Cloning and Transplantation of the Mesoplasma florum Genome

Abstract

Cloning and transplantation of bacterial genomes is a powerful method for the creation of engineered microorganisms. However, much remains to be understood about the molecular mechanisms and limitations of this approach. We report the whole-genome cloning of Mesoplasma florum in Saccharomyces cerevisiae, and use this model to investigate the impact of a bacterial chromosome in yeast cells. Our results indicate that the cloned M. florum genome is subjected to weak transcriptional activity, and causes no significant impact on yeast growth. We also report that the M. florum genome can be transplanted into Mycoplasma capricolum without any negative impact from the putative restriction enzyme encoding gene mfl307. Using whole-genome sequencing, we observed that a small number of mutations appeared in all M. florum transplants. Mutations also arose, albeit at a lower frequency, when the M. capricolum genome was transplanted into M. capricolum recipient cells. These observations suggest that genome transplantation is mutagenic, and that this phenomenon is magnified by the use of genome donor and recipient cell belonging to different species. No difference in efficiency was detected after three successive rounds of genome transplantation, suggesting that the observed mutations were not selected during the procedure. Taken together, our results provide a more accurate picture of the events taking place during bacterial genome cloning and transplantation.

Inferring the Minimal Genome of Mesoplasma florum by Comparative Genomics and Transposon Mutagenesis

“…Technical advances now also enable the exploration of the M. florum minimal genome. The development of oriC -based plasmids and antibiotic selection markers ( 27 ) constituted the basic steps that led to the whole-genome cloning of M. florum in yeast ( 29 ). This was followed by the establishment of a genome transplantation protocol for M. florum and by the investigation of the impact of phylogenetic distance on this procedure ( 28 ).…”

“…Furthermore, the complete genome of M. florum L1 has also been cloned in yeast and transplanted into a recipient Mycoplasma capricolum subsp. capricolum strain ( 28 , 29 ), which will enable sophisticated modifications and reengineering of the M. florum chromosome. This combination of low cell complexity, ease of manipulation, and the availability of genome engineering methods makes M. florum an interesting model for systems biology and synthetic genomics.…”

Designing minimal genomes using whole-cell models

“…Another approach, building novel organisms from the bottomup, is currently infeasible in the majority of bacteria due to technological and economic constraints 7 . Megabase sized genomes can be constructed within yeast 5,8 , but one of the most promising approaches, genome transplantation, has only been demonstrated in a subset of Mycoplasma species [9][10][11] and is mutagenic 10 .…”

La transplantation de génomes

“…Ces deux accomplissements phares de la génomique synthétique marquent le point culminant de plus de vingt ans de recherche. D'un point de vue technique, deux difficultés ont dû être surmontées : (1) [30]. De nouvelles techniques ont donc dû être développées pour extraire, manipuler, et transférer des génomes entiers dans un environnement favorable à leur expression.…”