FIGURE 4.

Analysis of Cac Nt-AS and Ct-AS asparagine synthetase activities in vitro and in vivo. A, complementation of the Asn auxotroph E. coli ER strain (asnA⁻, asnB⁻) by the Cac Nt-AS_pET15b and the Cac Ct-AS_pET15b constructs. The E. coli ER strain was transformed with either one of the two recombined pET15b vectors or with both recombinant vectors (Ct/Nt-AS_pET15b). Transformants were grown on minimal M9 medium agar plates supplemented with ampicillin and 0.5 mm IPTG in the absence (−) of Asn. B, asparagine synthetase activity assay using Cac protein extracts (S100). Reactions were carried out using a standard amidation mixture (see “Experimental Procedures”) and 100 μg of Sce (lane 1), Tth (lane 2), and Cac (lanes 3–8) S100. Six different Cac S100 extracts were analyzed for their asparagine synthetase activities. Lanes 3–8, S100 extracts taken from cells grown until acidogenesis (Ac.) or solventogenesis (S.) phase. −aa, no amino acids were added for the culture; +Asn, addition of 1 mm Asn; +Asp, addition of 1 mm Asp.