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 ΔtopA, confer lethality in derivatives of wild-type E. coli strain MG1655. Viability in the 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 has been reported to alleviate the deleterious consequences of RNAP backtracking and transcription-replication conflicts. Four phenotypes related to cSDR were identified for topA mutants: (i) one of the topA alleles rescued ΔdnaA 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 ΔdnaA. We propose that the terminal lethal consequence of hypernegative DNA supercoiling in E. coli topA mutants is RNAP backtracking during transcription elongation and associated R-loop formation, which in turn leads to transcription-replication conflicts and to cSDR. IMPORTANCE In all life forms, double-helical DNA exists in a topologically supercoiled state. The enzymes DNA gyrase and topoisomerase I act, respectively, to introduce and to relax negative DNA supercoils in Escherichia coli. That gyrase deficiency leads to bacterial death is well established, but the essentiality of topoisomerase I for viability has been less certain. This study confirms that topoisomerase I is essential for E. coli viability and suggests that in its absence, aberrant chromosomal DNA replication and excessive transcription-replication conflicts occur that are responsible for lethality.

R-loops in bacterial transcription: their causes and consequences.

Nascent untranslated transcripts in bacteria are prone to generating RNA-DNA hybrids (R-loops); Rho-dependent transcription termination acts to reduce their prevalence. Here we discuss the mechanisms of R-loop formation and growth inhibition in bacteria.

Rho-dependent transcription termination is essential to prevent excessive genome-wide R-loops in Escherichia coli.

Two pathways of transcription termination, factor-independent and -dependent, exist in bacteria. The latter pathway operates on nascent transcripts that are not simultaneously translated and requires factors Rho, NusG, and NusA, each of which is essential for viability of WT Escherichia coli. NusG and NusA are also involved in antitermination of transcription at the ribosomal RNA operons, as well as in regulating the rates of transcription elongation of all genes. We have used a bisulfite-sensitivity assay to demonstrate genome-wide increase in the occurrence of RNA-DNA hybrids (R-loops), including from antisense and read-through transcripts, in a nusG missense mutant defective for Rho-dependent termination. Lethality associated with complete deficiency of Rho and NusG (but not NusA) was rescued by ectopic expression of an R-loop-helicase UvsW, especially so on defined growth media. Our results suggest that factor-dependent transcription termination subserves a surveillance function to prevent translation-uncoupled transcription from generating R-loops, which would block replication fork progression and therefore be lethal, and that NusA performs additional essential functions as well in E. coli. Prevention of R-loop-mediated transcription-replication conflicts by cotranscriptional protein engagement of nascent RNA is emerging as a unifying theme among both prokaryotes and eukaryotes.

Two pathways for RNase E action in Escherichia coli in vivo and bypass of its essentiality in mutants defective for Rho-dependent transcription termination.

The endonuclease RNase E of Escherichia coli is essential for viability, but deletion of its C-terminal half (CTH) is not lethal. RNase E preferentially acts on 5'-monophosphorylated RNA whose generation from primary transcripts is catalysed by RppH, but ΔRppH strains are viable. Here we show that the RNase E-ΔCTH ΔRppH combination is lethal, and that the lethality is suppressed by rho or nusG mutations impairing Rho-dependent transcription termination. Lethality was correlated with defects in bulk mRNA decay and tRNA processing, which were reversed by the rho suppressor. Lethality suppression was dependent on RNase H1 or the helicase UvsW of phage T4, both of which act to remove RNA-DNA hybrids (R-loops). The rho and nusG mutations also rescued inviability of a double alteration R169Q (that abolishes 5'-sensing) with ΔCTH in RNase E, as also that of conditional RNase E deficiency. We suggest that the ΔCTH alteration leads to loss of a second 5'-end-independent pathway of RNase E action. We further propose that an increased abundance of R-loops in the rho and nusG mutants, although ordinarily inimical to growth, contributes to rescue the lethality associated with loss of the two RNase E cleavage pathways by providing an alternative means of RNA degradation.