Development of -Based Plasmids for Mesoplasma florum.

The near-minimal bacterium constitutes 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 of to common antibiotics and developed the first generation of artificial plasmids able to replicate in this bacterium. Selected regions of the predicted chromosomal origin of replication () 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 both and intergenic regions, interspaced or not with a copy of the gene, resulted in a frequency of ∼4.1 × 10 transformants per viable cell and were stably maintained throughout multiple generations. In contrast, plasmids containing only one intergenic region or the heterologous region of , , or failed to produce any detectable transformants. We also developed alternative transformation procedures based on electroporation and conjugation from , reaching frequencies up to 7.87 × 10 and 8.44 × 10 transformants per viable cell, respectively. Finally, we demonstrated the functionality of antibiotic resistance genes active against tetracycline, puromycin, and spectinomycin/streptomycin in Taken together, these valuable genetic tools will facilitate efforts toward building an -based near-minimal cellular chassis for synthetic biology. constitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. is closely related to the mycoides cluster of mycoplasmas, which has become a model for whole-genome cloning, genome transplantation, and genome minimization. However, shows higher growth rates than other , 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 of in order to modify its genome. To address this issue, we have evaluated the susceptibility of to 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.