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While most of the ribosome-targeting antibiotics are bacteriostatic, some members of the macrolide class demonstrate considerable bactericidal activity. We previously showed that an extended alkyl-aryl side chain is the key structural element determining the macrolides' slow dissociation from the ribosome and likely accounts for the antibiotics' cidality. In the nontranslating Escherichia coli ribosome, the extended side chain of macrolides interacts with 23S ribosomal RNA (rRNA) nucleotides A752 and U2609, that were proposed to form a base pair. However, the existence of this base pair in the translating ribosome, its possible functional role, and its impact on the binding and cidality of the antibiotic remain unknown. By engineering E. coli cells carrying individual and compensatory mutations at the 752 and 2609 rRNA positions, we show that integrity of the base pair helps to modulate the ribosomal response to regulatory nascent peptides, determines the slow dissociation rate of the extended macrolides from the ribosome, and increases their bactericidal effect. Our findings demonstrate that the ability of antibiotics to kill bacterial cells relies not only on the chemical nature of the inhibitor, but also on structural features of the target., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Antibiotics can cause dormancy (bacteriostasis) or induce death (cidality) of the targeted bacteria. The bactericidal capacity is one of the most important properties of antibacterial agents. However, the understanding of the fundamental differences in the mode of action of bacteriostatic or bactericidal antibiotics, especially those belonging to the same chemical class, is very rudimentary. Here, by examining the activity and binding properties of chemically distinct macrolide inhibitors of translation, we have identified a key difference in their interaction with the ribosome, which correlates with their ability to cause cell death. While bacteriostatic and bactericidal macrolides bind in the nascent peptide exit tunnel of the large ribosomal subunit with comparable affinities, the bactericidal antibiotics dissociate from the ribosome with significantly slower rates. The sluggish dissociation of bactericidal macrolides correlates with the presence in their structure of an extended alkyl-aryl side chain, which establishes idiosyncratic interactions with the ribosomal RNA. Mutations or chemical alterations of the rRNA nucleotides in the drug binding site can protect cells from macrolide-induced killing, even with inhibitor concentrations that significantly exceed those required for cell growth arrest. We propose that the increased translation downtime due to slow dissociation of the antibiotic may damage cells beyond the point where growth can be reinitiated upon the removal of the drug due to depletion of critical components of the gene-expression pathway., Competing Interests: Conflict of interest statement: In the previous years, A.S.M. had grants from Cempra, Inc. and Melinta Therapeutics, which were involved in the development of macrolide antibiotics.