Xfluor4

To measure β-galactosidase activity, strains 2499, 2499RR and 2499RS were grown oxically in PSY medium, collected by centrifugation at 8,000× g for 10 min at 4°C, washed twice with BS or BSN medium and cultured anoxically or under 2% initial O₂ in the same medium for 48 h (OD₆₀₀ of about 0.5). Activity was determined with permeabilized cells from at least three independently grown cultures assayed in triplicate for each strain and condition. β-Galactosidase assays were performed essentially as previously described [30] . The absorbance data for A₄₂₀, A₅₅₀, and A₆₀₀ were determined for all samples in a plate reader (SUNRISE Absorbance Reader, TECAN, Männedorf, Switzerland) using the XFluor4 software (TECAN). Data were then transferred to Microsoft Excel to calculate the specific activities in Miller units.

Expression of fixNOQP’-‘lacZ and fixK₂′-‘lacZ fusions in B. diazoefficiens cells grown under microoxic conditions was analyzed by measuring β-Galactosidase activity. Cells cultivated for 48 h were first permeabilized and subsequently used for the assays, as previously described [55 ,57 (link)]. The absorbance at 420 nm of the enzymatic reactions and at 600 nm of the cultures was recorded in a plate reader (SUNRISE Absorbance Reader; TECAN, Männedorf, Switzerland) using the XFluor4 software (TECAN, Männedorf, Switzerland). These data were used to calculate the specific activity of β-Galactosidase in Miller units (MU).

The extent of cell proliferation was assessed using the colorimetric CellTiter 96 AQ _ueous Non-Radioactive Cell Proliferation Assay (Promega). Cells were cultured in a 96-well plate (BD Falcon) and incubated with the appropriate treatments in a total of 100 μl of serum-free media. At the 48 h experiment end-point 1 ml of PMS [PMS (0.92 mg/ml) in Dulbecco’s PBS (DPBS); KCl (0.2 g/l), NaCl (8.0 g/l), KH₂PO₄ (0.2 g/l), Na₂HPO₄ (1.15 g/l), MgCl₂ (100 mg/l)·6H₂O, CaCl₂ (133 mg/l)·2H₂O; pH 7.35] Solution was added to 20 ml MTS Solution and 20 μl of this mixture was then combined with each well containing cells, thereby producing final concentrations of MTS (333 μg/ml) and PMS (25 μM). The cells were then incubated for 2 h at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. Thereafter, the absorbance at 490 nm was measured with the Sunrise microplate absorbance reader (Tecan) using the accompanying XFluor4 software (v4.51; Tecan); the amount of the red formazan product is directly proportional to the number of living cells. Results were blanked against control wells without cells containing 100 μl serum-free media and 20 μl combined MTS/PMS solution.

The capacity of the protein fractions to inhibit ACE-1 activity was quantified using the ACE-i assay with furanacroloyl-Phe-Glu-Glu (FAPGG) as the synthetic substrate for the ACE-1 enzyme. The substrate solution (150 μL of 0.5 mM FAPGG substrate) and samples (10 μL of 0.2% w/w protein concentration) were incubated at 37 °C for 5 min in the microplate reader, according to XFluor4 software instructions (Tecan, Ltd, Männedorf, Switzerland). After the incubation, the ACE-1 enzyme (50 μL of 15 mU enzyme) was added to the sample/substrate mixture and the kinetic reaction started. The kinetic reaction was monitored for the following 30 min using the microplate reader at 340 nm with the use of captopril as a positive control [13 (link)]. Hydrolysis of FAPGG by ACE-1 enzyme resulted in a decrease in absorbance at 340 nm. A 100% ACE-1i activity would indicate complete inhibition of the enzyme. All experiments were performed in duplicate:
Absno sample is the absorbance of the enzyme-substrate mixture in the absence of protein fraction, and Abssample is the absorbance of the enzyme-substrate mixture in the presence of protein fraction. ACE-1 enzyme, FAPGG and all other reagents were supplied from Sigma Aldrich (Dorset, UK).