Enzymatic digest

For extraction of compounds, the bacteria were cultivated in 1000-mL flasks containing 300 mL DVR1 medium (6.7 g malt extract (Sigma), 11.1 g peptone from casein, enzymatic digest (Sigma), 6.7 g yeast extract (Sigma), 0.5 L filtered sea water, and 0.5 L ddH₂O) cultures for 16 days, at 10 °C and 130 rpm. A total of 12 flasks were inoculated, giving 3.6 L of culture. The medium was autoclaved for 30 min at 120 °C prior to inoculation. Cultures were started by loop inoculation from the non-axenic glycerol stock solution.
Extraction of metabolites from the liquid media was done with Diaion^® HP-20 resin (Supelco, Bellefonte, PA, USA). The resin was activated by incubation in methanol for 30 min, followed by washing with ddH₂O for 15 min, and added to the cultures (40 g/L). The cultures were incubated with resin for 3 days prior to compound extraction. For extraction, the resin beads were separated from the liquid by vacuum filtration through a cheesecloth mesh (Dansk Hjemmeproduktion, Ejstrupholm, Denmark), the resin was washed with ddH₂O, and finally extracted two times with methanol. The extract was vacuum filtered through a Whatman No. 3 filter paper (Whatman plc, Maidstone, UK), and dried under reduced pressure at 40 °C.

Compound 1 was isolated from bacterial cultures started from a non-axenic glycerol stock. The bacterial glycerol stock originally contained a Leifsonia sp. isolate. The stock was found to be contaminated with both Shewanella sp. and Serratia sp. after several steps of cultivation and production of new glycerol stock solutions. The non-axenic glycerol stock was inoculated onto three different agar plates, in order to gather information of the different isolates present. Originally, the Leifsonia sp. isolate was provided as an axenic culture by The Norwegian Marine Biobank (Marbank, Tromsø, Norway) (Reference number: M10B719). The bacterium was isolated from the intestine/stomach of an Atlantic hagfish (Myxine glutinosa) collected by benthic trawl in Hadselfjorden (Norwegian Sea, 16th of April, 2010). The bacterium was grown in liquid FMAP medium (15 g Difco Marine Broth (Becton Dickinson and Company, Franklin Lakes, NJ, USA), 5 g peptone from casein, enzymatic digest (Sigma, St. Louis, MS, USA), 700 mL ddH₂O, and 300 mL filtrated sea water) until sufficient turbidity, and cryo-conserved at −80 °C with 30% glycerol (Sigma). Filtration of sea water was done through a Millidisk^® 40 Cartridge with a Durapore^® 0.22-µm filter membrane (Millipore, Burlington, MA, USA).

About 1 g of locally collected dead fruit flies (Drosophila melanogaster) was melted in a pestle, suspended in 10 mL of physiological saline and allowed to settle down at +4°C overnight. 5–50 μL aliquots of supernatant were plated on agarized (1.5%) lysogeny broth (LB): 10 g/L peptone from casein, enzymatic digest (Sigma-Aldrich), 5 g/L yeast extract (Fluka), 10 g/L NaCl, and 15 g/L granulated agar (Difco), prepared in distilled water, with further incubation at room temperature (RT) for 3 days. Individual colonies of morphologically different types (about 10) were picked up and purified by subculturing and suspended in 5 mL of LB, incubated stationary at RT to obtain the indicator cultures.