Bisphosphonates examined for relaxase inhibition. Chemicals examined for inhibition of TraI activity and F conjugation and for toxicity versus E. coli strains. Boxed chemicals were potent in vitro TraI inhibitors. In cell testing showed that PNP and PBENP were most effective at decreasing F⁺ population, CLODRO and ETIDRO were most effective at decreasing transconjugant population, and PCP and PCNCP were effective at both.

F TraI N300 Y16F bound to the scissile thymidine and a two-path model of F-like bacterial conjugation. (A) N300 Y16F active site with a metal ion (blue sphere) chelated by three histidines and the −1 Thy 3′-hydroxyl. Y16F occludes a fifth octahedral coordination site. (B) Cleavage by the first tyrosine forms a covalent phosphotyrosine intermediate (red circle) on the T (red) strand. Transfer with CPR diverges from simple transfer when the 3′-hydroxyl left by initial cleavage becomes a substrate for replication (blue strand). The newly created oriT requires a second cleavage event and second phosphotyrosine (purple circle).

Effects of F TraI inhibitors on E. coli survival and conjugation. Color coding for lines, bars, and symbols: orange, F⁻; red, F⁺/TraI⁺; cyan, F⁺/TraI⁻; green, F⁺/TraI 4Y-F (inactive relaxase); blue, F⁻/TraI⁺; yellow, transconjugants (DNA transfer). (A) Colony counts after PNP incubation (normalized versus uninhibited controls; averaged). Error bars represent standard errors (n ≥ 5). (B) EC₅₀ values from relative cell counts. Colored bars indicate averaged curve EC₅₀ values. Error bars represent ± 1 SD envelope EC₅₀ values (SI Fig. 10). Potent in vitro inhibitors are located above a strong black line, with negative controls below. Selectivities are EC₅₀ ratios for given inhibitors and strains. (C) Relative cell counts with median EC₅₀ values (drop arrows; colored by strain). Error bars represent the standard error (n ≥ 3).

Relaxase inhibition by PNP. (A) N300 oriT ssDNA cleavage velocity (v₀) inhibited by PNP (error bars represent compounded standard errors from time course parameter estimates; n ≥ 3). Competitive/uncompetitive inhibition constants (K_ic/K_iu) and uninhibited Michaelis constant/maximum velocity (K_m/V_max) are from nonlinear regression and Cornish-Bowden/Eisenthal direct linear plot analyses. Velocities for 100 and 10,000 nM PNP, estimated from low signal, were excluded from calculations. (B) N300 Y16F active sites with PNP (red) and without (orange). Only one PNP moiety was observed (purple). Y16F occludes the sixth coordination site. Residues 236–263 and 266 were disordered (small orange spheres). (C) Dual phosphotyrosine intermediate conformation model (red) constructed from the N300+PNP structure. Helix αA was rotated ≈120° about the helical axis to match that of TrwC structure 1OMH, and helicity was extended through αA′ and kinked about histidine-146. Important side chains (red sticks), PNP (purple sticks), a hypothetical second phosphate (purple circle), the bound metal (blue sphere), and the scissile thymidine (from 1OMH; blue) are shown over the TraI active-site cleft molecular surface.