DNA breaks-mediated cost reveals RNase HI as a new target for selectively eliminating antibiotic resistance

Roberto Balbontín; Nelson Frazão; Isabel Gordo

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . Figure 1A . Error bars represent mean ± standard deviation of independent biological replicates (n≥3). The black line in panels A-C represents the linear regression if the costs were identical D. Fitness cost of sensitive bacteria and Str R and/or Rif R mutants in the presence of the RNase HI inhibitor RHI001 (red bars). For comparison, the corresponding values in the absence of the inhibitor (data from the experiments shown in Figure 1A ) or in a ΔrnhA background (data from the experiments shown in panel A and Figure S4A ) are represented as white and black bars, respectively. Error bars represent mean ± standard deviation of independent biological replicates (n=3). N.S. non-significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (two-tailed Student's t test). See also Figures S4, S5 The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/756767 doi: bioRxiv preprint Figure S2 . A. Area under the curve (24h in LB broth) of sensitive and double resistant bacteria harboring an expression vector either empty (left) or carrying the dksA gene in the absence (circles) or presence (triangles) of the inducer. Error bars represent mean ± standard deviation of independent biological replicates (n =6). N.S. nonsignificant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (two-tailed Student's t test). B. Correlation between the fitness cost (y axis) and the SOS induction (x axis) at 8h in M9 broth supplemented with 0.4% glucose (data represented in figure 3C ). The blue line represents the logarithmic regression line, and the grey area represents the 95% confidence interval. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/756767 doi: bioRxiv preprint Figure S4 . Depletion of RNase HI increases fitness cost and SOS induction. A. SOS induction (blue bars) and fitness cost (red bars), of sensitive bacteria and Str R and/or Rif R mutants (labels represent the corresponding allele/s) in a ΔrnhA background. White bars represent the corresponding values in bacteria with an intact rnhA gene (data from the experiments shown in Figure 1A ), for comparison. Error bars represent mean ± standard deviation of independent biological replicates (n=6). N.S. non-significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (two-tailed Student's t test). B. Growth curves of RpsL K43N RpoB H526Y double mutant in the presence of either DMSO (blue lines) or RHI001 (orange lines). Points and error bars represent mean ± standard deviation of independent biological replicates (n=3). C. Growth curves of sensitive bacteria in the presence of either DMSO (blue lines) or RHI001 (orange lines). Points and error bars represent mean ± standard deviation of independent biological replicates (n=3). Figure S5 . Mild overproduction of RNase HI can ameliorate both cost and SOS induction. SOS induction (top) and fitness cost (bottom), of sensitive bacteria (left), two single resistant mutants (center) and the double mutant combining these two alleles (right) in LB broth at 4h (A) or 24h (B) in a background harboring an additional chromosomal copy of the gene encoding the RNase HI (rnhA) under the control of the arabinose promoter, either in the absence (red bar