Galactose

Before addition to cells, pairs of lectins and PE-conjugated streptavidin (for detection of biotin-conjugated MAAII or PNA) were combined at 10 μg/mL each in PBS (lectin working solution). For inhibition of lectin binding to cells by galactose and fetuin (Sigma Aldrich, Saint Louis, MO, USA), lectin working solution was prepared in 100 mM galactose or 0.1 mM fetuin in PBS. Thirty-five microliters of lectin working solution was then added to cells and incubated for 60 min at 4 °C. Cells were washed once with 1 mL of PBS, resuspended in 0.5 mL of the same buffer, and analyzed immediately in an Attune™ flow cytometer (blue/red lasers) from Thermo Fisher Scientific (Carlsbad, CA, USA). For neuraminidase treatment, 30 μL of neuraminidase type V from Clostridium perfringens (Sigma Aldrich) stock solution (6.1 U/mL) was added to the corresponding cells and incubated for 1.5 h at 37 °C, 5% CO₂. Cells were washed with 1 mL of PBS before staining.
Unlabeled cells, single-green and single-red labeled cells were used to set up voltage and compensation settings. Double-labeled cells were then analyzed under the same conditions. Regions of viable cells in FSC-H vs. SSC-H were depicted, and green and red fluorescence was determined.

The fermentation capacities were analyzed quantitatively by ethanol determination. One isolated strain (Mi4) was inoculated in culture media (3.0 g/L of yeast extract (Difco Laboratories, Detroit, MI, USA) and 5.0 g/L of proteose peptone No. 3 (Difco Laboratories, Detroit, MI, USA) with different carbon sources: galactose (1%) or lactate (1%) plus galactose (1%) or sucrose (1%) (Sigma Aldrich Chemie, Steinheim, Germany). After seven days the ethanol produced was measured with Enzytec fluid Ethanol purchased from R-Biopharm, Darmstadt, Germany (Cat. No. E5340), following the instructions supplied by the manufacturer.

Distilled sterile water was used for water-soluble molecules, including glutathione (Sigma-Aldrich), galactose (Sigma-Aldrich), and succinic acid (VWR Chemicals). galactose, L-aspartic acid, and acylases I were dissolved in 0.9 M NaCl, 2 M NaOH, and 100 mM potassium phosphate buffer, pH 7, respectively, according to the manufacturer's recommendations (Sigma-Aldrich). Concentrated solutions of the compounds were prepared and applied to ASM cultures to obtain a final concentration of 20 mM of aspartic acid, succinic acid, and galactose, 10 mM of glutathione, and 5 and 15 mg/L of acylases I as described in literature (Xu et al., 2003 (link); Sauer et al., 2004 (link); Klare et al., 2016 (link); Bahamondez-Canas and Smyth, 2018 (link)). All stock solutions were freshly prepared before their application on ASM. Three different concentrations of ciprofloxacin, two subinhibitory concentrations and one inhibitory concentration according to the MIC values obtained for the P. aeruginosa strains, were combined with the compounds.

YPAD media consisted of 1% Yeast Extract (BD, USA-212750), 2% Peptone (BD, USA-244620), 2% Dextrose (Fisher, USA-50-99-7), and 35 mg/L Adenine (Sigma, USA-A9126). Strains transformed with plasmid were grown in selective SD media containing 0.67% Yeast nitrogen base (BD, USA-233520), 2% Dextrose as a carbon source with required amino acids, 100 mg/L Adenine. For the galactose inducible system, SGal media containing 2% galactose (Sigma, USA-G0625) and 2% Raffinose (Sigma, USA-R0250) as carbon sources was used. For induction, the cells were grown in SD media until log phase, washed three times with sterile distilled water, and sub-cultured in SGal media. For induction of autophagy, transformants were grown in selective SD media until mid-log phase, washed 3–4 times with sterile distilled water, and transferred into media lacking ammonium sulfate (Sigma, USA-A4915).
For the yeast transformation experiments, all required strains were grown in liquid media until mid-log phase (~0.8 O.D._600nm). The cell density was normalized using O.D._600nm, and approximately-equal number of cells were used for transformation. Approximately 500 ng of plasmid was transformed using the PEG/LiAc method. Similar conditions were maintained for all strains during transformation. The images of transformants shown are representative of the entire plate.