Extended Data Fig. 2: In vivo characterization of Sir2/Ago systems.
a, Efficiency of plating (EOP) of 4 phages infecting E. coli cells with and without the GsSir2/Ago system from Geobacter sulfurreducens, where the GsSir2/Ago system exhibits no defence activity. The x-axis represents the number of p.f.u. Shown are the means of two replicates in the absence and in the presence of the inducer L-arabinose (L-Ara), with individual data points superimposed. Grey bars represent efficiency of plating (EOP) on pAgo-lacking cells and black bars are EOP in pAgo-containing cells. b, Left - qualitative characterization of plasmid restriction capabilities of PgSir2-Ago system in E. coli strain DH10B. Top: comparison of cell viability in the presence or absence of plasmid-borne PgSir2-Ago expression. Bottom: comparison of plasmid transformation efficiencies in the presence or absence of plasmid-borne PgSir2-Ago expression. Right - qualitative characterization of plasmid restriction capabilities of CcSir2/Ago system in E. coli strain BL21-AI: top - comparison of cell viability in the presence or absence of plasmid-borne CcSir2/Ago expression. Bottom - comparison of plasmid transformation efficiencies in the presence or absence of plasmid-borne CcSir2/Ago expression. c, Quantification of transformation efficiencies for pCDF plasmid with CcSir2/Ago system (three independent replicates, the red line represents average transformation efficiency). d, Cell viability control of BL21-AI, expressing GsSir2/Ago and mutants. e, Control for Fig. 2e – cells contain the pCDF plasmid, however, expression of the GsSir2/Ago system is not induced. f, Qualitative evaluation of pCDF plasmid transformation efficiency in E. coli cells carrying GsSir2/Ago mutants (GsSir2APAZ/Ago, GsSir2/AgoMID and GsSir2/AgoPIWI) of the putative surface of the interaction with nucleic acids. g, Expression analysis of GsSir2/Ago assayed by Western blot. Top: semiquantitative Western blot of the wt GsSir2/Ago complex and its mutants. Numbers above the lanes indicate which part of control protein amount is loaded. The red star shows the lane where the His-tag is on the C-terminus of Ago, rather than the N-terminus of Sir2. GAPDH, loading control. Bottom: Expression analysis of GsSir2/Ago mutants of the putative surface of the interaction with nucleic acids. Three replicates.
