period was performed as previously described (Uehara et�al. 2019) (link), using an ITbM chemical library (Ziadi et�al. 2017 , Toh et�al. 2018 (link)), in which all molecules were dissolved in dimethyl sulfoxide (DMSO, Molecular biology grade, Nacalai, Japan), CCA1:LUC transgenic seedlings and an automated luminescence monitoring system (CL96, Churitsu). Molecules dissolved in DMSO at 1 mM were diluted with half strength of MS media to 50 �M, and dropped on 4-day-old seedling grown under LD. Period length of CCA1:LUC was determined by CL96-attached software (Churitsu, Japan), as described previously (Kamioka et�al. 2016) (link). After the first screening, the hit molecule (B-AZ) was tested in different concentrations to period-lengthening effects of CCA1:LUC and TOC1:LUC. Other hit molecules discovered in this project will be described in future. B-AZ was also purchased from SINOVA Inc., Maryland. 7-Azaindole, 3-bromo-7-Azaindole and 4-bromo-7-Azaindole were purchased from Sigma-Aldrich, Tokyo Kasei and Fujifilm-Wako, respectively. The sensitivity of B-AZ in prr5-11 CCA1:LUC, toc1-2 CCA1:LUC and prr5-11 toc1-2 CCA1:LUC was performed by the same method. Period lengths were normalized to the period length of each genotype treated with DMSO solvent control, since period length of the mutants was shorter than that of the wild type.

X. eapokensis was grown in liquid SF-900 II SFM (Thermo Fisher Scientific) in presence of 2% Amberlite^™ XAD-16 (Sigmal Aldrich) or SF-900 supplemented with 1 mM of 2-amino-3-chlorobenzoic acid, 2-amino-5-chlorobenzoic acid, 2-amino-3-methylbenzoic acid, 2-amino-5-methylbenzoic acid, 2-amino-3-methoxybenzoic acid, 2-amino-5-methoxybenzoic acid, anthranilic acid, 3-hydroxy anthranilic acid, 3-methyl proline, 3-benzyl proline, or 4-hydroxy proline, pipecolic acid, and 2 mM of 5-fluoro indole, 5-phenyl indole, or 5-methyl indole and 3 mM of 7-azaindole (all chemicals were purchased from Sigma Aldrich). The cultures were cultivated at 30°C for three days with a starting OD₆₀₀ of 0.1 and cultures were extracted as described below.