Molecular Characterization of
            <i>Clostridium tetani</i>
            Strains by Pulsed-Field Gel Electrophoresis and Colony PCR

Plourde-Owobi, Lucile; Seguin, Delphine; Baudin, Marie-Anne; Moste, Catherine; Rokbi, Bachra

doi:10.1128/aem.71.9.5604-5606.2005

Cited by 16 publications

(10 citation statements)

References 19 publications

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“…Cells were subsequently transferred onto agar plates containing 5 g fructose/liter, 10 mg trimethoprim/liter for counterselection of E. coli, and antibiotics for selection of the plasmid and group II intron insertion (15 mg thiamphenicol/ml and 5 mg clarithromycin/ml, respectively). Single colonies were restreaked and then screened using colony PCR (50).…”

Section: Construction Of Strains With Disrupted Genesmentioning

confidence: 99%

Energy Conservation Associated with Ethanol Formation from H ₂ and CO ₂ in Clostridium autoethanogenum Involving Electron Bifurcation

et al. 2015

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Most acetogens can reduce CO 2 with H 2 to acetic acid via the Wood-Ljungdahl pathway, in which the ATP required for formate activation is regenerated in the acetate kinase reaction. However, a few acetogens, such as Clostridium autoethanogenum, Clostridium ljungdahlii, and Clostridium ragsdalei, also form large amounts of ethanol from CO 2 and H 2 . How these anaerobes with a growth pH optimum near 5 conserve energy has remained elusive. We investigated this question by determining the specific activities and cofactor specificities of all relevant oxidoreductases in cell extracts of H 2 /CO 2 -grown C. autoethanogenum. The activity studies were backed up by transcriptional and mutational analyses. Most notably, despite the presence of six hydrogenase systems of various types encoded in the genome, the cells appear to contain only one active hydrogenase. The active [FeFe]-hydrogenase is electron bifurcating, with ferredoxin and NADP as the two electron acceptors. Consistently, most of the other active oxidoreductases rely on either reduced ferredoxin and/or NADPH as the electron donor. An exception is ethanol dehydrogenase, which was found to be NAD specific. Methylenetetrahydrofolate reductase activity could only be demonstrated with artificial electron donors. Key to the understanding of this energy metabolism is the presence of membrane-associated reduced ferredoxin:NAD ؉ oxidoreductase (Rnf), of electron-bifurcating and ferredoxin-dependent transhydrogenase (Nfn), and of acetaldehyde:ferredoxin oxidoreductase, which is present with very high specific activities in H 2 /CO 2 -grown cells. Based on these findings and on thermodynamic considerations, we propose metabolic schemes that allow, depending on the H 2 partial pressure, the chemiosmotic synthesis of 0.14 to 1.5 mol ATP per mol ethanol synthesized from CO 2 and H 2 . IMPORTANCEEthanol formation from syngas (H 2 , CO, and CO 2 ) and from H 2 and CO 2 that is catalyzed by bacteria is presently a much-discussed process for sustainable production of biofuels. Although the process is already in use, its biochemistry is only incompletely understood. The most pertinent question is how the bacteria conserve energy for growth during ethanol formation from H 2 and CO 2 , considering that acetyl coenzyme A (acetyl-CoA), is an intermediate. Can reduction of the activated acetic acid to ethanol with H 2 be coupled with the phosphorylation of ADP? Evidence is presented that this is indeed possible, via both substrate-level phosphorylation and electron transport phosphorylation. In the case of substrate-level phosphorylation, acetyl-CoA reduction to ethanol proceeds via free acetic acid involving acetaldehyde:ferredoxin oxidoreductase (carboxylate reductase).

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Section: Construction Of Strains With Disrupted Genesmentioning

confidence: 99%

Energy Conservation Associated with Ethanol Formation from H ₂ and CO ₂ in Clostridium autoethanogenum Involving Electron Bifurcation

et al. 2015

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“…tetani specific primers targeting a fragment of the tetX gene (1354 bp) were used to amplify the DNA extracted from the sample [8]. The PCR reaction contained 10 nM each primer (Eurofins), 200 M each deoxynucleoside triphosphate (dNTP) (Genei), 1 U Taq polymerase (Genei) in the appropriate reaction buffer, and 50 ng and 100 ng of DNA extracts as the templates.…”

Section: Pcr Amplification and Sequencingmentioning

confidence: 99%

Detection of Clostridium tetani in human clinical samples using tetX specific primers targeting the neurotoxin

Ganesh

Sheikh

Shah

et al. 2016

Journal of Infection and Public Health

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Tetanus resulting from ear injury remains an important health problem, particularly in the developing world. We report the successful detection of Clostridium tetani using tetX specific primers targeting the Cl. tetani neurotoxin. The sample was obtained from an ear discharge of a case of otogenic tetanus in a 2-year-old male child. Based on the culture results of the ear discharge, Gram staining and virulence testing by genotyping, a diagnosis of tetanus was confirmed. This is the first report from India on the successful detection of Cl. tetani in a human clinical sample using tetX specific primers targeting the Cl. tetani neurotoxin.

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“…The multiplex PCR is a sensitive assay with the ability to detect a mean of one to two genome copy units per 100 μl clinical sample and allows nonculture-based diagnosis of the disease-causing organism within 3 h. A fluorescence-based multiplex PCR was automated for the simultaneous detection of Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae in clinical samples from patients with suspected meningitis (Smith et al, 2004). Pulsed-field gel electrophoresis and PCR were applied for the first time to the molecular characterization of Clostridium tetani (Plourde-Owobi et al, 2005).…”

Section: Polymerase Chain Reactionmentioning

confidence: 99%

“…Pulsed-field gel electrophoresis and colony PCR were applied for the first time by Plourde-Owobi et al (2005) for the molecular characterization of C. tetani . Among five strains tested, one turned out to contain a mixture of two genetically different clones and two (D11 and G761) to contain bacteria differing by the presence or absence of the 74-kb plasmid harboring the tetX gene.…”

Section: Polymerase Chain Reactionmentioning

confidence: 99%

Gene Technology: Applications and Techniques

2012

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Molecular Characterization of Clostridium tetani Strains by Pulsed-Field Gel Electrophoresis and Colony PCR

Cited by 16 publications

References 19 publications

Energy Conservation Associated with Ethanol Formation from H ₂ and CO ₂ in Clostridium autoethanogenum Involving Electron Bifurcation

Energy Conservation Associated with Ethanol Formation from H ₂ and CO ₂ in Clostridium autoethanogenum Involving Electron Bifurcation

Detection of Clostridium tetani in human clinical samples using tetX specific primers targeting the neurotoxin

Gene Technology: Applications and Techniques

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Molecular Characterization of Clostridium tetani Strains by Pulsed-Field Gel Electrophoresis and Colony PCR

Cited by 16 publications

References 19 publications

Energy Conservation Associated with Ethanol Formation from H 2 and CO 2 in Clostridium autoethanogenum Involving Electron Bifurcation

Energy Conservation Associated with Ethanol Formation from H 2 and CO 2 in Clostridium autoethanogenum Involving Electron Bifurcation

Detection of Clostridium tetani in human clinical samples using tetX specific primers targeting the neurotoxin

Gene Technology: Applications and Techniques

Contact Info

Product

Resources

About

Energy Conservation Associated with Ethanol Formation from H ₂ and CO ₂ in Clostridium autoethanogenum Involving Electron Bifurcation

Energy Conservation Associated with Ethanol Formation from H ₂ and CO ₂ in Clostridium autoethanogenum Involving Electron Bifurcation