Cargando…

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

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Suzuki, Yo, Assad-Garcia, Nacyra, Kostylev, Maxim, Noskov, Vladimir N., Wise, Kim S., Karas, Bogumil J., Stam, Jason, Montague, Michael G., Hanly, Timothy J., Enriquez, Nico J., Ramon, Adi, Goldgof, Gregory M., Richter, R. Alexander, Vashee, Sanjay, Chuang, Ray-Yuan, Winzeler, Elizabeth A., Hutchison, Clyde A., Gibson, Daniel G., Smith, Hamilton O., Glass, John I., Venter, J. Craig
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory Press 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4352883/
https://www.ncbi.nlm.nih.gov/pubmed/25654978
http://dx.doi.org/10.1101/gr.182477.114
_version_ 1782360519705886720
author Suzuki, Yo
Assad-Garcia, Nacyra
Kostylev, Maxim
Noskov, Vladimir N.
Wise, Kim S.
Karas, Bogumil J.
Stam, Jason
Montague, Michael G.
Hanly, Timothy J.
Enriquez, Nico J.
Ramon, Adi
Goldgof, Gregory M.
Richter, R. Alexander
Vashee, Sanjay
Chuang, Ray-Yuan
Winzeler, Elizabeth A.
Hutchison, Clyde A.
Gibson, Daniel G.
Smith, Hamilton O.
Glass, John I.
Venter, J. Craig
author_facet Suzuki, Yo
Assad-Garcia, Nacyra
Kostylev, Maxim
Noskov, Vladimir N.
Wise, Kim S.
Karas, Bogumil J.
Stam, Jason
Montague, Michael G.
Hanly, Timothy J.
Enriquez, Nico J.
Ramon, Adi
Goldgof, Gregory M.
Richter, R. Alexander
Vashee, Sanjay
Chuang, Ray-Yuan
Winzeler, Elizabeth A.
Hutchison, Clyde A.
Gibson, Daniel G.
Smith, Hamilton O.
Glass, John I.
Venter, J. Craig
author_sort Suzuki, Yo
collection PubMed
description 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.
format Online
Article
Text
id pubmed-4352883
institution National Center for Biotechnology Information
language English
publishDate 2015
publisher Cold Spring Harbor Laboratory Press
record_format MEDLINE/PubMed
spelling pubmed-43528832015-09-01 Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling Suzuki, Yo Assad-Garcia, Nacyra Kostylev, Maxim Noskov, Vladimir N. Wise, Kim S. Karas, Bogumil J. Stam, Jason Montague, Michael G. Hanly, Timothy J. Enriquez, Nico J. Ramon, Adi Goldgof, Gregory M. Richter, R. Alexander Vashee, Sanjay Chuang, Ray-Yuan Winzeler, Elizabeth A. Hutchison, Clyde A. Gibson, Daniel G. Smith, Hamilton O. Glass, John I. Venter, J. Craig Genome Res Method 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. Cold Spring Harbor Laboratory Press 2015-03 /pmc/articles/PMC4352883/ /pubmed/25654978 http://dx.doi.org/10.1101/gr.182477.114 Text en © 2015 Suzuki et al.; Published by Cold Spring Harbor Laboratory Press http://creativecommons.org/licenses/by-nc/4.0/ This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
spellingShingle Method
Suzuki, Yo
Assad-Garcia, Nacyra
Kostylev, Maxim
Noskov, Vladimir N.
Wise, Kim S.
Karas, Bogumil J.
Stam, Jason
Montague, Michael G.
Hanly, Timothy J.
Enriquez, Nico J.
Ramon, Adi
Goldgof, Gregory M.
Richter, R. Alexander
Vashee, Sanjay
Chuang, Ray-Yuan
Winzeler, Elizabeth A.
Hutchison, Clyde A.
Gibson, Daniel G.
Smith, Hamilton O.
Glass, John I.
Venter, J. Craig
Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling
title Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling
title_full Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling
title_fullStr Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling
title_full_unstemmed Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling
title_short Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling
title_sort bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling
topic Method
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4352883/
https://www.ncbi.nlm.nih.gov/pubmed/25654978
http://dx.doi.org/10.1101/gr.182477.114
work_keys_str_mv AT suzukiyo bacterialgenomereductionusingtheprogressiveclusteringofdeletionsviayeastsexualcycling
AT assadgarcianacyra bacterialgenomereductionusingtheprogressiveclusteringofdeletionsviayeastsexualcycling
AT kostylevmaxim bacterialgenomereductionusingtheprogressiveclusteringofdeletionsviayeastsexualcycling
AT noskovvladimirn bacterialgenomereductionusingtheprogressiveclusteringofdeletionsviayeastsexualcycling
AT wisekims bacterialgenomereductionusingtheprogressiveclusteringofdeletionsviayeastsexualcycling
AT karasbogumilj bacterialgenomereductionusingtheprogressiveclusteringofdeletionsviayeastsexualcycling
AT stamjason bacterialgenomereductionusingtheprogressiveclusteringofdeletionsviayeastsexualcycling
AT montaguemichaelg bacterialgenomereductionusingtheprogressiveclusteringofdeletionsviayeastsexualcycling
AT hanlytimothyj bacterialgenomereductionusingtheprogressiveclusteringofdeletionsviayeastsexualcycling
AT enriqueznicoj bacterialgenomereductionusingtheprogressiveclusteringofdeletionsviayeastsexualcycling
AT ramonadi bacterialgenomereductionusingtheprogressiveclusteringofdeletionsviayeastsexualcycling
AT goldgofgregorym bacterialgenomereductionusingtheprogressiveclusteringofdeletionsviayeastsexualcycling
AT richterralexander bacterialgenomereductionusingtheprogressiveclusteringofdeletionsviayeastsexualcycling
AT vasheesanjay bacterialgenomereductionusingtheprogressiveclusteringofdeletionsviayeastsexualcycling
AT chuangrayyuan bacterialgenomereductionusingtheprogressiveclusteringofdeletionsviayeastsexualcycling
AT winzelerelizabetha bacterialgenomereductionusingtheprogressiveclusteringofdeletionsviayeastsexualcycling
AT hutchisonclydea bacterialgenomereductionusingtheprogressiveclusteringofdeletionsviayeastsexualcycling
AT gibsondanielg bacterialgenomereductionusingtheprogressiveclusteringofdeletionsviayeastsexualcycling
AT smithhamiltono bacterialgenomereductionusingtheprogressiveclusteringofdeletionsviayeastsexualcycling
AT glassjohni bacterialgenomereductionusingtheprogressiveclusteringofdeletionsviayeastsexualcycling
AT venterjcraig bacterialgenomereductionusingtheprogressiveclusteringofdeletionsviayeastsexualcycling