Disodium succinate

The bacteria were grown in mineral media, as reported before (Hartmans et al., 1989 (link)), containing the following compounds (per liter demineralized water): 7 g Na₂HPO₄ × 2 H₂O; 2.8 g KH₂PO₄; 0.5 g NaCl; 0.1 g NH₄Cl; 0.1 g MgSO₄ × 7 H₂O; 10 mg FeSO₄; 5 mg MnSO₄; 6.4 mg ZnCl₂; 1 mg CaCl₂ × 6 H₂O; 0.6 mg BaCl₂; 0.36 mg CoSO₄ × 7 H₂O; 0.36 mg CuSO₄ × 5 H₂O; 6.5 mg H₃BO₃; 10 mg EDTA; 146 μl HCl (37%). The nitrogen-deficient mineral media did not contain NH₄Cl. As sole source of carbon and energy either 4 g/l disodium succinate (Sigma-Aldrich), 2 mM 2,4-TDA (Sigma-Aldrich) or 3 g/l PU oligomer (Sigma-Aldrich, dihydroxy-functional oligomer, aliphatic urethane of proprietary composition) was added. For growth on solid media 3.5% of agar was added. Cells were cultivated in 50 ml shaking cultures at 30°C at 150 rpm. All chemicals used were reagent grade and obtained from commercial sources. Optical density was measured at a wavelength of 560 nm (Perkin Elmer, Lambda 2S). Toluene, benzene, aniline, 2,4-dihydroxytoluene (4-methylresorcinol), methylsuccinate, sodium benzoate, 2-aminobenzoate (anthranilate), phenol, o-xylene, catechol, 4-methylcatechol and benzene-1,2,4-triol (hydroxyhydroquinone) were tested if they serve as sole source of carbon and energy for the isolated bacteria in 100, 200, and 300 mg/l concentrations and OD at 560 nm was measured to evaluate growth.

IPEC-J2 cell line was treated with 20 mM disodium succinate (Cat#S2378, Sigma-Aldrich) for 24 h and then samples were collected for the detection of protein expression. RAW264.7 cell line was treated with 50 mM disodium succinate for 24 h and/or TAK-242 (Cat#S7455, Selleck) for 30 min and then samples were collected for the detection of protein expression.

Succinic acid with a purity of 99.9% and disodium succinate with a purity of 99% were obtained from Sigma Aldrich. Hydroxide solution was prepared from deionized water and dry pellets with a purity of 99% (Sigma Aldrich, Taufkirchen, Germany).
NP030 membrane samples from Microdyn Nadir (Wiesbaden, Germany) in DIN A4 format were purchased directly from the manufacturer and were used without any pretreatment. Membrane properties are shown in Table 1.

SDH activity was assayed in purified mitochondria resuspended in 35 mM potassium phosphate monobasic buffer (pH 7.3; ref. 53 (link)). Disodium succinate (10 mM; Sigma) and 40 μM mM 2,6-dichlorophenolindophenol sodium salt hydrate (DCPIP; Sigma) was added to the mitochondria at 37 ^oC, and DCPIP extinction was measured at 610 nM as described in ref. 53 (link).

BacSp222 or suc-K20-BacSp222 were incubated at 37 °C for 3 h (unless otherwise indicated) in PBS or in Britton–Robinson buffer with a 3-carboxypropanoyl group donor such as succinyl coenzyme A sodium salt (Santa Cruz Biotechnology) or disodium succinate (Sigma) at molar ratios of 1:100 (for succinylation studies) or independently with the bacterial lysate or/and with 25 mM NAD⁺ (Roanal) (in case of desuccinylation studies). The pH and ionic strength of the Britton–Robinson buffer were adjusted by KOH and NaCl, respectively. Compounds containing amino groups i.e., glycine (Sigma), lysine (Sigma, St. Louis, MO, USA), and human lysozyme (Sigma, St. Louis, MO, USA) and the BacSp222 variants were used at compound:donor molar ratios of 1:100 or 1:500. The concentration of the BacSp222 forms was determined by RP-HPLC using a Discovery Bio Wide Pore C8 250 × 4.6 mm column (Sigma, St. Louis, MO, USA) and a linear gradient from 0 to 100% of buffer B over 15 min developed at 40 °C and a flow rate of 1 mL/min. The other details of the separations were identical to those described in Section 3.3. The results are presented as a percentage of the succinylated form in the pool of all BacSp222 forms (for succinylation studies) or a percentage of detected suc-K20-BacSp222 relative to the control suc-K20-BacSp222 sample (in the desuccinylation studies).