Bacto peptone

Bacteria were isolated from the slick SML of site #1 by plating 100 µl undiluted and diluted (10⁻¹–10⁻⁴) sample on Zobell Agar (1 g yeast extract (BD), 5 g bacto-peptone (BD), 15 g bacto agar (BD), 800 ml Baltic Sea water, 200 ml Milli-Q water). Plates were incubated at room temperature (~22 °C). Bacteria of different color and morphology assumed to represent different species were pure-cultured from single colonies thrice before they were inoculated in Zobell medium (1 g yeast extract (BD), 5 g bacto-peptone (BD), 800 ml Baltic Sea water, 200 ml Milli-Q water) over night, and stored as glycerol stocks (600 µl 50% glycerol (Sigma) and 900 µl bacterial culture) at −80 °C.

High molecular weight chitosan (CAS 9012-76-4; 310000-375000 Da) was purchased from Hangzhou Simit Chemical Technology Co., Ltd. (Hangzhou, China). ε-polylysine (CAS 25104-18-1), phosphate buffer (for microbiology, APHA, pH 7.2), ethyl acetate (CAS 141-78-6; ≥99.5%), and citric acid (CAS 77-92-9; ≥99.5%) were supplied by Sigma-Aldrich Química S.A. (Madrid, Spain). Neutrase^® 0.8L enzyme was supplied by Novozymes (Bagsvaerd, Denmark). Potato dextrose agar (PDA), yeast extract, and Bacto^TM Peptone were purchased from Becton, Dickinson and Company (Franklin Lakes, NJ, USA). Starch casein agar (SCA), Mueller Hinton agar, and malt extract agar (MEA) came from Oxoid Ltd. (Hampshire, UK). Molasses were supplied by ACOR, Sociedad Cooperativa General Agropecuaria (Castilla y León, España).
The three fungal isolates under study, viz. Diplodia seriata (ITACYL_F079), Neofusicoccum parvum (ITACYL_F111), and Botryosphaeria dothidea (ITACYL_F141), were supplied by ITACYL, Instituto Tecnológico Agrario de Castilla y León (Castilla y León, España).
The two Streptomyces spp. strains from which secondary metabolites were produced, Streptomyces lavendofoliae (DSM 40217) and Streptomyces rochei (DSM 41729) were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen; Braunschweig, Germany).

The two bacteria of the genus Streptomyces, viz. Streptomyces rochei (DSM 41729) and Streptomyces lavendofoliae (DSM 40217), were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen; Braunschweig, Germany). The Botrytis cinerea (CECT 20973) isolate came from CECT (Spanish Type Culture Collection, Valencia, Spain).
High molecular weight chitosan, (CAS 9012-76-4; 310,000-375,000 Da), was purchased from Hangzhou Simit Chemical Technology Co., Ltd. (Hangzhou, China). Phosphate buffer (for microbiology, APHA, pH 7.2), ethyl acetate (CAS 141-78-6), and citric acid (CAS 77-92-9) were supplied by Sigma-Aldrich Química S.A. (Madrid, Spain). Neutrase^® 0.8 L enzyme was supplied by Novozymes (Bagsvaerd, Denmark). Potato dextrose agar (PDA), potato dextrose broth (PDB), yeast extract, and Bacto^TM Peptone were purchased from Becton, Dickinson and Company (Franklin Lakes, NJ, USA). Tryptone soy broth (TSB), starch casein agar (SCA), Mueller–Hinton agar, and malt extract agar (MEA) came from Oxoid Ltd. (Hampshire, UK). Molasses was supplied by ACOR, Sociedad Cooperativa General Agropecuaria (Castilla y León, Spain).
The organic farming cv. ‘Red Globe’ and ‘Timpson’ grapes used in ex situ assays were supplied by FRUAMO, Las Cabezuelas Sociedad Cooperativa (Murcia, Spain).

S. cerevisiae BY4741 (MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0) and the derived deletion mutants pdr18Δ and snq2Δ were obtained from EUROSCARF collection. C. glabrata BPY55 (clinical isolate) and the derived deletion mutant snq2Δ built using the SAT1 flipper system were kindly provided by Professor Dominique Sanglard, Institut de Microbiologie, Centre Hospitalier Universitaire Vaudois (CHUV), Lausanne, Switzerland.
Cultivation of S. cerevisiae strains was performed in MM4 medium, containing 1.7 g/L yeast nitrogen base without amino acids and ammonium sulfate (Difco, Detroit, MI, United States), 20 g/L glucose (Merck, Darmstadt, Germany), 2.65 g/L (NH₄)₂SO₄ (Panreac AppliChem, CT, United States), 20 mg/L L-methionine, 20 mg/L L-histidine (both from Merck, Darmstadt, Germany), 60 mg/L L-leucine and 20 mg/L L-uracil (both from Sigma, St. Louis, MO, United States). C. glabrata strains were cultivated in MM medium, with the composition of MM4 medium, without supplementation with amino acids and uracil. YPD medium contained 20 g/L glucose, 20 g/L Bacto^TM Peptone and yeast extract (both from BD Biosciences, Franklin Lakes, NJ, United States). Solid media were prepared by the addition of 20 g/L agar (Iberagar, Barreiro, Portugal) to the different liquid media. Media pH were adjusted to 4.5 with HCl. Growth in liquid media was performed at 30°C with orbital agitation (250 rpm).

Saccharomyces cerevisiae strains BY4742 and BY4743 (EUROSCARF) were grown at 30 °C in yeast extract peptone dextrose (YPD) media (10 g/l BactoYeast extract, 20 g/l Bacto^TM peptone (BD), 2% w/v glucose). Cells were grown to log phase (OD₆₀₀ of 0.6), harvested by centrifugation at 1600 × g for 10 min at 4 °C, washed with cold Milli-Q water and then collected again by centrifugation at 10,000 × g for 5 min at 4 °C. Cells were lysed in 1% sodium deoxycholate, 10 mm TCEP, 40 mm CAA in 100 mm Tris pH 8.5, boiled for 10 min at 95 °C and sonicated for 3 min at 30% duty cycle and output control 3 (Branson Ultrasonics sonifier; model 250). Protein concentrations were determined by tryptophan fluorescence emission assay. Cell lysates were diluted 1:2 with Milli-Q water and digested by adding LysC (Wako Chemicals GmbH, ratio 1 μg LysC:50 μg sample protein) for 4 h at 37 °C, followed by adding again LysC (ratio 1:50) overnight at 37 °C. An equal volume of ethyl acetate acidified with 1% TFA was added to the solution, samples were vortexed for 2 min and digested peptides were purified with SDB-RPS StageTips as described in Kulak et al. (19 (link)). Peptide concentrations were determined using a NanoDrop spectrophotometer.

For the detection of lipolytic activity, a medium containing 0.5% Bacto^TM peptone (BD Biosciences, San Jose, CA, USA), 0.3% Bacto^TM yeast extract (BD Biosciences, USA), 1% arabic gum (Acros Organics, Somerville, NJ, USA), 1% (v/v) glyceryl tributyrate (Sigma-Aldrich, St. Louis, MO, USA), and 1.3% Bacto^TM agar (BD Biosciences, USA) was used [56 (link),57 (link),58 (link),59 (link)], supplemented with 0.02% arabinose and 12.5 µg/mL chloramphenicol. After addition of glyceryl tributyrate an emulsion was obtained by using a blender for 5 min.

Escherichia coli One Shot™ MAX Efficiency™ DH5α-T1^R (Thermo Fisher Scientific, Waltham, MA, USA) was used as bacterial host for transformation and propagation of the recombinant plasmids. LB medium (0.5% (w/v) yeast extract (BD Biosciences, San Jose, CA, USA), 1% (w/v) Bacto^TM Tryptone (BD Biosciences), and 1% (w/v) sodium chloride (BDH Prolabo, Leicestershire, England)) supplemented with 100 μg/mL ampicillin (PanReac AppliChem, Darmstadt, Germany) was used for selection. Bacterial cultures were maintained at 37 °C.
Yeast BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) (Thermo Fisher Scientific) was used as a background strain for yeast chromosomal gene modifications. YPD medium (1% (w/v) yeast extract, 2% (w/v) Bacto^TM Peptone (BD Biosciences), and 2% (w/v) dextrose (Sigma-Aldrich, St. Louis, MO, USA)) was used for general yeast cultures. G418 (PanReac AppliChem) (400 μg/mL final concentration) and hygromycin B (Merck, Darmstadt, Germany) (200 μg/mL final concentration) were used to select yeast transformants as indicated. All yeast strains were maintained at 30 °C.
All primers used in PCR-based, site-directed mutagenesis, amplification of DNA cassettes for transformation of yeast, and yeast strain verification by PCR and DNA sequencing are shown in Table S1.