Meat extract

Methylobacterium extorquens AM1 was cultivated on LB plates supplemented with 0.1% meat extract (Sigma), 0.1% methanol and rifampicin (50 μg/ml). E. coli and B. subtilis strains were grown on LB plates. Bacteria were scraped off plates and resuspended in the Tris-glucose medium (0.1 M Tris pH 7.4, 0.1 mM KH₂PO₄, sodium citrate (0.42 mg/ml), MgSO₄ × 7H₂0 (0.21 mg/ml), (NH₄)₂SO₄ (1 mg/ml), FeCl₃ (0.32 μg/ml), glucose (0.2%), and the following amino acids: lysine, proline, glycine, alanine, glutamic acid, aspartic acid, arginine, at 100 μg/ml; cysteine, methionine, tyrosine, tryptophan, and phenylalanine at 40 μg/ml) (Nishino et al., 1979 (link)). Nucleotides were labeled by the addition of [P³³] – phosphoric acid to 5 μCi/ml and incubated with shaking at 30°C for 45 min. For E. coli strains carrying pUC19 derivatives, 0.1 mM IPTG was added. Reactions were stopped by the addition of an equal volume of 23.6 M formic acid, and followed by three freeze-thaw cycles in liquid nitrogen. Extracts were centrifuged (14,000 × g, 10 min, room temperature) and the supernatants were spotted onto PEI-Cellulose plates. For two-dimensional TLC separation, first dimension buffer contained 1 M LiCl and 4 M formic acid; this was followed by soaking plates for 15 min in methanol and a second dimension run in 0.85 M KH₂PO₄ (unadjusted pH). Autoradiograms were visualized as before.

The sanitizers used in this study were chlorous acid water (Honbusankei Co., Ltd.) and NaClO (Nankai Chemical Co., Ltd.). Sodium thiosulfate, sodium chlorite, KH₂PO₄, K₂HPO₄, NaOH, N,N-diethyl-p-phenylenediamine (DPD) and sodium sulfate were purchased from Wako Pure Chemical Industries, Ltd. 3,3',5,5'-tetramethylbenzidine (TMB) was purchased from Tokyo Chemical Industry Co. Ltd. BSA (35% in PBS), polypeptone and meat extract were purchased from Sigma, Sumitomo Dainippon Pharma Co., Ltd. and Nacalai Tesque Inc., respectively. BSA, polypeptone and meat extract were added to microbicidal assays as an organic matter load.

Nonomuraea jiangxiensis strain A7611 was cultured in 5 mL SV2 media, (For 1 L, add 15 g glucose (1st BASE, Singapore, Singapore), 15 g glycerol (VWR, Radnor, PA, USA), 15 g soya peptone (Oxoid, Basingstoke, Hampshire, UK), and 1 g calcium carbonate (Sigma-Aldrich, St. Louis, MO, USA), pH was adjusted to 7.0) for 3 days at 28 °C with shaking performed at 200 rpm. Saturated seed cultures were diluted in fresh fermentation media: CA09LB (For 1 L, add 10 g meat extract (Sigma-Aldrich, St. Louis, MO, USA), 4 g yeast extract (BD Biosciences, Franklin Lakes, NJ, USA), 20 g glucose (1st BASE, Singapore, Singapore), and glycerol 3 g (VWR, Radnor, PA, USA), pH was adjusted to 7.0) in a 1:20 volume ratio and fermented with 200 rpm shook at 28 °C in the dark. The cultures were pelleted after 9 days followed by lyophilization of the separated biomass and supernatant. The dried samples were extracted by MeOH then filtered through filter paper (Whatman Grade 4, Maidstone, Kent, UK). MeOH was removed under reduced pressure to give a crude extract of a combined weight of 15.70 g. The crude extract consists of broth extract 84.4% (13.25 g) and biomass extract 15.6% (2.45 g).