BotR/A and TetR are alternative RNA polymerase sigma factors controlling the expression of the neurotoxin and associated protein genes in Clostridium botulinum type A and Clostridium tetani

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Abstract
Summary: Clostridium botulinum and Clostridium tetani, respectively, produce potent toxins, botulinum neurotoxin (BoNT) and tetanus neurotoxin (TeTx), which are responsible for severe diseases, botulism and tetanus. Neurotoxin synthesis is a regulated process in Clostridium. The genes botR/A in C. botulinum A and tetR in C. tetani positively regulate expression of BoNT/A and associated non‐toxic proteins (ANTPs), as well as TeTx respectively. The botR/A gene lies in close vicinity of the two operons which contain bont/A and antps genes in C. botulinum A, and tetR immediately precedes the tetX gene in C. tetani. We show that BotR/A and TetR function as specific alternative sigma factors rather than positive regulators based on the following results: (i) BotR/A and TetR associated with target DNAs only in the presence of the RNA polymerase core enzyme (Core), (ii) BotR/A and TetR directly bound with the core enzyme, (iii) BotR/A‐Core recognized −35 and −10 regions of ntnh‐bont/A promoter and (iv) BotR/A and TetR triggered in vitro transcription from the target promoters. In C. botulinum A, bont/A and antps genes are transcribed as bi‐ and tricistronic operons controlled by BotR/A. BotR/A and TetR are seemingly related to a new subgroup of the σ70 family that includes TcdR and UviA, which, respectively, regulate production of toxins A and B in C. difficile and bacteriocin in C. perfringens. Sequences of −35 region are highly conserved in the promoter of target toxin genes in C. botulinum, C. tetani, C. difficile and C. perfringens. Overall, a common regulation mechanism probably controls toxin gene expression in these four toxigenic clostridial species.