Origins Left, Right, and Centre: Increasing the Number of Initiation Sites in the Escherichia coli Chromosome

The bacterium E. 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 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-dnaA followed by deep sequencing to show that DNA replication preferentially initiates at a site ~0.6 Mb clockwise of oriC. Initiation from this site 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 however would utilise only a miniscule 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.

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“…They found that this mutant carried a ~0.8 Mb inversion spanning the ter (J. Dimude et al 2018). However, this mutant grew slowly.…”

Section: Resultsmentioning

confidence: 99%

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

Veetil

Malhotra

Dubey

et al. 2019

Preprint

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“…In the initial analysis, the doubling time of oriC + oriZ + and wild type cells was found to be similar (Wang et al, 2011). However, when we measured the doubling times for MG1655 and oriC + oriZ + constructs in direct comparison, we found that oriC + oriZ + cells grew slightly slower in two independent studies (∼21 min in comparison to ∼20 min in wild type cells) (Ivanova et al, 2015;Dimude et al, 2018b).…”

Section: Introducing a Second Origin Into The E Coli Chromosomementioning

confidence: 87%

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

et al. 2020

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“…Whereas the previous studies by Ivanova et al and Dimude et al, (45,46) isolated structural variations while creating the parent strain, we were able to isolate our suppressors only after 4 to 8 days of selection in a laboratory evolution experiment.…”

Section: Discussionmentioning

confidence: 70%

“…Dimude et al (46) placed a second ori, termed oriX, counterclockwise of oriC. They found that this mutant carried an ϳ0.8-Mb inversion spanning the ter (46). However, this mutant grew slowly.…”

Section: Discussionmentioning

confidence: 99%

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

Veetil

Malhotra

Dubey

et al. 2020

mSphere

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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.

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Origins Left, Right, and Centre: Increasing the Number of Initiation Sites in the Escherichia coli Chromosome

Cited by 31 publications

References 55 publications

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

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

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

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

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