Bacto peptone

The yeast culture and transformation were performed as previously described [63 ]. We used two types of medium: YPD and Synthetic Complete (SC) medium. YPD included 10 g/L Bacto Yeast extract (BD, USA), 20 g/L Bacto Peptone (Gibco, USA), and 20 g/L D-glucose. The SC medium included 6.7 g/L Yeast Nitrogen Base with Ammonium Sulfate (MP, USA), 0.65 g/L DO supplement-HisLeuUra (Clontech, USA), and 20 g/L D-glucose, or where appropriate, 20 mg/L Histidine, 8 mg/L Uracil, and 100 mg/L Leucine. The D-glucose solution was added to the medium after autoclaving. Milli-Q water (Merck, Germany) was used to condition the medium. In Fig 6D and 6E, YPD and 1 M NaCl/YPD were diluted four times with sterile water or 1 M NaCl solution. We used Shio (Shiojigyo, Japan) and Setonohonjio (Ajinomoto, Japan) as the table salt and crude salt representatives.

Kluyveromyces marxianus CBS 6556 ura3Δ his3Δ was used as a starting strain for all experiments described in this work. All constructed strains are listed in Table S2. Synthetic defined (SD) media was used for all plasmid-based expression experiments. The SD-U medium is defined as 6.7 g/L BD Difco™ Yeast Nitrogen Base without amino acids, 1.92 g/L Yeast Synthetic Drop-out Medium Supplements without uracil, and 20 g/L d-glucose. SD-H and SD-H-U are similar defined but with 0.75 g/L of CSM-His and CSM-His-Ura, respectively. For all pathway refactoring experiments and 2-PE biosynthesis analysis, K. marxianus strains were cultivated rich YPD medium (YPD: 10 g/L Gibco™ Bacto™ Yeast Extract, 20 g/L Gibco™ Bacto™ Peptone, 20 g/L d-glucose). 20 g/L agar was added to make solid agar plates. All yeast cultures were conducted in 250 mL baffled shake flasks containing 25 mL of appropriate media. Culturing was conducted in an INFORS HT Multitron incubation shaker with temperature control set to 30, 37 and 45 °C as needed.

The S. cerevisiae strains used in this study were W303-1B (WT) cells (MATα ade2 leu2 his3 trp1 ura3) and derivatives hap4∆ (hap4∆::KanMX4), rtg2∆ (rtg2∆::LEU2), and hap4∆rtg2∆ (rtg2∆::LEU2 hap4∆::KanMX4) obtained as described in [10 (link)]. Cells were grown at 30 °C in YPD (1% yeast extract, 2% bactopeptone (GIBCO, Life Technologies, Waltham, MA, USA), and 2% glucose (Sigma-Aldrich, St. Louis, MO, USA) with 2% agar (Invitrogen, Life Technologies Waltham, MA, USA) for solid medium in the absence or in the presence of 0.8 M sodium chloride (NaCl). Cell growth was monitored qualitatively on YPD agar plates and quantitatively by measuring optical density (600 nm) on liquid YPD medium cultures grown either in micro-well plates or in flasks. Mitochondrial respiratory competence was assessed by spotting equal amounts of serially diluted cells treated or not with NaCl on YPD and YPGlycerol solid [30 (link)].

HEK-293 cells (ATCC), HEK-293FT cells (Thermo Fisher), HeLa cells (ATCC), and Vero 2.2 cells (Massachusetts General Hospital) were maintained in Dulbecco’s modified Eagle media (DMEM) containing 4.5 g/L glucose, L-glutamine, and sodium pyruvate (Corning) supplemented with 10% fetal bovine serum (FBS, from VWR). CHO-K1 cells (ATCC) were grown in F12-K media containing 2 mM L-glutamine and 1500 mn/L sodium bicarbonate (ATCC) supplemented with 10% FBS. U2OS cells (ATCC) were grown in McCoy’s 5A media with high glucose, L-glutamine, and bacto-peptone (Gibco) supplemented with 10% FBS. All cell lines used in the study were grown in a humidified incubator at 37 °C and 5% CO₂. All cell lines tested negative for mycoplasma.

Naegleria amoebae (strain NEG) and their food source Aerobacter aerogenes (a gift from the laboratory of Chandler Fulton, Brandeis University) were routinely cultured following previously established protocols.¹² Briefly, A. aerogenes were regularly streaked from a frozen glycerol stock, and single colonies were grown stationary at room temperature in penassay broth (Difco antibiotic medium 3). Liquid cultures were used to grow lawns of A. aerogenes overnight on NM plates (2 g/L Gibco Bacto peptone, 2 g/L glucose, 1.5 g/L K2HPO4, 1 g/L KH2PO4, 20 g/L agar). Lawns were inoculated with a loopful of NEG amoebae or cysts to create an edge plate (from a previous edge or cyst plate). Plates were sealed with parafilm, inverted, and incubated for 1–3 days at 28 °C. For starvation-induced differentiation (Figures 1B and S3A), cells were shocked with ice cold 2 mM Tris, and transferred to a shaking flask at 28 °C for 1 h.
Axenic Naegleria gruberi amoebae (strain NEG-M) were grown in M7 medium (0.362 g/L KH2PO4, 0.5 g/L Na2HPO4, 5.4 g/L glucose, 5 g/L yeast extract (Difco), 45 mg/L L-methionine, 10% fetal bovine serum) at 28 °C without shaking in 25 cm² plug-seal tissue culture flasks (CellTreat Cat#229330).

The strains used are listed in Table 1. YPD medium consisted of 2% glucose, 2% BactoPeptone (Gibco) and 1% yeast extract (Gibco). SC medium consisted of 2% glucose, 1X Drop-out Mix Synthetic minus uracil, without yeast nitrogen base (USBiological Life Sciences), 0.5% ammonium sulfate, 1X BD Difco Yeast Nitrogen Base without ammonium sulfate and amino acids, and 100 μg/ml uridine. SC medium was adjusted to pH 6.6 to 6.8 except were indicated otherwise. CFU determinations were performed using SC medium solidified with 2% agar.
For media-conditional tests, strains were inoculated into SC medium from stock cultures stored at -80°C and incubated 24 h at 30°C. Cell density was determined by hemocytometer count and cells inoculated at an initial density of 1 x 10⁵ cells/ml in 10 ml of test media in 125 ml flasks with foil caps incubated at 30°C in a rotary water bath shaking at approximately 250 RPM. After 24 h, cell viability was assessed by trypan blue staining.
SC5314 cells for toxicity tests were cultured in YPD. Log-phase cells were collected by centrifugation at a density of 1 x 10⁷ cells/ml, stationary-phase cells at a density of 3 x 10⁸ cells/ml. Cells were washed once with 1/10^th volume of PBS and suspended in fresh PBS prior to inoculation of test media. Test media (10 ml) was inoculated to an initial cell density of 5 X 10⁶ cells/ml and incubated as above.