A universal mariner transposon system for forward genetic studies in the genus Clostridium.

DNA transposons represent an essential tool in the armoury of the molecular microbiologist. We previously developed a catP-based mini transposon system for Clostridium difficile in which the expression of the transposase gene was dependent on a sigma factor unique to C. difficile, TcdR. Here we have...

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Main Authors: Ying Zhang, Alexander Grosse-Honebrink, Nigel P Minton
Format: Article
Language:English
Published: Public Library of Science (PLoS) 2015-01-01
Series:PLoS ONE
Online Access:http://europepmc.org/articles/PMC4383383?pdf=render
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spelling doaj-644db27e36414e32a8c1697e88904dc12020-11-24T22:16:54ZengPublic Library of Science (PLoS)PLoS ONE1932-62032015-01-01104e012241110.1371/journal.pone.0122411A universal mariner transposon system for forward genetic studies in the genus Clostridium.Ying ZhangAlexander Grosse-HonebrinkNigel P MintonDNA transposons represent an essential tool in the armoury of the molecular microbiologist. We previously developed a catP-based mini transposon system for Clostridium difficile in which the expression of the transposase gene was dependent on a sigma factor unique to C. difficile, TcdR. Here we have shown that the host range of the transposon is easily extended through the rapid chromosomal insertion of the tcdR gene at the pyrE locus of the intended clostridial target using Allele-Coupled Exchange (ACE). To increase the effectiveness of the system, a novel replicon conditional for plasmid maintenance was developed, which no longer supports the effective retention of the transposon delivery vehicle in the presence of the inducer isopropyl β-D-1-thiogalactopyranoside (IPTG). As a consequence, those thiamphenicol resistant colonies that arise in clostridial recipients, following plating on agar medium supplemented with IPTG, are almost exclusively due to insertion of the mini transposon into the genome. The system has been exemplified in both Clostridium acetobutylicum and Clostridium sporogenes, where transposon insertion has been shown to be entirely random. Moreover, appropriate screening of both libraries resulted in the isolation of auxotrophic mutants as well as cells deficient in spore formation/germination. This strategy is capable of being implemented in any Clostridium species.http://europepmc.org/articles/PMC4383383?pdf=render
collection DOAJ
language English
format Article
sources DOAJ
author Ying Zhang
Alexander Grosse-Honebrink
Nigel P Minton
spellingShingle Ying Zhang
Alexander Grosse-Honebrink
Nigel P Minton
A universal mariner transposon system for forward genetic studies in the genus Clostridium.
PLoS ONE
author_facet Ying Zhang
Alexander Grosse-Honebrink
Nigel P Minton
author_sort Ying Zhang
title A universal mariner transposon system for forward genetic studies in the genus Clostridium.
title_short A universal mariner transposon system for forward genetic studies in the genus Clostridium.
title_full A universal mariner transposon system for forward genetic studies in the genus Clostridium.
title_fullStr A universal mariner transposon system for forward genetic studies in the genus Clostridium.
title_full_unstemmed A universal mariner transposon system for forward genetic studies in the genus Clostridium.
title_sort universal mariner transposon system for forward genetic studies in the genus clostridium.
publisher Public Library of Science (PLoS)
series PLoS ONE
issn 1932-6203
publishDate 2015-01-01
description DNA transposons represent an essential tool in the armoury of the molecular microbiologist. We previously developed a catP-based mini transposon system for Clostridium difficile in which the expression of the transposase gene was dependent on a sigma factor unique to C. difficile, TcdR. Here we have shown that the host range of the transposon is easily extended through the rapid chromosomal insertion of the tcdR gene at the pyrE locus of the intended clostridial target using Allele-Coupled Exchange (ACE). To increase the effectiveness of the system, a novel replicon conditional for plasmid maintenance was developed, which no longer supports the effective retention of the transposon delivery vehicle in the presence of the inducer isopropyl β-D-1-thiogalactopyranoside (IPTG). As a consequence, those thiamphenicol resistant colonies that arise in clostridial recipients, following plating on agar medium supplemented with IPTG, are almost exclusively due to insertion of the mini transposon into the genome. The system has been exemplified in both Clostridium acetobutylicum and Clostridium sporogenes, where transposon insertion has been shown to be entirely random. Moreover, appropriate screening of both libraries resulted in the isolation of auxotrophic mutants as well as cells deficient in spore formation/germination. This strategy is capable of being implemented in any Clostridium species.
url http://europepmc.org/articles/PMC4383383?pdf=render
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