title: Development oforiC-Based Plasmids for Mesoplasma florum.
abstract: The near-minimal bacteriumMesoplasma florumconstitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. However, the lack of genetic engineering tools for this microorganism has limited our capacity to understand its basic biology and modify its genome. To address this issue, we have evaluated the susceptibility ofM. florumto common antibiotics and developed the first generation of artificial plasmids able to replicate in this bacterium. Selected regions of the predictedM. florumchromosomal origin of replication (oriC) were used to create different plasmid versions that were tested for their transformation frequency and stability. Using polyethylene glycol-mediated transformation, we observed that plasmids harboring bothrpmH-dnaAanddnaA-dnaNintergenic regions, interspaced or not with a copy of thednaAgene, resulted in a frequency of ∼4.1 × 10-6transformants per viable cell and were stably maintained throughout multiple generations. In contrast, plasmids containing only oneM. florumoriCintergenic region or the heterologousoriCregion ofMycoplasma capricolum,Mycoplasma mycoides, orSpiroplasma citrifailed to produce any detectable transformants. We also developed alternative transformation procedures based on electroporation and conjugation fromEscherichia coli, reaching frequencies up to 7.87 × 10-6and 8.44 × 10-7transformants per viable cell, respectively. Finally, we demonstrated the functionality of antibiotic resistance genes active against tetracycline, puromycin, and spectinomycin/streptomycin inM. florumTaken together, these valuable genetic tools will facilitate efforts toward building anM. florum-based near-minimal cellular chassis for synthetic biology.IMPORTANCEMesoplasma florumconstitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology.M. florumis closely related to the mycoides cluster of mycoplasmas, which has become a model for whole-genome cloning, genome transplantation, and genome minimization. However,M. florumshows higher growth rates than otherMollicutes, has no known pathogenic potential, and possesses a significantly smaller genome that positions this species among some of the simplest free-living organisms. So far, the lack of genetic engineering tools has limited our capacity to understand the basic biology ofM. florumin order to modify its genome. To address this issue, we have evaluated the susceptibility ofM. florumto common antibiotics and developed the first artificial plasmids and transformation methods for this bacterium. This represents a strong basis for ongoing genome engineering efforts using this near-minimal microorganism.
authors.affiliation: Département de Biologie, Université de Sherbrooke, Sherbrooke, Québec, Canada.
authors.forename: Dominick
authors.initials: D
authors.lastname: Matteau
authors.affiliation: Département de Biologie, Université de Sherbrooke, Sherbrooke, Québec, Canada.
authors.forename: Marie-Eve
authors.initials: ME
authors.lastname: Pepin
authors.affiliation: Département de Biologie, Université de Sherbrooke, Sherbrooke, Québec, Canada.
authors.forename: Vincent
authors.initials: V
authors.lastname: Baby
authors.affiliation: Département de Biologie, Université de Sherbrooke, Sherbrooke, Québec, Canada.
authors.forename: Samuel
authors.initials: S
authors.lastname: Gauthier
authors.affiliation: Département de Biologie, Université de Sherbrooke, Sherbrooke, Québec, Canada.
authors.forename: Mélissa
authors.initials: M
authors.lastname: Arango Giraldo
authors.affiliation: Ginkgo Bioworks, Boston, Massachusetts, USA.
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Knight
authors.affiliation: Département de Biologie, Université de Sherbrooke, Sherbrooke, Québec, Canada sebastien.rodrigue@usherbrooke.ca.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Rodrigue
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics; D021901:DNA, Intergenic; D004268:DNA-Binding Proteins / Q000235:genetics; D024901:Drug Resistance, Multiple, Bacterial; D045622:Entomoplasmataceae / Q000187:drug effects / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D009174:Mycoplasma / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D011995:Recombination, Genetic; D018741:Replication Origin; D058615:Synthetic Biology; D014169:Transformation, Bacterial
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D021901:DNA, Intergenic; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria
keywords: Mesoplasma; antibiotic markers; chromosomal origin of replication; plasmids; synthetic biology; transformation methods