The ClosTron: Mutagenesis in Clostridium refined and streamlined

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Abstract

The recent development of the ClosTron Group II intron directed mutagenesis tool for Clostridium has advanced genetics in this genus, and here we present several significant improvements. We have shown how marker re-cycling can be used to construct strains with multiple mutations, demonstrated using FLP/FRT in Clostridium acetobutylicum; tested the capacity of the system for the delivery of transgenes to the chromosome of Clostridium sporogenes, which proved feasible for 1.0 kbp transgenes in addition to a marker; and extended the host range of the system, constructing mutants in Clostridium beijerinckii and, for the first time, in a B1/NAP1/027 ‘epidemic’ strain of Clostridium difficile. Automated intron design bioinformatics are now available free-of-charge at our website http://clostron.com; the out-sourced construction of re-targeted intron plasmids has become cost-effective as well as rapid; and the combination of constitutive intron expression with direct selection for intron insertions has made mutant isolation trivial. These developments mean mutants can now be constructed with very little time and effort for the researcher. Those who prefer to construct plasmids in-house are no longer reliant on a commercial kit, as a mixture of two new plasmids provides unlimited template for intron re-targeting by Splicing by Overlap Extension (SOE) PCR. The new ClosTron plasmids also offer blue–white screening and other options for identification of recombinant plasmids. The improved ClosTron system supersedes the prototype plasmid pMTL007 and the original method, and exploits the potential of Group II introns more fully.

Introduction

The eubacterial genus Clostridium encompasses organisms of both medical and industrial importance. These include the pathogens Clostridium tetani, Clostridium perfringens, Clostridium botulinum (Hatheway, 1990) and Clostridium difficile (Brazier, 2008); and the solvent-producers Clostridium acetobutylicum (Papoutsakis, 2008) and Clostridium thermocellum (Lynd et al., 2005). Research both to counter pathogens and exploit useful strains has been hindered by a history of limited genetic methods, especially for mutagenesis. A modest number of directed mutants of Clostridium strains had been reported prior to 2007, almost all constructed by homologous recombination (summarized in Heap et al., 2009a). Of these, most were either ‘single-crossover’ strains in which an integrated plasmid serves as an insertional mutagen, or ‘double crossover’ strains in which an introduced alternative allele is exchanged with the wild-type allele. The former type of mutant is inherently unstable, while the latter type has proven difficult to isolate, partly because the use of negative selection markers, commonly relied upon to facilitate allele exchange in other organisms, has not been established in Clostridium.

To address the need for reliable mutagenesis, we recently developed the ClosTron (Heap et al., 2007), a Group II intron directed mutagenesis system for Clostridium. Bacterial Group II introns are a relatively newly-characterized type of mobile element (Lambowitz and Zimmerly, 2004) which can be used for the directed construction of stable mutants thanks to two key properties. Firstly, intron target specificity is determined mainly by base-pairing between the target site DNA and intron RNA, which can be rationally modified. Secondly, intron mobility requires the presence of an intron-encoded protein (IEP), which can be provided transiently during mutagenesis and subsequently removed to ensure the stability of the strain; a strategy analagous to the stabilization of mini-transposon insertions by removal of the transposase. Like most other Group II intron mutagenesis systems, the ClosTron plasmid pMTL007 contains a mini-intron derivative of the Ll.LtrB intron from Lactococcus lactis, ideal for rational intron re-targeting and loss of the IEP gene ltrA (Karberg et al., 2001). Other elements of pMTL007 facilitate conjugal transfer of the plasmid into Clostridium spp., its subsequent replication and maintenance, expression of the intron and IEP, and specific selection of clones containing an insertion.

The ClosTron system has transformed our research, and we have distributed pMTL007 to many other laboratories, some of which have already begun to publish studies using ClosTron mutants (Emerson et al., 2009, Kirby et al., 2009, Twine et al., 2009, Underwood et al., 2009). Despite its usefulness, the system and procedures we described previously did not fully exploit the potential of Group II introns, and were limited in the range of host strains and applications for which they were suitable. Here we addressed these limitations and also thoroughly optimized the mutagenesis procedure. The result is a notably faster, less labor-intensive, more flexible, and more broadly-applicable approach to directed mutagenesis in Clostridium.

Section snippets

Materials and methods

Details of plasmid construction including oligonucleotides are provided in the Supplementary material online.

A modular ClosTron plasmid design facilitates construction of derivatives

In order to test and improve the scope and utility of ClosTron mutagenesis, we needed to construct several new plasmid variants. The four types of change likely to be most useful in constructing ClosTron plasmid variants are replacement of the Gram-positive origin of replication, replacement of the plasmid marker, replacement or removal of the intron marker, and addition of ‘cargo’ sequence to be delivered by the intron. None of these alterations can be easily made to the prototype ClosTron

Discussion

A substantial body of work on the characterization and exploitation of bacterial Group II introns has been produced in the few years since their discovery. The Lambowitz group in particular demonstrated the potential of these elements as directed mutagens (Mohr et al., 2000, Karberg et al., 2001). This potential remains to be fully realized, for two main reasons. Firstly, the novelty and relative complexity of designing introns with altered target specificity may be unappealing to researchers

Acknowledgements

The authors wish to acknowledge the financial support of BBSRC grants BB/F003390/1 (SysMO Project COSMIC), BB/G016224/1, BB/E021271/1 and BB/D522289/1; and MRC grant number G0601176.

References (33)

  • Y. Chen et al.

    Construction of an alpha toxin gene knockout mutant of Clostridium perfringens type A by use of a mobile group II intron

    Appl. Environ. Microbiol.

    (2005)
  • J.E. Emerson et al.

    A novel genetic switch controls phase variable expression of CwpV, a Clostridium difficile cell wall protein

    Mol. Microbiol.

    (2009)
  • H. Guo et al.

    Group II introns designed to insert into therapeutically relevant DNA target sites in human cells

    Science

    (2000)
  • L.M. Harris et al.

    Northern, morphological, and fermentation analysis of spo0A inactivation and overexpression in Clostridium acetobutylicum ATCC 824

    J. Bacteriol.

    (2002)
  • M.G.N. Hartmanis et al.

    Intermediary metabolism in Clostridium acetobutylicum: levels of enzymes involved in the formation of acetate and butyrate

    Appl. Environ. Microbiol.

    (1984)
  • C.L. Hatheway

    Toxigenic clostridia

    Clin. Microbiol. Rev.

    (1990)
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