Construction of an Alpha Toxin Gene Knockout Mutant of
            <i>Clostridium perfringens</i>
            Type A by Use of a Mobile Group II Intron

Chen, Yue; McClane, Bruce A.; Fisher, Derek J.; Rood, Julian I.; Gupta, Phalguni

doi:10.1128/aem.71.11.7542-7547.2005

Cited by 127 publications

(143 citation statements)

References 16 publications

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“…Since the DNA target site is recognized primarily by base pairing of intron RNA and a few IEP recognition spots, the intron targeting sequences can be modified to insert into a specific DNA target site (Lambowitz and Zimmerly, 2004;Perutka et al, 2004;Yao et al, 2006;Zhong, Karberg, and Lambowitz, 2003). We recently developed a Clostridium-specific targetron donor plasmid and used it to successfully inactivate the plc gene in the C. perfringens' chromosome (Chen et al, 2005).…”

Section: Resultsmentioning

confidence: 99%

“…To construct a pfoA/plc double knockout mutant producing p27, we used our previously constructed plc knockout mutant of C. perfringens ATCC3624 (Chen et al, 2005), which is naturally enterotoxin-and β-2 toxin-negative. Vegetative cultures of all C. perfringens isolates were grown for 9 h at 37C in fluid thioglycolate broth (FTG) (Difco).…”

Section: Bacterial Strain and Growth Conditionsmentioning

confidence: 99%

“…plc TargeTron donor plasmid pJIR750ai (Chen et al, 2005) was used in this study to construct a pfoA targetron for inactivation of pfoA gene in bacterial chromosome. The Sigma TargeTron Design web site (http://www.sigma-genosys.com/targetron/checksequence/), predicts 10 targetron insertion sites in the pfoA gene open reading frame (ORF).…”

Section: Theta Toxin Gene Targetron Donor Plasmid Constructionmentioning

confidence: 99%

“…The C. perfringens type A transformant used for the delivery vehicle is negative for enterotoxin and β-2 toxin and considered safe for oral administration Smedley et al, 2004). However, to ensure the safety of the C. perfringens based vehicle, the alpha toxin gene (plc) in C. perfringens chromosome has been inactivated using mobile group II intron based targetron technology (Chen et al, 2005). In these recombinant clostridia, Simian Immunodefiency Virus (SIV) p27 antigens are expressed from intracellular plasmids carrying antibiotic resistance genes for selective pressure.…”

Section: Introductionmentioning

confidence: 99%

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Disruption of a toxin gene by introduction of a foreign gene into the chromosome of Clostridium perfringens using targetron-induced mutagenesis

Chen

Caruso

McClane

et al. 2007

Plasmid

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Section: Resultsmentioning

confidence: 99%

Section: Bacterial Strain and Growth Conditionsmentioning

confidence: 99%

Section: Theta Toxin Gene Targetron Donor Plasmid Constructionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Disruption of a toxin gene by introduction of a foreign gene into the chromosome of Clostridium perfringens using targetron-induced mutagenesis

Chen

Caruso

McClane

et al. 2007

Plasmid

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“…The mobile group II intron, originating from the Lactococcus lactis L1.LtrB intron, has been successfully used in a wide range of bacteria including Clostridium perfringens [5]. Without a proper replicon and/or promoter, the targetron plasmid pJIR750ai for C. perfringens from Sigma Aldrich was not applicable for gene disruption in the C. acetobutylicum directly (data not shown).…”

Section: Dear Editormentioning

confidence: 99%

Targeted gene disruption by use of a group II intron (targetron) vector in Clostridium acetobutylicum

et al. 2007

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Dear Editor:Clostridium acetobutylicum, a gram-positive, anaerobic, spore-forming bacterium, is capable of using a wide variety of carbon sources to produce acetone, butanol and ethanol. To improve solvent productivity of C. acetobutylicum, metabolic engineering is considered as a useful tool in developing strains with industrially desirable characteristics. However, to date, there are few useful methods for genetic manipulation of C. acetobutylicum, especially for gene disruption. To our knowledge, two types of vectors, including non-replicative and replicative integrative plasmids, have been developed for gene-inactivation in C. acetobutylicum. By using non-replicative integrative plasmids, buk and solR genes of C. acetobutylicum were inactivated [1,2]. However, due to their low frequencies of transformation and recombination, the non-replicative integrative plasmids are usually transformed at less than 1 integrative transformant per mg plasmid DNA. To obtain the integrative mutant, it may require higher transformation frequencies up to 10 5 , but the typical transformation frequencies were reported at 10 3 [3]. Harris et al. described the construction of a replicative integrative plasmid pETSPO and its application in the disruption of gene spo0A which could not be inactivated by using the non-replicative integrative plasmid [4]. With the functional replication origin in C. acetobutylicum, pETSPO increases opportunity for homologous recombination, but it is still time-consuming to screen for double crossover integration. Therefore, a more efficient tool for targeted gene inactivation in the C. acetobutylicum is much needed.Recently, a new strategy was developed to construct gene inactivation mutants by using group II intron-based Targetron technology. The mobile group II intron, originating from the Lactococcus lactis L1.LtrB intron, has been successfully used in a wide range of bacteria including Clostridium perfringens [5]. Without a proper replicon and/or promoter, the targetron plasmid pJIR750ai for C. perfringens from Sigma Aldrich was not applicable for gene disruption in the C. acetobutylicum directly (data not shown). Therefore, a modified targetron plasmid pSY6 was created by cloning the L1.LtrB group II intron fragment into the pIMP1-ptb, which was an E. coli-C. acetobutylicum shuttle vector containing a ptb (phosphotransbutyrylase) promoter [6].The gene buk, encoding the butyrate kinase, catalyzes the production of butyrate, and the gene solR located on the megaplasmid of the strain, encodes a putative repressor of solvent formation genes [7,8]. pSY6-buk and pSY6-solR vectors, constructed based on pSY6 ( Figure 1A and Supplementary information, Figure S1), were electroporated into C. acetobutylicum ATCC 824, respectively. Then, the cells were incubated overnight to induce the intron invasion (See Supplementary information, Materials and Methods). The overnight cultures were spread onto CGM medium (25 μg/ml erythromycin) and the transformants were analyzed

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LiveClostridia: A Powerful Tool in Tumor Biotherapy

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2010

Emerging Cancer Therapy

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Construction of an Alpha Toxin Gene Knockout Mutant of Clostridium perfringens Type A by Use of a Mobile Group II Intron

Cited by 127 publications

References 16 publications

Disruption of a toxin gene by introduction of a foreign gene into the chromosome of Clostridium perfringens using targetron-induced mutagenesis

Disruption of a toxin gene by introduction of a foreign gene into the chromosome of Clostridium perfringens using targetron-induced mutagenesis

Targeted gene disruption by use of a group II intron (targetron) vector in Clostridium acetobutylicum

LiveClostridia: A Powerful Tool in Tumor Biotherapy

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