ACC deaminase activity

To determine ACC deaminase activity, A. cicer and A. glycyphyllos symbionts were grown in 5 ml of TY medium (Beringer 1974) at 30 °C for 2–3 days until they reached the stationary phase. The bacterial cells were centrifuged and washed twice with 0.1 M Tris–HCl (pH 7.5). Next, the bacteria were suspended in 2 ml of M9 minimal medium supplemented with ACC (final concentration of 5 mM) and incubated at 30 °C with shaking for 36 h. ACC deaminase activity was determined according to the method described before (Ma et al. 2003; Penrose and Glick 2003). ACC deaminase activity was determined by measuring the production of α-ketobutyrate (Honma and Shimomura 1978).

The protein concentration in the cell extracts was determined by the method of Bradford (1976) using the Bio-Rad protein reagent (Bio-Rad; Protein Assay Dye Reagent Concentrate #500-0006).