FIG 7 .

Mutational analysis of the three invariant EarP residues D13, D17, and E273. (A) In vitro rhamnosylation of EF-P_Ppu by single-amino-acid exchange variants, specifically, D13A^EarP, D17A^EarP, and E273A^EarP variants. EF-P (2.5 µM) and TDP-Rha (1 mM) were incubated together with the EarP_Ppu variants (0.5 µM) and sampled at different time points. Rhamnosylated EF-P_Ppu (EF-P^Rha) was detected after Western blotting using 0.25 µg/ml anti-Arg^Rha. (B) Overlay of ¹H-¹⁵N HSQC spectra of wild-type EarP_Ppu that was free and bound to TDP-Rha. (C) Overlay of ¹H-¹⁵N HSQC spectra of the free and TDP-Rha-bound D13A^EarP variant. (D) Overlay of ¹H-¹⁵N HSQC spectra of the free and TDP-Rha-bound D17A^EarP variant. The color coding is indicated in the upper left corner of each spectrum. The titrations are described in detail in Materials and Methods. (E) Zoom into the overlaid spectra shown in panels B, C, and D. The position of the zoom is indicated by a black frame in the corresponding original overlay. Peak assignments are shown. The movement of G16 and G19 upon TDP-Rha titration is indicated by dashed arrows. (F) Bacterial two-hybrid analysis of protein-protein interactions between EarP_Ppu, the D13A^EarP, D17A^EarP, and E273A^EarP variants, and the protein acceptor EF-P_Ppu in E. coli BTH101. The blue color of colonies results from cleavage of X-Gal by β-galactosidase and indicates protein-protein interaction between coexpressed hybrids. (G, top) Analysis of in vivo activities of EarP_Ppu, D13A^EarP, D17A^EarP, and E273A^EarP. In vivo EarP_Ppu activities were determined by measuring the β-galactosidase activities of an E. coli MG1655 P_cadBA::lacZ Δefp strain heterologously expressing efp_Ppu together with the wild-type or mutant earP_Ppu genes from o/n cultures in LB (pH 5.8). Means of three independent measurements are shown. Standard deviations from three independent experiments were determined. (Bottom) Western blot analysis of o/n cultures of E. coli MG1655 P_cadBA::lacZ Δefp heterologously expressing efp_Ppu together with the wild-type or earP_Ppu mutants. Rhamnosylated EF-P_Ppu (EF-P^Rha) was detected using 0.25 µg/ml anti-Arg^Rha.