S 5 adenosyl l methionine p toluenesulfonate salt sam

Folic acid (FA), calcium d,l-5-methyltetrahydrofolate (5-Me-THF), d,l-homocysteine (Hcy), and S-(5′-Adenosyl)-l-methionine p-toluenesulfonate salt (SAM) were purchased from Sigma-Aldrich (Darmstadt, Germany). For cell culture applications, stock solutions of FA (5 mg/mL in DMSO) and 5-Me-THF (5 mg/mL in dH₂O) were diluted in cell culture medium to the appropriate concentration, prior to the addition to cells.
The isotopically labelled compounds used as internal standards were levomeFolic acid-13C,d3 from Santa Cruz Biotechnology (Heidelberg, Germany) and d,l-homocysteine-d4 from Toronto Research Chemicals (North York, ON, Canada). HPLC-grade acetonitrile ChromasolV^®, acetic acid SupraPur^®, perchloric acid, ascorbic acid, citric acid monohydrate, and perchloric acid were from Sigma-Aldrich (Darmstadt, Germany). Methanol and 1,4-dithiotreitol were provided by Merck (Darmstadt, Germany) and ultra-pure water was produced by an ELGA PureLab Flex LabWater purification system (UK MilliQ water; A10 Advantage, Millipore, Burlington, MA, USA).

Listeria monocytogenes (wild type, ΔagrA, and ΔagrD) cells were centrifuged, and the pellets were diluted to 10⁷ CFU/ml based on plate enumeration. A 200‐μl aliquot of each strain was inoculated into 96‐well polystyrene microtiter plates (CELLTREAT) with BHI broth or 10% BHI broth containing 250 and 500 μM membrane‐permeable S‐(5'‐adenosyl)‐l‐methionine p‐toluenesulfonate salt (SAM; Sigma). For RNA extraction from biofilm cultures, a 5‐ml aliquot of each strain was inoculated in 6‐well polystyrene microtiter plates. The plates were incubated statically at 37°C for 24 hr.