Laboratory Evolution Experiments Help Identify a Predominant Region of Constitutive Stable DNA Replication Initiation

Abstract

The bacterium Escherichia coli can initiate replication in the absence of the replication initiator protein DnaA and/or the canonical origin of replication oriC in a ΔrnhA background. This phenomenon, which can be primed by R-loops, is called constitutive stable DNA replication (cSDR). Whether DNA replication during cSDR initiates in a stochastic manner through the length of the chromosome or at specific sites and how E. coli can find adaptations to loss of fitness caused by cSDR remain inadequately answered. We use laboratory evolution experiments of ΔrnhA-ΔdnaA strains followed by deep sequencing to show that DNA replication preferentially initiates within a broad region located ∼0.4 to 0.7 Mb clockwise of oriC. This region includes many bisulfite-sensitive sites, which have been previously defined as R-loop-forming regions, and includes a site containing sequence motifs that favor R-loop formation. Initiation from this region would result in head-on replication-transcription conflicts at rRNA loci. Inversions of these rRNA loci, which can partly resolve these conflicts, help the bacterium suppress the fitness defects of cSDR. These inversions partially restore the gene expression changes brought about by cSDR. The inversion, however, increases the possibility of conflicts at essential mRNA genes, which would utilize only a minuscule fraction of RNA polymerase molecules, most of which transcribe rRNA genes. Whether subsequent adaptive strategies would attempt to resolve these conflicts remains an open question. IMPORTANCE The bacterium E. coli can replicate its DNA even in the absence of the molecules that are required for canonical replication initiation. This often requires the formation of RNA-DNA hybrid structures and is referred to as constitutive stable DNA replication (cSDR). Where on the chromosome does cSDR initiate? We answer this question using laboratory evolution experiments and genomics and show that selection favors cSDR initiation predominantly at a region ∼0.6 Mb clockwise of oriC. Initiation from this site will result in more head-on collisions of DNA polymerase with RNA polymerase operating on rRNA loci. The bacterium adapts to this problem by inverting a region of the genome including several rRNA loci such that head-on collisions between the two polymerases are minimized. Understanding such evolutionary strategies in the context of cSDR can provide insights into the potential causes of resistance to antibiotics that target initiation of DNA replication.

Too Much of a Good Thing: How Ectopic DNA Replication Affects Bacterial Replication Dynamics

“…In recent studies, replication profiles revealed in more detail the locations where cSDR is initiated, which are reasonably well-defined, including one particularly strong site roughly 500-600 kb clockwise from oriC at ∼4.5 Mbp, as well as a peak of synthesis in the termination area (Maduike et al, 2014;Dimude et al, 2015;Veetil et al, 2020). Despite a detailed analysis of the locations of initiation sites, the precise molecular mechanism that triggers the initiation of DNA synthesis in these defined locations is not fully understood (Maduike et al, 2014;Dimude et al, 2015;Veetil et al, 2020).…”

Replication-dependent organisation constrains positioning of long DNA repeats in bacterial genomes

“…On homology-based recombination, long repeats lead to deletion or duplication of the genomic segment or inversion of the region flanked by them. Laboratory evolution studies from various groups in different genomes of Escherichia, Bacillus , and Salmonella have shown that these structural variations affect bacterial fitness in different stresses and can be adaptive in nature under different conditions (Veetil et al 2020; Srinivasan et al 2015; Rocha et al 1999; Sonti & Roth 1989; Maharjan et al 2013; Achaz et al 2003). Different types of structural variations in the genome impact bacteria differently.…”

“…Changes in the position of a gene can lead to changes in their dosage and thus expression; this might further hamper bacterial growth (Bryant et al 2014). Alternatively, any shift in the position of the oriC or a disruption of oriC functioning, can impact gene expression on a global scale and might be substantially deleterious for the bacteria (Veetil et al 2020; Dimude et al 2018; Bryant et al 2014; Duigou & Boccard 2017). Changes in gene orientation, caused by inversions, might result in head-on collisions of DNA and RNA polymerase.…”

“…Despite organisational features of the genome being shared across bacteria (Couturier & Rocha 2006; Khedkar & Seshasayee 2016; Tamames 2001), the events that generate chromosome variations are inevitable in nature and large rearrangements are one of the major contributors to these variations. Chromosome rearrangements like deletions, duplications and inversions are caused by cellular processes like homologous recombination and transpositions under different genetic and environmental conditions (Bishop 2000; Veetil et al 2020; Srinivasan et al 2015; Sonti & Roth 1989). DNA repeats are one of the major players mediating such events (Achaz et al 2003; Bi & Liu 1996; Lambert et al 1999; Treangen et al 2009).…”

“…Previous studies have reported such events of bacterial rearrangements under different stresses. The rearrangements observed were associated with repeat elements like insertion sequences, and were in turn advantageous or disadvantageous in different environments (Veetil et al 2020; Repar et al 2017; Srinivasan et al 2015; Sonti & Roth 1989; Adler et al 2014; Maharjan et al 2013). Since these repeats-associated changes in genome organisation play a role in affecting fitness, there might be selection on the positioning of such repeats on the chromosome.…”