Inferring the Minimal Genome of
            <i>Mesoplasma florum</i>
            by Comparative Genomics and Transposon Mutagenesis

Baby, Vincent; Lachance, Jean-Christophe; Gagnon, Jules; Lucier, Jean-François; Matteau, Dominick; Knight, Tom; Rodrigue, Sébastien

doi:10.1128/msystems.00198-17

Cited by 20 publications

(31 citation statements)

References 61 publications

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“…Going forward, it is easy to suggest that Mesoplasma florum , E. coli , Saccharomyces cerevisiae or other well-studied or model minimal organisms may serve as useful next genomes to consider formally completing (15, 44–46). However, extending the approaches developed here to such organisms will be challenging.…”

Section: Discussionmentioning

confidence: 99%

Definitive demonstration by synthesis of genome annotation completeness

Jaschke

Dotson

Hung

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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We develop a method for completing the genetics of natural living systems by which the absence of expected future discoveries can be established. We demonstrate the method using bacteriophage øX174, the first DNA genome to be sequenced. Like many well-studied natural organisms, closely related genome sequences are available—23 Bullavirinae genomes related to øX174. Using bioinformatic tools, we first identified 315 potential open reading frames (ORFs) within the genome, including the 11 established essential genes and 82 highly conserved ORFs that have no known gene products or assigned functions. Using genome-scale design and synthesis, we made a mutant genome in which all 11 essential genes are simultaneously disrupted, leaving intact only the 82 conserved but cryptic ORFs. The resulting genome is not viable. Cell-free gene expression followed by mass spectrometry revealed only a single peptide expressed from both the cryptic ORF and wild-type genomes, suggesting a potential new gene. A second synthetic genome in which 71 conserved cryptic ORFs were simultaneously disrupted is viable but with ∼50% reduced fitness relative to the wild type. However, rather than finding any new genes, repeated evolutionary adaptation revealed a single point mutation that modulates expression of gene H, a known essential gene, and fully suppresses the fitness defect. Taken together, we conclude that the annotation of currently functional ORFs for the øX174 genome is formally complete. More broadly, we show that sequencing and bioinformatics followed by synthesis-enabled reverse genomics, proteomics, and evolutionary adaptation can definitely establish the sufficiency and completeness of natural genome annotations.

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Section: Discussionmentioning

confidence: 99%

Definitive demonstration by synthesis of genome annotation completeness

Jaschke

Dotson

Hung

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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“…We then estimated the intracellular levels of M. florum nucleic acid and protein species using our macromolecular biomass quantification data, starting with the DNA fraction. In M. florum L1, the genome is organized as a single and circular chromosome of 793,224 bp (Baby et al, 2013(Baby et al, , 2018. Based on its sequence, this chromosome has a predicted molecular weight of 489,954 kDa.…”

Section: Estimation Of Intracellular Levels Of Protein and Nucleic Acmentioning

confidence: 99%

“…First described in 1984 as Acholeplasma florum (McCoy et al , 1984), the near‐minimal bacterium Mesoplasma florum constitutes another member of the Mollicutes class particularly well suited for synthetic genomics and systems biology studies. While closely related to M. mycoides , M. florum however has a smaller genome, shows faster growth rates, and has no pathogenic potential (Sirand‐Pugnet et al , 2007; Gibson et al , 2010; Matteau et al , 2015; Baby et al , 2018). The genome of the L1 type strain, for example, comprises only 793 kb and 720 predicted genes (GenBank: AE017263.1), while the genome of the M. mycoides capri LC GM12 accounts for 1.09 Mb and 879 genes (GenBank: CP001621.1).…”

Section: Introductionmentioning

confidence: 99%

Integrative characterization of the near‐minimal bacterium Mesoplasma florum

Matteau

Lachance

Grenier

et al. 2020

Molecular Systems Biology

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The near‐minimal bacterium Mesoplasma florum is an interesting model for synthetic genomics and systems biology due to its small genome (~ 800 kb), fast growth rate, and lack of pathogenic potential. However, fundamental aspects of its biology remain largely unexplored. Here, we report a broad yet remarkably detailed characterization of M. florum by combining a wide variety of experimental approaches. We investigated several physical and physiological parameters of this bacterium, including cell size, growth kinetics, and biomass composition of the cell. We also performed the first genome‐wide analysis of its transcriptome and proteome, notably revealing a conserved promoter motif, the organization of transcription units, and the transcription and protein expression levels of all protein‐coding sequences. We converted gene transcription and expression levels into absolute molecular abundances using biomass quantification results, generating an unprecedented view of the M. florum cellular composition and functions. These characterization efforts provide a strong experimental foundation for the development of a genome‐scale model for M. florum and will guide future genome engineering endeavors in this simple organism.

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“…To the best of our knowledge, studies regarding essentiality in Mycoplasmas have only been published so far for M. genitalium , 8 M. pneumoniae , 6 Mycoplasma pulmonis , 47 Mycoplasma bovis 10 or Mesoplasma florum. 48 However, while for M. pneumoniae almost 350,000 unique transposon insertion mutants have been tracked, the other studies analysed a substantially lower number of clones. For instance, the M. bovis study only obtained 319 mutants, representing one insertion every 3,145 bp of the total genome, 10 and the study in M. genitalium analysed around 3,300 mutants, showing an insertional coverage of one disruption over every 175 bp 8 .…”

Section: Resultsmentioning

confidence: 99%

SynMyco transposon: engineering transposon vectors for efficient transformation of minimal genomes

et al. 2019

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Mycoplasmas are important model organisms for Systems and Synthetic Biology, and are pathogenic to a wide variety of species. Despite their relevance, many of the tools established for genome editing in other microorganisms are not available for Mycoplasmas. The Tn4001 transposon is the reference tool to work with these bacteria, but the transformation efficiencies (TEs) reported for the different species vary substantially. Here, we explore the mechanisms underlying these differences in four Mycoplasma species, Mycoplasma agalactiae, Mycoplasma feriruminatoris, Mycoplasma gallisepticum and Mycoplasma pneumoniae, selected for being representative members of each cluster of the Mycoplasma genus. We found that regulatory regions (RRs) driving the expression of the transposase and the antibiotic resistance marker have a major impact on the TEs. We then designed a synthetic RR termed SynMyco RR to control the expression of the key transposon vector elements. Using this synthetic RR, we were able to increase the TE for M. gallisepticum, M. feriruminatoris and M. agalactiae by 30-, 980- and 1036-fold, respectively. Finally, to illustrate the potential of this new transposon, we performed the first essentiality study in M. agalactiae, basing our study on more than 199,000 genome insertions.

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

Cited by 20 publications

References 61 publications

Definitive demonstration by synthesis of genome annotation completeness

Definitive demonstration by synthesis of genome annotation completeness

Integrative characterization of the near‐minimal bacterium Mesoplasma florum

SynMyco transposon: engineering transposon vectors for efficient transformation of minimal genomes

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