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

Abstract

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.

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

“…Genomic DNA from the ATCC 3502 wild-type strain and codY mutant and the pMTL007C-E2 plasmid DNA were digested overnight with HindIII (New England BioLabs) and subjected to Southern blot analysis with the Ll.LtrB-derived intron-specific probe as previously described (23).…”

“…DNase I footprinting was performed in multiple replicates using a modification described in reference 52. The PntnhbotA probe was amplified using 6-carboxyfluorescein (FAM)-labeled for-ward primer and HEX-labeled reverse primer as previously described (23). Binding reactions were performed as described for EMSA except that 2 mM GTP, 10 nM 5=-FAM-labeled probe, and 5 M the CodY protein were used.…”

“…To investigate whether CodY regulates the transcription of the neurotoxin gene cluster directly, an EMSA was performed to examine the binding of recombinant CodY to a probe encompassing the upstream region of ntnh containing the promoter of the ntnh-botA operon (Pntnh-botA probe) (23). The presence of an increasing concentration of CodY caused a shift in the mobility of the Pntnh-botA probe (Fig.…”

“…Also, the CLC_1093/CLC_1094, CLC_1914/CLC_1913, and CLC_0661/ CLC_0663 two-component signal transduction systems (TCSs) were proposed to positively regulate the neurotoxin synthesis (22). The first report on negative regulation of neurotoxin synthesis demonstrated the CBO0787/CBO0786 TCS to repress neurotoxin synthesis by the CBO0786 response regulator directly binding to the conserved Ϫ10 site of the core promoter of ntnh-botA and ha operons and blocking BotR-directed transcription (23).…”

The CLO3403/CLO3404 Two-Component System of Clostridium botulinum E1 Beluga Is Important for Cold Shock Response and Growth at Low Temperatures

“…Primer Sequence (5=¡3=) Use (45,46). The resulting pMTL82151::clo3404-clo3401 was sequenced, transformed into E. coli CA434, and further conjugated into the mutants and the WT strain.…”