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. 2013 Jan 1;288(8):5426–5442. doi: 10.1074/jbc.M112.431569

FIGURE 5.

FIGURE 5.

Analysis of NR and TMAOR activities after overexpression of the ISC operon, TusA, and IscU. A, plasmid encoding the A. vinelandii ISC operon was introduced into BW25113 (DE3), and the cells were grown for 8 h at 37 °C under aerobic (white bars) and anaerobic (black bars) conditions in the presence of either 20 mm nitrate or 20 mm TMAO, respectively. NR and TMAOR activities were determined in cell lysates. Reduced benzyl viologen was used as the electron donor. The activity of the wild type strain containing an arabinose-induced ampicillin-resistant plasmid was set to 100% for each condition. The activities were related to total protein concentration in the crude extract. Error bars are derived from three independent measurements within a standard deviation of 10% at maximum. B, plasmids encoding IscU (pJD54), TusA (pJD34), and pET15b (−) were introduced into BW25113 (DE3), and the cells were grown for 8 h at 37 °C in the presence of either 20 mm nitrate or 20 mm TMAO under aerobic conditions. TMAOR activities (white bars) and NR activities (black bars) were determined in cell lysates as described for Table 5. C, plasmids encoding IscU (pJD54), TusA (pJD35), and pACYCDuet (−) were introduced into BW25113 (DE3), and the cells were grown for 8 h at 37 °C under aerobic conditions. Cleared cell lysates were incubated with 500 μm l-cysteine for 10 min at 30 °C, and l-cysteine desulfurase activity was measured by determining total sulfide produced using methylene blue as reported previously (29). D, plasmids encoding TusA (pJD34), TusA-C19S (pJD49), and pET15b (−) were introduced into ΔtusA(DE3) and ΔmoaD, respectively, and the cells were grown for 8 h at 37 °C in 20 mm nitrate containing LB under aerobic conditions with (black bars) and without (white bars) the addition of IPTG. Quantification of cPMP concentration was performed after oxidation to its stable fluorescent derivative, compound Z. Fluorescence was monitored with excitation at 370 nm and emission at 450 nm. The amount of compound Z of the ΔmoaD strain was set to 100%. The concentrations were related to protein concentration in crude extract. n.d., not determined.