Targeting the Bacterial Orisome in the Search for New Antibiotics

Grimwade, Julia E.; Leonard, Alan C.

doi:10.3389/fmicb.2017.02352

Cited by 12 publications

(12 citation statements)

References 79 publications

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“…Though cSDR may not necessarily be a physiological or natural phenomenon in E. coli, with the possible exception of its manifestation as nSDR in stationary phase, it has been argued that this could be a potential primordial mechanism of DNA replication initiation (11). Further, cSDR can provide the bacterium avenues for the development of resistance to new antibiotics targeting initiation of DNA replication (47,48). positions in these genomes were predicted for all E. coli strains by performing blastn searching using the E. coli K-12 MG1655 (NC_000913.3) oriC region as query.…”

Section: Discussionmentioning

confidence: 99%

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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Section: Discussionmentioning

confidence: 99%

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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“…Due to their statistical properties, models employed in gammaBOriTax will only identify motifs as relevant for classification that are both specific for a given taxon and evolutionarily conserved in this taxon. Therefore, the proteins that bind to and interact with these motifs ideal potential targets for novel antibiotics 65 .…”

Section: Discussionmentioning

confidence: 99%

gammaBOriS: Identification and Taxonomic Classification of Origins of Replication in Gammaproteobacteria using Motif-based Machine Learning

Sperlea

Muth

Martin

et al. 2020

Sci Rep

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The biology of bacterial cells is, in general, based on information encoded on circular chromosomes. Regulation of chromosome replication is an essential process that mostly takes place at the origin of replication (oriC), a locus unique per chromosome. Identification of high numbers of oriC is a prerequisite for systematic studies that could lead to insights into oriC functioning as well as the identification of novel drug targets for antibiotic development. Current methods for identifying oriC sequences rely on chromosome-wide nucleotide disparities and are therefore limited to fully sequenced genomes, leaving a large number of genomic fragments unstudied. Here, we present gammaBOriS (Gammaproteobacterial oriC Searcher), which identifies oriC sequences on gammaproteobacterial chromosomal fragments. It does so by employing motif-based machine learning methods. Using gammaBOriS, we created BOriS DB, which currently contains 25,827 gammaproteobacterial oriC sequences from 1,217 species, thus making it the largest available database for oriC sequences to date. Furthermore, we present gammaBOriTax, a machine-learning based approach for taxonomic classification of oriC sequences, which was trained on the sequences in BOriS DB. Finally, we extracted the motifs relevant for identification and classification decisions of the models. Our results suggest that machine learning sequence classification approaches can offer great support in functional motif identification. Before every cell division, bacteria need to duplicate their genetic material to ensure that this information can faithfully be passed on to both daughter cells. This essential process, called DNA replication, initiates in a highly regulated manner at chromosomal sites called oriC and is coordinated with many other cellular processes 1,2. With notable exceptions as e.G. Vibrionales, usually, bacteria contain one or multiple copies of a single chromosome, which carries a single oriC sequence 3,4. Since many different proteins need to bind to and act upon oriC for initiation to occur, oriC contains many protein binding sites and DNA motifs 5,6. While there is a high level of variation between oriC sequences of different organisms, there are also nearly universally occurring DNA motifs in oriC sequences 7-9. Central among these are 9 bp short DNA motifs called DnaA boxes, which act as binding sites for the initiator protein DnaA, and exhibit differing protein binding characteristics depending on the exact sequence. Starting from these motifs, DnaA polymerizes and spreads across multiple DnaA boxes and DnaA trio motifs 10 , which then, in interplay with the protein IHF 11 , leads to double helix unwinding at a closely positioned AT-rich region called DNA unwinding element (DUE) so that the replication machinery can be loaded onto the DNA 12,13. As oriC contains binding sites for proteins that relay information on the status of the cell, it can be considered as a biological information compiler and processor 14,15. Taken together, these properties make oriC sequ...

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“…As can be seen from the construction of BOriS DB, this leads to a large amount of newly-identified oriC sequences, among which are sequences from organisms that are notoriously hard to sequence and impossible to culture. This enables the use of data-intensive cutting-edge methods such as deep learning (50) for the identification previously unknown initiation factors, which might, due to their high degree of taxonomic specificity, be good candidates for targets of new antibiotics (51). Furthermore, a deeper knowledge of the components of of oriC will make it possible to de novo design chromosomes with desired replication characteristics and synthetic oriC sequences (52).…”

Section: Discussionmentioning

confidence: 99%

γBOriS: Identification of Origins of Replication in Gammaproteobacteria using Motif-based Machine Learning

Sperlea

Muth

Martin

et al. 2019

Preprint

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The biology of bacterial cells is, in general, based on the information encoded on circular chromosomes. Regulation of chromosome replication is an essential process which mostly takes place at the origin of replication (oriC). Identification of high numbers of oriC is a prerequisite to enable systematic studies that could lead to insights of oriC functioning as well as novel drug targets for antibiotic development. Current methods for identyfing oriC sequences rely on chromosome-wide nucleotide disparities and are therefore limited to fully sequenced genomes, leaving a superabundance of genomic fragments unstudied. Here, we present γBOriS (Gammaproteobacterial oriCSearcher), which accurately identifies oriC sequences on gammaproteobacterial chromosomal fragments by employing motif-based DNA classification. Using γBOriS, we created BOriS DB, which currently contains 25,827 oriC sequences from 1,217 species, thus making it the largest available database for oriC sequences to date.

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Targeting the Bacterial Orisome in the Search for New Antibiotics

Cited by 12 publications

References 79 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

gammaBOriS: Identification and Taxonomic Classification of Origins of Replication in Gammaproteobacteria using Motif-based Machine Learning

γBOriS: Identification of Origins of Replication in Gammaproteobacteria using Motif-based Machine Learning

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