D alanine

E. coli NEB 10-beta (New England BioLabs (NEB), Ipswich, MA, #C3019I) was used for cloning. Kanamycin (5 μg/mL, GoldBio, St Louis, MO, #K-120-5) was used for liquid and solid media selection. DNA oligos and genes were ordered from Integrated DNA Technologies and Twist Biosciences. B. subtilis XPORT (JH642, trp-phe- ICEBs1+) [38 (link), 40 (link)] was used as the parent donor conjugation strain. B. subtilis XPORT were grown in LB media (Becton Dickinson (BD), Franklin Lakes, NJ, #244630), and 1.5% w/v agar plates (BD #247940). The P15a plasmid used to transform the GFP payload into the mini-ICEBs1 cassette of B. subtilis XPORT was based on pICEGFP05, described in Meng et al. [38 (link)] (S10 Fig in S1 File). Genetic part sequences are provided in S2 Table in S1 File. Plasmid DNA was introduced into B. subtilis XPORT through natural transformation. D-alanine (100 μg/mL) (Sigma, St. Louis, MO, A7377) was added to the LB media or MD media for counter selections. Tris-Spizzizen Salts (TSS) media was used for ICE conjugations on plates (per L: 2 g NH₄Cl (Fischer A649), 0.35 g K₂HPO₄∙3H₂O (VWR #0705), 6 g Tris base (Affymetrix, Santa Clara, CA, 75825); pH to 7.5 with HCl (EMD, Millipore, Burlington, MA, HX0603-3)). Mating plates were made with TSS, 125 mM MgCl₂ (Sigma 230391) and 1.5% w/v agar (BD 214010).

d-Alanine, d-Arginine, d-Asparticacid, d-Asparagine, d-Glutamicacid, d-Histidine, d-Isoleucine, d-Leucine, d-Lysine, d-Methionine, d-Phenylalanine, d-Proline, d-Serine, d-Threonine, d-Tryptophan, d-Tyrosine, d-Valine, KBr (spectral grade), bovine serum albumin (BSA), glutathione and 3,3′,5,5′-tetramethylbenzidine (TMB) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). d-Cystine and d-Glutarnine were purchased from Aladdin Reagent Co, Ltd. (Shanghai, China). All other chemicals and reagents were of analytical grade. All aqueous solutions were prepared with Milli-Q water.

As previously described, pyrophosphate released from the adenylation reaction is broken down into phosphate by pyrophosphatase
^{17 (link)}. The resulting phosphate was quantified by a dye solution containing 0.033% w/v Malachite Green, 1.3% w/v ammonium molybdate and 1.0 M HCl
^{36 (link)}. The 200 μL reaction solutions contained 5 μM BcDltA, 0.1 M KCl, 0.01 M MgCl
₂, 0.05 M Tris-Hepes buffer at pH 7.2, 5 unit/mL of inorganic pyrophosphatase from baker’s yeast (Sigma-Aldrich) and specified concentrations of D-alanine, ATP and CoA (Sigma-Aldrich). A volume of 25 μL of reaction solution was retrieved every 3 or 5 minutes and mixed thoroughly with 475 μL of the dye solution. The absorption at a wavelength of 620 nm was recorded after 90 seconds. The initial rates (1/2 of the phosphate concentration increase per minute) of the adenylation reaction were derived from the time courses of phosphate accumulation. The correlation between initial reaction rate and substrate concentration was fitted with Michaelis-Menten equation using the Prism software (GraphPad Software).

Retinal pigment epithelial cells (hTERT RPE-1, ATCC, CRL-4000) were cultured in DMEM-F12 (Gibco, 11320033), supplemented with 10% FBS (Bodinco), 100 Units Penicillin-Streptomycin (Sigma-Aldrich, P4458) and 2 mM L-glutamine (Sigma-Aldrich, G7513). Cells were grown at 37 °C under 6% CO₂ atmosphere. Human breast cancer cells (MCF7, ATCC, HTB-22) and human epithelial kidney cells (HEK293T, ATCC, CRL-3216) used for lentiviral production were cultured in DMEM-High Glucose (Sigma-Aldrich, DB6429) supplemented with 10% FBS, 100 Units Penicllin-Streptomycin and 2 mM L-glutamine. L-alanine (Sigma-Aldrich, A7626) and D-alanine (Sigma-Aldrich, A7377) were dissolved in PBS to create a 1 M stock solution and frozen in aliquots, only to be thawed once. Addition of alanine was always precluded by refreshing medium. For trapping cells in mitosis to visualize a G_0/1 arrest, 250 ng/ml nocodazole (Sigma-Aldrich, M1404) was used. For blocking ferroptosis, 500 nM ferrostatin (Sigma-Aldrich, SML0583) or 200 nM Liproxstatin (Sigma-Aldrich, SML1414) was added 1 h before treatment with L- or D-Ala. For inhibiting thioredoxin reductase, 1 µM Auranofin (Sigma-Aldrich, A6733) was used.

The strains used in this study are Aeromonas hydrophila HBNUAh01, which was isolated from sick Paralichthys olivaceus [22 ], and the alanine racemase gene (alr-2) knockout mutant (A.H.Δalr) of A. hydrophila HBNUAh01. A.H.Δalr mutant by deletion of alr-2 gene was constructed by homologous recombination using the suicide plasmid pK18mobsacB [19 , 20 (link)]. Strains were cultured in Luria–Bertani (LB, Oxoid; UK) medium at 30 °C. D-alanine was obtained from Sigma (St Louis, MO, USA). Counting of A. hydrophila was performed by serial dilution and spread plating on LB medium. The plates were incubated at 30 °C for 48 h and colonies were counted and expressed as colony forming units (CFU)/ml. For each of the three independent experiments, two plates per dilution were used to calculate the results.

Thiamine hydrochloride (99%, MW 337.27), biotin (>99%, MW 244.31), vitamin B12 (>98%, MW 1355.37), and α-Cyano-4-hydroxycinnamic acid (>99%, MW 189.17), 4-Nitrophenol (>99%, MW 139.11) were supplied by Sigma-Aldrich (St. Louis, MO, USA). Hydrogen peroxide (30%, MW 34.01), sodium nitrate (>99%, MW 84.99), sodium dihydrogen phosphate monohydrate (>99%, MW 137.99), sodium molybdate dihydrate (>99%, MW 241.95), manganese (II) chloride tetrahydrate (>99%, MW 197.91), and cobalt (II) chloride hexahydrate (>99%, DW 237.93) were obtained from Chempur^® (Piekary Śląskie, Poland). Zinc sulfate heptahydrate (>99%, MW 287.54), iron (III) chloride hexahydrate (>99%, MW 270.32), EDTA disodium dihydrate (>99%, MW 372.24), and copper (II) sulfate pentahydrate (>99%, MW 249.68) were purchased from Scharlab (Barcelona, Spain). Sodium metasilicate nonahydrate (44–47.5% total solids, MW 284.19) were supplied by Acros Organics, ThermoFisher Scientific (Waltham, MA, USA). Deionized water was obtained by using a Milli-Q^® purification system (Millipore Co., Bedford, MA, USA). Standards of D-glucose (>99%), D-alanine (>99%) and cesium triiodide (>99%) were purchased from Sigma-Aldrich (Steinheim, Germany). Standard mixture of triacylglycerols (TG internal standard, Ultimate SPLASH^TM) was purchased from Avanti Polar Lipids (Alabaster, AL, USA).

Tetraethylorthosilicate (TEOS), n-cetyltrimethyl ammonium bromide (CTABr), Curcumin, sodium hydroxide (NaOH), rhodamine B, tris(hydroxymethyl) aminomethane (TRIS), (3-aminopropyl) triethoxysilane, HSA, L-alanine, D-alanine, histidine, phenyl glycine, serine, valine, glutamine, and urea were purchased from Sigma Aldrich (Burlington, MA, USA). Curcumin was purchased from Tokyo Chemical Industry Co., Ltd. (TCI, Tokyo, Japan). Analytical-grade solvents were from Scharlab (Barcelona, Spain). All products were used as received.