Construction of a sequencedClostridium perfringens-Escherichia coli shuttle plasmid

Sloan, Joan; Warner, Tracy; Scott, Paul T.; Bannam, Trudi Leanne; Berryman, David; Rood, Julian I.

doi:10.1016/0147-619x(92)90023-4

Cited by 113 publications

(54 citation statements)

References 32 publications

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“…C. perfringens 13 (13) was used as a host for plasmid pJIR418 (24) derivatives. E. coli JM109 (30) was used for both pJIR418 and pUC19 (30) derivatives.…”

Section: Methodsmentioning

confidence: 99%

“…The mutant was thought to be defective in a coordinating regulatory factor for the expression of these toxin genes. A chromosomal DNA fragment which was cloned from the parent strain into an Escherichia coli-C. perfringens shuttle vector, pJIR418 (24), gave rise to the production of these toxins in strain SI112. Deletion and sequence analysis revealed that the cloned fragment carried a trans-acting regulatory gene (virR) for the production of these toxins and that the virR gene appears to be a response regulator of the two-component signal transduction systems characteristic of many bacteria.…”

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confidence: 99%

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The virR gene, a member of a class of two-component response regulators, regulates the production of perfringolysin O, collagenase, and hemagglutinin in Clostridium perfringens

et al. 1994

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The perfringolysin O (theta-toxin) gene (pfoA) of Clostridium perfringens was cloned into an Escherichia coli-C. perfringens shuttle vector, and the pfoA gene was expressed in mutants of C. perfringens 13 which lacked the production of perfringolysin O. One group (SI117) could express the pfoA gene, and the other (SI112) could not. A mutation in the regulatory system for pfoA gene expression was suspected in SI112. A chromosomal DNA library constructed from strain 13 was transformed into strain SI112 to identify the regulatory gene(s) for the pfoA gene. Five strains of 10,000 transformants restored perfringolysin O production. All contained a 2.5-kb DNA fragment. This fragment activated the transcription of the pfoA gene and also restored the production of collagenase (kappa-toxin) and hemagglutinin in strain SI112. Deletion analysis showed that a 1.25-kb region was sufficient for the trans activity, and sequence analysis disclosed that open reading frame 2 (ORF2) was located in this region. A homology search for the deduced amino acid sequence revealed that ORF2 was homologous to a response regulator in a two-component signal transduction system. ORF2 was designated virR, and it is suggested that the virR gene plays an important role in the pathogenicity of C. perfringens.

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“…C. perfringens 13 (13) was used as a host for plasmid pJIR418 (24) derivatives. E. coli JM109 (30) was used for both pJIR418 and pUC19 (30) derivatives.…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

The virR gene, a member of a class of two-component response regulators, regulates the production of perfringolysin O, collagenase, and hemagglutinin in Clostridium perfringens

et al. 1994

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“…V. harveyi strain BB170 was cultured in AB medium at 30∞C with aeration for the AI-2 bioassay (Surette and Bassler, 1998). Plasmid pUC18 (Yanisch-Perron et al, 1985) was used for general cloning in E. coli, and pJIR418 (Sloan et al, 1992) was used as an E. coli-C. perfringens shuttle vector. Erythromycin (50 mg ml -1 ), chloramphenicol (25 mg ml -1 ) and ampicillin (100 mg ml -1 ) were added to the media for appropriate bacterial strains.…”

Section: Strains Plasmids and Growth Conditionsmentioning

confidence: 99%

The luxS gene is involved in cell–cell signalling for toxin production in Clostridium perfringens

Ohtani

Hayashi

Shimizu

2002

Molecular Microbiology

156

112

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Summary A Gram‐positive anaerobic pathogen, Clostridium perfringens, causes clostridial myonecrosis or gas gangrene in humans by producing numerous extracellular toxins and enzymes that act in concert to degrade host tissues. C. perfringens possesses a homologue of the luxS gene that is reported to be responsible for the production of autoinducer 2 (AI‐2), which participates in quorum sensing in bacteria. The luxS mutant was constructed using C. perfringens strain 13, and the role of the luxS gene in toxin production was examined. The cell‐free culture supernatant from wild‐type strain 13 greatly stimulated the luminescence of Vibrio harveyi BB170, whereas that from the luxS mutant caused no significant stimulation, indicating that the luxS gene is necessary for AI‐2 production in C. perfringens. The luxS mutant showed a reduced level of production of alpha‐, kappa‐ and theta‐toxins. In the luxS mutant, the transcription of the theta‐toxin gene (pfoA) was lower at mid‐exponential growth phase, whereas alpha‐ and kappa‐toxin gene transcription was not significantly affected. The production of toxins in the luxS mutant was stimulated by the addition of the culture supernatant from the wild‐type cells, possibly because of the presence of AI‐2. Moreover, the expression of the pfoA gene in the luxS mutant was apparently activated when the mutant cells were cultured in the presence of culture supernatants from the wild‐type C. perfringens, Escherichia coli DH5α carrying the luxS gene of C. perfringens. A deletion analysis of the luxS operon showed that the luxS gene alone is responsible for cell–cell signalling, and that the metB or cysK genes located upstream of luxS are not involved in regulating toxin production. Our results indicate that cell–cell signalling by AI‐2 plays an important role in the regulation of toxin production in C. perfringens.

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“…Clostridium perfringens strain 13 11) and its derivatives were cultured at 37 C in GAM medium (Nissui, Tokyo) under anaerobic conditions, as described by Shimizu et al 9) C. perfringens strains carrying plasmids derived from pJIR418 vector 12) were cultured in GAM medium containing 25 mg/ml of chloramphenicol. The plasmids were cloned in Escherichia coli strain JM109 (Takara Bio, Shiga, Japan), which was cultured in LB medium containing 50 mg/ml of ampicillin.…”

Section: Methodsmentioning

confidence: 99%

“…The PCR product (632 bp) was digested with BamHI and cloned into the BamHI site of a pUC18 vector to generate pUC2168. An erythromycin determinant gene (ermBP) was amplified by PCR from pJIR418 vector 12) using a primer set (F 5 0 -GGCCGAATTCCAGG-AAACAGCTATGACATG-3 0 and R 5 0 -GCCGGAATTCTTTCAAC-TTGCCCACTTCGA-3 0 ; EcoRI sites underlined). The PCR product (1,199 bp) was digested with EcoRI and cloned into the EcoRI site of y To whom correspondence should be addressed.…”

Section: Methodsmentioning

confidence: 99%

Effects of Depletion of RNA-Binding Protein Tex on the Expression of Toxin Genes inClostridium perfringens

Abe

Obana

Nakamura

2010

Bioscience, Biotechnology, and Biochemistry

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Construction of a sequencedClostridium perfringens-Escherichia coli shuttle plasmid

Cited by 113 publications

References 32 publications

The virR gene, a member of a class of two-component response regulators, regulates the production of perfringolysin O, collagenase, and hemagglutinin in Clostridium perfringens

The virR gene, a member of a class of two-component response regulators, regulates the production of perfringolysin O, collagenase, and hemagglutinin in Clostridium perfringens

The luxS gene is involved in cell–cell signalling for toxin production in Clostridium perfringens

Effects of Depletion of RNA-Binding Protein Tex on the Expression of Toxin Genes inClostridium perfringens

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