Casamino acid

To evaluate the bacterial swimming motility, a similar-sized colony of each isolate was spotted in the center of a 0.3% agar plate. The plates used to promote migration contained a low concentration of nutrients (2 g/ L (NH₄)₂SO₄, 6 g/ L Na₂HPO₄, 3 g/ L KH₂PO₄, 3 g/ L NaCl, 1 mM MgCl₂, 0.1 mM CaCl₂, 0.01 mM FeCl₃, 2.5 mg/ L thiamine, supplemented with 0.5% (wt/vol) glucose, and Casamino Acids (Merck)) (37 (link)). The plates were incubated at 37°C for 24 h, and the diameters of migration from the site of inoculation were measured.

The bacterial strains and plasmids used in this study are described in Supplementary file 1. Bacteria were routinely grown aerobically in lysogeny broth (LB) or on LB agar plates (1.5% agar) at 30°C or 37°C. Half-concentrated defined artificial seawater medium (0.5x DASW) containing HEPES and vitamins (Meibom et al., 2005 (link)) was used for growth on chitinous surfaces for chitin-induced T6SS killing and natural transformation experiments (as previously described; Marvig and Blokesch, 2010 (link); Borgeaud et al., 2015 (link)). Agar plates containing M9 minimal medium (Sigma-Aldrich) supplemented with vitamins (MEM vitamin solution; Gibco), 0.001% casamino acids (Merck), and 0.2% mannose were used to select V. cholerae strain A1552 and to exclude strain Sa5Y (to check the direction of transformation for comEC-positive prey). Thiosulfate-citrate-bile salts-sucrose (TCBS; Sigma-Aldrich) agar plates were used to counterselect E. coli strains after mating with V. cholerae. Antibiotics were used at the following concentrations whenever required: chloramphenicol (Cm), 2.5 µg/ml; kanamycin (Kan), 75 µg/ml; streptomycin (Strep), 100 µg/ml; ampicillin (Amp), 100 µg/ml; and rifampicin (Rif), 100 µg/ml.

Yeast strains were pre-grown on YNB supplemented with 5 g/L of casamino acids (Difco), 1 g/L of tryptophan (Sigma) and 50 g/L of d-maltose for 24 h. Cells were then harvested by centrifugation, washed three times with sterile water and resuspended to an OD₆₀₀ of 10. Fermentation experiments were performed aerobically in 250 mL Erlenmeyer flasks using 70 mL of YNB supplemented with 5 g/L of casamino acids, 1 g/L of tryptophan (Sigma) and 10 g/L of xylose. For simultaneous glucose and xylose co- fermentation, 10 g/L of both sugars were used. The cells were incubated at 30 °C/200 rpm. Experiments were performed in triplicate and samples were collected to measure optical density and for HPLC analysis.

Both minimal and rich media were used in parallel for the culturing fungal organisms at 37 °C. These were the Czapek Dox (CD) minimal medium and the Potato Dextrose (CD) rich medium (both from Difco, Detroit, MI, USA). Although PD medium is typically used for the culturing of filamentous fungi, we used the same medium for yeasts to facilitate better proteomics comparisons between the genera. The inoculum size was 10⁷ spores or yeast cells for the molds Aspergillus and Mucor, or the yeasts Candida, Cryptococcus, and Saccharomyces, respectively. Cultures were harvested in the late exponential growth phase, 16–24 h after inoculation for PD medium and 2–5 days for CD medium, depending on the species. For C. albicans, it was necessary to supplement CD medium with 1 mg/mL casamino acids (Sigma Chemical Co., St. Louis, MO, USA) to ensure optimal growth (designated as CD+ medium). C. posadasii could not be cultured well in the media listed above and was therefore grown in a rich broth of 2% glucose and 1% yeast extract for 5 days at ambient temperature on a gyratory shaker at 150 rpm. Culturing and sample preparation of the hyphal form of C. posadasii was done in a BSL3 facility at the California Institute for Medical Research.

To determine if the chitin-degrading genotype resulted in a phenotype, the 50 strains from the Galathea collection were tested for chitin degradation. Strains were grown with aeration at 25°C and 200 rpm overnight in marine broth (MB 2216; Difco BD). Two microliters of each culture was inoculated in duplicate on chitin agar, consisting of 1.5% agar, 2% sea salt (catalog number S9883; Sigma), 0.3% Casamino Acids, and 0.2% colloidal chitin (catalog number C7170; Sigma). colloidal chitin was prepared as described previously (20 (link)). Plates were inspected daily for 10 days, and chitin degradation was determined by the appearance of a clearing zone around the colonies.

Caco-2 cells seeded at 1 × 10⁴ cells/ml were grown in 96-well tissue culture plates containing Dulbecco’s modified Eagle’s medium (DMEM), at 37 °C in 5% CO₂ statically. H10407 strain of ETEC was grown overnight at 37 °C in ETEC medium containing 1% Casamino Acids (Sigma) and 0.15% yeast extract (Fisher Bioreagents) plus 0.005% MgSO₄ (Sigma) and 0.0005% MnCl₂ (Sigma). The next day, bacteria were resuspended in PBS and diluted until an OD₆₀₀ nm of 0.4 was reached. Antibody dilutions were set up in a deep well plate. Antibody dilutions and bacteria were combined at a 1:10 ratio and allowed to shake at 300 rpm for 1 h at room temperature. Meanwhile, Caco-2 cells were washed and incubated in antibiotic free DMEM containing 500 µg/ml of a nanobody. After incubation, 0.035 ml of the mixture of antibody and bacteria was added to each well containing Caco-2 cells. The cells were then incubated statically for 3 h at 37 °C. The cells were then washed four times with 1 ml PBS to remove non adherent ETEC cells and intensity of luciferase signal was determined.