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A Family of Single Copy repABC-Type Shuttle Vectors Stably Maintained in the Alpha-Proteobacterium Sinorhizobium meliloti

[Image: see text] A considerable share of bacterial species maintains segmented genomes. Plant symbiotic α-proteobacterial rhizobia contain up to six repABC-type replicons in addition to the primary chromosome. These low or unit-copy replicons, classified as secondary chromosomes, chromids, or megap...

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Autores principales: Döhlemann, Johannes, Wagner, Marcel, Happel, Carina, Carrillo, Martina, Sobetzko, Patrick, Erb, Tobias J., Thanbichler, Martin, Becker, Anke
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2017
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610768/
https://www.ncbi.nlm.nih.gov/pubmed/28264559
http://dx.doi.org/10.1021/acssynbio.6b00320
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author Döhlemann, Johannes
Wagner, Marcel
Happel, Carina
Carrillo, Martina
Sobetzko, Patrick
Erb, Tobias J.
Thanbichler, Martin
Becker, Anke
author_facet Döhlemann, Johannes
Wagner, Marcel
Happel, Carina
Carrillo, Martina
Sobetzko, Patrick
Erb, Tobias J.
Thanbichler, Martin
Becker, Anke
author_sort Döhlemann, Johannes
collection PubMed
description [Image: see text] A considerable share of bacterial species maintains segmented genomes. Plant symbiotic α-proteobacterial rhizobia contain up to six repABC-type replicons in addition to the primary chromosome. These low or unit-copy replicons, classified as secondary chromosomes, chromids, or megaplasmids, are exclusively found in α-proteobacteria. Replication and faithful partitioning of these replicons to the daughter cells is mediated by the repABC region. The importance of α-rhizobial symbiotic nitrogen fixation for sustainable agriculture and Agrobacterium-mediated plant transformation as a tool in plant sciences has increasingly moved biological engineering of these organisms into focus. Plasmids are ideal DNA-carrying vectors for these engineering efforts. On the basis of repABC regions collected from α-rhizobial secondary replicons, and origins of replication derived from traditional cloning vectors, we devised the versatile family of pABC shuttle vectors propagating in Sinorhizobium meliloti, related members of the Rhizobiales, and Escherichia coli. A modular plasmid library providing the elemental parts for pABC vector assembly was founded. The standardized design of these vectors involves five basic modules: (1) repABC cassette, (2) plasmid-derived origin of replication, (3) RK2/RP4 mobilization site (optional), (4) antibiotic resistance gene, and (5) multiple cloning site flanked by transcription terminators. In S. meliloti, pABC vectors showed high propagation stability and unit-copy number. We demonstrated stable coexistence of three pABC vectors in addition to the two indigenous megaplasmids in S. meliloti, suggesting combinability of multiple compatible pABC plasmids. We further devised an in vivo cloning strategy involving Cre/lox-mediated translocation of large DNA fragments to an autonomously replicating repABC-based vector, followed by conjugation-mediated transfer either to compatible rhizobia or E. coli.
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spelling pubmed-76107682021-05-13 A Family of Single Copy repABC-Type Shuttle Vectors Stably Maintained in the Alpha-Proteobacterium Sinorhizobium meliloti Döhlemann, Johannes Wagner, Marcel Happel, Carina Carrillo, Martina Sobetzko, Patrick Erb, Tobias J. Thanbichler, Martin Becker, Anke ACS Synth Biol [Image: see text] A considerable share of bacterial species maintains segmented genomes. Plant symbiotic α-proteobacterial rhizobia contain up to six repABC-type replicons in addition to the primary chromosome. These low or unit-copy replicons, classified as secondary chromosomes, chromids, or megaplasmids, are exclusively found in α-proteobacteria. Replication and faithful partitioning of these replicons to the daughter cells is mediated by the repABC region. The importance of α-rhizobial symbiotic nitrogen fixation for sustainable agriculture and Agrobacterium-mediated plant transformation as a tool in plant sciences has increasingly moved biological engineering of these organisms into focus. Plasmids are ideal DNA-carrying vectors for these engineering efforts. On the basis of repABC regions collected from α-rhizobial secondary replicons, and origins of replication derived from traditional cloning vectors, we devised the versatile family of pABC shuttle vectors propagating in Sinorhizobium meliloti, related members of the Rhizobiales, and Escherichia coli. A modular plasmid library providing the elemental parts for pABC vector assembly was founded. The standardized design of these vectors involves five basic modules: (1) repABC cassette, (2) plasmid-derived origin of replication, (3) RK2/RP4 mobilization site (optional), (4) antibiotic resistance gene, and (5) multiple cloning site flanked by transcription terminators. In S. meliloti, pABC vectors showed high propagation stability and unit-copy number. We demonstrated stable coexistence of three pABC vectors in addition to the two indigenous megaplasmids in S. meliloti, suggesting combinability of multiple compatible pABC plasmids. We further devised an in vivo cloning strategy involving Cre/lox-mediated translocation of large DNA fragments to an autonomously replicating repABC-based vector, followed by conjugation-mediated transfer either to compatible rhizobia or E. coli. American Chemical Society 2017-03-06 2017-06-16 /pmc/articles/PMC7610768/ /pubmed/28264559 http://dx.doi.org/10.1021/acssynbio.6b00320 Text en Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Döhlemann, Johannes
Wagner, Marcel
Happel, Carina
Carrillo, Martina
Sobetzko, Patrick
Erb, Tobias J.
Thanbichler, Martin
Becker, Anke
A Family of Single Copy repABC-Type Shuttle Vectors Stably Maintained in the Alpha-Proteobacterium Sinorhizobium meliloti
title A Family of Single Copy repABC-Type Shuttle Vectors Stably Maintained in the Alpha-Proteobacterium Sinorhizobium meliloti
title_full A Family of Single Copy repABC-Type Shuttle Vectors Stably Maintained in the Alpha-Proteobacterium Sinorhizobium meliloti
title_fullStr A Family of Single Copy repABC-Type Shuttle Vectors Stably Maintained in the Alpha-Proteobacterium Sinorhizobium meliloti
title_full_unstemmed A Family of Single Copy repABC-Type Shuttle Vectors Stably Maintained in the Alpha-Proteobacterium Sinorhizobium meliloti
title_short A Family of Single Copy repABC-Type Shuttle Vectors Stably Maintained in the Alpha-Proteobacterium Sinorhizobium meliloti
title_sort family of single copy repabc-type shuttle vectors stably maintained in the alpha-proteobacterium sinorhizobium meliloti
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610768/
https://www.ncbi.nlm.nih.gov/pubmed/28264559
http://dx.doi.org/10.1021/acssynbio.6b00320
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