Development of oriC -Based Plasmids for Mesoplasma florum

Abstract

The near-minimal bacterium Mesoplasma florum 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 M. florum to common antibiotics and developed the first generation of artificial plasmids able to replicate in this bacterium. Selected regions of the predicted M. florum chromosomal origin of replication (oriC) were used to create different plasmid versions that were tested for their transformation frequency and stability. Using polyethylene glycolmediated transformation, we observed that plasmids harboring both rpmH-dnaA and dnaA-dnaN intergenic regions, interspaced or not with a copy of the dnaA gene, resulted in a frequency of ϳ4.1 ϫ 10 Ϫ6 transformants per viable cell and were stably maintained throughout multiple generations. In contrast, plasmids containing only one M. florum oriC intergenic region or the heterologous oriC region of Mycoplasma capricolum, Mycoplasma mycoides, or Spiroplasma citri failed to produce any detectable transformants. We also developed alternative transformation procedures based on electroporation and conjugation from Escherichia coli, reaching frequencies up to 7.87 ϫ 10 Ϫ6 and 8.44 ϫ 10 Ϫ7 transformants per viable cell, respectively. Finally, we demonstrated the functionality of antibiotic resistance genes active against tetracycline, puromycin, and spectinomycin/streptomycin in M. florum. Taken together, these valuable genetic tools will facilitate efforts toward building an M. florum-based near-minimal cellular chassis for synthetic biology.IMPORTANCE Mesoplasma florum constitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. M. florum is closely related to the mycoides cluster of mycoplasmas, which has become a model for whole-genome cloning, genome transplantation, and genome minimization. However, M. florum shows higher growth rates than other Mollicutes, 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 M. florum in order to modify its genome. To address this issue, we have evaluated the susceptibility of M. florum 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.

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 ).…”

“… M. florum strains were grown at 34°C in ATCC 1161 medium ( 27 ). Genomic DNA (gDNA) extraction was performed with the Quick-gDNA MiniPrep kit (Zymo Research) for the preparation of Illumina libraries and with Puregene Yeast/Bact.…”

“…The transposon DNA was obtained from the digestion of the pTT01 plasmid with restriction enzyme PvuII and conferred tetracycline resistance. The transposomes were transformed via electroporation as described by Matteau et al ( 27 ) with a voltage of 2.5 kV. Insertion mutants were selected on ATCC 1161 solid medium supplemented with 15 µg/ml tetracycline.…”

“…The genomes of two M. florum strains, L1 and W37 ( 26 ), have been completely sequenced, revealing a single circular chromosome of ~800 kbp and positioning this species among the simplest free-living organisms. Basic genetic manipulation tools comprising antibiotic resistance genes, plasmids, and transformation methods have recently been developed for M. florum ( 27 ). Furthermore, the complete genome of M. florum L1 has also been cloned in yeast and transplanted into a recipient Mycoplasma capricolum subsp.…”

Integrative characterization of the near‐minimal bacterium Mesoplasma florum

“…As for most Mollicutes, M. florum also uses an alternative genetic code ( Mycoplasma/Spiroplasma code) that limits the exchange of genetic material from and to other microorganisms (Navas‐Castillo et al , 1992). Importantly, genetic manipulation tools have recently been developed specifically for this bacterium, including procedures for whole‐genome cloning in yeast and genome transplantation (Matteau et al , 2017; Baby et al , 2017). Gene conservation and essentiality analyses have also showed that 57 putatively essential M. florum genes have no homolog in the synthetic JCVI‐syn3.0 strain, suggesting that different minimal genome compositions and configurations probably exist, even within closely related species (Baby et al , 2018).…”