The ClosTron: A universal gene knock-out system for the genus Clostridium
Introduction
Clostridium is a large genus composed of obligately anaerobic, gram-positive, endospore-forming, rod-shaped bacteria. Although it encompasses some notorious pathogens (such as Clostridium botulinum, Clostridium perfringens and Clostridium difficile) the vast majority of the genus are entirely benign, and include solventogenic species useful in the industrial production of biofuels (such as Clostridium acetobutylicum and Clostridium beijerinckii) as well as strains with potential as anti-cancer delivery systems, such as Clostridium sporogenes and Clostridium novyi (Dürre, 2005). Representative genome sequences of all of the major species have now been determined, and various DNA transfer systems developed. However, effective exploitation of the amassed genome data has been precluded by a woeful inadequacy in current procedures for insertionally inactivating genes. The targeted inactivation of clostridial genes has been reported in relatively few species, most notably C. acetobutylicum, C. beijerinckii, C. perfringens and most recently C. difficile. Mutants have been almost exclusively confined to single crossover knockouts brought about by Campbell-like integration through homologous recombination of a replication-defective plasmid (Shimizu et al., 1994, Wilkinson and Young, 1994, Green et al., 1996, Green and Bennett, 1996, Liyanage et al., 2001, O'Connor et al., 2006). Such integrants are segregationally unstable, as plasmid sequences at the target site are flanked by two directly repeated copies of the DNA segment directing integration. Despite the preference for integrants resulting from allelic exchange, such double crossover mutants have only ever been reported in C. perfringens (Awad et al., 1995, Bannam et al., 1995). These rare integration events can be detected in C. perfringens as a consequence of the high frequency with which DNA can be transformed into this organism. Gene transfer into most clostridial species is far less efficient, making integration reliant upon host-mediated, homologous recombination very difficult to detect.
Recently, a completely novel way to achieve directed insertional inactivation of genes using a bacterial group II intron has been described (Karberg et al., 2001). Comprehensive analyses of the mobile group II intron from the ltrB gene of Lactococcus lactis (Ll.ltrB) have revealed that such elements propagate into their specific target site via an RNA-mediated ‘retrohoming’ mechanism. During this process, target site recognition is achieved primarily by base-pairing between the excised intron lariat RNA and the target site DNA (Mohr et al., 2000). Intron target specificity can therefore be rationally re-programmed by altering the DNA sequence encoding the appropriate part of the intron. Furthermore, since most activities required to effect the Ll.ltrB intron's site-specific integration are encoded within the intron, its mobility is essentially independent of host factors. The Ll.ltrB intron's host range is proving to be broad in the extreme, and variants have been used in both gram-negative and gram-positive bacteria (Karberg et al., 2001, Frazier et al., 2003, Yao et al., 2006) including C. perfringens (Chen et al., 2005), where inactivation of the alpha toxin gene (plc) was demonstrated.
Intron integration frequencies vary widely between target sites, and can make the screening effort required to isolate a mutant prohibitively laborious, particularly if no simple phenotypic screen for gene inactivation is available. Zhong and co-workers (Zhong et al., 2003) devised an ingenious solution to this problem by introducing into the group II intron an antibiotic resistance gene, which is itself interrupted by a self-splicing group I intron. The three elements are arranged such that only after successful insertion of the group II intron into its target, when the nested group I intron will have spliced out, is the integrity of the antibiotic resistance gene restored. Acquisition of antibiotic resistance is thereby strictly coupled to integration, and can be used to positively select for integration events. Such a marker, referred as a Retrotransposition-Activated Marker (RAM), overcomes potentially low integration frequencies and represents an important refinement for those seeking to use re-targeted introns as general gene inactivation tools.
The generation of the plc mutant of C. perfringens did not make use of a RAM element, but instead relied upon a simple visual phenotypic screen (Chen et al., 2005). In the present study we wished to develop a clostridial RAM which would allow positive selection for integration of group II intron elements into any gene, without recourse to a phenotypic screen, and in clostridial species in which, unlike C. perfringens, mutants have seldom or never previously been obtained.
Section snippets
Bacterial growth, conjugation and transformation
C. difficile 630Δerm (Hussain et al., 2005) was grown in BHI broth or on BHI agar (Purdy et al., 2002), C. acetobutylicum ATCC824 was grown in CGM broth or on CGM agar (Hartmanis and Gatenbeck, 1984) and C. sporogenes NCIMB 10696 and C. botulinum ATCC3502 were grown in TYG broth or on TYG agar (Purdy et al., 2002). The C. acetobutylicum pyrF mutant was grown in CGM broth or on CGM agar supplemented with 50 μg/L uracil. All growth media were pre-reduced by overnight incubation in an anaerobic
Results
At the heart of the system devised by Zhong et al. (2003) was an IPTG-inducible T7 promoter for controlled production of the group II intron RNA, and a RAM element based on a trimethoprim resistance gene to positively select integrants. The Sigma-Aldrich derivative pACD4K-C is almost identical, but features a RAM derived from a kanamycin-resistance gene. The T7 promoter does not function in clostridia. Moreover, clostridia tend to be naturally resistant to both trimethoprim and kanamycin,
Discussion
To date the generation of mutants in a range of clostridial species, other than C. perfringens, using classical homologous recombination has proven difficult. Thus, only five mutations have ever been made in C. acetobutylicum. Four (butK, CAC3075; pta, CAC1742; aad, CACP0162; and solR, CACP061) were made by single cross-over integration of replication deficient plasmids (Green et al., 1996, Green and Bennett, 1996, Harris et al., 2002) while a fifth in spo0A (CAC2071) was isolated by a strategy
Acknowledgements
We thank C. Cooksley, D. Burns, J. Scott and B. Blount for help with mutant characterisation. This work was supported by the BBSRC (grants BB/D522797/1 and BD/D522289/1) and Morvus Technology Ltd.
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These authors contributed equally to the described studies.