Trehalose assay kit k treh

Supernatant and crude cells grown on 50 ml MSB media were separated by ultracentrifugation (3000 r.p.m., 20 min). Then, intracellular trehalose was extracted by boiling the cell pellet at 90°C for 20 min. Trehalose levels of supernatant and crude cells were measured using a Trehalose assay kit K‐TREH (Megazyme International Ireland, Bray, Ireland) according to the manufacturer's instructions. All samples were standardized by the Bradford assay, and triplicates were used.

For isolation of compatible solutes, cells were grown in mineral medium with 300 mM NaCl until they reached an OD₆₀₀ of 0.9. Additionally, solutes were determined directly after osmotic upshift. Therefore, cells were grown in mineral medium to the exponential growth phase (OD₆₀₀ of 0.4 to 0.5), and hyperosmotic stress was applied by the addition of 300 mM NaCl. Cells were incubated (37°C, 130 rpm) and partly harvested over a time period of 3 h. Solute extraction was performed with chloroform and methanol as described before (36 (link)). Mannitol, trehalose, and glutamate were determined enzymatically (trehalose assay kit K‐TREH, l‐glutamic acid assay kit K‐GLUT, and d-mannitol assay kit from Megazyme, Bray, Ireland) (36 (link)).