Effects of cephalexin treatment on DNA replication dynamics in Escherichia coli

Abstract

Background: In bacteria, DNA replication and chromosome segregation are tightly coordinated with cell division. The replication run-out method, which combines inhibition of transcription initiation by rifampicin with inhibition of cell division by cephalexin, is widely used to analyse replication dynamics by allowing ongoing replication forks to complete in the absence of new initiations. While this approach is generally robust, the physiological consequences of the individual components of the method, particularly cephalexin, are less well characterised.

Results: Here, we investigated the effects of cephalexin treatment on DNA replication in Escherichia coli. Cephalexin treatment resulted in rapid filamentation and, when applied alone, led to extensive cell lysis. Concomitantly, a subpopulation of cells displayed an increase in the number of replisome-associated DnaN foci, which was accompanied by amplification of both origin- and terminus-proximal chromosomal loci. Genetic analysis demonstrated that this response was largely dependent on the replication initiator DnaA, whereas mutants impaired in replication restart pathways did not show a corresponding reduction, arguing against a major contribution from pathways such as recombination-dependent replication. Importantly, co-treatment with rifampicin largely suppressed the increase in replication activity observed following cephalexin treatment and strongly reduced cell lysis, revealing a physiological antagonism between the two drugs under these conditions.

Conclusions: Our findings show that cephalexin treatment alone is not physiologically neutral with respect to DNA replication and can result in DnaA-dependent replication activity in a subset of cells. While the classical rifampicin-cephalexin replication run-out approach largely mitigates these effects, residual heterogeneity remains and may be exacerbated in sensitised genetic backgrounds. These results highlight the importance of considering antibiotic-induced physiological responses, including antagonistic drug interactions, when interpreting replication phenotypes in both single- and combination-treatment experiments.