Figure 7. Activity of Nuc/Nuc2 chimeric proteins.

Chimeras consisting of combinations of Nuc and Nuc2 N- and C-terminal protein domains were constructed. Each chimera was constructed to include a His₆-tag on the C-terminal protein domain. A. Schematic showing the Nuc/Nuc2 chimeric proteins constructed, with Nuc domains colored in teal and Nuc2 domains colored in purple (see Fig. 1A for additional description of domains and processing sites). Chimeras constructed include, from the top down: Nuc control (pMK12), Nuc2 control (pMK13), Nuc signal sequence fused to the Nuc2 active domain (pMK8), Nuc signal sequence and NucB leader fused to the Nuc2 active domain (pMK9), Nuc2 membrane anchor fused to NucA (pMK10) and Nuc2 membrane anchor fused to NucB (pMK11). Each construct was expressed in a S. aureus nuc mutant (strain AH1680) for 15 hr, and culture supernatant was saved and membranes purified. Anti-His immunoblots were performed to detect the presence of C-terminal His₆-tag on chimeric proteins in culture supernatants (B) or membrane (C) fractions. FRET activity assays were performed to detect nuclease activity in culture supernatants (D) and membrane (E) fractions. The nuc mutant strain was included as a negative control (called “no plasmid”).