Fig. 2. Antibiotics did not significantly increase conjugation efficiency.

(a) A synthetic conjugation system (see Table S1). The recipient (R⁻) carries an immobile plasmid expressing cm^R and an mCherry reporter. The donor (G⁺) carries a mobilizable plasmid expressing kan^R and a GFP reporter. When mixed, G⁺ transfers a copy of its plasmid to R⁻, generating transconjugant Y. Transconjugants can be quantified by selective plating or by fluorescence imaging.

(b) Quantifying conjugation efficiency (η_C) for R⁻ and G⁺ (see Table S2 and Methods for details) with 10 antibiotics. The y-axis is the conjugation efficiency normalized with the efficiency in the absence of antibiotics, η_C0 The x-axis is antibiotic concentration [A] normalized with each IC₅₀ measured in plate readers (Fig. S2). There was no significant increase in η_C0 amongst all antibiotics and concentrations tested (P >0.15, one-tailed t-test, Table S3a). G⁻ was mated with R⁻ as control; no transconjugants were detected (CFU data not shown, demonstrated by Video S2).

(c) Left: Physiological state of the cells significantly influenced the conjugation efficiency. G⁺ and R^−F cells were grown into exponential phase for 2 hours (See Methods). All four combinations of R^−F and G⁺ from either stationary (s) or exponential (e) phase were tested, from top to bottom, G_e and R_s (crosses), G_e and R_e (pluses), G_s and R_s (circles), and G_s and R_e (squares). Str was used for these experiments, at concentrations of 0, 2, and 4 µg/mL. Differences in η_C between combinations are statistically significant (P<5×10⁻⁴, two-tailed t-test).

Right: Glucose significantly increases η_C. Crosses indicate without and boxes indicate with 2µg/mL Kan. Here, η_C0 is for the standard M9 conditions (glucose = 0.4%, Fig. 2b), since η_C under these conditions insignificantly changes between 0 and 2µg/mL Kan (P>0.71, one-tailed t-test).

(d) Antibiotics did not significantly increase the conjugation efficiency for five native self-transmissible conjugation systems, including F(i), RP4(ii), R388(iii), R6K(iv), and pESBL-283(v) from five incompatibility groups (IncF, IncP, IncW, IncX, and IncI), or for four ESBL-producing clinical E. coli isolates donors with unknown conjugation machinery (vi–ix) (P>0.15, one-tailed t-test. Donors and recipients are labeled in each panel (Table S1 and Methods).

Error bars for all data in Fig. 2 indicate mean ± standard deviation from four-six replicates.