Final Report Summary - BMC (Bacteria with Multiple Chromosomes: Interplay between genome architecture and cell physilogy) Recently, the first artificial cell was generated by chemically synthetizing the whole DNA of a Mycoplasma mycoides and transplanting it into a recipient cell. This study was the proof of principle that genomes can be designed in silico, chemically synthetized and introduced into a cell to replace the endogenous genome. This opens the possibility to generate microorganism à la carte. However, the basic requirements of bacterial genome architecture are not yet understood. Such knowledge is needed before being able to design novel life forms. In this vein, increasing evidence indicates that nucleoid spatiotemporal organization is crucial for bacterial physiology since these microorganism lack compartmentalized nucleus. Compounding the problem, at least 10% of known bacteria harbor multiple chromosomes. The evolutionary advantage of this trait remains obscure. Additionally, it is still unclear how bacterial gene order within the chromosome can influence cell physiology. The in silico analysis of an ever-increasing number of complete bacterial genome sequences permitted the detection of correlations between gene positioning and global growth control. However, very few studies were able to address this issue experimentally.We designed a project to tackle these issues. We used Vibrio cholerae as working organism since it the model of bacteria with multiple chromosomes (BMC) and it has been extensively studied using genetic and molecular biology tools. Importantly, Vibrio cholerae is a globally important pathogen. We developed novel recombineering tools that allowed us to precisely relocate a locus harboring half of the ribosomal protein genes to different regions within the Vibrio cholerae genome without widely altering its structure. We coined the term “positional genetics” to describe this approach as it does not assess gene-function relationships but rather how gene location influences bacterial physiology. We generated a set of isogenic mutants that displayed different phenotypes. First, increasing distance between this locus and the origin of replication resulted in slower growth rates. Second, relocation of this major cluster of ribosomal protein genes far away from its original location impaired the ability of these Vibrio cholerae mutants to infect the model organism Drosophila melanogaster. We showed that replication-linked RP gene dosage reduction is the main mechanism behind these phenotypes, imposing strong constraints on their genomic location. We establish that the genomic position of RP genes is linked to bacterial growth rate, suggesting that this is a common rule for genes involved in the expression of genetic information in bacteria. We were able to rationally tune bacterial growth rate by the relocation of RP genes. Simultaneously, we provide insight into the evolution of bacteria with multiple chromosomes. Application of similar strategies using other bacterial models and targeting different genes will provide insights into the rules of genome organization. In this vein, understanding the genomic factors affecting GR would permit to reprogram bacterial growth, help to predict the behavior of more complex biological systems, and develop better theoretical models, thus promising a deep impact in genome design, bioengineering and biotechnology.
