Quantitative Effects of Carbohydrates and Aromatic Amino Acids on &lt;i&gt;Clostridium botulinum &lt;/i&gt;Toxin Gene Expression Using a Rapid Competitive RT/PCR Assay

Sharkey, Freddie H.; Dooley, James; Haylock, R. W.

doi:10.1159/000088144

Cited by 7 publications

(7 citation statements)

References 65 publications

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“…The expression pattern of plc could potentially reflect an evolutionary strategy to increase substrate availability in crowded environments by increasing toxin (phospholipase) production. A similar up-shift in transcription of the gene encoding botulinum neurotoxin (cntA) has been observed in Clostridium botulinum (Sharkey et al, 2005;Shin et al, 2006). A difference in transcript levels between strains T9 and S3 by up to a factor 10 was shown in Fig.…”

Section: Transcript Levels Of Plc and Production Of A-toxinsupporting

confidence: 53%

Dynamics of plc gene transcription and α-toxin production during growth of Clostridium perfringens strains with contrasting α-toxin production

Abildgaard¹,

Schramm²,

Rudi³

et al. 2009

Veterinary Microbiology

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The aim of the present study was to investigate transcription dynamics of the alpha-toxin-encoding plc gene relative to two housekeeping genes (gyrA and rplL) in batch cultures of three Clostridium perfringens strains with low, intermediate, and high levels of alpha-toxin production, respectively. The plc transcript level was always low in the low alpha-toxin producing strain. For the two other strains, plc transcription showed an inducible pattern and reached a maximum level in the late exponential growth phase. The transcription levels were however inversely correlated to alpha-toxin production for the two strains. We propose that this discrepancy is due to differences in plc translation rates between the strains and that strain-specific translational rates therefore must be determined before alpha-toxin production can be extrapolated from transcript levels in C. perfringens.

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Section: Transcript Levels Of Plc and Production Of A-toxinsupporting

confidence: 53%

Dynamics of plc gene transcription and α-toxin production during growth of Clostridium perfringens strains with contrasting α-toxin production

Abildgaard¹,

Schramm²,

Rudi³

et al. 2009

Veterinary Microbiology

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“…Understanding the activity of bacterial virulence genes at low temperatures is a key to establishing novel strategies to ensure the safety of refrigerated foods. To date, all published studies on type E neurotoxin gene expression have been conducted at 30°C or higher (3,18,22,23). However, to the best of our knowledge, no report is available on the type E neurotoxin gene expression at low temperatures or on the relative expression of the six type E neurotoxin cluster genes.…”

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confidence: 99%

Quantitative Real-Time Reverse Transcription-PCR Analysis Reveals Stable and Prolonged Neurotoxin Cluster Gene Activity in a Clostridium botulinum Type E Strain at Refrigeration Temperature

Chen

Korkeala

Lindén

et al. 2008

Appl Environ Microbiol

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The relative expression levels of six botulinum neurotoxin cluster genes in a group II Clostridium botulinum type E strain grown at 10 or 30°C were investigated using quantitative real-time reverse transcription-PCR. An enzyme-linked immunosorbent assay was used to confirm neurotoxin expression. Distinct mRNA and toxin production patterns were observed at the two temperatures. The average relative mRNA levels at 10°C were higher than (ntnh and p47), similar to (botE), or lower than (orfx1, orfx2, orfx3) those at 30°C. The maximum botE expression levels and average neurotoxin levels at 10°C were 45 to 65% of those at 30°C. The relative mRNA levels at 10°C declined generally slowly within 8 days, as opposed to the rapid decline observed at 30°C within 24 h. Distinct expression patterns of the six genes at the two temperatures suggest that the type E neurotoxin cluster genes are transcribed as two tricistronic operons at 30°C, whereas at 10°C monocistronic (botE or orfx1 alone) and bicistronic (ntnh-p47 and orfx2-orfx3) transcription may dominate. Thus, type E botulinum neurotoxin production may be involved with various temperature-dependent regulatory events. In light of group II C. botulinum type E being a dangerous food-borne pathogen, these findings may be important in terms of the safety of refrigerated packaged foods of extended durability.Botulinum neurotoxins ingested with food and drink can cause a potentially lethal paralysis to humans and animals. The neurotoxins are mainly produced by Clostridium botulinum and appear in seven distinct serotypes (A through G) (7,20,21). Upon secretion, the neurotoxins are associated with nontoxic proteins forming progenitor toxin complexes of differing structures and molecular sizes, depending on serotype and strain (7,8,21).The genes encoding the nontoxic-neurotoxin-associated proteins are located immediately upstream of the neurotoxin gene and vary in structure, composition, and arrangement between different serotypes and different strains (7). All types of the neurotoxin gene clusters share the same organization of bot (also called bont, cnt, and ntx) and ntnh at the 3Ј end but differ at their 5Ј ends (20). Type A1, B, C, D, and G neurotoxin gene clusters are associated with three hemagglutinin-encoding genes (ha70, ha17, and ha33), whereas types A2 to A4 and E and F toxin clusters are associated with p47 and orfx1, orfx2, and orfx3, encoding proteins with unknown function (2,4,6,11,20). In addition, all other types of toxin gene clusters, with the exception of type E, carry botR that encodes a sigma factor responsible for positive regulation of the neurotoxin gene (16).The role of the nontoxic-neurotoxin-associated proteins has not been fully identified. It has been proposed that they play a role in protecting the neurotoxin against the acidity of the gastrointestinal tract (19), and the hemagglutinin components seem to assist in the absorption of ingested neurotoxin from the gastrointestinal tract (17). Nothing is known about the activities of orfx1, orfx2, and orfx...

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“…It is possible that this is a growth rate phenomenon, where toxin production is enhanced due to a lowered growth rate, as reported for Clostridium difficile under stress conditions (30). Sharkey et al (39), however, indicated that for nonproteolytic C. botulinum type E, an increased growth rate was associated with an increased expression of cntE, while the presence of sorbic acid and sodium nitrite reduced both the growth rate and expression of cntE (40). Lövenklev et al (27,28) also reported an inhibitory effect of nitrite on cntB expression.…”

Section: Discussionmentioning

confidence: 95%

“…However, little is known about the direct regulation of the neurotoxin gene (cnt) or of neurotoxin formation by nonproteolytic C. botulinum type E. While a positive regulator, cntR, has been found in other types of C. botulinum, it appears to be absent in C. botulinum type E (9,24,36). Several in vitro methods have been developed for monitoring cnt expression in proteolytic C. botulinum and nonproteolytic C. botulinum, including a gene reporter system (12), competitive reverse transcription (RT)-PCR (29,39,40), and quantitative RT-PCR (qRT-PCR) (10,23,27,28,42). Experiments using these methods found a peak in neurotoxin gene expression in late exponential or early stationary phase.…”

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confidence: 99%

Effects of Carbon Dioxide on Neurotoxin Gene Expression in NonproteolyticClostridium botulinumType E

Artin

Carter

Holst

et al. 2008

Appl Environ Microbiol

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Carbon dioxide is an antimicrobial gas commonly used in modified atmosphere packaging. In the present study, the effects of carbon dioxide on the growth of and neurotoxin production by nonproteolytic Clostridium botulinum type E were studied during the growth cycle. Quantitative reverse transcription-PCR and an enzyme-linked immunosorbent assay were used to quantify expression of the type E botulinum neurotoxin gene (cntE) and the formation of type E neurotoxin. The expression levels of cntE were similar in two strains, with relative expression peaking in the transition between exponential phase and stationary phase. In stationary phase, cntE mRNA expression declined rapidly. The cntE mRNA half-life was calculated to be approximately 9 minutes. Neurotoxin formation occurred in late exponential phase and stationary phase. High carbon dioxide concentrations delayed growth by increasing the lag time and decreasing the maximum growth rate. The effects of carbon dioxide concentration on relative neurotoxin gene expression and neurotoxin formation were significant. Expression of cntE mRNA and the formation of extracellular neurotoxin were twofold higher with a headspace carbon dioxide concentration of 70% (vol/vol) compared to 10% (vol/vol). This finding sheds a new, cautionary light on the potential risks of botulism associated with the use of modified atmosphere packaging.

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Quantitative Effects of Carbohydrates and Aromatic Amino Acids on <i>Clostridium botulinum </i>Toxin Gene Expression Using a Rapid Competitive RT/PCR Assay

Cited by 7 publications

References 65 publications

Dynamics of plc gene transcription and α-toxin production during growth of Clostridium perfringens strains with contrasting α-toxin production

Dynamics of plc gene transcription and α-toxin production during growth of Clostridium perfringens strains with contrasting α-toxin production

Quantitative Real-Time Reverse Transcription-PCR Analysis Reveals Stable and Prolonged Neurotoxin Cluster Gene Activity in a Clostridium botulinum Type E Strain at Refrigeration Temperature

Effects of Carbon Dioxide on Neurotoxin Gene Expression in NonproteolyticClostridium botulinumType E

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