Direct Genome Transfer from Acholeplasma laidlawii to Yeast

Cloning bacterial
genomes in yeast or other hosts is a crucial
step in workflows for creating highly engineered strains. The genome
of Acholeplasma laidlawii PG-8A can
be cloned in yeast if a toxic gene is removed, but this deletion has
not been tested in live bacteria. Here, we demonstrate cloning the
1.46 Mb genome of A. laidlawii strain
DN-E by integrating a yeast vector into the toxic gene and transferring
the genome directly to yeast by using cell fusion. Genomic integration
was possible only when A. laidlawii was complemented with a functional recA gene. Following
cell fusion, 13 out of 20 screened yeast colonies contained the A. laidlawii genome, as determined by multiplex PCR.
This is the first demonstration of cloning entire A.
laidlawii genomes by simply mixing donor and recipient
cells in fusion buffer, bringing us one step closer to creating fully
synthetic Acholeplasma strains.