Fig. 2.
In vivo activity tests for Enterobacteria phage 9g dG+ pathway genes. Source data are provided as a Source Data file. a Northern blot of an acrylamide electromobility gel shift assay showing the tRNA-Q complementation of E. coli mutants by Enterobacteria phage 9g orthologs. The WT strain modifies tRNAAsp with Q and is shifted in its migration (Q line), but the E. coli mutant strains (ΔfolE, ΔqueD, ΔqueE, ΔqueC, and Δtgt) are not modified and migrate further (no Q line). In each mutant, the orthologs of Enterobacteria phage 9g are expressed in trans. The complementation of Δtgt by E. coli tgt is shown as a positive control of complementation. b Agarose gel of uncut (0) or EcoRI-cut (D) pGH39/pGH66 extracted from a WT strain of E. coli; expression from the plasmids was repressed in 0.4% glucose (Glu) or induced in 0.4% arabinose (Ara). White arrows indicate the undigested plasmids. c Agarose gel of EcoRI digestion of plasmids extracted from different strains of E. coli (WT, ΔqueC, ΔqueD, Δtgt) carrying variants of pBAD33 and pBAD24 (empty plasmid, 0; encoding Enterobacteria phage 9g dpdA, A; or encoding Enterobacteria phage 9g gat-queC, C). EcoRI cuts pBAD24 once (4542-bp fragment) and pBAD33 twice (2479 and 2873-bp fragments). The resulting sizes for the digestion of pBAD24 are 5971 and 5509 bp when gat-queC or dpdA is inserted, respectively. For pBAD33, the 2873-bp fragment remains unchanged, but the 2479-bp fragment shifts to 3911 when gat-queC is inserted and 3449 bp when dpdA is inserted. When plasmids are undigested, they can be seen in the white rectangle zone.