Replication Control in Bacteria with Multiple Chromosomes – RepliChroms
Replication Control in Bacteria with Multiple Chromosomes
The RepliChroms project aims to identify fundamental mechanisms responsible for chromid domestication in bacteria with multipartite genomes, with a detailed description of their replication process.
Transition from a plasmid to a chromosomal mode of replication.
The main hypothesis driving this proposal is that the usual plasmid-type machineries, which are suitable for maintenance of multiple copy-number plasmids, firing their replication randomly in time, evolved towards systems which are able to sustain replication of large, low-copy-number replicons with respect to the cell cycle. Chromosomes usually replicate at a fixed time once per cell cycle while plasmids generally initiate replication several times over the entire bacterial cell cycle. In contrast, megaplasmids and secondary chromosomes, i.e. RepABC megaplasmids of a-proteobacteria and secondary chromosome of Vibrio cholerae (Chr2), replicate once per cell cycle. In V. cholerae, Chr2 has integrated a very sophisticated initiation regulation system originally acquired from plasmids and further improved to curb Chr2 replication to once per cell cycle and at particular time of the cell cycle. However, knowledge of the mechanisms coordinating the replication of its two chromosomes is largely incomplete and much remains to be learned about their functional interrelationships. The RepliChroms project aims at understanding how was operated the transition from a plasmid to a chromosomal mode of replication. <br />The three specific objectives of RepliChroms are:<br />1. To provide a deeper understanding of the mechanisms synchronizing the replication of V. cholerae’s secondary chromosome (Chr2) with the cell cycle. <br />2. To extend our knowledge to other Vibrio chromids (Chr3) to obtain have a better understanding of their domestication process.<br />3. To define how far the Vibrio paradigm applies to other Chromid-Containing Bacteria (CCB). <br />The understanding of how Chr2 and Chr3 replication is fully integrated within the Vibrio cell cycle, combined with the work on other multi-chromosome taxa will yield a generalized model of plasmid domestication and on the evolution of bacterial genomes.
We will use a combination of cutting-edge genome-wide studies (i.e. chromosome conformation capture “3C”, marker frequency analysis “MFA” and Chromatin Immunoprecipitation sequencing “ChIP-Seq”). The advantage of these techniques is that they can be universally applied to any cultivable organism with the least fitness perturbations.
To improve the resolution of our analysis, we will confront our results obtained from genome-wide and population analyses to single-cell studies. We will use live-cell fluorescence microscopy and digital PCR. This should provide complementary results such as cell-to-cell fluctuations or the spatial and temporal choreography of replication factors. Beyond what has already been developed in our lab, we will (1) improve live-cell fluorescence microscopy in Vibrio to simultaneously track three loci and (2) implement single-cell digital PCR analysis to unmask heterogeneous phenotypes in individual cells. All the combined results will help us to draw a more comprehensive picture of the cellular mechanisms that coordinate the replication of multiple replicons.
- crtS acts as an anti-inhibitory site by preventing inhibitory sites in ori2 from repressing initiation of Chr2.
- The coordinated replication of Vibrio cholerae's two chromosomes required the acquisition by the RctB initiator of a unique domain.
- The extreme C-terminal tail of RctB is crucial for the control exerted by crtS.
RepliChroms aims at providing a precise model of plasmid adaptation and their domestication as secondary chromosomes in host with pathogenic and environmental importance. This project will provide a detailed description of cell-cycle replication coordination, which is an important transitional step in the domestication of plasmids into chromosomes. Our research will describe the molecular mechanism of a new replication checkpoint control in Vibrio. This will greatly impact our fundamental knowledge of DNA replication and genome maintenance in bacteria. Bacteria with multiple replicons might share similar, evolutionarily conserved, strategies of temporal replication coordination. Our results will establish a model for replication coordination in bacteria with multipartite genomes.
- manuscript under revision
The RepliChroms project aims at decrypting plasmid adaptation mechanisms in their bacterial host. Bacterial genomes are composed of two types of replicons: chromosomes, which are by definition essential, and plasmids, which are dispensable. Bacteria commonly have one single circular chromosome, and current models of genome dynamics are based on this assumption. However, 10% of bacteria also have secondary chromosomes which originate from megaplasmids. Secondary chromosomes and megaplasmids also called “chromids” confer specific traits to their host (e.g. pathogenicity, resistance). Incoming plasmids are often poorly adapted to their new hosts and their stabilization requires integration with the host’s cellular mechanisms in a process termed domestication. The RepliChroms project will focus on the identification of the fundamental mechanisms driving chromid domestication in bacteria with a detailed description of their replication process.
Vibrios are an important and abundant group of bacteria in aquatic ecosystems, comprising both symbionts and pathogens of many aquatic living organisms. All Vibrios carry one chromosome (Chr1) and one chromid (Chr2). A large portion of the RepliChroms project work will be undertaken in the globally-important pathogen, Vibrio cholerae and the conclusions of our experiments are expected to be valid and generalized for all Vibrio species. We recently discovered how Chr2 synchronizes its replication with Chr1. Chr2 replication initiation is triggered by the replication of an intergenic sequence (crtS) located on Chr1. This checkpoint mechanism is an elegant and cost-effective way for chromids to harmonize their replication with the well-established replication regulatory system of the host chromosome. RepliChroms will provide a deeper understanding of the molecular mechanisms synchronizing the replication of Chr2 in V. cholerae.
In the last decade, new virulent Vibrio isolates with additional chromids (Chr3) have emerged. RepliChroms will seek how well the replication of Vibrio’s Chr3 are integrated within the bacterial cell cycle. This will help understanding the mechanisms of genome evolution that contribute to the emergence of new Vibrio pathogens. RepliChroms will further investigate the replication mode of other bacterial models with multipartite genomes (e.g. Burkholderia and Agrobacterium) and provide a detailed description of the coordinated replication of their chromids. The fact that additional chromids are conserved through speciation across many bacterial genera as additional chromosomes, indicates a fundamental characteristic of evolution, adaptation and pathogenicity of many bacteria. RepliChroms will integrate modern techniques, including digital PCR, fluorescence microscopy and high-throughput sequencing, to significantly advance our understanding of multipartite genome dynamics and will pave the way for new perspectives.
Project coordination
Marie-Eve KENNEDY-VAL (INSTITUT PASTEUR)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
Partner
IP INSTITUT PASTEUR
Help of the ANR 263,409 euros
Beginning and duration of the scientific project:
November 2019
- 36 Months
