Threonine

Stock solutions of Casamino Acids (Difco Laboratories, Detroit, MI), alanine (Merck, Darmstadt, Germany), aspartate (Merck), glutamate (Merck), glycine (Sigma-Aldrich, Taufkirchen, Germany), leucine (AppliChem, Darmstadt, Germany), threonine (Merck), tyrosine (Merck), valine (Merck), ribose (Sigma-Aldrich), formate (Sigma-Aldrich), succinate (Sigma-Aldrich), and glucose (AppliChem) were prepared with anoxic sodium phosphate buffer (36 mM, pH 7 [pH was adjusted with NaOH]). Solutions were filter sterilized (0.22-μm pore size, cellulose-acetate membrane) into sterile anoxic 100-ml serum vials that were crimp sealed with sterile butyl-rubber stoppers (Glasgerätebau Ochs Laborfachhandel e.K., Bovenden, Germany [product number 102049]); the vials were then flushed 10 min with sterile argon (100%).

Hydrogen peroxide, ferric sulfate, ferric chloride, catalase, the Hydrogen peroxide assay kit, and the phosphorothioate-modified dinucleotides dGsA and dGA were purchased from Sangon Biotech (Shanghai) Co., Ltd. Trimethoprim, thymine, α-(4-pyridyl-N-oxide)-N-tert-butylnitrone (POBN), diethylenetriaminepentaacetic acid (DTPA), Hanks’ Balanced Salt Solution (HBSS, without phenol red, calcium chloride and magnesium sulfate), threonine, NAD⁺ and Chelex-100 were purchased from Sigma. The DNeasy Tissue kit was purchased from QIAGEN. The Cycle-Pure Kit, Bacterial DNA Kit, and Gel Extraction Kit were purchased from OMEGA Bio-Tech. Luria-Bertani (LB) broth contained 10 g/L of BactoTryptone, 5 g/L yeast extract, and 10 g/L sodium chloride unless otherwise noted. The modified marine broth medium 2216E contained 5 g/L tryptone, 1 g/L yeast extract, 0.1 g/L FePO₄, and 34 g/L sodium chloride. K medium contained A salts, 0.2% glucose, 1 mM MgCl₂, 0.5 mM amino acids and 5 mg/L thiamine. The media were supplemented with the following concentrations of antibiotics (unless otherwise noted): ampicillin (Amp) 100 μg/ml and chloramphenicol (Cml) 12.5 μg/ml. Solid medium was supplemented with 1.5% (w/v) agar-A.

HPLC-grade formic acid was obtained from Sigma-Aldrich (USA). HPLC/MS-grade formic acid, ammonium formate, and acetonitrile were acquired from Fisher Scientific (USA). The internal standards Labeled Carnitine Standards (L-carnitine (N-trimethyl-D₉), L-acetylcarnitine (N-methyl-D₃), L-propionylcarnitine (Nimethyl-D₃), L-butyrylcarnitine (N-methyl-D₃), L-isovalerylcarnitine (N-trimethyl-D₉), L-octanoylcarnitine (N-methyl-D₃), L-myristoylcarnitine (N-trimethyl-D₉), L-palmitoylcarnitine (N-methyl-D₃)) were attained from Cambridge Isotope Laboratories. Seventeen amino acid standards (alanine, valine, leucine, isoleucine, phenylalanine, methionine, proline, glycine, serine, threonine, cysteine, tyrosine, histidine, lysine, arginine, aspartic acid, glutamic acid) were purchased from Sigma-Aldrich. Eight amino acid standards (tryptophan, asparaginate, glutamine, ornithine, taurine, citrulline, cysteine, γ-aminobutyric acid) were gained from J&K Scientific. Ultrahigh purity water was prepared by Millipore-Q Water Purification System (Millipore, Germany).