PRJNA670792
Topoisomerase I Essentiality, DnaA-independent Chromosomal Replication, and Transcription-Replication Conflict in Escherichia coli
Topoisomerase I (Topo I) of Escherichia coli, encoded by topA, acts to relax negative supercoils in DNA. Topo I deficiency results in hypernegative supercoiling, formation of transcription-associated RNA-DNA hybrids (R-loops), and DnaA- and oriC-independent constitutive stable DNA replication (cSDR), but some uncertainty persists as to whether topA is essential for viability in E. coli and related enterobacteria. Here we show that several topA alleles, including delta topA, confer lethality in derivatives of wild-type E. coli strain MG1655. Viability in absence of Topo I was restored with two perturbations, neither of which reversed the hypernegative supercoiling phenotype: (i) in a reduced-genome strain MDS42, or (ii) by an RNA polymerase (RNAP) mutation rpoB*35 that alleviates the deleterious consequences of RNAP backtracking and transcription-replication conflicts. Absence of Topo I in MDS42 was synthetically lethal with compromised Rho-dependent transcription termination, which was also alleviated by rpoB*35. Four phenotypes related to cSDR were identified for topA mutants: (i) One of the topA alleles rescued dnaA knockout lethality; (ii) in dnaA+ derivatives, Topo I deficiency generated a characteristic copy number peak in the terminus region of the chromosome; (iii) topA was synthetically lethal with rnhA (encoding RNase HI, whose deficiency also confers cSDR); and (iv) topA rnhA synthetic lethality was itself rescued by dnaA knockout. We propose that transcription-replication conflicts from two sources, namely those arising from RNAP backtracking during transcription elongation and at ribosomal RNA operons during cSDR, are responsible for topA inviability in E. coli.