Lysogeny broth

ClearColi BL21 (DE3) (Lucigen) were transformed with a pET20b(+)apoA1–IL4 expression vector. Transformed bacteria were inoculated in 40 ml lysogeny broth (Sigma-Aldrich) supplemented with 100 μg l⁻¹ ampicillin and grown overnight at 37 °C. Subsequently, the overnight culture was inoculated in 2YT medium (16 g l⁻¹ peptone, 10 g l⁻¹ yeast extract and 10 g l⁻¹ NaCl) supplemented with 100 µg l⁻¹ ampicillin and grown at 37 °C. At the point that absorbance at 600 nm reached >1.5, 1.0 mM isopropyl β-d-thiogalacopyranoside was added to induce pET20b(+)apoA1–IL4 expression, and cells were incubated overnight at 20 °C. Cells were collected by centrifugation before preparation of lysates and purification.

Soil samples were collected from cattle farm sites in South Korea. Organic solvent-stable bacteria were isolated from soil samples according to established methods (10 (link)). Briefly, 1 g of soil sample was suspended in 10 mL of sterile water by shaking, and 5 mL of this suspension were added to 250 mL bottles containing 25 mL of Lysogeny broth (Sigma, USA) supplemented with toluene and benzene (2.5% v/v each). Culture vessels were sealed with chloroprene rubber stoppers to prevent evaporation of organic solvents and then incubated at 37°C for 72 h on a shaker at 180 rpm. Next, 5-mL aliquots of culture were transferred into fresh media and cultured again under the same conditions. These cultures were diluted and plated onto skim milk agar media (1 g/L yeast extract, 20 g/L agar, 1% skim milk) lacking organic solvents, and then incubated at 37°C for 36 h to screen for protease-producing strains. These strains were purified and screened again on skim milk agar plates for further confirmation.

Bacterial strains were kept as stocks at −80°C in 15% glycerol. Before bacteria were used, growth curves were generated to correlate optical density (OD) with colony‐forming units (CFUs). E. coli (ATCC 25922) and S. aureus (ATCC 25923) were cultured in lysogeny broth (Sigma‐Aldrich, St Louis, MO) and L. monocytogenes (inlA) in tryptic soy broth (TSB) (Sigma‐Aldrich), at 37°C under shaking incubator (200 rpm). S. pneumoniae strain D39 (R6) was cultured in Todd Hewitt Broth and yeast extract (0.5%) at 37°C without shaking. Bacterial strains were cultured and harvested at a midpoint of the log‐growth phase (OD: 0.3‐0.6) for cell stimulations.

We tested if E. coli inoculation prior to marker injection can affect phagocytosis. To this end, we contrasted three treatment groups that all were injected with latex beads: one group with prior injection of 1μL of E.coli/Grace’s insect medium solution (strain ATTC 25404 K-12), a first control with prior injection of 1μL Grace’s insect medium only and a second control without prior injection. E.coli was grown overnight in Lysogeny broth (Sigma-Aldrich, Saint Louis, Missouri, USA) at 37°C on a shaker. Cell counts were diluted to approximately 800.000 per μL. Cells were centrifuged at 5200 rpm for 5min, washed twice and re-suspended to the same concentration in Grace’s insect medium. Prior to latex bead injection, honeybees were incubated for 2h. After latex bead injection, the honeybees were incubated for additional 2h as before, in total 4h.

Human urine from volunteers was prepared using an established urine metabolomics protocol [18 (link)]. Urine was aliquoted into 200 µL samples and prepared, including the depletion of urea with urease, precipitation of urease with methanol and extraction of metabolites. The plant metabolome extract was generated from seagrass leaves (Posidonia oceanica, collected in the Mediterranean sea, the island of Elba) and was prepared using the method for global plant metabolomics [6 (link)]. Briefly, shock-frozen leaf tissue was ground with a mortar and pestle (all cooled with liquid N₂), 100 mg powder was extracted with methanol and strong agitation, chloroform and water were added and the sample mixed. After phase-separation, the upper layer was taken for metabolite analysis. Lysogeny broth (Sigma Aldrich) was prepared after the manufacturer’s recipe, excluding the addition of salts, and 100 µL was used as a sample for the yeast cell metabolite extract. Animal tissue (common earthworm, Lumbricus terrestris) was collected and extracted as described previously [19 (link)]. Briefly, the shock-frozen whole animal was ground with a mortar and pestle (all liquid N₂ cooled), 50 mg powder was extracted as described above for the plant tissue. All final extracts were dried under vacuum (Extractor Plus^®, Agilent; settings: 30 °C, 2 h, 1000 rpm, VAQ).