RT Journal
A1 Suzuki, Yo
A1 Assad-Garcia, Nacyra
A1 Kostylev, Maxim
A1 Noskov, Vladimir N.
A1 Wise, Kim S.
A1 Karas, Bogumil J.
A1 Stam, Jason
A1 Montague, Michael G.
A1 Hanly, Timothy J.
A1 Enriquez, Nico J.
A1 Ramon, Adi
A1 Goldgof, Gregory M.
A1 Richter, R. Alexander
A1 Vashee, Sanjay
A1 Chuang, Ray-Yuan
A1 Winzeler, Elizabeth A.
A1 Hutchison, Clyde A.
A1 Gibson, Daniel G.
A1 Smith, Hamilton O.
A1 Glass, John I.
A1 Venter, J. Craig
T1 Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling
JF Genome Research 
JO Genome Research 
YR 2015 
FD March 01 
VO 25 
IS 3 
SP 435 
OP 444 
DO 10.1101/gr.182477.114 
UL http://genome.cshlp.org/content/25/3/435.abstract 
AB The availability of genetically tractable organisms with simple genomes is critical for the rapid, systems-level understanding of basic biological processes. Mycoplasma bacteria, with the smallest known genomes among free-living cellular organisms, are ideal models for this purpose, but the natural versions of these cells have genome complexities still too great to offer a comprehensive view of a fundamental life form. Here we describe an efficient method for reducing genomes from these organisms by identifying individually deletable regions using transposon mutagenesis and progressively clustering deleted genomic segments using meiotic recombination between the bacterial genomes harbored in yeast. Mycoplasmal genomes subjected to this process and transplanted into recipient cells yielded two mycoplasma strains. The first simultaneously lacked eight singly deletable regions of the genome, representing a total of 91 genes and ∼10% of the original genome. The second strain lacked seven of the eight regions, representing 84 genes. Growth assay data revealed an absence of genetic interactions among the 91 genes under tested conditions. Despite predicted effects of the deletions on sugar metabolism and the proteome, growth rates were unaffected by the gene deletions in the seven-deletion strain. These results support the feasibility of using single-gene disruption data to design and construct viable genomes lacking multiple genes, paving the way toward genome minimization. The progressive clustering method is expected to be effective for the reorganization of any mega-sized DNA molecules cloned in yeast, facilitating the construction of designer genomes in microbes as well as genomic fragments for genetic engineering of higher eukaryotes.