Extended Data Fig. 9: Mutation verification of Gp5.7 and rifampicin treatment.
From: Bacteria deplete deoxynucleotides to defend against bacteriophage infection
(A) Verification of the absence of Gp5.7 in T7 mutant n. 5 using mass spectrometry. Peptide fragments of Gp5.7 identified by protein mass spectrometry of cells 15 minutes post infection by WT T7 and T7 mutant n.5 (MOI = 2). Multiple peptides of Gp5.7 are observed in the WT T7, but no peptides are detected in the mutant T7, supporting that the mutant does not express Gp5.7. Peptide fragments are as follows: peptide 1: GHISCLTTSGR, peptide 2: NGGAWEITASGTR, peptide 3: NNASLVAEAASR, peptide 4: TFQSNYVR. Bar graphs represent average of two biological replicates, with individual data points overlaid. (B) As control to the measurements in panel A, shown are peptide fragments of the T7 RNA polymerase identified by protein mass spectrometry in cells 15 minutes post infection. The T7 RNA polymerase is readily identified in both WT and mutant phages. Peptide 1: EQLALEHESYEMGEAR, peptide 2: MNTINIAK, peptide 3: SVMTLAYGSK, peptide 4: VLAVANVITK. Bar graphs represent average of two biological replicates, with individual data points overlaid. (C-H) Concentrations of dNTP nucleotides in cell lysates extracted from rifampicin-treated cells, as measured by LC-MS with synthesized standards. X axis represents minutes post treatment, with zero representing non-treated cells. (C-E) Each panel shows data acquired for dCTP deaminase-expressing cells or for control cells that contain an empty vector. Bar graphs represent average of three biological replicates, with individual data points overlaid. (F-H) Each panel shows data acquired for dGTPase-expressing cells or for control cells that express GFP. Bar graphs represent average of three biological replicates, with individual data points overlaid. In panels D-F, 60 minute data represent the average of two biological replicates.
