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

Abstract

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...

Temperature Exerts Control of Bacillus cereus Emetic Toxin Production on Post-transcriptional Levels

“…At higher temperatures, transcription was declining but still detectable up to the upper growth boundary. In comparison, studies on the effect of temperature on the botulinum toxin complex L-TC have shown higher mRNA levels at lower temperatures and lower transcript levels as well as less stability at high temperatures (Couesnon et al, 2006; Chen et al, 2008). Therefore, considering the differential regulation of CesB protein and cereulide, it is tempting to speculate that temperature sensing of the cell triggers a response of cereulide synthesis on a translational and/or post-translational level rather than on a transcriptional level.…”

Two-Component Signal Transduction System CBO0787/CBO0786 Represses Transcription from Botulinum Neurotoxin Promoters in Clostridium botulinum ATCC 3502

“…Botulinum neurotoxin was quantified by using a commercial type A neurotoxin ELISA kit (Tetracore) in three independent WT and mutant culture supernatants collected at mid-exponential, late exponential, and early stationary growth phases [24], [26], [28]. The plates were read at 405 nm (Multiskan Ascent, Thermo Fisher).…”

“…A peak in the level of neurotoxin gene cluster expression in late-exponential to early-stationary phase cultures [19], [24] suggests that neurotoxin production is tightly regulated. To date only positive regulatory elements have been implicated in this control.…”

Positive Regulation of Botulinum Neurotoxin Gene Expression by CodY in Clostridium botulinum ATCC 3502

“…Transcription of the neurotoxin gene cluster is increased in the exponential growth phase, peaks at the transition from lateexponential-to early-stationary-phase cultures, and is drastically decreased during the stationary phase (24)(25)(26). Botulinum neurotoxin production is also affected by the availability of certain carbon and nitrogen sources (27)(28)(29).…”