Blocking neurotransmission, botulinum neurotoxin is the most poisonous biological substance known to mankind. Despite its infamy as the scourge of the food industry, the neurotoxin is increasingly used as a pharmaceutical to treat an expanding range of muscle disorders. Whilst neurotoxin expression by the spore-forming bacterium Clostridium botulinum appears tightly regulated, to date only positive regulatory elements, such as the alternative sigma factor BotR, have been implicated in this control. The identification of negative regulators has proven to be elusive. Here, we show that the two-component signal transduction system CBO0787/CBO0786 negatively regulates botulinum neurotoxin expression. Single insertional inactivation of cbo0787 encoding a sensor histidine kinase, or of cbo0786 encoding a response regulator, resulted in significantly elevated neurotoxin gene expression levels and increased neurotoxin production. Recombinant CBO0786 regulator was shown to bind to the conserved −10 site of the core promoters of the ha and ntnh-botA operons, which encode the toxin structural and accessory proteins. Increasing concentration of CBO0786 inhibited BotR-directed transcription from the ha and ntnh-botA promoters, demonstrating direct transcriptional repression of the ha and ntnh-botA operons by CBO0786. Thus, we propose that CBO0786 represses neurotoxin gene expression by blocking BotR-directed transcription from the neurotoxin promoters. This is the first evidence of a negative regulator controlling botulinum neurotoxin production. Understanding the neurotoxin regulatory mechanisms is a major target of the food and pharmaceutical industries alike.

ABSTRACTA key survival mechanism ofClostridium botulinum, the notorious neurotoxic food pathogen, is the ability to form heat-resistant spores. While the genetic mechanisms of sporulation are well understood in the model organismBacillus subtilis, nothing is known about these mechanisms inC. botulinum.Using the ClosTron gene-knockout tool,sigK, encoding late-stage (stage IV) sporulation sigma factor K inB. subtilis, was disrupted inC. botulinumATCC 3502 to produce two different mutants with distinct insertion sites and orientations. Both mutants were unable to form spores, and their elongated cell morphology suggested that the sporulation pathway was blocked at an early stage. In contrast,sigK-complemented mutants sporulated successfully. Quantitative real-time PCR analysis ofsigKin the parent strain revealed expression at the late log growth phase in the parent strain. Analysis ofspo0A, encoding the sporulation master switch, in thesigKmutant and the parent showed significantly reduced relative levels ofspo0Aexpression in thesigKmutant compared to the parent strain. Similarly,sigFshowed significantly lower relative transcription levels in thesigKmutant than the parent strain, suggesting that the sporulation pathway was blocked in thesigKmutant at an early stage. We conclude that σKis essential for early-stage sporulation inC. botulinumATCC 3502, rather than being involved in late-stage sporulation, as reported for the sporulation model organismB. subtilis. Understanding the sporulation mechanism ofC. botulinumprovides keys to control the public health risks that the spores of this dangerous pathogen cause through foods.

Botulinum neurotoxin, produced mainly by the spore-forming bacterium Clostridium botulinum, is the most poisonous biological substance known. Here, we show that CodY, a global regulator conserved in low-G؉C Gram-positive bacteria, positively regulates the botulinum neurotoxin gene expression. Inactivation of codY resulted in decreased expression of botA, encoding the neurotoxin, as well as in reduced neurotoxin synthesis. Complementation of the codY mutation in trans rescued neurotoxin synthesis, and overexpression of codY in trans caused elevated neurotoxin production. Recombinant CodY was found to bind to a 30-bp region containing the botA transcription start site, suggesting regulation of the neurotoxin gene transcription through direct interaction. GTP enhanced the binding affinity of CodY to the botA promoter, suggesting that CodY-dependent neurotoxin regulation is associated with nutritional status. Clostridium botulinum is a Gram-positive, spore-forming anaerobic bacterium that produces botulinum neurotoxin, which is the most poisonous biological substance known to mankind. Botulinum neurotoxin blocks neurotransmission in cholinergic nerves (1, 2) in humans and animals to cause botulism, a potentially lethal flaccid paralysis. Despite its extreme toxicity, botulinum neurotoxin is widely utilized as a powerful therapeutic agent to treat numerous neurological disorders (3, 4).Seven antigenically distinct botulinum neurotoxin types (A to G), and several subtypes therein, have been identified (5-9). Moreover, a novel toxin type H was recently proposed (10) and awaits further characterization (11). Type A1 neurotoxin is well characterized as a consequence both of its frequent involvement in human botulism worldwide and of its use as a therapeutic agent (12). Type A1 neurotoxin is produced as a complex containing the neurotoxin itself and associated nontoxic proteins (ANTPs) that comprise a nontoxic nonhemagglutinin protein (NTNH) and three hemagglutinin proteins (HAs; HA17, HA33, and HA70) (13-15). The NTNH protects the neurotoxin from low pH-and protease-induced inactivation in the gastrointestinal tract (16), while the HAs assist the neurotoxin absorption, probably by interacting with oligosaccharides and E-cadherin on intestinal epithelial cells (17).In C. botulinum type A1, the genes encoding the neurotoxin (botA) and ANTPs (ntnh, ha17, ha33, ha70) are located in a gene cluster and are organized in two operons, namely, the ntnh-botA and ha operons (18). Within the neurotoxin gene cluster, botR, located between the two operons, encodes an alternative sigma factor that is a member of group 5 of the sigma 70 family, including Clostridium difficile TcdR, Clostridium perfringens UviA, and Clostridium tetani TetR. BotR directly controls the transcription of both the ntnh-botA and ha operons (19,20). An Agr-like quorum sensing system was found to be involved in positive regulation of the neurotoxin production (21), suggesting that the cell density-dependent signals control neurotoxin production. Also, the CLC_10...

In October 2011 in Finland, two persons fell ill with symptoms compatible with botulism after having eaten conserved olives stuffed with almonds. One of these two died. Clostridium botulinum type B and its neurotoxin were detected in the implicated olives by PCR and mouse bioassay, respectively. The olives were traced back to an Italian manufacturer and withdrawn from the market. The public and other European countries were informed through media and Europe-wide notifications.

bThe role of the two-component system (TCS) CBO0366/CBO0365 in the cold shock response and growth of the mesophilic Clostridium botulinum ATCC 3502 at 15°C was demonstrated by induced expression of the TCS genes upon cold shock and impaired growth of the TCS mutants at 15°C.