Bactiter glo microbial cell viability assay

ATP contained in MVs was measured by a BacTiter-Glo Microbial Cell Viability Assay (Promega). Isolated MVs were retrieved from exponential growing cultures as described above and samples were stored at −80°C to avoid ATP degradation. Aliquots of each MV preparation from the three assayed strains were inoculated into the corresponding fresh culture media and incubated overnight at 37°C to check for bacterial contamination. 100 μl of isolated MVs were processed according to the manufacturer’s instructions and the luminescence was measured in Modulus Microplate Multimode Reader (Turner Biosystems). The standard curve was prepared from 1 μM of Adenosine 5’-triphosphate sodium salt (Promega) initial solution and 10-fold serial dilutions were done (1 μM to 10 pM). ATP concentrations in MV samples were determined by comparing the signal emitted with the ATP standard curve for each assay. The protein concentration of each sample was determined. Each experiment was performed in duplicate and the ATP content was normalized by protein concentration.

Bradyrhizobium diazoefficiens bacteroids were isolated from 3 weeks post‐inoculated G. max root nodules. Nodules (1 g wet weight) were crushed in PBS (10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 137 mM NaCl, 2.7 mM KCl, pH 7.4) and filtered through three layers of gauze to remove debris. Bacteroid suspension was pelleted by centrifugation at 2,500 g, and the supernatant (nodule extract) was saved for later usage. Bacteroid suspension (5 × 10⁸} cells) was washed twice with PBS and resuspended in 1 ml of induction media (2 μM biotin, 1 mM MgSO4, 42.2 mM Na₂HPO₄, 22 mM KH₂PO₄, 8.5 mM NaCl, 21 nM CoCl₂, 1 μM NaMoO₄ pH 7.0). To avoid ATP generation from aerobic respiration, the bacteroid suspension was placed under anaerobic conditions (92% N₂, 8% H₂) to perform the rest of the procedure. Bacteroids (200 μL) were incubated in 2 mL of nodule extract (obtained at the beginning of the protocol) or induction media without supplements or supplemented with either 7.4 mM succinate or 5 mM arginine or both substrates. ATP content was determined for each sample (using a 1:10 dilution) through ATP‐dependent luciferase reaction (BacTiter‐Glo Microbial Cell Viability Assay, Promega). Luminescence from luciferase activity was quantified with Victor3 multilabel plate counter (PerkinElmer).

To evaluate the effect of 3C expression in bacteria, pSNAP FLAG-3C plasmids were transformed into T7 Express competent E. coli (New England BioLabs) and incubated in 10 ml of 100 μg/ml carbenicillin Terrific broth on a shaker for 3 h at 37°C. After incubation, viable E. coli cells were quantified using the BacTiter-Glo microbial cell viability assay (Promega). For all samples, equivalent numbers of viable E. coli cells were mixed in 200 μl containing 100 μg/ml carbenicillin in Terrific broth, and 75 μl was plated on LB plates containing either 100 μg/ml carbenicillin or 100 μg/ml carbenicillin, 0.1 mM IPTG, and 60 μg/ml X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside). Plates were incubated at 37°C overnight and examined for growth.