D glucose

Biofilm formation was determined in flat-bottom 96-well polystyrene microplates (Corning, New York, NY, USA) according to Stepanović et al. with some modifications [56 (link)]. A total of 200 μL of bacterial suspension 0.5 McFarland (1.5 × 10⁸ CFU/mL) in Tryptic Soy Broth medium (Becton Dickinson, Franklin Lakes, NJ, USA) with 1% (w/v) D-(+)-glucose (TSBG) was inoculated in wells and incubated at 37 °C for 24 h. After the incubation period, the wells were discharged and washed three times with 200 μL of phosphate-buffered saline (PBS). 200 μL of 1% (v/v) crystal violet (CV) (Merck, Damm, Germany) was added per well for 15 min and after the wells were discharged and washed three times with 200 μL of PBS. The microplates were air-dried and the biofilm-bound CV was dissolved adding 200 μL of 96% (v/v) ethanol per well. O.D. was determined at λ = 570 nm [56 (link),57 (link),58 (link)]. Bacterial strains were categorized as non-adherent (O.D. ≤ 0.120), weakly adherent (O.D. > 0.120) and strongly adherent (O.D. > 0.240) [30 (link)]. Results are expressed as the mean of three experiments.

Isoxanthohumol was prepared by chemical cyclization in aqueous NaOH solution at 0 °C as described by Stevens et al. [4 (link)], and also by a microbial transformation method using the fungus R. oryzae KCTC 6946 as previously reported by Kim and Lee [19 (link)]. Isoxanthohumol prepared by both methods was extracted with EtOAc and then purified by chromatographic methods including silica gel and reversed-phase C₁₈ MPLC. The spectroscopic data of isoxanthohumol (1) were in good agreement with data in the literature [1 (link)] and its structure was also confirmed by 2D NMR experiments. Optical rotation and CD measurements revealed that the substrate isoxanthohumol was a racemic mixture of (2S)- and (2R)-isoxanthohumol. Ingredients for media including D-glucose, peptone, malt extract, yeast extract, and potato dextrose medium were purchased from Becton, Dickinson and Co. (Sparks, MD, USA), and sucrose was purchased from Sigma-Aldrich Co. (St Louis, MO, USA).

Growth experiments were performed in defined M9 minimal media (M9MM) (Fluka) to confirm the observed phenotype. Single isolated colonies of SMG 9 and EPI300::pCC1FOS were grown overnight in M9MM (containing final concentrations of; D-glucose (0.4%), Bacto™ casamino acids (w/v 0.2%) (Becton, Dickinson and Co, Sparks, MD, USA), magnesium sulfate (MgSO₄) (2 mM), calcium chloride (CaCl₂) (0.1 mM) and 12.5 μg/ml Cm). Reagents were purchased from Sigma Aldrich (St. Louis, MO, USA) unless otherwise stated. Cells were harvested by centrifugation, washed in ¼ strength Ringers solution and resuspended in fresh M9MM. A 2% v/v inoculum was sub-cultured in fresh M9MM containing various concentrations of sodium chloride (0–8% w/v NaCl) and 1 mM of L-carnitine when required. Triplicate wells of a 96-well micro-titre plate were inoculated with 200 μl of the appropriate cell suspension. Plates were incubated at 37°C for 24–48 h in an automated spectrophotometer (Tecan Genios) which recorded the optical density at 595 nm (OD_{595 nm}) every hour. After 48 h the data was retrieved and analyzed using the Magellan 3 software program and graphs were created with Sigma Plot 10.0 (Systat Software Inc, London, UK). Results are presented as the average of triplicate experiments, with error bars being representative of the standard error of the mean (SEM).

(−)-α-Bisabolol (1) was purchased from Sigma-Aldrich (St. Louis, MO, USA). All the ingredients for microbial media including D-glucose, peptone, yeast extract, malt extract, and potato dextrose medium were purchased from Becton, Dickinson and Company (Sparks, MD, USA).
All the microorganisms were obtained from the Korean Collection for Type Cultures (KCTC). The microorganisms used for preliminary screening were as follows: Absidia coerulea KCTC 6936, Alternaria alternata 6005, Aspergillus fumigatus 6145, Cunninghamella elegans var. elegans 6992, Filobasidium neoformans 7902, Fusarium merismoides 6153, Gliocladium deliquescens 6173, Glomerella cingulata 6075, Hormoconis resinae 6966, Kluyveromyces marxianus 7155, Microbacterium lacticum 9230, Mortierella ramanniana var. angulispora 6137, Mucor hiemalis 26779, Penicillium chrysogenum 6933, Trichoderma koningii 6042.
Fermentation experiments were performed in three types of media: A. coerulea, A. alternata, A. fumigatus, M. hiemalis, P. chrysogenum and T. koningii were incubated on malt medium (malt extract 20 g/L, D-glucose 20 g/L, peptone 1 g/L); F. neoformans, K. marxianus, and M. lacticum were cultured on yeast-malt medium (D-glucose 10 g/L, peptone 5 g/L, malt extract 3 g/L, and yeast extract 3 g/L); other microorganisms were cultured on potato dextrose medium (potato dextrose broth 24 g/L).

All yeast strains used in this work are derived from the prototrophic FY4 (MATa) and FY5 (MATα) parental strains (47 (link)). Strains are created by transformation using the standard lithum acetate transformation protocol (48 (link)). Yeast strains and oligonucleotide sequences are provided in Supplemental Tables. Unless otherwise noted, all strains are grown in YPD (2% w/v Bacto Peptone, BD Biosciences #211677; 2% w/v D-glucose, BD Biosciences #215520; 1% w/v Yeast Extract, BD Biosciences #212750) at 30°C.

All yeast strains used in this work are derived from the prototrophic FY4 (MATa) and FY5 (MATα) parental strains (47 (link)). Strains are created by transformation using the standard lithum acetate transformation protocol (48 (link)). Yeast strains and oligonucleotide sequences are provided in Supplemental Tables. Unless otherwise noted, all strains are grown in YPD (2% w/v Bacto Peptone, BD Biosciences #211677; 2% w/v D-glucose, BD Biosciences #215520; 1% w/v Yeast Extract, BD Biosciences #212750) at 30°C.