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

Roberto Balbontín; Nelson Frazão; Isabel Gordo

Sensitive and RpsL K43N RpoB H526Y strains carrying the expression vector pCA24N -gfp, either empty or carrying the dnaA gene (93) were streaked individually onto LB agar plates supplemented with the appropriate antibiotic (to select for the presence of the plasmid), and incubated overnight at 37°C. The next day, three independent colonies from each strain were inoculated separately in LB broth supplemented with the appropriate antibiotic (150 µl per well) in a 96-well plate and incubated overnight at 37°C with shaking (700 r.p.m). The next day, the OD600 of the overnight cultures was measured using a Thermo Scientific Multiskan Go spectrophotometer, bacteria were appropriately diluted in 1X PBS to generate solutions containing approximately 10 10 , 10 9 , 10 8 , 10 7 , 10 6 , 10 5 Error bars represent mean ± standard deviation of independent biological replicates (n≥5). N.S. non-significant; *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) representing all the data points from A. The blue line represents the logarithmic regression line, and the grey area represents the 95% confidence interval. C. SOS induction (blue bars) and fitness cost (red bars) of sensitive bacteria and Erm R mutants in LB broth at 4 hours. The dashed line indicates no SOS induction. 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). The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/756767 doi: bioRxiv preprint 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). B. Fitness cost (red bars), and SOS induction (blue bars) of sensitive, ancestral RpsL K43T RpoB H526Y and nine compensated clones in LB broth at 4 hours. The black dashed line indicates no SOS induction. The grey dashed lines mark the cost/SOS of the ancestral double mutant. 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). 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 1A . B. Schematic representation of DNA replication of E. coli during fast (left) or slow (right) growth. Black dots represent the origin of replication, and red lines represent transcription forks; green arrows mark regions of potential replication-transcription conflicts. C. Fitness cost (red bars), and SOS induction (blue bars) of sensitive bacteria and Str R and/or Rif R mutants in minimal medium at 8 hours. The strains are ordered from lower to higher cost (top to bottom). The dashed line indicates no SOS induction. 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). D. Aliquots of serial dilutions (approximately from 5x10 7 to 5 cells) of sensitive and RpsL K43N All rights reserved. No reuse allowed without permission.