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Cloning, Stability, and Modification of Mycoplasma hominis Genome in Yeast
Fabien Rideau1,2, Chloé Le Roy1,2, Elodie C T Descamps1,2
1Univ. Bordeaux , USC-EA3671 Mycoplasmal and Chlamydial Infections in Humans, F-33000 Bordeaux, France.
Abstract:
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.
Insights
Researchers successfully cloned the Mycoplasma hominis PG21 genome in yeast, enabling genetic manipulation. They then used CRISPR/Cas9 to delete the vaa gene, advancing studies on this human pathogen.
Area of Science:
- Microbiology
- Genetics
- Molecular Biology
Background:
- Mycoplasma hominis is a human pathogen causing genital and neonatal infections.
- Lack of efficient genetic tools hinders research into M. hominis pathogenicity and metabolism.
- Cloning and engineering of other mycoplasma genomes in yeast offers a potential solution for genetically intractable organisms.
Purpose of the Study:
- To establish a method for genetically manipulating the Mycoplasma hominis PG21 genome.
- To investigate the stability of the cloned M. hominis genome in yeast.
- To demonstrate the feasibility of genome editing in M. hominis using CRISPR/Cas9 in yeast.
Main Methods:
- Transformation-associated recombination (TAR) cloning was used to clone the M. hominis PG21 genome in Saccharomyces cerevisiae.
- The stability of the cloned genome was assessed over successive generations in selective media.
- CRISPR/Cas9 gene editing was employed in yeast to modify the cloned M. hominis genome.
Main Results:
- The M. hominis PG21 genome was successfully cloned in yeast, maintaining a conserved size at low passages.
- Large degradation events were observed after approximately 60 generations, indicating instability of the heterologous DNA.
- CRISPR/Cas9 editing allowed for the efficient deletion of the adhesion-related vaa gene from the M. hominis PG21 genome.
Conclusions:
- Yeast-based TAR cloning provides a viable, albeit time-limited, platform for manipulating the Mycoplasma hominis genome.
- Minimizing host propagation is crucial for maintaining the integrity of large heterologous DNA molecules in yeast.
- This study successfully generated M. hominis PG21 genomes lacking the vaa gene, paving the way for future functional studies.
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