A Fork Trap in the Chromosomal Termination Area Is Highly Conserved across All Escherichia coli Phylogenetic Groups

Abstract

Termination of DNA replication, the final stage of genome duplication, is surprisingly complex, and failures to bring DNA synthesis to an accurate conclusion can impact genome stability and cell viability. In Escherichia coli, termination takes place in a specialised termination area opposite the origin. A ‘replication fork trap’ is formed by unidirectional fork barriers via the binding of Tus protein to genomic ter sites. Such a fork trap system is found in some bacterial species, but it appears not to be a general feature of bacterial chromosomes. The biochemical properties of fork trap systems have been extensively characterised, but little is known about their precise physiological roles. In this study, we compare locations and distributions of ter terminator sites in E. coli genomes across all phylogenetic groups, including Shigella. Our analysis shows that all ter sites are highly conserved in E. coli, with slightly more variability in the Shigella genomes. Our sequence analysis of ter sites and Tus proteins shows that the fork trap is likely to be active in all strains investigated. In addition, our analysis shows that the dif chromosome dimer resolution site is consistently located between the innermost ter sites, even if rearrangements have changed the location of the innermost termination area. Our data further support the idea that the replication fork trap has an important physiological role that provides an evolutionary advantage.

Phage N15-Based Vectors for Gene Cloning and Expression in Bacteria and Mammalian Cells

“…16 The results revealed that the host cell viability (in respect of growth rate and cellular morphology) and stability of genome structure were not influenced by the topological changes when the linearization of the chromosome was designed at the region apart from the bacterial ori site. 16 This is in agreement with a later report which found that an E. coli chromosome linearized by TelN at a tos sequence inserted near the chromosome dimer resolution site dif (located within terminus region of the chromosome 72 ) does not delay the growth rate of wild-type E. coli. 73 Surprisingly, linearization of the circular E. coli chromosome almost did not alter the genome-wide gene expression, as only three genes were found to exhibit different gene expression levels among the 4300 genes analyzed.…”

Delineation of the Ancestral Tus-Dependent Replication Fork Trap

“…However, it now appears that the type II replication fork trap, which is mostly found in Enterobacteriaceae, is more of an exception or even an anomaly with respect to its many redundant Ter sites and their wide spread around the chromosome. It seems that the complexity of the type II fork trap in E. coli and closely related species [46] has merely distracted scientists from capturing the elegance and simplicity of the type I system in other Enterobacterales. Nevertheless, the work on the E. coli Tus-Ter complex was instrumental to decipher the unique Tus-Ter-lock mechanism [9], which still stands true and is seemingly conserved in all tus-harbouring bacteria.…”

Interplay between chromosomal architecture and termination of DNA replication in bacteria

“…The highly conserved ter core (6, 8–18) is highlighted by a darker grey. Tus binding ( Toft et al, 2021 ), in vivo fork blocking observed by 2D gel electrophoresis ( Duggin and Bell, 2009 ) and analysis whether a ter site is part of an open reading frame ( Duggin and Bell, 2009 ; Goodall et al, 2021 ) are indicated (see text for further details).…”

“…However, within the limits of this resolution, our data demonstrate that the fork fusion point, as determined by LOESS regression, is in precisely the same location in the presence and absence of Tus protein ( Rudolph et al, 2013 ; Dimude et al, 2016 ). The only change observed is a mild distortion of the marker frequencies in the vicinity of the fusion point (see Dimude et al (2016) and Goodall et al (2021) for a direct comparison of the replication profiles in the presence and absence of Tus terminator proteins). In contrast, in cells in which the replichore arrangement is artificially skewed by an additional ectopic origin, the fork fusion point is significantly shifted if the tus gene is deleted, as expected ( Ivanova et al, 2015 ; Dimude et al, 2018b ; Syeda et al, 2020 ).…”

“…Indeed, when we analyzed the sequence conservation of all ter sites across all phylogenetic groups in E. coli , we found a revealing trend: for the innermost ter sites A–D , which clearly have a role in blocking forks ( Duggin and Bell, 2009 ; Toft et al, 2021 ), strict conservation was observed for all bases that make contact with Tus. However, especially at bases 1–5 and 7 numerous variations were found, which are unlikely to affect Tus binding ( Goodall et al, 2021 ). Interestingly, the same pattern was observed for terG ( Goodall et al, 2021 ), the one ter site which is not part of the inner fork trap, but for which Tus binding and significant fork blocking activity was demonstrated ( Duggin and Bell, 2009 ; Midgley-Smith et al, 2018 ; Toft et al, 2021 ).…”