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Title: Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling

Journal Article · · Genome Research
 [1];  [2];  [1];  [2];  [3];  [1];  [1];  [2];  [1];  [1];  [1];  [4];  [1];  [2];  [2];  [5];  [1];  [1];  [1];  [6] more »;  [6] « less
  1. J. Craig Venter Inst., La Jolla, CA (United States). Synthetic Biology Group
  2. J. Craig Venter Inst., Rockville, MD (United States). Synthetic Biology Group
  3. J. Craig Venter Inst., La Jolla, CA (United States). Synthetic Biology Group; Univ. of Missouri, Columbia, MO (United States). Dept. of Molecular Microbiology and Immunology
  4. J. Craig Venter Inst., La Jolla, CA (United States). Synthetic Biology Group; Univ. of California, San Diego, CA (United States). School of Medicine
  5. Univ. of California, La Jolla, CA (United States). School of Medicine
  6. J. Craig Venter Inst., La Jolla, CA (United States). Synthetic Biology Group; J. Craig Venter Inst., Rockville, MD (United States). Synthetic Biology Group
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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 in this paper 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.

Research Organization:
J. Craig Venter Inst., La Jolla, CA (United States)
Sponsoring Organization:
USDOE; US Defense Advanced Research Projects Agency (DARPA)
Grant/Contract Number:
FC02-02ER63453; EE0006109; N66001-12-C-4039
OSTI ID:
1344872
Journal Information:
Genome Research, Vol. 25, Issue 3; ISSN 1088-9051
Publisher:
Cold Spring Harbor Laboratory PressCopyright Statement
Country of Publication:
United States
Language:
English
Citation Metrics:
Cited by: 18 works
Citation information provided by
Web of Science

References (45)

Disruption of the S41 Peptidase Gene in Mycoplasma mycoides capri Impacts Proteome Profile, H2O2 Production, and Sensitivity to Heat Shock journal December 2012
Basic Local Alignment Search Tool journal October 1990
An activator/repressor dual system allows tight tetracycline-regulated gene expression in budding yeast [published erratum appears in Nucleic Acids Res 1998 Apr 1;26(7):following 1855] journal February 1998
Cloning whole bacterial genomes in yeast journal March 2010
Designer deletion strains derived fromSaccharomyces cerevisiae S288C: A useful set of strains and plasmids for PCR-mediated gene disruption and other applications journal January 1998
BLAST+: architecture and applications journal January 2009
Recovery and potential utility of YACs as circular YACs/BACs journal September 2000
Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems journal March 2013
ICEA of Mycoplasma agalactiae : a new family of self-transmissible integrative elements that confers conjugative properties to the recipient strain: Mycoplasma  ICE on the move journal August 2013
Construction and Use of Derivatives of Transposon Tn4001 That Function in Mycoplasma pulmonis andMycoplasma arthritidis journal August 2000
Methionine sulfoxide reductases in prokaryotes journal January 2005
Functional profiling of the Saccharomyces cerevisiae genome journal July 2002
Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome journal February 2008
Enzymatic assembly of DNA molecules up to several hundred kilobases journal April 2009
Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome journal May 2010
Essential genes of a minimal bacterium journal January 2006
Three new dominant drug resistance cassettes for gene disruption inSaccharomyces cerevisiae journal October 1999
Systematic artifacts in metagenomes from complex microbial communities journal July 2009
The Chemical Genomic Portrait of Yeast: Uncovering a Phenotype for All Genes journal April 2008
Global Transposon Mutagenesis and a Minimal Mycoplasma Genome journal December 1999
From The Cover: Combining two genomes in one cell: Stable cloning of the Synechocystis PCC6803 genome in the Bacillus subtilis 168 genome journal October 2005
Cloning the Acholeplasma laidlawii PG-8A Genome in Saccharomyces cerevisiae as a Yeast Centromeric Plasmid journal January 2012
Direct transfer of whole genomes from bacteria to yeast journal March 2013
Assembly of eukaryotic algal chromosomes in yeast journal January 2013
Transferring whole genomes from bacteria to yeast spheroplasts using entire bacterial cells to reduce DNA shearing journal March 2014
Plasmid transformation of Mycoplasma mycoides subspecies mycoides is promoted by high concentrations of polyethylene glycol journal September 1991
Simultaneous non-contiguous deletions using large synthetic DNA and site-specific recombinases journal June 2014
Host specificity of mollicutes oriC plasmids: functional analysis of replication origin journal November 2003
Creating Bacterial Strains from Genomes That Have Been Cloned and Engineered in Yeast journal August 2009
Tandem repeat coupled with endonuclease cleavage (TREC): a seamless modification tool for genome engineering in yeast journal March 2010
Assembly of Large, High G+C Bacterial DNA Fragments in Yeast journal May 2012
Shrimp Pm-fortilin inhibits the expression of early and late genes of white spot syndrome virus (WSSV) in an insect cell model journal April 2011
Structure and mitotic stability of minichromosomes originating in yeast cells transformed with tandem dimers of CEN11 plasmids journal June 1984
Saccharomyces cerevisiae Genome Shuffling through Recursive Population Mating Leads to Improved Tolerance to Spent Sulfite Liquor journal July 2011
Yeast colony size reflects YAC copy number journal May 1997
Emergent Properties of Reduced-Genome Escherichia coli journal May 2006
Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein journal November 2004
In vivo site-directed mutagenesis using oligonucleotides journal August 2001
PCR-mediated repeated chromosome splitting in Saccharomyces cerevisiae journal June 2005
Knocking out multigene redundancies via cycles of sexual assortment and fluorescence selection journal January 2011
Sequence analysis of a complete 1.66 Mb Prochlorococcus marinus MED4 genome cloned in yeast journal August 2012
Pathogenic mycoplasmas: cultivation and vertebrate pathogenicity of a new spiroplasma journal March 1977
New heterologous modules for classical or PCR-based gene disruptions inSaccharomyces cerevisiae journal December 1994
Distinctive Repertoire of Contingency Genes Conferring Mutation- Based Phase Variation and Combinatorial Expression of Surface Lipoproteins in Mycoplasma capricolum subsp. capricolum of the Mycoplasma mycoides Phylogenetic Cluster journal July 2006
High-Efficiency Transformation of Plasmid DNA into Yeast book January 2001

Cited By (7)

Tn-sequencing of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis mutant libraries reveals non-essential genes of porcine mycoplasmas differing in pathogenicity. text January 2019
Comparative chemical genomics reveal that the spiroindolone antimalarial KAE609 (Cipargamin) is a P-type ATPase inhibitor journal June 2016
Tn-sequencing of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis mutant libraries reveals non-essential genes of porcine mycoplasmas differing in pathogenicity journal July 2019
Design and synthesis of a minimal bacterial genome journal March 2016
Host-Pathogen Interactions of Mycoplasma mycoides in Caprine and Bovine Precision-Cut Lung Slices (PCLS) Models journal June 2019
Host-Pathogen Interactions of Mycoplasma mycoides in Caprine and Bovine Precision-Cut Lung Slices (PCLS) Models. text January 2019
Rewriting the blueprint of life by synthetic genomics and genome engineering journal June 2015

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59 BASIC BIOLOGICAL SCIENCES