RIDEAU, Fabien; LE ROY, Chloé; DESCAMPS, Elodie; RENAUDIN, Hélène; LARTIGUE-PRAT, Carole; BEBEAR, Cécile

doi:10.1021/acssynbio.6b00379

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RIDEAU, Fabien
USC-EA3671 Mycoplasmal and Chlamydial Infections in Humans

LE ROY, Chloé
USC-EA3671 Mycoplasmal and Chlamydial Infections in Humans

DESCAMPS, Elodie
USC-EA3671 Mycoplasmal and Chlamydial Infections in Humans

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ACS Synthetic Biology. 2017, vol. 6, n° 5, p. 891-901

American Chemical Society

Résumé en anglais

Mycoplasma hominis is a minimal human pathogen that is responsible for genital and neonatal infections. Despite many attempts, there is no efficient genetic tool to manipulate this bacterium, limiting most investigations of its pathogenicity and its uncommon energy metabolism that relies on arginine. The recent cloning and subsequent engineering of other mycoplasma genomes in yeast opens new possibilities for studies of the genomes of genetically intractable organisms. Here, we report the successful one-step cloning of the M. hominis PG21 genome in yeast using the transformation-associated recombination (TAR) cloning method. At low passages, the M. hominis genome cloned into yeast displayed a conserved size. However, after ~60 generations in selective media, this stability was affected, and large degradation events were detected, raising questions regarding the stability of large heterologous DNA molecules cloned in yeast and the need to minimize host propagation. Taking these results into account, we selected early passage yeast clones and successfully modified the M. hominis PG21 genome using the CRISPR/Cas9 editing tool, available in Saccharomyces cerevisiae. Complete M. hominis PG21 genomes lacking the adhesion-related vaa gene were efficiently obtained.< Réduire

Mots clés

bactérie pathogène

mollicute

santé humaine

Mots clés en anglais

human health

pathogenic bacteria

transformation-associated recombination (TAR) cloning

Mycoplasma hominis

CRISPR/Cas9

genome stability

Vaa

yeast centromeric plasmid