Figure 3

FLAG-tagged MoaX is functional and cleaved in M. smegmatis . (A) Nitrate assimilation in the ΔmoaD2 ΔmoaE2 mutant with a C-terminal FLAG-tagged derivative of moaX. In all cases, genes were expressed constitutively from the tetO operator. (B) Western blot analysis of protein extract from ΔmoaD2 ΔmoaE2 carrying C-terminally FLAG-tagged moaX showing cleavage of the fused MPT synthase at the Gly82 residue. Incorporation of the FLAG tag did not interrupt the function of MoaX, evidenced by the retained ability of the FLAG-tagged derivative to restore growth of ΔmoaD2 ΔmoaE2 in MPLN. (C) Peptide fragments of MoaX identified by mass spectrometry. Shown are two peptide fragments for each fraction. Red indicates the terminal Gly residues. Peptide reads all stopped at the Gly82 position, indicating cleavage at this point.