title: Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling.
abstract: 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.
authors: Synthetic Biology Group, J. Craig Venter Institute, La Jolla, California 92037, USA; ysuzuki@jcvi.org.
authors: Yo
authors: Y
authors: Suzuki
authors: Synthetic Biology Group, J. Craig Venter Institute, Rockville, Maryland 20850, USA;
authors: Nacyra
authors: N
authors: Assad-Garcia
authors: Synthetic Biology Group, J. Craig Venter Institute, La Jolla, California 92037, USA;
authors: Maxim
authors: M
authors: Kostylev
authors: Synthetic Biology Group, J. Craig Venter Institute, Rockville, Maryland 20850, USA;
authors: Vladimir N
authors: VN
authors: Noskov
authors: Synthetic Biology Group, J. Craig Venter Institute, La Jolla, California 92037, USA; Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, Missouri 65212, USA;
authors: Kim S
authors: KS
authors: Wise
authors: Synthetic Biology Group, J. Craig Venter Institute, La Jolla, California 92037, USA;
authors: Bogumil J
authors: BJ
authors: Karas
authors: Synthetic Biology Group, J. Craig Venter Institute, La Jolla, California 92037, USA;
authors: Jason
authors: J
authors: Stam
authors: Synthetic Biology Group, J. Craig Venter Institute, Rockville, Maryland 20850, USA;
authors: Michael G
authors: MG
authors: Montague
authors: Synthetic Biology Group, J. Craig Venter Institute, La Jolla, California 92037, USA;
authors: Timothy J
authors: TJ
authors: Hanly
authors: Synthetic Biology Group, J. Craig Venter Institute, La Jolla, California 92037, USA;
authors: Nico J
authors: NJ
authors: Enriquez
authors: Synthetic Biology Group, J. Craig Venter Institute, La Jolla, California 92037, USA;
authors: Adi
authors: A
authors: Ramon
authors: Synthetic Biology Group, J. Craig Venter Institute, La Jolla, California 92037, USA; University of California, San Diego, School of Medicine, La Jolla, California 92093, USA.
authors: Gregory M
authors: GM
authors: Goldgof
authors: Synthetic Biology Group, J. Craig Venter Institute, La Jolla, California 92037, USA;
authors: R Alexander
authors: RA
authors: Richter
authors: Synthetic Biology Group, J. Craig Venter Institute, Rockville, Maryland 20850, USA;
authors: Sanjay
authors: S
authors: Vashee
authors: Synthetic Biology Group, J. Craig Venter Institute, Rockville, Maryland 20850, USA;
authors: Ray-Yuan
authors: RY
authors: Chuang
authors: University of California, San Diego, School of Medicine, La Jolla, California 92093, USA.
authors: Elizabeth A
authors: EA
authors: Winzeler
authors: Synthetic Biology Group, J. Craig Venter Institute, La Jolla, California 92037, USA;
authors: Clyde A
authors: CA
authors: Hutchison
authors: Synthetic Biology Group, J. Craig Venter Institute, La Jolla, California 92037, USA;
authors: Daniel G
authors: DG
authors: Gibson
authors: Synthetic Biology Group, J. Craig Venter Institute, La Jolla, California 92037, USA;
authors: Hamilton O
authors: HO
authors: Smith
authors: Synthetic Biology Group, J. Craig Venter Institute, La Jolla, California 92037, USA; Synthetic Biology Group, J. Craig Venter Institute, Rockville, Maryland 20850, USA;
authors: John I
authors: JI
authors: Glass
authors: Synthetic Biology Group, J. Craig Venter Institute, La Jolla, California 92037, USA; Synthetic Biology Group, J. Craig Venter Institute, Rockville, Maryland 20850, USA;
authors: J Craig
authors: JC
authors: Venter
mesh_terms: D001419:Bacteria / Q000235:genetics*; D004251:DNA Transposable Elements; D022761:Gene Transfer, Horizontal; D016680:Genome, Bacterial; D005810:Multigene Family; D017384:Sequence Deletion; D015003:Yeasts / Q000235:genetics*
chemical_list: D004251:DNA Transposable Elements
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