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

Roberto Balbontín; Nelson Frazão; Isabel Gordo

To test that hypothesis, we influenced the occurrence of replication-transcription conflitcs through two strategies: negatively by growing our set of mutants in minimal medium, where DNA replication is slower (57) , or positively it by overexpressing the DNA replication initiator protein DnaA, which causes simultaneous initiation of multiple replication forks (58) . Our hypothesis predicts that DNA breaks should be reduced in minimal medium, where replication-transcription conflicts are less pronounced ( Figure 3B ). In agreement with our hypothesis, the SOS induction is smaller in minimal than in rich media ( Figure 3C , right panel), and the correlation with the cost is much weaker in minimal medium ( Figure S2B ). Coherently, the number of resistant mutants showing a fitness cost is smaller in minimal medium (8, compared to 13 in rich medium), with four of them even showing a higher fitness than sensitive bacteria ( Figure 3C , left panel). Moreover, some of the most costly mutations in rich medium (e. g. RpsL K43N RpoB H526Y and RpsL K43T RpoB H526Y ) generate a much smaller cost in minimal medium (compare Figure 1A with Figure 3C ). Besides this general trend, however, there are resistant mutants (notably, those carrying the allele RpoB S531F ) that conserve or even increase their fitness cost in minimal medium (compare Figure 1A with Figure 3C ), evidencing the existence of additional factors contributing to the cost of resistance (such as the known defects in protein synthesis). Conversely, our hypothesis also anticipates that increasing the occurrence of replication-transcription conflicts by overexpressing DnaA should particularly affect resistant bacteria. As hypothesized, overexpression of dnaA severely compromises the viability of double resistant bacteria, whereas sensitive bacteria remains largely unaffected ( Figure 3D ). Altogether, these results demonstrate the involvement of replication-transcription conflicts in the generation of the cost of antibiotic resistance, also corroborating the key contribution of DNA breaks to it.